From genetic constraints to process hurdles: A review of Kluyveromyces marxianus for efficient bio-manufacturing and bio-processing in food industry

Abstract

Kluyveromyces marxianus (K. marxianus) has emerged as a prominent microbial platform owing to its exceptional intrinsic properties, demonstrating significant potential for biomanufacturing. A literature search with the keyword ‘Kluyveromyces marxianus’ in title in the PubMed database indicated that the yeast has been developed as a microbial cell factory for lignocellulosic biomass valorization, heterologous protein production, flavor and fragrance molecule synthesis during the past 10 years. However, the most recent review of K. marxianus application in food industry was 5 years ago. The relatively slow progression is partially due to the paucity of molecular tools and the lagging advancements in process optimization strategies. This review summarizes the physiological characteristics of K. marxianus and its potential industrial applications. Addressing the requirements of industrial-scale production, key limiting factors in high-cell-density fermentation, including carbon source utilization efficiency, dissolved oxygen regulation, and inhibition by metabolic byproducts were analyzed. An integrated approach combining dynamic feeding, metabolic pathway engineering, and fermentation parameter optimization was proposed. Considering the previous reviews, the present work updated the application of K. marxianus in the food industry, including the production of enzymes, sugars, aromatic compounds, single-cell protein, and transformation of food waste, as well as the treatment of wastewater during food processing, and aims to make a comprehensive discussion. Through the systematic construction of the research paradigm from molecular constraints to process hurdles, the work may help the transition of K. marxianus for efficient manufacturing and processing in the food industry.

Graphical abstract

Highlights

• •The specific advantages of K. marxianus are presented.
• •The high-density fermentation of K. marxianus and optimization strategies are summarized.
• •Biological toolkits for constructing K. marxianus cell factories are proposed.
• •Emerging applications of K. marxianus in food are summarized.

1. Introduction

With the continuous development of biotechnology in the direction of sustainability and environmental friendliness, microbial chassis cells have been widely used in the food, medical, agricultural, and chemical industries due to the advantages of a high metabolic rate, simple process control, and easy large-scale production (Komera et al., 2023). As prototypical eukaryotic biofactories, Saccharomyces cerevisiae and Pichia pastoris have developed well in the green manufacturing of natural products and biofuels. However, due to its growth in the medium temperature range of about 30 °C, relatively narrow substrate spectrum, and preferential fermentation metabolism, S. cerevisiae has a high cost in large-scale high-cell-density fermentation production, which limits its application in industrial production (Pereira et al., 2021). Pichia pastoris is a better chassis for protein expression, but with codon bias and refolding may be required. When the codon usage preference of a foreign gene does not match the host endogenous tRNA library, it may lead to blocked translational extension, misfolded protein accumulation, and the formation of insoluble aggregates, which significantly increases production complexity. (Kulagina et al., 2021). Using molecular biology techniques to develop new eukaryotic chassis cells suitable for industrial recombinant protein production has become a new trend in the field of research. Kluyveromyces marxianus is a food-grade heat-tolerant yeast that is one of the most potentially valuable candidates strains due to its diverse substrate utilization, high-cell-density aerobic fermentation capabilities, and broad tolerance to temperature and pH variations (Karim et al., 2020; Zhang et al., 2023). In recent years, it has developed into a hot topic in the field of microbial synthetic biology research and has broad application prospects in the fields of food, feed, and agricultural waste utilization (Karim et al., 2020; Qiu et al., 2023).

K. marxianus has many potential advantages, including the inherent ability and heat resistance of fermented inulin, and as well as weak glucose inhibition of sucrose, raffinose, and inulin (Wang et al., 2023). Unlike S. cerevisiae, the majority of K. marxianus strains exhibit Crabtree-negative behavior, which prioritizes aerobic respiration over aerobic alcoholic fermentation. This property is a significant advantage in aerobic culture processes where biomass or target products such as heterologous proteins need to be maximized, as it effectively avoids the accumulation of ethanol as a competitive byproduct (Dekker et al., 2021). In addition, due to its special physiological properties and the ability to yield heterologous proteins, K. marxianus can utilize a diverse array of inexpensive raw materials, including cheese whey and byproducts from the dairy industry (Drezek et al., 2023; Gombert et al., 2016). Currently, numerous applications of K. marxianus have been realized at both laboratory and industrial scales. These applications include the synthesis of diverse heterologous proteins, aromatic compounds, and biological components; the reduction of lactose content in food products; the production of ethanol under anaerobic or oxygen-limited conditions or single-cell proteins; as well as bioremediation efforts (Koukoumaki et al., 2024; Martínez et al., 2018; Zhang et al., 2023). Despite its tendency to respiration metabolism under aerobic conditions, K. marxianus demonstrates superior ethanol production capabilities under anaerobic or microanaerobic conditions. Its remarkable capacity for ethanol production combined with heat tolerance has contributed to advancements in ethanol high-temperature fermentation (HTF) and simultaneous saccharification and fermentation (SSF) technologies (Nurcholis et al., 2020).

It has become commonplace to use techniques in synthetic biology and approaches in metabolic engineering to maximize the biomanufacturing of yeast chassis cells. With the completion of genome sequences, versatile genetic toolkits, including promoters, terminators, resistance markers and reporter genes, have been developed (Lang et al., 2020; Lee et al., 2013; Lertwattanasakul et al., 2015). The improvement of efficient methods for transformation and gene editing, encompassing systems for gene inactivation and overexpression, has also expedited the advancement of K. marxianus (Nurcholis et al., 2020). Therefore, it is very timely and meaningful to summarize the fermentation strategy, engineering transformation and progress in the industrial production of K. marxianus to provide the latest research status in this field. In this paper, the physiological characteristics and potential industrial applications of K. marxianus are reviewed. Aiming at the requirements of industrial production, the key limiting factors such as carbon source utilization efficiency, dissolved oxygen regulation, and metabolic by-products inhibition in high-cell-density fermentation processes were analyzed. Moreover, the present work proposes a comprehensive solution based on a dynamic feeding strategy, metabolic pathway reconstruction, and fermentation parameter optimization. At the same time, the application breakthroughs of new gene editing tools such as CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-Associated Protein 9) in the genetic transformation of the strain were summarized, which effectively improved the genetic plasticity and environmental adaptability of the strain. In the field of food application, its industrialization prospects as a new type of fermentation strain in natural enzyme synthesis, prebiotic preparation, functional protein development, and food waste conversion were discussed. Through the systematic construction of the research paradigm from molecular mechanism analysis to industrial scene application, it builds a bridge between basic research and industrial transformation for the efficient biological manufacturing using K. marxianus in the food industry.

2. Basic characteristics of K. marxianus

2.1. Strong growth capacity under higher temperature

The ability of microorganisms to cope with environmental changes that are not conducive to their own growth is one of the key factors to be considered in industrial production. Kluyveromyces marxianus can resist high-temperature environments. As a potential industrial species, high-temperature fermentation gives heat-resistant yeast many advantages. At first, using K. marxianus has the potential to lower expenses associated with cooling, distillation, and separation processes in the production process due to its high temperature resistance (Zhang et al., 2016). Secondly, higher temperatures improve the catalytic efficiency of enzymes (such as cellulase and hemicellulose), which often need to be added in fermentation, to completely decompose the substrate into monosaccharides that can be directly utilized. The optimal temperature of these commercial enzymes is often 48–52 °C, and at the fermentation temperature of 50 °C in K. marxianus, these enzymes can effectively play a role, increase the fermentation speed and shorten the fermentation time (De Brabander et al., 2023; Sharma et al., 2016). Thirdly, the high temperature can inhibit the growth of most microorganisms and reduce contamination from other bacteria during the fermentation process.

Unlike S. cerevisiae (optimum ∼30 °C) and P. pastoris (optimum ∼28–30 °C), the fermentation conditions of K. marxianus up to 52 °C are one of its most significant advantages. This thermotolerant trait may be attributed to the elevated levels of trehalose present in its cells, which confer a degree of protection against heat shock damage (Mejia-Barajas et al., 2017). Thermotolerance endows K. marxianus with unique advantages for co-cultivation. During the simultaneous saccharification and fermentation (SSF) process, the optimal temperatures for growth and activity of many hydrolases (e.g., cellulase) are highly matching, thereby improving the overall efficiency (Nurcholis et al., 2020). During glucose utilization under aerobic conditions at 40 °C, the growth rate of K. marxianus is still as high as 0.86–0.99/h. Transcriptome analysis of K. marxianus at 45 °C revealed that genes associated with the pentose phosphate pathway (PPP) were markedly increased. The PPP serves as the primary source of NADPH. This suggests that high levels of NADPH were positively correlated with the antioxidant capacity of cells, which also explained the reason for the ability of K. marxianus to withstand high temperatures (Gao et al., 2015; Lertwattanasakul et al., 2015). Untargeted metabolomics research has demonstrated that K. marxianus yeast is capable of adapting to elevated temperatures by extending the average length of its fatty acid chains. High temperatures induce the biosynthesis of purine, pyridoxine, and riboflavin, among other metabolic pathways. The induction of oxidative stress and the subsequent antioxidant responses are crucial for the adaptation of thermotolerant yeast to higher ambient temperatures (Li et al., 2021, Li et al., 2021).

2.2. High growth rate

Kluyveromyces marxianus is recognized as one of the most rapidly proliferating eukaryotic organisms documented, exhibiting a peak growth rate of 0.99/h at 40 °C (Groeneveld et al., 2009; Rocha et al., 2011) and a cell density of 120 g/L during fermentation (Yang et al., 2021) (Table 1). In industrial production, only a certain number of organisms can achieve high-cell-density fermentation, thereby increasing the content of fermentation products. The growth rate of cells determines the time to achieve high-cell-density production (Dubencovs et al., 2021). An increased rate of cell proliferation and a reduced fermentation duration can reduce the fermentation cost and better meet the needs of industrial production. K. marxianus is considered not only to have the shortest doubling time (52 min), but also to be one of the fastest growing eukaryotes. This may shorten the time required for genetic or evolutionary engineering, which is of great interest for strain construction and improvement (Gombert et al., 2016; Groeneveld et al., 2009). Moreover, compared to many conventional microorganisms such as S. cerevisiae, the thermotolerant nature and rapid growth rate of K. marxianus typically lead to reduced cooling energy demands during fermentation. This decrease in energy consumption is directly linked to lower greenhouse gas emissions in the production phase, which constitutes a critical factor in life cycle assessment affecting environmental footprints such as the global warming potential.

Table 1.

Summary of the growth performance of different K. marxianus under various culture conditions.

Strains	Carbon source (g/L)	pH	Temperature (°C)	Rotation speed (rpm)	Volume (L)	Fermentation method	Specific growth rate (/h)	Biomass dry cell weight (g/L)	References
K. marxianus CCT 7735 (UFV-3)	Glucose		37	180	0.25	Batch fermentation	0.673		Tinôco and Silveira (2021)
K. marxianus CBS 6556	Glucose	5.5	42	600	2	Batch fermentation	0.5		Longhi et al. (2004)
K. marxianus Fim-1ΔURA3	Glucose	5.5	30	220	5	Fed-batch fermentation		120	Yang et al. (2021)
K. marxianus DSM 5422	Glucose	5.5	30	800	5.4	Fed-batch fermentation		70	Dubencovs et al. (2021)
K. marxianus DSM 5422	Glucose	5	30	800	3.6	Fed-batch fermentation	0.5		Dabros et al. (2010)
K. marxianus MTCC 4139	Glucose	5	30	150	0.5	Fed-batch fermentation	0.2	5	Leelaram et al. (2016)
K. marxianus CBS 6556	Glucose	5	37	700	4	Fed-batch fermentation	0.49		Fonseca et al. (2013)
K. marxianus CBS 6556	Glucose	4.5	40	650	0.5	Batch fermentation		4.5	Hoekstra et al. (1994)
K. marxianus CBS 6556	Glucose	4.5	40	800	2	Batch fermentation	0.6	2	Groeneveld et al. (2009)
K. marxianus KM-526	Glucose	4.5	28	450	10	Fed-batch fermentation		108	Zhang et al. (2019)
K. marxianus IWBT Y885	Glucose, fructose	5.5	25	120	0.25	Fed-batch fermentation	0.25		Labuschagne et al. (2021)
K. marxianus MTCC 5933	Glucose, xylose	5.5	45	150	0.5	Fed-batch fermentation	0.24	5	Sharma et al. (2016)
K. marxianus IMB4	Glucose, molasses	5.5	40	250	0.5	Batch fermentation	0.99		Banat et al. (1992)
K. marxianus 6C17	Galactose	5	37	150	3	Batch fermentation	0.37	3.82	Beniwal et al. (2017)
K. marxianus ATCC 10022	Sucrose	5	30	300	5	Batch fermentation	0.27	5.5	Cazetta et al. (2010)
K. marxianus ATCC 16045	Sucrose	4.5	30	300	2	Fed-batch fermentation		18.17	Manera et al. (2008)
K. marxianus MTCC1288	Lactose	4.5	34	500		Batch fermentation	0.157		Zafar and Owais (2005)
K. marxianus FII 510700	Lactose	5	30	180	5	Fed-batch fermentation	0.27	105	Lukondeh et al. (2005)
K. marxianus KMS2	Cheese whey		37	200		Batch fermentation	0.34	5.5	Júnior et al. (2001)
K. marxianus NBRC1777	Xylose		42	450	2.5	Batch fermentation		30(OD600)	Zhang et al. (2020)
K. marxianus NIRE-K1	Xylose		45	150	0.2	Batch fermentation	0.225	4.21	Sharma et al. (2016)

2.3. Broad-spectrum substrates

In addition to high temperature tolerance, strong stress resistance, and high growth ability, the broad substrate profiles are also one of the advantages of K. marxianus. Unlike S. cerevisiae, which can only efficiently utilize six-carbon sugars such as glucose and sucrose, K. marxianus can naturally utilize lactose (the main component of whey) and inulin (plant sources such as Jerusalem artichoke), and some strains can also use xylose. Furthermore, its capability to efficiently utilize a variety of low-cost and abundant carbon sources, including whey, lignocellulosic hydrolysates, and food processing by-products, not only reduces production costs but also realizes value-added use of waste at the beginning of the life cycle, reducing the environmental burden caused by traditional waste disposal methods, and aligns with the principles of a circular economy (Zhang et al., 2024). Due to the expression of β-galactosidase, K. marxianus can use lactose as a sole carbon source. The ability of K. marxianus to convert lactose from different types of whey into ethanol also provides a new direction to utilize whey byproducts in the food sector for the use of whey waste in the food industry (Ohstrom et al., 2023). With cellulose, lignocellulose, and inulin as substrates, K. marxianus possesses the potential to generate glycerol and glycerol derivatives. For example, when fermented at 42 °C, 40.32, 41.84, and 18.64 g/L glycerol with yields of 0.84, 0.50, and 0.22 g/L/h and no by-product formation were achieved when 80 g/L glucose, fructose and xylose were used (Zhang et al., 2020a, Zhang et al., 2020b). This indicates that K. marxianus can produce high-added products using cheap lignocellulosic biomass. K. marxianus is also considered to be a good protein expression host and has been used for the production of different enzymes, like β-galactosidase, inulinase, pectinase, and lipase (Mao et al., 2019; Silva et al., 2015). At the same time, K. marxianus is a biosafe microorganism; it has been widely used in both food and pharmaceutical industries (Fig. 1).

Fig. 1.

Primary sources, biological characteristics, and optimization strategies of K. marxianus. (1) Strain selection and metabolism optimization: e.g., selecting thermotolerant, osmotolerant, or ethanol stress-resistant strains through adaptive laboratory evolution (ALE) or CRISPR-based genome editing; (2) Media & process: e.g., selecting low-cost carbon sources and optimizing the carbon-to-nitrogen (C/N) ratio via mixed organic/inorganic nitrogen supplementation; maintaining low residual sugars with exponential feeding or feedback control to avoid substrate inhibition and ethanol overproduction. (3) Metabolic regulation: overexpress TCA cycle enzymes (e.g., isocitrate dehydrogenase) and optimize respiratory chain components (e.g., coenzyme Q synthase) to boost oxidative phosphorylation efficiency. (4) Scale-up model optimization: maintain DO >30 % using oxygen-enriched ventilation and microbubble dispersion to enhance oxygen transfer. Retain highly active cells via hollow fiber membranes or centrifugal recycling, coupled with continuous fresh medium feeding and in situ product separation.

2.4. Intense resistance to stresses during fermentation

K. marxianus generally shows good tolerance to stress conditions in a variety of industrial settings. For example, it tolerates lactic acid better than S. cerevisiae, which is essential for downstream recovery during lactic acid production (Gosalawit et al., 2023). In terms of ethanol tolerance, it is generally inferior to S. cerevisiae, which has been domesticated for thousands of years. Pichia yeast has good adaptability to high osmolality (e.g., high concentration of glycerol/methanol) (Soyaslan Calik., 2011). Excellent stress resistance enhances the robustness of the co-culture system. In the production of organic acids, K. marxianus can be used as a tolerance carrier to co-culture high-acid-producing strains to maintain the stability of the system in low pH and high-acid environments and improve the final yield (Gosalawit et al., 2023; Stergiou et al., 2012). When used to produce recombinant proteins, yeast undergoes emergency reactions similar to those at high temperatures, e.g., the thermostable mutant strain CYR1N1546K of K. marxianus reduced adenylate cyclase activity and cAMP production. RNA-seq analysis showed that the decrease in cAMP levels induced by the CYR1 mutation could stimulate cells to improve their energy supply system, optimize material synthesis and enhance stress resistance (Ren et al., 2024). As a substrate used for fermentation, lignocellulosic biomass produces several toxic inhibitors during pretreatment, including weak acids, furans, and phenolic compounds. The KmYME protein, located in the mitochondrial matrix, can improve the tolerance of K. marxianus to inhibitors such as acetic acid and furfural. In addition, it was reported that K. marxianus improves its resistance to stresses such as ethanol, temperature, and osmolality by upregulating the expression of transcriptional levels. It was found that this inhibition is mainly achieved by disrupting the intracellular CoASH reservoir to diminish the synthesis of NAD(P)+, NAD(P)H, and ATP (Wu et al., 2020). Moreover, tolerance to inhibitors can be significantly enhanced by the overexpression of a transcriptional regulator KmMsr in K. marxianus, and this overexpression also enhances tolerance to high temperatures, ethanol, and high concentrations of NaCl and glucose (Zhang et al., 2024).

Alongside the aforementioned strategies for rational modification aimed at specific genes, techniques like adaptive laboratory evolution (ALE) and chemical mutagenesis serve as effective approaches to enhance the overall stress resistance of K. marxianus. Through direct evolution or random mutagenesis, in conjunction with high-throughput screening in simulated industrial fermentation stress conditions (including high inhibitor concentration, elevated temperatures, and high osmosis), mutant strains exhibiting markedly improved tolerance to multiple stresses can be rapidly acquired (de Lima et al., 2021; Mo et al., 2019; Ren et al., 2022). Following a period of 100 days of adaptive evolution of K. marxianus in the presence of 6 % (v/v) ethanol, the organism's tolerance to ethanol was observed to rise to 10 % (v/v). This increase was not attributed to changes in ploidy or significant mutations, but rather to a comprehensive reprogramming of transcription across the genome. Furthermore, cell viability assessments have demonstrated that the evolved strain KM-100d may exhibit resistance to additional stressors, including heat, osmotic pressure, and oxidative conditions (Mo et al., 2019). Compared with native strains, adapted strains exhibit a threefold increase in xylose uptake rate and achieve significant reduction in lag phase duration (Sharma et al., 2016). On the medium without the addition of L-phenylalanine (L-Phe), the strains resistant to p-fluoro-DL-phenylalanine (PFP) were screened by ultraviolet (UV) irradiation, among which the Km_PFP41 mutant strain had the highest yield of 2-phenylethanol (2-PE), and its DAHP synthase activity was higher than that of the parent Kluyveromyces marxianus CCT 7735 strain (de Lima et al., 2021). Compared with transgenic strains, the core advantage of adaptive strains is that they achieve non-GMO (Non-Genetically Modified Organism) phenotypic optimization through spontaneous mutations in the endogenous genome, circumventing strict transgenic regulatory hurdles. Under long-term environmental stress domestication, the strains could simultaneously enhance multi-pathway synergistic adaptability (such as stress tolerance, substrate utilization and product secretion) and obtain genetic background stability (Fiedurek et al., 2011; Sharma et al., 2016). This systematic evolution strategy is particularly suitable for biomanufacturing scenarios that need to cope with complex fermentation environments and are subject to regulatory restrictions, which not only accelerates the development of highly tolerant industrial strains, but also improves cell activity, fermentation process stability, and target product yield in high-cell-density fermentation (Ren et al., 2022; Sharma et al., 2016).

3. High-cell-density fermentation of K. marxianus and its improvement strategy

High-cell-density fermentation represents a crucial and well-established process strategy within the realm of industrial biotechnology. The primary objective is to enhance the accumulation of microbial or cell culture biomass within a constrained bioreactor volume by means of meticulous process control and optimization, consequently leading to a substantial increase in yield per unit volume and the efficiency of target product synthesis (Cheng et al., 2025; Dubencovs et al., 2021). Compared with traditional fermentation processes, high-cell-density fermentation breaks through multiple physiological and engineering constraints such as nutrient substrate limitations, dissolved oxygen transport bottlenecks, inhibition of harmful metabolic by-products (such as acetic acid and lactic acid), and osmotic stress, which are commonly faced during cell growth, representing the advanced level of biological process enhancement technology (Zhang et al., 2024). While high-cell-density fermentation can successfully address numerous constraints, its effective application in K. marxianus is significantly dependent on precise regulation of the culture conditions (e.g., dissolved oxygen, pH, and temperature) and the employment of sophisticated feeding methods (e.g., continuous feeding and batch fermentation) that can satisfy their elevated metabolic requirements (Bilal et al., 2022; Leelaram et al., 2016; Tomas-Pejo et al., 2009).

3.1. The dissolved oxygen

The concentration of dissolved oxygen has been identified as the most influential parameter that impacts the growth and target product accumulation of K. marxianus. For aerobic microorganisms, anaerobic conditions can lead to a decrease in biomass and product yield, as well as the formation of by-products (Dekker et al., 2021). Under aerobic conditions, Kluyveromyces species can participate in the fermentation of galactose, xylose, maltose among others. Without anaerobic alcoholic fermentation, Kluyveromyces species can produce many compounds with high added value. This phenomenon is known as the Kluyver effect, and this “respiration-dependent” species is called Kluyver effect positive. This effect is usually defined as the inability of yeast to ferment certain disaccharides (or other specific sugars) under respiratory defect conditions, but to ferment their corresponding constituent monosaccharides. Researchers have found that this “respiratory dependent” positive species can be converted to negative by introducing related sugar transporter genes (Fukuhara, 2003).

The majority of K. marxianus strains exhibit clear Crabtree-negative traits or possess characteristics of aerobic respiration, making them unlikely to engage in alcoholic fermentation. This phenotype is anticipated for large-scale biosynthetic processes linked to biomass production, as the generation of ethanol, an undesirable by-product, can be circumvented in aerobic environments (Bilal et al., 2022; Dekker et al., 2021). Transcriptomics and metabolomics analysis of K. marxianus under different oxygen restriction conditions showed that the lack of a functional sterol-uptake mechanism was a key factor in the aerobic process of K. marxianus. The results showed that the heterologous expression of a squalene-tetrahymanol cyclase could restore the anaerobic growth of K. marxianus on glucose at 45 °C, which expanded the fermentation of heat-tolerant yeast strains in anaerobic environments (Dekker et al., 2021).

K. marxianus is generally sensitive to anaerobic conditions. It was found that K. marxianus acetyl-CoA biosynthesis and the circulating flux of reduced and oxidized Tricarboxylic Acid Cycle (TCA) were significantly enhanced, while the hexokinase activity decreased in an aerobic state. These effects resulted in a significant reduction of ethanol and acetic acid production during fermentation, highlighting variations in the essential metabolic profiles of yeast engaged in respiration versus fermentation (Sakihama et al., 2019). In fact, the appropriate amount of dissolved oxygen will greatly increase the yield of microbial biomass and target products, and save production costs, during the production of industrial enzymes and recombinant proteins. However, oxygen fluctuations often occur during high-cell-density fermentation, and the whole fermentation changes from hyper-oxygen to an anaerobic environment. When yeast is exposed to a low-oxygen environment, the biomass reduces and metabolic by-products accumulate, resulting in a significant reduction in the performance of large-scale fermentation. Studies have evaluated the effect of dissolved oxygen on yeast biomass, e.g., the amount of dissolved oxygen between 0 and 20 % results in a significant increase in the specific activity of lactase compared to a constant amount of dissolved oxygen in the same range, without affecting the maximum biomass of K. marxianus (Cortes et al., 2004). The optimization of the fermentation process of inulinase production in K. marxianus found that changing the airflow rate and stirring rate could affect the oxygen transfer rate, thereby affecting the viability and protein expression level of yeast cells. At the stirring rate ≤100 rpm, the oxygen transfer rate was small, and the inulinase was increased by about 2 times at the airflow rate of 1 lpm and the stirring rate of 150 rpm and finally reached 20.96 IU/mL (Santharam et al., 2019). During industrial fermentation, the growth rate of microorganisms can be controlled by adjusting the amount of dissolved oxygen, which in turn can regulate the production of recombinant proteins. Limiting the feed rate of the substrate allows yeast cells to grow at a lower growth rate, resulting in lower oxygen and energy requirements, and minimizing cell rupture during fermentation (Cortes et al., 2004; Dekker et al., 2021). These results lead to a significant reduction in the various lytic enzymes released into the matrix, resulting in a large accumulation of target proteins.

3.2. pH

The pH level of the medium serves as a crucial regulatory factor throughout the fermentation process, greatly influencing the yeast growth rate, the stability of target proteins, and the catalytic efficiency of essential enzymes. Consequently, the determination of the ideal pH for fermentation is not set in stone but should be established in close alignment with the traits of the particular host strain and the intended outcome of the final application, whether it be the aim for high biomass or the production of specific metabolites. In high-cell-density fermentation, it is necessary to introduce significant amounts of acids or bases to sustain microbial growth at the optimal pH. Studies have shown that the optimal pH range varies for different target products. For example, when the goal is to maximize biomass accumulation, K. marxianus achieved high-density growth over a wide pH range (3.5–5.0) (Gosalawit et al., 2023). While when the goal is to optimize ethanol production, a higher ethanol concentration (12.98 g/L) was observed at pH 5.5 (López-Domínguez et al., 2019). This clearly indicates that the fermentation pH must be optimized in accordance with the specific aims of fermentation. In general, low pH conditions lead to the inactivation of proteases and reduced proteolytic activity. In the optimal range for the growth of microorganisms, the fermentation process can be improved by optimizing the pH value to reduce the hydrolysis of the protein. After optimization, the yield of lipase produced by K. marxianus increased by 65 times after fermentation for 65 h at pH 6.5 (Stergiou et al., 2012). Growth conditions at pH 6 increased the expression of recombinant lignin peroxidase in P. pastoris by more than 10-fold compared to standard growth conditions (Biko et al., 2023). The concentration of recombinant erythropoietin produced by P. pastoris yeast at pH 4.5 increased by 50 % compared to pH 5.0 (Soyaslan and Calik, 2011).

3.3. Temperature

K. marxianus demonstrates an ability to grow at temperatures reaching or exceeding 50 °C. This feature can be used to synthesize heterologous proteins in non-sterile bioprocessing, reducing production costs. For example, when the temperature is raised from 37 °C to 42 °C, the production of xylitol by K. marxianus through the synergistic use of glucose and xylose at high temperatures increases xylitol yield by 30 % and unit productivity by 70 % (Zhang et al., 2020a, Zhang et al., 2020b). K. marxianus DMKU 3–1042 has been repeatedly cultured for a long time, with a gradual increase in temperature, enhanced resistance to ethanol, furfural, hydroxymethylfurfural and vanillin, and shows higher ethanol yields in media containing 16 % glucose at elevated temperatures and can even grow at 48 °C (Zhang et al., 2023).

Extremely high temperatures may reduce cell viability and lead to the accumulation of acetate, as yeasts tend to accumulate more reactive oxygen species (ROS) in addition to temperature stress, thus damaging the cells. A combination of multiple stresses, such as acetic acid, elevated temperature, ROS, ethanol, and other by-products, is thought to affect viability (Pattanakittivorakul et al., 2022). In addition, high temperatures lead to reduced protease activity during the expression of recombinant proteins. Studies have reported a 50 % reduction in the concentration of ergosterol produced by K. marxianus when the temperature increased from 25 to 30 °C. When K. marxianus IF 00288 was used for the production of extracellular α-amylase, it was found that this yeast strain produced α-amylase at temperatures ranging from 20 to 40 °C and exhibited maximum α-amylase yield and cell growth at 30 °C, while at temperatures above 40 °C, the extracellular amylolysis activity was almost completely lost (Stergiou et al., 2014). Therefore, it is necessary to balance temperature during the fermentation process to achieve both high-cell-density fermentation and maximum yield of the target protein as soon as possible.

3.4. Substrate feeding strategy

3.4.1. Continuous cultivation

Continuous cultivation can improve the industrial efficiency of the fermentation process and reduce production costs. This method provides a very promising strategy for the industrial production of recombinant proteins in yeast, as it is highly self-controlled and easy to automate the operation with a variety of instruments. In addition, the product quality is stable, and the fermentation process is efficient. Because it simplifies the process of loading, cleaning and sterilization, the production time is shortened, the utilization rate of the equipment is increased, and the cost competitiveness of the product is improved. For example, recent studies have shown that continuous culture can increase the ability of K. marxianus to produce 2-phenylethanol by 60 % compared to the traditional fed-batch mode (Drezek et al., 2021). Furthermore, continuous culture technology can significantly increase the potential of K. marxianus to produce single-cell protein, and the protein recovery through precipitation has been found to increase protein productivity by 45 % and the chemical oxygen demand removal rate from 80 % to 93 % (Yadav et al., 2014a). However, microorganisms are prone to degradation and may undergo a certain degree of mutation during long-term consecutive replication, so it is necessary to ensure that the strains before fermentation have a high degree of genetic stability, which is a major challenge for continuous culture technology (Fig. 2). Besides, the utilization rate of nutrients in continuous culture is lower than that of batch culture and fed-batch culture, and the requirements for an aseptic environment in the fermentation process are higher, facing a high potential for miscellaneous bacterial contamination. Research on the production of recombinant proteins in yeast with continuous fermentation cultures is still limited. The lack of such a process is possible due to its immaturity in practice and relatively insufficient development (De Brabander et al., 2023).

Fig. 2.

The key regulation processes and strategies of high-cell-density fermentation of K. marxianus. First, mutant strains with enhanced tolerance are selected under stress conditions such as elevated temperature and high osmotic pressure to improve the stability of microbial cultures in industrial production. Secondly, dynamic adjustment of C/N ratios and phased temperature regulation combined with low pH stress to activate secondary metabolic pathways. Thirdly, control fermentation with process intensification and intelligent feedback. Apply the Monod and Luedeking-Piret models for feeding, and engineer metabolism (e.g., NAD⁺/NADH, ATP) to synchronize glycolysis with product synthesis. Finally, the challenges and solutions faced by K. marxianus yeast in achieving high-cell-density fermentation in the current research are highlighted. UV, Ultraviolet.

3.4.2. Fed-batch cultivation

To obtain high biomass and product yield, the fed-batch strategy is supplemented. The results of high-cell-density culture of K. marxianus with cheese whey as the basal medium indicated that the ongoing supplementation of lactose to the culture could enhance the activity of β-galactosidase by 50 % within 25 h (Rech et al., 2007). Using intermittent substrate-fed interruptions, during which the yeast is deprived of any carbon source, higher recombinant protein expression efficiency can be achieved by creating conditions of applied carbon starvation stress (Martinez-Avila et al., 2019). K. marxianus was cultured in batches on different concentrations of lactose medium to prolong lactose feeding, and a biomass of 105 g/L was achieved on lactose-based medium under intermittent fed conditions (Lukondeh et al., 2005). In order to achieve high-cell-density fermentation of K. marxianus and avoid the Crabtree effect, different fed-batch methods were used to maintain the glucose concentration at a low level, such as constant flow rate, intermittent feeding, and exponential rate addition. Feeding by constant concentration theoretically ensures the supply of glucose in the fermentation medium at a certain level, but the concentration of glucose is often not easy to determine in industrial fermentation. The biomass of K marxianus FII 510700 can reach 105 g/L in a fed-batch culture through prolonged lactose feeding (Lukondeh et al., 2005).

Controlling the feeding rate during the fermentation process can maintain the balance between the supply and demand of various substances, which can not only satisfy the ongoing requirements for microbial growth and product synthesis but also mitigates the potential regulatory responses triggered by excessive substrate, thereby maximizing enzyme yield (de et al., 2016). A study of inulinase production by fermentation of K. marxianus found that linear feeding increased the yield of inulinase by 17 times compared with batch feeding, and it was speculated that maintaining a specific growth rate was the main factor for maximizing enzyme yield. Further exponential feeding appears to be a better option, increasing inulinase activity by 50 % (Leelaram et al., 2016). Because the reaction matrix in the whole fermentation process is controlled at a low level, the generation of harmful substances is greatly reduced. The K. marxianus increased exponentially at a certain specific growth rate, which maximized the expression of exogenous proteins while making the K. marxianus grow stably. In real industrial fermentation, the concentration of restricted nutrients in fermentation can be adjusted through online monitoring and adjustment, and the feed supply in the fermentation process can be maintained at a certain rate, and the performance of yeast fermentation can be improved, which of course facilities the high-cell-density cultivation of K. marxianus.

3.5. Strategies and challenges of bioprocess scaling

Achieving high-cell-density fermentation of K. marxianus and subsequently scaling up the process to industrial levels are critical steps toward its commercial application. Although laboratory studies have provided valuable data on the optimization of dissolved oxygen, pH, temperature, and substrate feeding strategies, the scale-up process still faces several challenges, primarily including oxygen transfer efficiency, heat removal issues, and contamination control (Dekker et al., 2021). These factors often exhibit significantly different behaviors in large bioreactors than at the laboratory scale, directly affecting yeast growth, product synthesis, and the economic feasibility of the overall process (Bilal et al., 2022; Dekker et al., 2021). Therefore, systematically studying and solving engineering problems in the scale-up process is essential for efficient K. marxianus fermentation.

Oxygen transfer efficiency becomes a critical bottleneck during scale-up. In large-scale reactors, reduced mixing efficiency, increased hydrostatic pressure, and altered bubble behavior make it challenging to maintain a high volumetric oxygen transfer coefficient (kLa). This often results in dissolved oxygen (DO) limitations, thereby restricting the high-density aerobic growth of the yeast. To enhance oxygen transfer, integrated strategies are required, such as optimizing impeller configuration (e.g., combining Rushton turbines with pitched-blade impellers), increasing agitation speed, improving sparger design, and employing oxygen-enriched aeration or pressurized operation (De Brabander et al., 2023).

Kluyveromyces marxianus has vigorous growth and metabolism, high optimal temperature (usually >30 °C), and large heat production. However, the decrease in surface area/volume ratio in large-scale reactors makes the jacket heat transfer insufficient. This can easily cause local overheating, which may lead to enzyme inactivation, cell stress and by-product accumulation, therefore reduce the yield of biomass (Dekker et al., 2021). Therefore, the scale-up process requires the integration of efficient cooling systems, such as external heat exchangers or internal cooling coils, and the establishment of thermodynamic models to predict heat loads. At the same time, reducing heat generation by regulating the growth rate of bacteria (e.g., based on substrate feeding strategies) is also a feasible strategy.

The risk of contamination increases substantially with scale-up, particularly in the production of fermented foods, where a balance must be struck between economic feasibility and sterility assurance. This can be addressed through equipment design (e.g., incorporation of Cleaning in Place, CIP/Sterilization in Place, SIP systems), strict operational protocols (such as maintaining positive pressure and employing sterile inoculation/sampling techniques), and leveraging inherent strain properties (e.g., thermotolerance and acid tolerance) to exert selective environmental pressure against contaminants (Zhang et al., 2020, 2023). Bioprocess scale-up represents a central challenge in transitioning high-cell-density fermentation of K. marxianus from laboratory research to industrial application. By systematically addressing key issues such as oxygen transfer, heat removal, and contamination control through multidisciplinary engineering strategies, it is possible to develop efficient and scalable bioprocesses that provide robust technical support for yeast-based food production (De Brabander et al., 2023).

4. Genetic elements for K. marxianus chassis engineering

4.1. Counter-selectable markers

To further design and engineer K. marxianus and carry out genetic manipulation, appropriate screening markers must be selected, so that cells modified by gene knockout, insertion, and overexpression can be quickly screened. Currently, genes are associated with antibiotic resistance and auxotrophic labels are the most used screening markers in systems for genetic engineering of yeast. Specifically, the fungal antibiotics G418 and bleomycin are extensively utilized in K. marxianus (Bragança et al., 2015; Iborra, 1993). Due to the high cost of antibiotics in eukaryotes and their strong toxicity to animals and plants, the use of antibiotics in food and pharmaceutical related products is limited. Therefore, the application of auxotrophic screening markers has been extensively explored. The trophic markers TRP1 (Tryptophan biosynthesis gene 1), LEU2 (Leucine biosynthesis gene 2), and URA3 (Uracil biosynthesis gene 3), which are widely used in S. cerevisiae, are also ideal markers in K. marxianus for the screening of positive transformants (Yang et al., 2022; Zhang et al. 2023, 2024). Bartkeviciute et al. (2000) successfully expressed the endopolygalacturonase gene (EPG1) in K. marxianus yeast using the URA3 screening marker of yeast. (Bartkeviciute et al., 2000). Zhang et al. (2024) successfully constructed TRP1, LEU2, and URA3 auxotroph, and overexpressed transcriptional regulators to improve the ability of K. marxianus to simultaneously saccharification and co-ferment corncob (Zhang et al., 2024). Construction of multiple screening tags is thought to be useful. With multiple selectable markers, it is convenient for multi-gene knockout and indefinitely accelerates reconstructing metabolic pathways and the creation of super microbial cell factories.

4.2. Promoters and terminators

In the metabolic engineering of microorganisms, appropriate transcriptional regulatory elements can regulate the expression of various genes. As one of the effective regulatory elements, the promoter can achieve precise regulation of genes and provide a viable approach to enhance the production of exogenous products in K. marxianus. (Table 2). Promoters can be generally divided into constitutive promoters and inducible promoters. Endogenous promoters of K. marxianus, including P_KmINU1 (inulinase promoter), P_KmGAL1, P_KmTDH3, P_KmPGK1, and P_KmADH1, are utilized for the expression of recombinant proteins. The activation of P_KmINU1 and P_KmGAL1 is contingent upon the carbon source. Research indicates that the native promoter of K. marxianus demonstrates greater strength compared to the homologous promoter from S. cerevisiae when glucose is used as the carbon source during growth conditions (Kumar et al., 2021). Lang et al. (2020) defined P_NC1 and P_TEF3 as strong promoters that drive gene expression up to 87-fold in response to glucose metabolism and more than 17.8-fold in the presence of xylose as a carbon source. P_HHF1 and P_PGK were used as medium-level promoters, and P_SSA3 and P_ADH1 were used as low-strength promoters (Lang et al., 2020). Yang et al. (2015) evaluated six constitutive promoters (P_TDH3, P_PGK and P_ADH1) in S. cerevisiae and K. marxianus by heterologous expression of versatile genes under different conditions. Real-time PCR and enzyme activity assays showed that the promoter activity was reduced to a certain extent at high temperatures, and the promoter activity was P_KmPGK > P_KmTDH3 > P_ScPGK > P_ScTDH3 > P_KmADH1 > P_ScADH1. When using xylose as the only carbon source, the order of promoter strength is P_KmPGK > P_ScPGK > P_KmADH1 > P_ScADH1 > P_ScTDH3 > P_KmTDH3 (Yang et al., 2015). Lee et al. (2013) also reported that the strength of promoters cloned from S. cerevisiae in K. marxianus is P_GPD > P_ADH ≈ P_TEF ≫ P_CYC (Lee et al., 2013). In a recent study, a novel synthetic carbon-responsive promoter was developed that fused the carbon-source-responsive element of the native ICL1 promoter to the strong S. cerevisiae TDH3 or native NC1 promoter core, which resulted in an increase in protein expression capacity by more than 50 % (Bassett and Da Silva, 2024).

Table 2.

Common plasmids and genetic elements in K. marxianus.

Strains	Plasmids	Transformation methods	Genetic elements	References
K. marxianus FIM-1ΔU	pLHZ626	PEG-mediated protoplast transformation	The ARS1/CEN5 sequence derived from K. marxianus; the ARSH4/CEN6 sequence obtained from S. cerevisiae; ADH1, TEF promoter and terminator; three screening markers, uracil auxotrophy (URA3), hygromycin resistance (HphMX4), and kanamycin resistance (KanMX6).	Lyu et al. (2021)
K. marxianus UFV-3	pKLAC2	Electroporation	The α-mating factor signal peptide was derived from Kluyveromyces lactis; ADH1 promoter; aminoglycoside antibiotics G418 and kanamycin resistance.	Bragança et al. (2015)
K. marxianus BKM Y-719	pKDU7	Lithium acetate	The circular plasmid pKD1, measuring 1.6 μm, from Kluyveromyces drosophilarum; the URA3 gene derived from K. marxianus; and the pUC19 sequences that encompass cloned endopolygalacturonase.	Bartkeviciute et al. (2000)
K. marxianus BKM Y-719	pJSKM316	Electroporation	Constitutive promoters (PCYC, PTEF, PGPD, PADH) and terminator (TCYC1) were from S.cerevisiae; URA3 auxotrophic marker; GFP reporter gene.	Lee et al. (2013)
K. marxianus ATCC 12424	pKL2	Electroporation	A replication origin KARS2 of Kluyveromyces lactis, the URA3 gene of S. cerevisiae and G418 resistance label.	Iborra (1993)
K. marxianus Fim-1ΔURA3	pUKD-S-PIT	Lithium acetate	The self-replicating sequence from plasmid pKD1; inulinase promoter and terminator; URA3 screening markers.	Yang et al. (2021)
K. marxianus CBS 6556	pE1	Lithium acetate	Episomal vectors derived from the K. drosophilarum 2-μm-like endogenous plasmid pKD1	Antunes et al. (2000)
Kluyveromyces marxianus NBRC1777	pMTU-DO-URA	Lithium acetate	Centromeric plasmid with GFP drop-out; ScURA3 KmARS/CEN7;	Rajkumar and Morrissey (2020)
Kluyveromyces marxianus NBRC1777	pI1-MTU-DO-HIS	Lithium acetate	Integrative plasmid with GFP drop-out targeting LAC4; ScHIS3;	Rajkumar and Morrissey (2020)
Kluyveromyces marxianus NBRC1777	pI4-MTU-DO-G418	Lithium acetate	Integrative plasmid with GFP drop-out, kanMX, targeting integration site I4	Rajkumar and Morrissey (2020)
Kluyveromyces marxianus NBRC1777	pI6-MTU-DO-URA	Lithium acetate	Integrative plasmid with GFP drop-out targeting ARO3; ScURA3;	Rajkumar and Morrissey (2020)

For temperature-inducible promoters, Rajkumar et al. (2019) reported many heat-inducible promoters, like P_HSP104, P_SSA2, P_TSA1, and P_HSP150. P_HSP150 maintains relatively stable expression at high temperature. P_TSA1 exhibits the highest fold induction at a temperature of 42 °C, while P_SSA2 increases its strength at a medium to high level with the increase of temperature. For xylose-inducible promoters, P_XYL2 has the strongest induction among P_XYL1, P_XYL2, and P_ALD4. The lactose-inducible promoter P_LAC4 induces 10-fold more expression in media with lactose as the only carbon source than glucose does (Rajkumar et al., 2019). The capacity to modulate biochemical pathways through the adjustment of enzyme and protein expression levels in yeast is essential for creating yeast strains tailored for specific applications. Consequently, the innovation of new promoters designed for various environmental conditions will enhance the potential of K. marxianus as a robust microbial cell factory (Kumar et al., 2021; Lang et al., 2020).

Alongside promoters, terminators significantly contribute to protein expression by affecting the stability of mRNAs, thereby offering an additional method for achieving precise regulation of gene expression. LAC4t is a naturally occurring terminator commonly utilized in Kluyveromyces. The use of terminators of ScADH1 and ScPGK1 increased the gene expression level in K. marxianus by about 2 times (Rajkumar et al., 2019). Terminators can be utilized to minimize background, keep basal expression levels, and to smooth expression changes under different conditions when expressing heterologous genes. The utilization of T_KmIMTT1 and T_KmIMTT2 has effectively enhanced the production of β-galactosidase in K. marxianus. Additional frequently utilized terminators comprise T_TDH3, T_INUI (terminator of the inulinase gene), T_PGK, T_PDC, and T_ADH1 (Kumar et al., 2021; Lee et al., 2013). Anyhow, promoters and terminators available for protein production and metabolic engineering applications in K. marxianus are still limited. Further identification of novel promoters and terminators is required to regulate the ideal biochemical pathways for efficient expression of proteins, enzymes and metabolic engineering in K. marxianus. Alternatively, the design of synthetic promoters and terminators which exhibit strong efficacy and stable performance will push the development of K. marxianus as a chassis cell, and finetune yeast cell in dynamic regulation and efficient biomanufacturing (Kumar et al., 2021; Lang et al., 2020).

4.3. Plasmid vectors

The construction of expression vectors is a major step for the expression of exogenous genes in the yeast. The discovery of the pKD1 plasmid in K. drosophilarum is a breakthrough in the molecular biology of Kluyveromyces (Chen et al., 1986). Later, researchers introduced the kanamycin resistance gene, the URA3 gene of S. cerevisiae, and the starting point of replication of Escherichia coli into the pKD1 plasmid, and constructed a pGL2 plasmid that can efficiently replicate autonomously in K. marxianus (Fonseca et al., 2008; Gombert et al., 2016; Iborra, 1993). Bartkeviciute et al. (2000) constructed a multifunctional shuttle vector pKDU7 by combining elements from the circular plasmid pKD1, the URA3 gene of K. marxianus, and the pUC19 plasmid (Fig. 3). The vector can be used for heterologous gene expression in a variety of Kluyveromyces and yeast strains (Bartkeviciute et al., 2000). Soares Braganca et al. (2015) constructed an integrated vector of pKLAC2 and successfully expressed dengue virus type 1 nonstructural protein 1 (NS1) in K. marxianus (Bragança et al., 2015). The efficient expression of inulinase gene in K. marxianus was achieved by ligating the INU1 gene to the expression plasmid pINA1317 (Zhao et al., 2010).

Fig. 3.

Genetic elements and molecular manipulation of K. marxianus. (a) Selection of counter-selectable markers during K. marxianus transformation; (b) Promoters and terminators driving high-level gene expression; (c) Representative plasmid expression vectors; (d) Genome editing technologies. The discovery of pKD1 plasmid is an important breakthrough in the molecular biology research of K. marxianus. Based on the findings, the pGL2 plasmid and multi-functional plasmid shuttle vector system pKDU7 were constructed, which can be efficiently replicated in K. marxianus. Gene editing tools that have been established in K. marxianus include traditional homologous recombination and Crispr-cas9 gene editing techniques. LEU: Leucine; TRP: Tryptophan; URA: Uracil; HIS: Histidine; ADE: AdenIne; 5-FOA: 5-Fluoroorotic Acid.

Plasmids that utilize autonomous replication sequences (ARSs) are extensively employed in Kluyveromyces. These ARSs are in the intergenic sequence of the genome and usually contain the origin of replication (ORI), centromere (CEN), and linker sequences of different lengths. The results showed that the ARSs in K. marxianus are usually in the 50 bp sequence, at least 21 bp, which can induce the formation of circular DNA and replicate effectively in the cell (Abdel-Banat et al., 2021). The application of plasmid vectors often has defects instability during passaging, so subsequent studies need to find a balance between copy number and plasmid stability to achieve high yield of recombinant proteins.

5. Development of gene editing technologies

Gene editing represents a method of making precise changes to a specific nucleic acid sequence. Early gene editing in K. marxianus relied on conventional homologous recombination (HR)-based techniques, which suffered from low efficiency and dependence on lengthy homologous arms (generally >500 bp) (Liao et al., 2024). The recent advancements in CRISPR/Cas9 technology have rendered gene editing more efficient, user-friendly, and customizable, which also provides favorable technical support for the adaptive assembly and optimization of functional components within K. marxianus chassis cells (Chu, 2022). The CRISPR-Cas system was initially identified as a defense mechanism for archaea and bacteria, enabling them to protect themselves against invading bacteriophages (viruses) and mobile genetic elements. Presently, it has become a widely utilized resource for the genetic engineering of yeast strains. Wang et al. (2024) designed the K. marxianus NBRC 104275 strain by damaging non-homologous end joining and enhancing homologous recombination mechanisms, thereby improving the effective homologous directional repair of homologous arms up to 40 bp in length (Wang et al., 2024). Kwon et al. (2019) used the CRISPR-Cas9 system to knock out the PHO13 gene in K. marxianus, and the xylose consumption and xylose ethanol yield of the mutated strain increased by 18.29 % and 21.28 %, respectively (Kwon et al., 2019). The YZB392 strain constructed by integrating Utr1 into the Ypr1 site of the strain overexpressing NcXyl1 and CiGxf1 and carrying the destroyed Xyl2 by CRISPR-Cas9 in K. marxianus can increase the glycerol utilization rate by 20 % (Ren et al., 2022). CRISPR-Cas9 technology can also successfully integrate multiple genes into K. marxianus without destroying the function of endogenous genes. It laid a solid foundation for the long-term stable and continuous expression of recombinant protein in K. marxianus (Zhou et al., 2024).

In addition to modifying essential metabolic genes, numerous studies have shown that the CRISPR-Cas9 system can be utilized to generate stable haploid isolates exhibiting enhanced characteristics. During the isolation process of K. marxianus haploid cells, the alleles MATa and MATα were easily converted, and the cells were transformed from haploid cells to diploid cells. The stability of haploid cells is contingent upon the function of the α3 transposase, which cleaves the MAT locus, allowing the conversion of the MAT alpha type to the MATa type. By knocking out the MATα3 gene in the diploid background by CRISPR/Cas9, it is possible to avoid mating type switching, thus obtaining stable haploid strains (Lee et al., 2018). An exceptionally efficient CRISPR-Cas9 genome editing toolkit was created to design the K. marxianus NBRC 104275 strains. The system stably integrates Cas9 nuclease into the genome in advance and designs a library of gRNA expression plasmids with different screening markers, realizing "plug and play" iterative editing, and only needs to replace the gRNA plasmid for multiple rounds of operations, eliminating the tedious plasmid removal step. gRNA expression is expressed using efficient and easy-to-assemble tRNA-gRNA arrays, which significantly improve processing efficiency compared to hammerhead ribozyme and support multiple editing of at least four targets. The tool has been successfully applied to complex operations such as scar-free in vivo assembly and label-free integration of large fragments (12 kb), greatly simplifying the K. marxianus genetic processing process significantly enhances its engineering capabilities as a chemical production platform (Wang et al., 2024).

CRISPR interference (CRISPRi) is a molecular tool for precise control over gene activity. It regulates gene expression by turning genes off or down, unlike traditional gene editing that alters the genetic code. CRISPRi uses a modified Cas9 protein called deactivated Cas9 (dCas9). This dCas9 protein is engineered to eliminate its DNA-cutting activity while retaining its ability to bind to specific DNA sequences (Lobs et al., 2018). Besides, studies have reported that highly multiplexed CRISPRi inhibits respiratory function to enhance mitochondrial local ethyl acetate biosynthesis in K. marxianus. By modulating the expression of genes in the TCA cycle and electron transport chain, ethyl acetate was produced at a level 3.8-fold higher the native strain, which already has a high capacity (Lobs et al., 2018). In addition, it has been reported that xylose conversion efficiency was significantly improved by using a synergistic strategy of knockout and overexpression (Zhang et al., 2022). The gene editing technology of K. marxianus is based on CRISPR-Cas9 as the core, combined with homologous recombination optimization and multiple editing strategies, which enables a full range of operations from single-gene knockout to the integration of complex metabolic pathways. These advances not only enhance their potential for application as industrial chassis bacteria but also provide important tools for synthetic biology and green manufacturing. In the future, with the further optimization of editing tools, its application in the fields of biomedicine, energy and environmental protection will be more extensive.

6. Application of K. marxianus in the food industry

6.1. Production of food enzymes

The production of enzymes by K. marxianus is one of the most important sectors of its application in modern industrial biotechnology (Table 3). Due to its exceptional resistance to high temperatures, rapid growth rate, broad range of suitable substrates, and capability to utilize various inexpensive substrates and inducers like whey and corn steep liquor, it offers distinct advantages in enzyme production (Fig. 4). Inulinase is sufficient to hydrolyze β-2,1 fructosidic bonds, which is widely used in the manufacturing of fructooligosaccharides and high fructose syrup, in addition to its applications in the production of ethanol from Jerusalem artichoke and other areas. (Liu et al., 2013). Silva et al. (2015) compared the inulinase production capacity of different yeasts, among which K. marxianus ATCC 36907 demonstrated stability for 60 min at a pH of 4.0 and a high temperature of 45 °C, indicating significant potential for inulinase production (Silva et al., 2015). On the other hand, K. marxianus CBS 6556 displayed enhanced characteristics compared to other strains regarding high temperature tolerance, substrate specificity, and inulinase synthesis (Rouwenhorst et al., 1988). It was observed that the expression of the inulinase gene in K. marxianus was inhibited by glucose and fructose, while it was stimulated by inulin and sucrose (Liu et al., 2013). Deglucose inhibition and overexpression of native glycanase genes are essential for inulinase production. The transcriptional stress factor MIG1, encoded by MIG1, is a core component of glucose-sugar inhibition. When the MIG1 gene is disrupted, K. marxianus produces more inulinase than the wild-type strain, e.g., activity of 101.7 units/ml v.s. 84.3 units/ml. The inulinase production of this K. marxianus may be further enhanced by overexpressing the natural inulinase-encoding gene INU1, and inulin could be effectively hydrolyzed (Zhou et al., 2014). In addition, purified inulinase can also be used in feed, pharmaceutical, and chemical industries. β-galactosidase hydrolyzes lactose in dairy products, solves problems with whey processing, and also produces a functional food additive galacto - oligosaccharides (GOS). Natural inducers, including lactose and galactose, regulate the expression of β-galactosidase in K. marxianus. When subjected to higher concentrations of lactose or glucose, the inhibition effect superimposes the induction one, resulting in a decrease in β-galactosidase activity. Following the disruption of the MIG1 gene in K. marxianus, the production and expression of β-galactosidase were significantly enhanced, and 121.0 U/mL of β-galactosidase activity was produced within 60 h (Zhou et al., 2013). Also, the concentration of dissolved oxygen in the fermentation broth is crucial to produce β-galactosidase. Excessive oxygen pressure can lead to the formation of reactive oxygen species (ROS), which are highly toxic to microbial cells. In aerobic cultures, this can significantly inhibit growth and target product formation. It was found that the specific yield of β-galactosidase was increased by 60 % at an air pressure of 6 bar compared to the atmospheric state (Pinheiro et al., 2003). (Fig. 5)

Table 3.

Summary of different enzyme production of K. marxianus.

Bio-products	Strains name	Cultivation conditions	Enzyme activity	Regulate strategies	Reference
β-glucuronidase	K. marxianus YHJ010	Glucose, 42 °C	1.27 U/mg	At 42 °C, PGK had the highest promoter activity when glycerol or xylose was used as carbon source.	Yang et al. (2015)
Inulinase	K. marxianus KM-0	Inulin, 37 °C	896.1 U/mL	Optimization of inulinase expression by promoter engineering, codon optimization and high-cell-density fermentation.	Zhang et al. (2019)
β-galactosidase	K. marxianus YHJ010	Glucose, xylose, glycerin, 42 °C	30, 10, 17 U/mg	The PGK promoter has high expression activity when glucose is the only carbon source, and the expression activity decreases with increasing temperature.	Yang et al. (2015)
Endo-polygalacturonase pectase	K. marxianus CDBB-L-278	Glucose, 30 °C	0.233 U/mg	The production of endo-PG under anaerobic conditions is the result of the regulation of EPG gene by dissolved oxygen saturation in the growth medium.	Rivera-Noriega et al. (2017)
β-mannanase	K. marxianus Fim-1ΔURA3	Glucose, 30 °C	159.8 U/mL	Coordinated expression of hemicellulolytic enzymes in K. marxianus by 2A-mediated ribosome hopping strategy.	Lan et al. (2021)
β-xylanase	K. marxianus Fim-1ΔURA3	Glucose, 30 °C	2210.5 U/mL	Coordinated expression of hemicellulolytic enzymes in K. marxianus by 2A-mediated ribosome hopping strategy.	Lan et al. (2021)
β-xylosidase	K. marxianus Fim-1ΔURA3	Glucose, 30 °C	1.25 U/mL	Coordinated expression of hemicellulolytic enzymes in K. marxianus by 2A-mediated ribosome hopping strategy.	Lan et al. (2021)
Lipase	K. marxianus L-2029	Glucose, 30 °C	3.47 U/mL	Avocado oil can be a good substrate for inducing lipase synthesis.	Martinez-Corona et al. (2020)
Protease	K. marxianus JY-1	Glucose, 28 °C	183.22 U/mL	K.marxianus JY-1 can highly hydrolyze the casein and promote the proliferation and cytokine production of RAW264.7 macrophages.	Li et al. (2023)
Serine protease	K. marxianus IFO 0288	Glucose, 30 °C	3.42 U/mg	Elevated Ca2+ and NaCl to 3.0 M promoted the protease activity of Protease-KM-IFO-0288-A.	Foukis et al. (2012)
Endo-1,4-β- glucanase RuCelA	K. marxianus FIM-1ΔU	Glucose, 30 °C	24 U/mL	Mutagenesis optimization of K. marxianus inulinase promoter and signal sequence can improve the expression of lignin cellulose hydrolase.	Zhou et al. (2018)
Tannase	K. marxianus NRRL Y-8281	Glucose, 30 °C	12711 U/mL	Under the condition of solid-state fermentation, K. marxianus can ferment olive residue as a solid carrier to produce tannase.	Mahmoud et al. (2018)

Fig. 4.

Metabolic pathways of K. marxianus and the formation of various commercial metabolites using glucose, lactose, and xylose as substrates. These commercial metabolites include xylitol, glycerol, acetate, ethyl acetate, ethanol, lactate, aldehydes, higher alcohols, acetate esters. GALK1: galactokinase 1; HXK1: Hexokinase-1; ZWF1: Glucose-6-phosphate 1dehydrogenase; GND1, 6-phosphogluconate dehydrogenase; RPE1: Ribulose-phosphate 3-epimerase; PDC: Pyruvate decarboxylase; LDH: Lactate dehydrogenase; ADH: Alcohol dehydrogenase; AAT, alcohol O-acyltransferase; ALDH: Acetaldehyde dehydrogenase; ACS, acetyl-CoA synthetase; PPP: Pentose phosphate pathway; TCA: tricarboxylic acid cycle.

Fig. 5.

Applications for food biomanufacturing. (a) Production of enzymes: K. marxianus can produce lactase, inulinase, protease, lipase, β-glucosidase, pectinase from a variety of carbon sources. (b) Used as a prebiotic: K. marxianus secretes inulinase to break down inulin to produce prebiotic fructooligosaccharides. (c) Generation of aroma compounds: Flavor compounds such as esters and aldehydes are generated through metabolic pathways, which are used in bread, cheese, Temphe and fruit wine fermentation. (d) Ethanol and single-cell protein production: K. marxianus can use whey, pomace, and agricultural waste as substrates to produce single-cell protein (SCP) or ethanol. Km: K. marxianus; SCP: single-cell protein.

Yeast pectinases have the potential to be utilized across various industries, including the processing of fruits and vegetables, citrus processing, brewing, and the fermentation of tea and coffee, owing to their distinctive advantages. Endo-polygalacturonases (EP), commonly known as pectinases, are mainly produced by plants and different microorganisms because of their ability to degrade cell walls and play an important role in fruit and vegetable processing. Factors affecting the synthesis of K. marxianus EP mainly focus on carbon source and dissolved oxygen (Serrat et al., 2002). The specific enzyme production under anaerobic conditions was five times higher than that under aerobic conditions, indicating that anaerobic conditions promoted EP synthesis in K. marxianus. Quantification of the EP gene showed 21.19-fold higher transcripts under anaerobic conditions, suggesting that the production of EP under anaerobic culture conditions was a result of dissolved oxygen saturation regulating the EP gene in the growth medium (Rivera-Noriega et al., 2017). Oxygen and galacturonic acid inhibit enzyme production, while glucose serves as a superior carbon source, yielding higher enzyme outputs compared to lactose. Most pectinases are induced by pectin and are subject to catabolic inhibition. EP of K. marxianus is noteworthy because its production is compositional, uninhibited by carbohydrates, and EP activity can be increased by the addition of pectin or its degradation products to the fermentation medium (Radoi et al., 2004). Pectinases from K. marxianus release soluble fragments of polygalacturonic acid, resulting in a drastic reduction in pectin viscosity, resulting in more free-flowing wines with richer volatile compounds (Rollero et al., 2018). Recombinant galacturonidase from K. marxianus led to a significant increase in the odor glycoside in Albarino wines, resulting in wines with richer aromas of citric acid, balsamic and spicy, especially floral (violet and rose) (Sieiro et al., 2014).

6.2. Production of fructose and fructooligosaccharides (FOS)

Fructose is a sweetening agent that can be up to 1.5 times sweeter than sucrose, offering fewer calories and functional components that improve flavor, color, and stability of products. Furthermore, the metabolism of fructose circumvents established glucose metabolic pathways, eliminating the need for insulin, making it suitable for individuals with diabetes (Rawat et al., 2016). Fructooligosaccharide (FOS) is often used as a food ingredient because it can maintain a good balance of the intestinal flora and promote the reproduction of the intestinal bifidobacteria community. (Bilal et al., 2022; Rawat et al., 2016). Remarkably, both substances can be derived through the enzymatic hydrolysis of inulin. It is mainly produced by two different enzymatic processes: fructose transferase (FTase) transfructosylation of sucrose; hydrolysis of inulin by inulinase, which randomly cleaves the β-2,1 bond of inulin to produce oligosaccharides (Struyf et al., 2017). However, the production of FOS and fructose is usually carried out at high temperatures (about 60 °C). Consequently, the isolation and characterization of thermostable inulinase holds considerable importance for the hydrolysis of inulin at elevated temperatures. K. marxianus is regarded as a promising candidate to produce FOS. This organism can utilize fructans like inulin, and inulinase may possess the capability to encapsulate fructans derived from plants. Certain researchers have identified the endogenous strain K. marxianus, extracted from aguamiel (the naturally fermented sugary juice of agave plants), as the primary strain exhibiting significant enzyme synthesis capabilities and inulinase activity. A laboratory-scale bioreactor-level investigation demonstrated that operating at 30.6 °C, 152 rpm, 1.3 VVM ventilation, and a pH of 6.3 optimized inulinase production, yielding a fructose-rich syrup containing 95 % fructose (Garcia-Aguirre et al., 2009). A high-performance engineered inulin endonuclease for the production of FOS from inulin was constructed by fusing an insulin-binding module (IBM) to the N-terminus or C-terminus of the inulinase endonuclease, producing FOS at 60 °C in a 10 L fermenter, and finally, obtaining FOS with a purity of 91.4 % (Mao et al., 2019). Using immobilized K. marxianus inulinase hydrolyzed glycans and oligosaccharides of plant extracts to produce fructose would be an effective and beneficial method for the production of commercial sugar. More than 39.2 g/L and 40.2 g/L fructose were obtained within 4 h using immobilized exoinulinase from K. marxianus YS-1 with raw and pure inulin from asparagus as substrate (Singh et al., 2007). In addition, to produce highly concentrated fructose syrup, the utilization of K. marxianus as a carbon source for xylose offers a potentially beneficial alternative for industrial applications (Hoshida et al., 2018).

6.3. Synthesis of aroma compounds

Kluyveromyces produces aromatic compounds in liquid fermentation, inciuding fruit esters, ketones, carboxylic acids, alcohols, furans, isoamyl acetate and monoterpene alcohols. Among these compounds, 2-phenylethanol (2-PE) and 2-phenylethyl acetate (2-PEA) with rose petal aroma have broad application prospects in food, cosmetics, aviation and medical fields. The most used biotechnology for the production of 2-PE and 2-PEA is the bioconversion of L-phenylalanine by food-grade yeast through the Ehrlich pathway. During this procedure, L-phenylalanine undergoes conversion to phenylpyruvate, followed by decarboxylation to phenylacetaldehyde, and is ultimately reduced to 2-PE through dehydrogenation. Subsequently, 2-PE can be esterified to form 2-PEA (Martínez et al., 2018). Using different yeast strains for efficient biotransformation of L-phenylalanine (L-phe) to 2-PE has become a hot research topic. Among them, K. marxianus is the most promising one due to its potential for the synthesis of 2-PE, fruit esters, alcohol, ketones, carboxylic acids furanone and aromatic hydrocarbons. The value of K. marxianus as a 2-PE production platform had been demonstrated using metabolic engineering strategies, resulting in the production of more than 800 mg/L of 2-PE (Rajkumar Morrissey., 2020). Using K. marxianus, the highest 2-PE 10.21 mg/g and 2-PEA 8.20 mg/g were obtained by solid-state fermentation of sugarcane bagasse with extra L-phenylalanine (Martínez et al., 2018). It has been reported that K. marxianus CCT7735 was the best strain to produce the maximum 2-PE (3.44 g/L) among 267 strains under the optimal conditions (de et al., 2018). Overproduction of phenylpyruvate to improve the biosynthetic pathway involving phenylpyruvate decarboxylase and reconstruct the shikimic acid metabolic pathway has been shown to be an effective way to increase 2-PE production (Li et al., 2021, Li et al., 2021).

K. marxianus can also be used to produce other aromatic compounds (see Table 4). Alcohol acetyl transferase Eat1 is a key enzyme in the production of ethyl acetate, isoamyl ester and phenethyl ester by K. marxianus. The biosynthesis of high esters depends on the mitochondrial localization of Eat1 (Lobs et al., 2018). A research study has established a dual-channel chemically regulated gene expression system capable of enhancing ethyl acetate production by concurrently inhibiting pyruvate dehydrogenase (PDA1) and activating pyruvate decarboxylase (PDC1) (Wei et al., 2024). The concurrent knockdown of ACO2b, SDH2, RIP1, and MSSSI led to a 3.8-fold enhancement in the production of ethyl acetate, surpassing the already elevated levels observed in K. marxianus (Lobs et al., 2018).

Table 4.

The food chemicals produced by K. marxianus.

Chemicals	Carbon source	Temperature (°C)	Transformation methods	Yield (g/L)	References
2-Phenylethanol	glucose	30	Electroporation	1.0	Kim et al. (2014)
Lactic acid	glucose	37	Electroporation	24′	Yarimizu et al. (2015)
Cytochrome P450 monooxygenase	glucose	28	Dimethyl sulfoxide		Theron et al. (2014)
Denguevirustype1 non-structural protein1(NS1)	galactose	37	Electroporation	1.2	Bragança et al. (2015)
Hexanoic acid	galactose	37	Electroporation	0.15	Cheon et al. (2014)
Xylitol	xylose	45	Lithium acetate	60.03	Zhang et al. (2014)
Ethanol	starch	48	Lithium acetate	36.88	Wang et al. (2014)

6.4. Used as probiotics and prebiotics

Kluyveromyces marxianus, a Generally Recognized as Safe (GRAS) microorganism, demonstrates significant dual potential in the functional food sector, particularly in the development of prebiotics and probiotics for regulating gut health. Its application value lies not only in its capacity as a potential probiotic microorganism but also in its cell wall components serving as highly effective prebiotics. Through synergistic action, it achieves multifaceted regulation of the intestinal microbiota. First, K. marxianus itself exhibits outstanding probiotic properties. This yeast demonstrates strong tolerance to the gastrointestinal environment, with certain strains capable of surviving digestion by gastric acid and bile salts while effectively colonizing the colon region to exert direct probiotic effects (Cho et al., 2018). Research indicates that viable K. marxianus can inhibit the adhesion and proliferation of intestinal pathogens such as Salmonella and pathogenic E. coli through competitive exclusion mechanisms (Díaz-Vergara et al., 2017). Furthermore, components like β-glucan in its cell wall act as immunomodulators, enhancing the intestinal mucosal immune response and boosting the body's defense against pathogenic microorganisms. These mechanisms collectively contribute to maintaining gut microbiota stability and strengthening host resistance. Notably, even when K. marxianus cells lose their activity during digestion, their cell wall structure retains biological activity and functions as a prebiotic. The cell wall, rich in mannan-oligosaccharides (MOS) and β-glucans, serves as specific metabolic substrates selectively utilized by beneficial gut microbiota (such as Bifidobacterium and Lactobacillus species), thereby stimulating their growth and metabolic activity. Research indicates that cell wall extracts from K. marxianus CBS 6556 significantly promote the proliferation of Bifidobacteria and Lactobacilli while inhibiting the growth of harmful bacteria like Clostridium difficile, demonstrating its potential for precisely regulating gut microbiota composition. These prebiotic components undergo microbial fermentation to produce short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate. These SCFAs further lower intestinal pH, inhibiting pathogenic bacteria survival while strengthening the intestinal epithelial barrier function. They also participate in systemic immune regulation and energy metabolism, exerting profound effects on maintaining intestinal homeostasis (Maccaferri et al., 2012). Additionally, K. marxianus exhibits significant synergistic effects when combined with other bioactive substances. The study found that the strain K. marxianus KU140723-02, isolated from kefir, exhibited significantly enhanced free radical scavenging capacity and approximately doubled antioxidant activity when co-administered with polyphenol-rich grape seed extract or grape seed flour (Cho et al., 2018). This suggests K. marxianus may interact with polyphenolic compounds to enhance their defense against oxidative stress, offering novel insights for developing functional composite formulations with anti-inflammatory and antioxidant properties.

6.5. Transformation of food waste into ethanol

The yeast K. marxianus has garnered significant attention in recent years for its application in ethanol production from food waste and lignocellulosic biomass, owing to its thermotolerance, broad substrate utilization spectrum, and rapid fermentation kinetics. In contrast to S. cerevisiae, K. marxianus is capable of directly assimilating diverse carbon sources such as lactose, xylose, and arabinose, making it particularly suitable for the bioconversion of complex substrates including dairy wastewater, fruit and vegetable residues, and molasses (Zhang et al., 2023). Madeira et al. (2018) used K. marxianus NCYC 3396 to produce ethanol from sugarcane at 48 °C, such temperature lows contamination rate, cooling cost, and reduces the final cost of ethanol production (Madeira-Jr Gombere., 2018). It was demonstrated that temperature is one of the most important factors in the production of ethanol from cheese whey by K. marxianus URM7404, followed by pH and lactose concentration (Murari et al., 2019). K. marxianus can directly use lactose in whey to produce ethanol, thereby solving the problem of wastewater discharge in dairy production.

However, under most conditions, not pure sugars, but various food waste containing large amounts of cellulosic biomass were used as substrates for biotransformation. The inhibitors generated by pretreatment seriously affect the activity and fermentation efficiency of the strains, so improving the tolerance of the strains to inhibitors in complex matrices is the prerequisite for achieving efficient fermentation. Studies have reported that inhibitors produced during the fermentation of cellulose by K. marxianus to ethanol include different weak acids, furans and phenolic compounds (Oliva et al., 2003). Overexpression of a transcriptional regulatory gene, KmMsr, in K. marxianus significantly improved tolerance to lignocellulosic biomass-derived inhibitors. This overexpression also enhances tolerance to elevated temperatures, ethanol, and high concentrations of NaCl and glucose (Zhang et al., 2024). On the other side, ethanol tolerance of K. marxianus could be improved by modifying the TATA-binding protein Spt15, which enables the differential expression of hundreds of genes (Li et al., 2018, Li et al., 2018). Moreover, as a promising process for cellulosic ethanol production, synchronous saccharification fermentation (SSF) reduces the inhibition effect of decomposed products from cellulase. Using K. marxianus as the host, the expression of various enzymes required for cellulose degradation, such as xylosidase and xylose transportase, and the construction of enhanced functional strains by surface display may be an effective way to improve cellulosic ethanol production in the future.

Though it was shown that recombinant K. marxianus DMB13 can rapidly convert xylose to ethanol, especially after glucose depletion, conversion of xylose to ethanol is generally inefficient (Suzuki et al., 2019). Solving the problem of inefficient xylose utilization is the key to improving ethanol yield. Strategies in metabolic engineering have been developed to tackle the issues arising from redox imbalances when xylose serves as a carbon source in oxygen-limited environments. The expression of NADPH-preferring xylose reductase (XR) from Cercera vulgaris (NcXR) and NADP + -preferring xylitol dehydrogenase (XDH) from S. vulgaris (SsXDH) in K. marxianus has been documented. Instead of changing the coenzyme's preference for NADH, the combination of NcXR and SsXDH greatly improved xylose fermentation capacity and redox balance. Overcoming other additional potential constraints of xylose assimilation and by-product disruption by targeting key genes in the downstream pathway allowed ethanol productivity reach 2.14 g/(Lh) in recombinant K. marxianus YZJ088, when utilizing a co-fermentation strategy of xylose and glucose (Zhang et al., 2015). Generally, through directed evolution and metabolic engineering targeting key aspects such as inhibitor tolerance and xylose utilization, combined with adaptive processes like simultaneous saccharification and fermentation (SSF), K. marxianus is poised to become an efficient microbial cell factory to produce cellulosic ethanol from food waste.

6.6. Treatment of wastewater during food processing

Wastewater from the processing of dairy raw materials is high-intensity wastewater due to its high chemical oxygen demand (COD) and biochemical oxygen demand (BOD) (Buathong et al., 2020). Using yeasts to treat wastewater is a promising technology with mild reaction conditions and simple processes. K. marxianus is a suitable strain to produce alcohol and feed yeast by fermentation of deproteinized whey concentrate. It was found that K. marxianus ATCC 8554 had a higher ethanol production capacity than Candida kefyr ATCC 14245 when the two yeasts were fermented with whey percolate (Koushki et al., 2012). Murari et al. (2017) found that when the initial whey was 18.8 g/L, fermentation with K. marxianus for 12 h reduced the COD content by 82.28 % (Murari et al., 2017). Cheese whey fermentation was performed to examine COD removal and to determine the fate of soluble whey proteins. After batch fermentation for 36 h, the biomass of K. marxianus increased from 2.0 g/L to 6.0 g/L, and the COD decreased by 55 %, while the soluble whey protein concentration decreased (Yadav et al., 2014b). Co-fermentation with K. marxianus and Candida krusei showed more pronounced effects, e.g., a maximum COD removal rate of 80.2 % was obtained at 24 h and the biomass productivity was 0.17 g/L/h (Yadav et al., 2014a). Furthermore, K. marxianus can be used for the recovery of heavy metals in wastewater from the agri-food industry. For example, K. marxianus demonstrated the capacity to accumulate significant quantities of copper from the feeding medium at elevated copper concentrations while maintaining its biological activity (DoÈnmez and Aksu, 1999).

6.7. Production of single-cell protein

Production of Single-Cell Protein (SCP) has been recognized as a pivotal solution for the future food and feed industries due to its exceptional advantages including enhanced resource utilization efficiency, shortened production cycles, and superior environmental sustainability. Yeast, owing to its high protein content, ease of processing, small cell size, and cost-effectiveness, stands out as a superior microbial candidate for single-cell protein (SCP). K. marxianus has demonstrated unique potential in the SCP domain due to its robust metabolic adaptability and efficient biomass yield under industrial fermentation conditions. For instance, the strain K. marxianus EXF-5288 achieved a biomass accumulation of 14.24 g/L and an SCP yield of 6.14 g/L under shake-flask cultivation at 20 °C, with its protein fraction exhibiting notably high contents of glutamic acid (15.5 mg/g), aspartic acid (12.0 mg/g), and valine (9.5 mg/g) based on dry biomass quantification. (Koukoumaki et al., 2024). Further investigation revealed that mixed-culture strategies could substantially enhance SCP production efficiency. For example, co-cultivation of K. marxianus with Candida krusei augmented biomass yield and volumetric productivity by 19 % and 33 % respectively, demonstrating the feasibility of synchronizing wastewater treatment with SCP biosynthesis under extreme operational conditions through microbial consortia engineering. Simultaneously, the research findings indicate that the SCP composition primarily consists of protein (43.4 % w/w), carbohydrates (33.6 % w/w), crude fiber (4.6 % w/w), lipids (6.4 % w/w), and ash (minerals) (8.4 % w/w). Lysine and sulfur-containing amino acids represent particularly noteworthy essential amino acid components (Yadav et al., 2014a). Similarly, in beet pulp-containing media, co-cultivation of K. marxianus with Trichoderma reesei enhanced SCP production by 54 % with a substrate-to-protein conversion efficiency of 41.8 %, while the resultant biomass demonstrated a complete essential amino acid profile with amino acids such as leucine, phenylalanine, threonine, valine, aspartic acid, glutamic acid, and proline abundant (Ghanem, 1992). Furthermore, under controlled operational parameters (30 °C, pH 6.5), co-cultivation of K. marxianus with S. cerevisiae for food-grade SCP production in fermented whey supernatant (FWS) medium demonstrated total protein recovery efficiencies of 84 % and 92 % in monoculture and co-culture systems, respectively, following process parameter optimization (Yadav et al., 2016).

K. marxianus exhibits remarkable substrate conversion capabilities, as evidenced by strain CC1 which efficiently utilizes diverse carbon sources through solid-state fermentation (SSF) to valorize food industry residues into high-value-added products containing 59.2 % combined lipids and proteins (w/w), demonstrating industrial symbiosis potential in circular bioeconomy frameworks (Aggelopoulos et al., 2014). Recent investigations have revealed that K. marxianus strain KM812 effectively hydrolyzes whey substrates to generate bioactive metabolites including L-isoleucine, ornithine, betaine, α-linolenic acid, and palmitoleic acid, thereby markedly enhancing the nutritional profile and functional characteristics of the bioconversion products (Gao et al., 2024).

K. marxianus exhibits high-efficiency bioconversion of industrial and agricultural waste streams (e.g., whey and sugar beet pulp), which not only substantially lower production costs but also establishes an eco-technological route for waste valorization through resource circularity (Ghanem, 1992). The integration of genetic engineering techniques has further amplified its potential in lipid biosynthesis and functional metabolite synthesis, while the strain's intrinsic capacity to enrich essential amino acids and nutritional cofactors significantly augments the application value of SCP in feed formulations and functional food systems (Aggelopoulos et al., 2014). By integrating cultivation parameter optimization, innovative microbial consortium engineering, and advanced metabolic flux modulation, K. marxianus is positioned to be a pivotal biological platform for sustainable protein provisioning (Li et al., 2018, Li et al., 2018). The technological convergence steers K. marxianus production toward a high-efficiency, low-carbon direction and provides a circular solution to global food security challenges.

7. Regulatory and safety considerations

As a microorganism with a wide range of application potential in the food and feed industry, the commercial promotion of K. marxianus depends not only on technical performance but also on clear regulatory status and comprehensive safety evaluation. At present, yeast has been recognized by the US Food and Drug Administration (FDA) as GRAS (Generally Recognized as Safe) and based on sufficient scientific evidence and a long history of safe use, it is allowed to be used in the food field as a starter or production additive. Similarly, the European Food Safety Authority (EFSA) has included it in the QPS (Qualified Presumption of Safety) list, providing safety presets for its use within the EU and significantly lowering policy and market entry barriers.

Non-genetically engineered (non-GMO) strains of wild type or obtained through conventional mutagenesis breeding can be commercialized by following a relatively simplified filing or notification procedure based on the above GRAS/QPS status (Baptista and Domingues, 2022). In contrast, engineered strains (GMOs) that have been genetically modified, such as gene editing and xenogene transfer, face a more stringent and complex regulatory framework for commercialization, although they have significant advantages in terms of product yield, substrate utilization range, or environmental tolerance. For example, in the European Union, such strains are required to comply with the regulation and submit a comprehensive safety dossier, including molecular characteristics, toxicity, allergenicity, potential for gene level transfer, and assessment of unintended effects, which is a long and costly approval process, which constitutes an important consideration in actual industrialization (Lane and Morrissey, 2010).

8. Conclusion and foresights

The yeast K. marxianus has proven to be a promising eukaryotic microorganism with promising applications in protein production, dairy fermentation, aromatic compounds and ethanol production, as well as wastewater treatment in the food industry. In the development of K. marxianus as a cell factory, the study of yeast genetic characteristics, the optimization of high-cell-density fermentation strategies, the development of genetic toolkits, and the utilization of renewable biomass raw materials provide the basis and impetus for the further development and application of the K. marxianus cell factory. In addition, the progress of K. marxianus in exogenous protein expression and genetic engineering has broadened its application prospects in industrial biotechnology. The latest breakthrough in genome sequencing of K. marxianus has enabled researchers to validate the exogenous protein secretion capacity of this yeast chassis cell through the integration of heterogeneous enzyme systems (including thermophilic enzymes and antigenic protein expression platforms), while innovatively achieving coordinated biosynthesis of target proteins with high-value-added metabolites such as xylitol. New powerful CRISPR/Cas9 genome editing and regulation technologies have been explored in K. marxianus. The use of food waste such as corn cob residues, Jerusalem artichoke and whey waste makes green industrial ethanol production reality.

However, the industrial development of K. marxianus also faces challenges. During anaerobic fermentation, the ethanol tolerance of K. marxianus was poor, and improving the ethanol tolerance of K. marxianus was the premise for the renewable utilization of cellulosic biomass. In the realm of heterologous recombinant protein expression and genetic engineering, K. marxianus continues to face multifaceted challenges. First, the scarcity of advanced genetic tools, coupled with low CRISPR-Cas9 editing efficiency, elevated off-target risks, and lack of high-efficiency promoters and stable multi-copy chromosomal integration systems, collectively results in suboptimal heterologous gene expression levels. Second, the inadequate secretory capacity, compounded by the absence of a systematic signal peptide screening platform and insufficient characterization of protein folding and secretion mechanisms, hinders the adaptation to the high-efficiency secretion demands of complex eukaryotic proteins. Third, the metabolic regulation is complex, the degradation of proteases is active at high temperatures, and the redundant metabolic network is easy to cause product shunts.

Therefore, future research breakthroughs should prioritize three key dimensions. (1) Developing an efficient and accurate genome editing and integration platform: although basic CRISPR tools have been applied in K. marxianus, low editing efficiency and high off-target risk remain the primary bottlenecks. Breakthroughs in the past five years, including base editing, prime editing, and various innovative editors, along with enhancements in sgRNA design and optimization of Cas protein variants, offer a definitive pathway for creating effective and high-fidelity editing tools for K. marxianus. Simultaneously, to address the challenge of multicopy integration, site-specific recombination system or transposase/microchromosome strategy successfully applied in other industrial microorganisms can be combined with CRISPR targeting to build a stable and controllable multi-copy chromosome integration platform, which will significantly improve the expression level and stability of heterologous proteins. (2) Systematic elucidation and optimization of protein secretion pathways: using the rapidly developing omics technology and bioinformatics methods (such as comparative genomics, transcriptomics, and secretomics) in recent years, it is possible to mine and identify K. marxianus endogenous high-performance signal peptide. Combined with high-throughput screening platforms, such as fluorescent reporters or surface-based systems, signal peptide performance can be efficiently evaluated and optimized. It is necessary to study secretion-related mechanisms such as protein folding, ERAD (Endoplasmic Reticulum-Associated Degradation), and vesicle transport unique to K. marxianus, and learn from the key regulatory factors revealed in model yeast (such as unfolded protein reaction UPR elements) to systematically optimize the entire secretory pathway and finally achieve high yield and high-fidelity secretion of complex proteins. (3) Integrating artificial intelligence and systems biology to decode metabolic networks: The explosion of genome-scale metabolic model (GEM) construction and artificial intelligence (AI)/machine learning (ML) algorithms in microbial metabolic engineering has been witnessed over the past five years. For instance, a high-quality genome-scale metabolic model (GEM) of K. marxianus can be constructed based on its published genome sequence, using platforms such as RAST or ModelSEED. This model can be integrated with machine learning algorithms to predict key gene targets for enhancing ethanol tolerance (e.g., ERG genes involved in sterol synthesis or membrane transporters) and identify optimal carbon-nitrogen ratios for efficient recombinant protein synthesis. Specifically, convolutional neural networks (CNNs) can be employed to analyze historical fermentation omics data, predicting key knockout targets, such as GPD1 or ALD genes, to suppress the formation of byproducts like glycerol or acetate (Marcišauskas et al., 2019; Oyetunde et al., 2018). Furthermore, deep reinforcement learning algorithms can be applied to design dynamic regulation circuits that precisely control metabolic flux at different stages of bioreactor operation, thereby maximizing the yield of the target product (Treloar et al., 2020). The development of a high-quality GEM for K. marxianus has been thoroughly integrated with AI-Powered Prediction Tools (e.g., pathway design, enzyme optimization, phenotypic prediction models). This integration allows for a systematic decoding of the critical nodes within its metabolic network, facilitating precise dynamic pathway optimization. It provides a strong rational design foundation for simultaneously improving ethanol tolerance, reducing by-product shunting, and enhancing target product synthesis. Of course, comprehensive functional annotation of genes and mechanistic elucidation of metabolic regulation will serve as fundamental pillars for rational strain design. By integrating synthetic biology and fermentation engineering, K. marxianus is expected to break through the bottlenecks and become an efficient and stress-resistant industrial-grade chassis, finally empowering innovation in the field of food biomanufacturing.

Author contributions

Huan Wang: Investigation; validation; writing—original draft. Zhongke Sun: Conceptualization; formal analysis; supervision; writing—review and editing. Zifu Ni: validation; methodology. Yanli Qi: methodology; visualization; Xianyang Feng: Formal analysis; Methodology; Hongkun Xiao: methodology; visualization; Le Wang: Conceptualization; validation; methodology; Jiong Hong: Conceptualization; visualization; Yongheng Liang: Conceptualization; methodology; Chengwei Li: Validation; investigation; writing—review and editing; project administration.

Ethics statement

Not applicable.

Funding

This study was partially supported by the National Key R&D Project (No. 2022YFD2101405).

Declaration of competing interest

We declare that the manuscript has neither been published nor been submitted simultaneously for publication elsewhere. We also confirm that no conflict of interest exists in the submission of this manuscript, and the manuscript is approved by all authors.

Handling Editor: Dr. Quancai Sun