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. 2024 Jun 3;14(7):172. doi: 10.1007/s13205-024-03998-5

Expression of microbial lipase in filamentous fungus Aspergillus niger: a review

Hongmei Nie 1, Yueting Zhang 1, Mengjiao Li 1, Weili Wang 1, Zhao Wang 1, Jianyong Zheng 1,✉
PMCID: PMC11147998  PMID: 38841267

Abstract

Lipase has high economic importance and is widely used in biodiesel, food, detergents, cosmetics, and pharmaceutical industries. The rapid development of synthetic biology and system biology has not only paved the way for comprehensively understanding the efficient operation mechanism of Aspergillus niger cell factories but also introduced a new technological system for creating and optimizing high-efficiency A. niger cell factories. In this review, all relevant data on microbial lipase enzyme sources and general properties are gathered and updated. The relationship between A. niger strain morphology and protein production is discussed. The safety of A. niger strain is investigated to ensure product safety. The biotechnologies and factors influencing lipase expression in A. niger are summarized. This review focuses on various strategies to improve lipase expression in A. niger. The summary of these methods and the application of the gene editing technology CRISPR/Cas9 system can further improve the efficiency of constructing the engineered lipase-producing A. niger.

Keywords: Aspergillus niger, Lipase, Biotechnology, Optimization, CRISPR/Cas9

Introduction

Lipase, widely recognized as triacylglycerol ester hydrolases (EC3.1.1.3), represents a form of serine hydrolase and pertains to the α/β foldase family. In addition to, its lipid hydrolysis capacity, lipase displays its versatility by enabling reactions, such as alcoholysis, esterification, transesterification, and amidation of triglycerides, as shown in Fig. 1 (Salihu and Alam 2015). Lipase plays a crucial role in the food industry and is widely utilized in various applications such as processing of oil, manufacturing of dairy, and the formulation of diverse food supplements (Moayedallaie et al. 2010). In medical and pharmaceutical applications, lipase is a vital marker enzyme that can predict the occurrence of various diseases (Johannsen et al. 2009; Al-Bahrani and Ammori 2005). In the chemical industry, lipase is widely used in the washing industry, because it has a strong hydrolysis effect on oil (Saisubramanian et al. 2006). Lipase can also be used in the production of biodiesel (Nie et al. 2006). In addition, microorganisms possess the characteristics of rich variety, fast reproduction, and short growth cycles; they are not affected by seasons, which makes them suitable for industrial large-scale production. Thus far, microorganisms that produce lipase divided into three categories: bacteria, yeast, and mold (Hasan et al. 2006).

Fig. 1.

Fig. 1

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The various reactions catalyzed by lipase

Although aspergillus lipase research started late, compared with animal and plant lipases, it has the advantages of broader temperature and pH range, wider substrate specificity, higher stability, and stronger resistance to organic solvents. Hence, the research on aspergillus lipase is developing rapidly (Brockerhoff and Jensen 1974; Jaeger and Reetz 1998). Aspergillus lipase products have shown excellent application performance and are an emerging research hotspot in green biomanufacturing technology. In summary, understanding Aspergillus lipase will not only help deeply understand the biological process of fat metabolism but also promote the development of industrial production and biotechnology. Among the filamentous fungi, A. niger is one of the representative Aspergillus and is a common industrial enzyme production strain. Whether it is self-fermentation to produce enzymes or the transformation and expression of homologous or heterologous genes, many local and international teams have tried both (Lie et al. 2018; Chen et al. 2012). As a result of the extensive utilization of A. niger in the food sector and safety assessments, numerous products containing A. niger are generally recognized as safe by the U.S. Food and Drug Administration (Putrid et al. 2020). The application of A. niger as a microbial cell factory has steadily advanced and transformed into a promising system for expressing enzymes (Kobayashi et al. 2014; Niu et al. 2016). Lipase derived from A. niger is widely used in food, feed, detergent, biofuel, and other fields.

A comprehensive evaluation of lipase expression in A. niger is lacking, and information about it is scattered. Hence, in this review, all the data and advancements concerning lipase synthesis in A. niger, encompassing lipase generation, safety, genetic engineering, and approaches to enhance lipase manifestation in A. niger, among other aspects, are gathered and revised. The current status and deep insights into the expression system of A. niger and future research needs are comprehensively discussed.

General information about microbial lipase

Sources of microbial lipase

Lipases are primarily found in microorganisms, constituting approximately 66% of the total (Patil et al. 2011). Lipase-producing strains have been isolated from various environments such as volcanoes, hot springs, alkaline lakes, polluted soils, wastewater, and activated sludge (Enespa and Singh 2023). Microbial lipase sources can be categorized into three: bacteria, yeast, and fungi. Bacterial sources of lipases include Bacillus subtilis (van Pouderoyen et al. 2001), Pseudomonas aeruginosa (Nardini et al. 2000), Staphylococcus aureus (Meier et al. 2007), Serratia marcescens, and Escherichia coli (Su et al. 2015). Yeast sources include Candida (Irani et al. 2013) and Saccharomyces cerevisiae (Aschauer et al. 2016). Fungal sources include A. niger, Penicillium, Rhizopus, Mucor, and Geotrichum albus (Nakajima-Kambe et al. 2012; Barrigon et al. 2013). Table 1 summarizes the expression of lipases from various microbial origins in A. niger over the past decade.

Table 1.

Expression of lipases from different microbial sources in A. niger in the last decade

Organism Host genotype Promoter Expression Expression level Culture condition Name Accession number References
U/mL U/g
Candida antarctica A. niger SH-1 PgpdA Heterologous 1100.28 Submerged fermentation CALB Z30645.1 Li et al. (2018)
Rhizopus chinensis A. niger CICC 2475 PgpdA Heterologous 71 Submerged fermentation RCL EF405962.2 Zhang et al. (2015)
Candida antarctica A. niger SH-1 PglaA Heterologous 440.3 Submerged fermentation CALB Z30645.1 Pan et al. (2014)
Candida antarctica A. niger HL-1 PnalI/tpi Heterologous 171 Submerged fermentation CALB Z30645.1 Zhang et al. (2019a, b)
A. niger A. niger 89 – Homologous 314.67 Submerged fermentation ANL AB570425.1 Zhu et al. (2020a, b)
A. niger A. niger MA PglaA Homologous 1.68 Submerged fermentation ANL-H3 XM_025600473.1 Zhao et al. (2022)
A. niger A. niger CICC2462 PglaA Homologous 1480 Submerged fermentation Anlip A 4,988,581 Lie et al. (2018)
A. niger A. niger MH078571.1 and A. niger MH079049.1 – Homologous 795.39 Submerged fermentation – – Alabdalall et al. (2021)
A. niger A. niger MTCC 872 – Homologous 212.20 ± 6.36 U/gds Solid-state fermentation MTCC lipase KF006848.1 Mandari et al. (2020)
A. niger A. niger CICC2169 PgpdA Homologous – – – ANL AB570425.1 Zhu et al. (2020a, b)
A. niger – – Homologous 282 Solid-state fermentation – – Putrid et al. (2020)
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General properties of microbial lipase

Distinct lipase sources display varying degrees of glycosylation and disparate conformations of the enzyme protein due to distinct mechanisms involved in post-translational modifications. These disparities result in variations in the structure and properties of lipase among sources. As an effective biocatalyst, lipase can participate in many reactions. The catalytic function of lipase is greatly influenced by several factors, including the pH level of the solution, the temperature of the reaction, and the presence of metal ions, organic solvents, and surfactants, which can all remarkably affect the enzymatic activity of lipase. The general properties of lipase from different microbial sources are summarized in Table 2.

Table 2.

General properties of lipase from different microbial sources

Organism Name Molecular
weight (kDa)		 pHopt Topt (℃) Thermal stability Metal ions
dependent activity		 Accession number References
A. niger CBS101883 H3 31.953 7.5 45 Stable K+, Mg2+ XM_025600473.1 Zhao et al. (2022)
Enterococcus durans NCIM5427 – – 4.6 30 Unstable Ba2+, Na+, Li+, K+ AGN12840 Ramakrishnan et al. (2015)
A. niger ANL 53 2.5 30 Stable below 50 °C – XP_001397501.1 Zhang et al. (2019a, b)
Lactiplantibacillus plantarum WCFS1 Lp_3562 30 7.0 40 Stable below 45 °C Mn2+ CCC80525.1 Esteban-Torres et al. (2015)
Haloferax mediterranei HML 45.011 7.0 60 Stable Ca2+, Mg2+, Fe2+, Cu2+ Akmoussi-Toumi et al. (2018)
Thermomyces lanuginosus TLL 58.6 8.5 65 Stable – ABV69592.1 Goncalves et al. (2014)
Bacillus licheniformis KM12 KM12 lipase 33 8.0 35 Stable Na+, Li+ TWK36414.1 Malekabadi et al. (2018)
Marine Streptomyces sp. strain W007 MAS1 29 7.0 40 Stable Cu2+, Ni2+, Ca2+, Mg2+ RPK40156.1 Yuan et al. (2016)
Staphylococcus capitis SCL 45 8.5 37 Stable Ca2+, Mg2+, K+ KP455742 Rmili et al. (2019)
Psychrotrophic fungal isolates BPF4 and BPF6

BPF4 and BPF6

lipase

 – 11.0 and 9.0 40 Stable Ca2+, Fe3+ KF247215 and KX462167 Sahay and Chouhan (2018)
Alkalibacillus salicaceous – – 8.0 40 Stable Fe3+, Fe2+, Ni2+, Co2+, Mg2+, Mn2+, Zn2+, Cu2+, Hg2+, Al3+ KF738177 Samaei-Nouroozi et al. (2015)
Pseudomonas stutzeri PS59 P. stutzeri PS59 lipase 55 8.5 20 Stable Ca2+, Mn2+ WP_169855134.1 Li et al. (2014)
Pseudomonas sp. DMVR46 HZ 32 8. 5 37 Stable Ca2+, Ba2+, Mg2+ QYY43100.1 Masomian et al. (2013)
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Properties of the filamentous fungus A. niger

Morphology of A. niger

Based on its morphology, A. niger can be divided into conidial A. niger and aconidial A. niger (Guo et al. 2010). Aconidial A. niger, also known as nonsporulating A. niger, lacks the cell wall regulatory gene prpA. This genetic characteristic leads to the destruction of its cell wall integrity, resulting in the organism's inability to produce spores. Aconidial A. niger can be obtained through mutation breeding in industrial production (Yin et al. 2014). Krijgsheld et al. (2013) discovered the flbA gene, which is responsible for the secretion of a protein related to sporulation. The flbA-deficient strain enhances protein secretion, potentially due to variations in the thickness of the hyphae's cell wall. In addition to its effect on sporulation, researchers have been interested in investigating how mycelial pellets influence protein expression in A. niger when cultivated in liquid culture (Zhang and Zhang 2016).

In individual mycelia, most proteins are secreted at the hyphal tip (Moukha et al. 1993; Wosten et al. 1991), but only some target proteins accumulate in the internal hyphal compartment (Talabardon and Yang 2005). Walisko et al. (2015) have shown that highly branched hyphae produce more enzymes than less branched hyphae. Bekker et al. (2011) found that the size of microspheres in liquid culture also affects the industrial fermentation of protein.

Safety of A. niger

A. niger is commonly perceived as a harmless microorganism, and numerous A. niger variants hold the GRAS designation, indicating their overall safety is acknowledged. The completion of A. niger genome has led to numerous studies showing that like other filamentous fungi, A. niger produces various secondary metabolites and small amounts of mycotoxins (Frisvad et al. 2018), which can be associated with allergies or fungal-related conditions. This finding highlights the importance of not overlooking safety concerns associated with A. niger simply because it has GRAS certification. Even though it has low mycotoxin content, conducting a safety assessment for A. niger strains approved for food enzyme production is still crucial. This assessment is necessary because different strains of A. niger can synthesize various secondary metabolites, including fumonisins and ochratoxin A, which are considered remarkable carcinogenic mycotoxins (Susca et al. 2014). In conclusion, A. niger strains should be tested for mycotoxins before they are used as food-grade hosts. Diminishing or eradicating the capacity of A. niger to produce mycotoxins is imperative to ensure the safety of food-grade strains.

Research progress of microbial lipase expression in A. niger

Biotechnology of lipase expression in A. niger

The five processes involved in creating lipase engineering strains of A. niger are illustrated in Fig. 2, namely, include plasmid synthesis, target gene transformation, transformant identification, isolation of individual spores, and fermentation-based protein expression.

Fig. 2.

Fig. 2

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Diagram of constructing engineering Aspergillus Niger for producing lipase

Three transformation methods are commonly used for A. niger agrobacterium-mediated transformation (AMT; Li et al. 2017), electroporation (Rivera et al. 2014), and polyethylene glycol (PEG)-mediated transformation (PMT Ballance et al. 1983; Tilburn et al. 1983). AMT is a method of cocultivating Agrobacterium tumefaciens and A. niger. The method is less demanding on the state and form of A. niger and is suitable for wild-type A. niger transformation (Michielse et al. 2005). One drawback of this transformation approach is the tedious procedures and the lengthy duration of the experimental cycle. Low-frequency transformation is the foundation of the electroporation, where strong electric fields induce the formation of transient reversible pores in the cellular membrane of A. niger spores once they have begun to germinate. This approach makes entering the cells easier for the targeted DNA (He et al. 2017). Electroporation of A. niger MGG029-aamA results in 50 transformants per microgram of DNA (Li et al. 2010). PMT uses divalent cation Ca2+ as a signal molecule to introduce exogenous target DNA into A. niger protoplasts. The key to improving PMT transformation efficiency is to prepare high-quality A. niger protoplasts (Li et al. 2020). Various enzymes have to enhanced the preparation efficiency of protoplasts by optimizing the enzymatic hydrolysis system and conditions. However, the delicate nature of the protoplasts makes preserving them impractical (Chen et al. 2017).

Using genetic markers to distinguish positive transformants from a large population of untransformed cells (protoplasts) is necessary because genetic transformation occurs infrequently. Tags often utilized in genetic modification screening encompass resistance markers such as Hph and Bar as well as auxotrophic markers such as pyrG (Sen et al. 1969), pyrA (Goosen et al. 1987), args (Lenouvel et al. 2002), hisB (Fiedler et al. 2017), along with nutritional tags such as amdS and ptrA. Common resistance gene markers for A. niger include oligomycin, bleomycin, and hygromycin B. Most fungi demonstrate higher sensitivity to hygromycin B compared with other antibiotics. Utilizing auxotrophy as a selection marker is superior to resistance selection (Nguyen et al. 2016; Navarrete et al. 2009) because it avoids the use of antibiotics and does not require the establishment of minimum drug inhibitory concentrations.

The screening of microorganisms for lipase production is usually performed using the pour plate method on a standard potato dextrose agar medium supplemented with tributyrin or olive oil. Microbial strains with the most comprehensive clear zones are positive (D’Annibale et al. 2006). Due to the thick cell wall of A. niger, identifying positive transformants through simple colony PCR (Polymerase Chain Reaction) can be challenging. Therefore, extracting the genome of A. niger and performing PCR identification can achieve more accurate results. In A. niger, performing a single spore isolation step is crucial to identify positive transformants because colonies of genetically modified filamentous fungi can be prone to contamination by unmodified hyphae. Microorganisms are currently a reliable source of lipase for industrial use, and the ability of microorganisms to produce lipase varies substantially with different strains and fermentation conditions. Lipase can be fermented through three distinct methods: liquid-state fermentation, solid-state fermentation, and immobilized cell fermentation. Patrick et al. (1969) utilized olive oil and peptone as carbon and nitrogen sources, and they observed that the lipase activity in supplemental batch fermentation increased sixfold compared with regular batch fermentation.

Optimization of lipase expression in A. niger

Four steps are involved in the term "extracellular proteins" in filamentous fungi: transcription, translation, processing, and secretion. The efficiency of each stage is influenced by numerous factors and remarkably affects the final output of extracellular proteins. Various methods have been used to enhance the expression of A. niger lipase including utilizing multicopy promoters and target genes, expressing gene fusion, optimizing codons, and incorporating molecular chaperones.

A. niger utilizes a diverse range of promoters, which are listed in Table 3. The A. niger glucoamylase promoter PglaA and the A. niger neutral amylase promoter PamyA are frequently utilized as inducible strong promoters in A. niger. These promoters have proven effective in driving gene expression and are frequently employed in biotechnological applications or genetic engineering experiments to enhance the production of desired proteins or enzymes. Zhao Shufan et al. (2022) and others cloned eight lipase genes in A. niger for homologous expression and used the strong promoter PglaA to achieve high expression. Li Jie et al. (2018) used six strong promoters PglaA and high-efficiency secretion signal peptide SglaA to control the expression of the A. niger lipase gene. The food-grade A. niger engineered bacteria has an enzyme activity of 1480 U·mL−1. The PgpdA promoter, derived from the gene encoding 3-phosphate glyceraldehyde dehydrogenase, demonstrates strong and constant activity, enabling continuous transcriptional activity throughout the cell cycle. Furthermore, protein expression regulated by PgpdA does not necessitate stringent cultivation conditions or specific medium components (Kluge et al. 2018).

Table 3.

Different promoters are employed in Aspergillus niger expression systems to produce recombinant proteins

Promoter Induction Accession number Product Gene source Yield References
glaA

Maltose,

starch

 XM_001390493.3 CALB Candida antarctica 440 U/mL Pan et al. (2014)
Glucoamylase-hIL6 synthetic construct 15 mg/L Broekhuijsen et al. (1993)
Human interleukin-6 (hIL6) Aspergillus niger 25 ng/ml Carrez et al. (1990)
Manganese peroxidase (MnP) Aspergillus niger 100 mg/L Conesa et al. (2000)
Human interleukin-6 (hIL-6) Aspergillus niger 5 mg/L Contreras et al. (1991)
Hen egg-white lysozyme (HEWL) Aspergillus niger 1 g/L Jeenes et al. (1993)
Feruloyl esterase A (FAEA), dockerin Aspergillus niger 1.7 nkat/mL Levasseur et al. (2004)
Active human mucus proteinase inhibitor((MPI)) Aspergillus niger 3 mg/L Mikosch et al. (1996)
Pyranose dehydrogenase (PDH) Aspergillus niger 55.9 U/mg Pisanelli et al. (2010)
Porcine pancreatic prophospholipase A2 (proPLA2) Aspergillus niger 10 μg/ml Roberts et al. (1992)
Humanized immunoglobulin G1(κ) antibodies Aspergillus niger 0.9 g/L Ward et al. (2004)
gpdA Constitutive XM_050611187.1 Hen eggwhite lysozyme (HEWL) Aspergillus nidulans 1 mg/L Jeenes et al. (1993)
Trametes versicolor laccase Aspergillus 2700 U/L Bohlin et al. (2006)
Laccase Aspergillus nidulans – Record et al. (2002)
Humanα1-proteinase inhibitor (α1-PI) – 12 mg/L Karnaukhova et al. (2007)
Pna II/tpi

Maltose,

starch

Genozymes ID:

Corse1p7_008350

 Trehalase Myceliophthora sepedonium 406.44 U/m L Dong et al. (2020a)
CALB Candida antarctica 117 U/mL Pan et al. (2014)
amdS

Acetamide,

acetate,

ω-amino acids

 XM_676954.2 Enterotoxin subunit B (LTB) Aspergillus nidulans 1.67 ± 0.12 (n = 3) ng / µg Turnbull et al. (1989)
A cell surface glycoprotein (Bm86) Aspergillus nidulans 1.8 mg /L Turnbull et al. (1990)
amy (taka)

Starch,

maltose,

maltodextrin

 – Lipase Aspergillus oryzae

1.60 ± 0.81 KU/g DW/h

(maltose-limited chemostats)

1.10 ± 0.41 KU/g DW/h

(glucose-limited chemostats)

 Prathumpaiet al. (2004)
aphA Phosphate XM_001396276.3 Human Interferon α-2 (hIFNα2) protein Aspergillus niger – MacRae et al. (1993)
sucA/pkiA Constitutive XM_050611187.1 Recombinant proteins Aspergillus niger – Roth and Dersch (2010)
alcA/aldA

Ethanol,

methionine

 – Peroxidase Pleurotus eryngii 412 U /L Eibes et al. (2009)
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Regarding heterologous protein expression in filamentous fungi, two commonly used signal peptides are the glaA signal peptide derived from A. niger and the cbhI signal peptide derived from Trichoderma reesei. However, in some studies, exogenous signal peptides are less effective compared with self-signal peptides (Xu et al. 2018). In many research investigations, the predominant strategy for improving expression involves augmenting the gene target's replication count (Nijland et al. 2010). Once the gene copy number reaches a saturation point, any subsequent amplification may lead to a decline in expression levels, because excessive copies of the gene can lead to competition for cellular resources and limitations in transcriptional machinery, ultimately impairing the overall expression of the gene. Therefore, optimizing the gene copy number is crucial to ensuring optimal expression levels in genetic engineering or biotechnological applications. Moreover, the random insertion of genes into various locations while maintaining the same copy number can have considerable effects on gene expression, such as the silencing of genes and the duplication of copies (Hammond and Keller 2005). In 2012, the German Meyer research group successfully acquired a strain of A. niger that contained multiple copies of the amylase gene. This genetic modification led to a remarkable increase in amylase production, which was seven times higher than that of the wild-type strain (Kwon et al. 2012).

The stability of mRNA is crucial in determining the expression level of heterologous genes. Fusion expression can be a beneficial strategy to enhance mRNA stability (Dunncoleman et al. 1991). When the integration position is close to an actively transcribed gene, it not only improves the strength of the target gene's mRNA but also overcomes the limitations of the early protein secretion pathway (Ward et al. 1995). The existence of introns in fungal strains promotes mRNA accumulation and controls gene transcription and post-transcriptional processes. Studies have proposed two possible mechanisms is intron-dependent mRNA accumulation, where the presence of introns provides binding sites, resulting in a more stable secondary structure and protecting precursor mRNA factors from degradation, such as oligo U-binding protein or heterogeneous nuclear ribonucleoprotein (Morello et al. 2002; Sivak et al. 1999). The other mechanism involves the assembly of partial elements to introns in pre-mRNA, which increases mRNA integrity or assists mRNA trafficking (Xu and Gong 2003). Approximately 87% of A. niger genes contain introns, averaging 2.6 per gene (Wang et al. 2009). In recent years, the effect of introns on A. niger protein expression has attracted attention. Studies have shown that intron deletion can remarkably inhibit the expression of endogenous acid lipase in A. niger (Zhu et al. 2020a, b). Despite progress, many non-fungal proteins in A. niger still show minimal or no efficacy (Lubertozzi and Keasling 2009).

Homologous and heterologous protein production in filamentous fungi is frequently hindered by the high expression levels of proteases, which can have a detrimental effect on the yield of the desired proteins. One cause of low yields is proteolytic degradation of the target proteins by host proteases (Wang et al. 2016). The degradation of heterologous proteins in A. niger is substantially influenced by additional proteases. The analysis of the protease spectrum from A. niger reveals the existence of four proteases responsible for acid production: aspartyl proteases (PEPA and PEPB) (Mattern 1992) and both serine carboxypeptidases (PEPF or CPDII, and PEPG or CPDI; Degan et al. 1992). PEPA protease comprised approximately 85% of the total protease activity (Mattern 1992). Yields were improved by deleting the major aspartyl protease (pepA) of the host. PEPB protease accounted for 6% of the total protease activity, and PEPE protease comprised approximately 68% of intracellular protease activity (van den Hombergh et al. 1995). Other than PepA, the degradation of heterologous proteins in A. niger is remarkably influenced by additional proteases, including PepAa, PepAb, PepAc, and PepAd (Mattern 1992). Van den Hombergh et al. (1995) successfully disrupted the function of the pepA, pepB, and pepE genes in the high-expression pectinase strain of A. niger, resulting in a considerable increase in pectinase activity (Fig. 3). This discovery presents compelling proof of the participation of the proteases encoded by these three genes in the degradation of pectinase in A. niger. MacKenzie et al. (2000) discovered that destroying pepA increases glucoamylase, while Moralejo et al. (2000) found that destroying pepA also increases thaumatin. Additionally, there are research results from multiple studies (Sun et al. 2004; Zhao et al. 2005; Wang et al. 2008) showing that destroying pepA, pepB, and pepD increases laccase activity.

Fig. 3.

Fig. 3

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Impact of A. niger protease deficiency on target proteins. The red words represented protease gene and NCBI gene number. The round with letters symbolizes secreted proteins. Proteins A, B, D, F, G and H are secreted outside the cell

The expression levels of 32 genes that encode proteases in A. niger HL-1, encompassing peptidases from the serine, aspartate, and aminopeptidase subclasses, along with carboxypeptidases, are under the control of the PrtT protein (Vanden Hombergh et al. 1997; Huang et al 2020). Subsequent deletion of the prtT gene resulted in a notable suppression of the expression of pepA, PepB, serine carboxypeptidase, and subtilisin-like serine protease in A. niger. Additionally, the 32 proteases' promoters and their activator, PrtT, comprise AmyR-binding sites. Overexpression of amyR could reduce protease levels by suppressing PrtT transcription and its associated proteases (Kamaruddin et al. 2018).

Compared with the natural genetic variation process, mutagenesis breeding can promote the high-frequency variation of organisms with the help of external factors, which is conducive to selecting target strains with excellent traits and is an efficient practical method in strain breeding (Han et al. 2020). Approaches to induce mutations encompass physical, chemical, and combined mutagenesis techniques. Commonly used chemical mutagens (Cheng et al. 2009) are nitrosoguanidine (NTG), diethyl sulfate (DES), ethyleneimine (EI), nitrosoethyl carbamate (NMU), methanesulfonate, and ethyl acetate. Jia et al. (2016) used A. niger G27 as the initial strain to prepare protoplasts. The prepared protoplasts were mutated with NTG, and the lipase activity of the P316 strain was (394.3 ± 12.9) U/mL, which was 22.9% higher than that of the original strain. Meng et al. (2010) obtained a high-yielding lipase-producing A. niger mutant strain CM2 with stable genetic traits through combined mutagenesis with diethyl sulfate and microwaves, and the enzyme activity was as high as 174.93 U/mL. Commonly used physical mutagenesis techniques include ultraviolet, laser, microwave, and atmospheric and room- temperature plasma (Toscano et al. 2011). Yu Long et al. (2017) screened Saccharomyces cerevisiae with high alcohol production by combined mutagenesis with ultraviolet light and ion implantation. Ji Yizhi et al. (2008) used protoplast laser mutagenesis of Rhizopus aureus to screen lipase-producing bacteria. After mutation, the lipase activity of the Rhizopus strain was 151 U/mL, 1.41 times higher than before mutagenesis. Zhao Xingxiu et al. (2015) employed ultraviolet and microwave mutagenesis techniques to isolate a highly lipase-producing strain. The resulting mutant strain showed an astounding 46.85% increase in lipase activity compared with the original. Compared with the traditional mutagenesis technique, this technique has a higher positive mutation rate and is more accessible to screen out strains that meet the research purpose.

The rapid advancements in gene editing have played a crucial role in enhancing our improvement of the operational mechanisms of A. niger cell factories. Consequently, the tools and knowledge for comprehensively analyzing and improving the functioning of these cell factories are available, leading to more efficient results. The promising technology of CRISPR/Cas9 has recently gained prominence (Wang et al. 2016). The complete capacity of A. niger cell factories can be unlocked, and their contributions to the biotechnology domain can be maximized. Initially, Kleinstiver et al. (2016) successfully identified the Cas9 mutant SpCas9-HF1, which exhibits heightened recognition specificity, consequently mitigating the risk of off-target effects. Second, editing efficiency can be substantially enhanced by opting for sgRNAs with maximum specificity and minimal off-target risks (Xie et al. 2014). Shen et al. (2019) reduced the risk of off-target effects by designing a precise artificial switch that self-regulates the expression of sgRNA and Cas9 protein. This genetic engineering technique has been used to modify the genetic makeup of filamentous fungi, such as Fusarium fujikuroi, Myceliophthora, A. niger, and others (Table 4).

Table 4.

Application of CRISPR–Cas9 in filamentous fungi hosts

Host Product Yield Improve CRISPR/Cas9 Expression Gene disruption References

thermophilic fungal

Myceliophthora

 Lignocellulase 1300 U/mL 13-fold U6 snRNA- promoter Heterologous

cre-1,

res-1,

gh1-1,

alp-1

 Liu et al. (2017)
Fusarium fujikuroi Gibberellic acids 4 and 7 716.37 mg/L 8.1-fold 5S rRNA-promoter Homologous

fcc1,

ura3,

ppt1

 Shi et al. (2019)
Aspergillus niger Thermostable trehalase 1698.83 U/mL 8.9-fold U6 snRNA- promoter Heterologous amyA Dong et al. (2020a, b)
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The gene kusA is accountable for nonhomologous end-joining. By interfering with the kusA gene in wild-type A. niger, the efficiency of homology directed repair remarkably improves. The homologous integration efficiency increases from 7% to more than 80% (Meyer et al. 2007). As a result, the heightened vulnerability to UV irradiation greatly facilitated the UV-induced mutagenesis of A. niger strains. Importantly, this robust gene knockout system will remarkably contribute to developing A. niger as commercially valuable cell factory. These strategies offer efficient solutions for mitigating the off-target effects of A. niger genome editing.

Conclusion

This article reviews the research progress on the production of homologous and heterologous lipases by A. niger cell factories. The initial construction and improvement strategies of the A. niger secretion cell factory is outlined. When A. niger heterologously expresses secreted lipase, the yield is often low. Therefore, genetically modifying the A. niger host and conducting in-depth research on the mechanism of A. niger expression, processing, and secretion of proteins are necessary. In the study of genetic modification technology of filamentous fungi, gene knockout technology (transposon queue and homologous recombination), gene editing technology (Cre/Loxp, FLP/FRT, and CRISPR/Cas), and gene knockdown technology have been applied (RNAi). The application of CRISPR systems to filamentous fungal cell factories is still in its infancy. The current research focus in this field is on establishing CRISPR/Cas9 technology systems in different filamentous fungal species and improving their editing efficiency. Thus far, no industrial strains constructed through CRISPR/Cas9 genome editing technology have been industrialized. In the future, establishing a universal editing technology system that is not limited to a certain fungal species and combining the CRISPR system with high-throughput technology for high-throughput editing and transformation is a good research direction. As the research progresses, the genetic modification of A. niger will become more successful. A. niger, as a cell factory, will undoubtedly achieve remarkable strides in secreted protein expression, which is critical to advancing the creation of industrial enzymes.

Acknowledgements

This research was financially supported by the Technology Innovation and Application Development Special Key Project of Chongqing (cstc2021jscx-jbgsX0002).

Author contribution

Conceptualization: JZ and ZW; writing original draft preparation: HN; writing review and editing: YZ, ML, WW and ML; funding acquisition: JZ; supervision: JZ.

Data availability

All relevant data are within the paper.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

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