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. 2024 Dec 22;17:149. doi: 10.1186/s13068-024-02596-0

Enhancing D-lactic acid production by optimizing the expression of D-LDH gene in methylotrophic yeast Komagataella phaffii

Yoshifumi Inoue 1, Ryosuke Yamada 1,✉, Takuya Matsumoto 1, Hiroyasu Ogino 1
PMCID: PMC11665205  PMID: 39710696

Abstract

Background

Currently, efficient technologies producing useful chemicals from alternative carbon resources, such as methanol, to replace petroleum are in demand. The methanol-utilizing yeast, Komagataella phaffii, is a promising microorganism to produce chemicals from methanol using environment-friendly microbial processes. In this study, to achieve efficient D-lactic acid production from methanol, we investigated a combination of D-lactate dehydrogenase (D-LDH) genes and promoters in K. phaffii. The yeast strain was constructed by integrating a gene cassette containing the identified gene and promoter into the rDNA locus of K. phaffii, followed by post-transformational gene amplification. Subsequently, D-lactic acid production from methanol was evaluated.

Results

Among the five D-LDH genes and eight promoters tested, the combination of LlDLDH derived from Leuconostoc lactis and CAT1 and FLD1 promoters was suitable for expression in K. phaffii. GS115_CFL/Z3/04, the best-engineered strain constructed via integration of LlDLDH linked to CAT1 and FLD1 promoters into the rDNA locus and post-transformational gene amplification, produced 5.18 g/L D-lactic acid from methanol. To the best of our knowledge, the amount of D-lactic acid from methanol produced by this engineered yeast is the highest reported value to date when utilizing methanol as the sole carbon source.

Conclusions

This study demonstrated the effectiveness of combining different enzyme genes and promoters using multiple promoters with different induction and repression conditions, integrating the genes into the rDNA locus, and further amplifying the genes after transformation in K. phaffii. Using our established method, other K. phaffii strains can be engineered to produce various useful chemicals in the future.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13068-024-02596-0.

Keywords: D-Lactic acid, Komagataella phaffii, Methanol, Metabolic engineering, Yeast

Background

Currently, technologies producing various useful chemicals from alternative carbon resources to replace petroleum are in demand [1]. Methanol has attracted considerable attention as an alternative carbon source as it is a liquid at room temperature, easy to transport, and stable [2]. In recent years, development of new production technologies with low environmental impact, such as the production of methanol from CO2 and methane, has increased [3, 4]. Currently, production of chemicals from methanol is performed using chemical processes with high environmental impact, and methanol is converted into various chemical products via ethylene and propylene.

Chemical processes require high-temperature and high-pressure conditions, exhibit low reaction specificities, and are not suitable for producing compounds with complex structures or high optical purity. In contrast, chemical production using microbial metabolism allows reactions to proceed under mild conditions and facilitates the selective production of compounds with high reaction specificity and optical purity. Because of these advantages, research on the production of useful chemicals from methanol using methanol-utilizing microorganisms has increased in recent years [5]. Many microorganisms can utilize methanol, including bacteria, such as Methylobacterium extorquens and Bacillus ethanolicus, and yeasts, such as Komagataella phaffii (formerly known as Pichia pastoris), Candida boidinii, and Ogataea polymorpha. These microorganisms metabolize methanol via the serine, ribulose phosphate, and xylose phosphate pathways [6–8]. These methylotrophic microorganisms can be used to produce useful compounds, such as mevalonic acid [9] and fatty alcohols [10], from methanol.

Lactic acid is used as a chemical in the food and pharmaceutical industries and as a raw material for polylactic acid, a biodegradable plastic [11–14]. Thermal stability of the polymer is improved when the enantiomers, poly(L-lactic acid) and poly(D-lactic acid), are mixed to form a stereocomplex structure [15]. Consequently, the demand for both L- and D-lactic acids is increasing [16], but the availability of D-lactic acid is lower than that of L-lactic acid in the market as D-lactic acid is more expensive. Therefore, an efficient method for D-lactic acid production is required.

K. phaffii, a type of methanol-utilizing yeast, is safe and used in well-established genetic engineering techniques as a host for protein production [17]. Recently, research on the development of recombinant K. phaffii capable of producing useful chemicals, such as malic acid [18] and lycopene [19], from methanol is attracting attention [20]. In K. phaffii, integration of a multicopy plasmid into the non-transcribed spacer of the ribosomal DNA (rDNA) locus leads to high mitotic stability and gene expression [21]. Additionally, a method for increasing gene expression based on the integration of genes into the rDNA locus and amplification of the gene copy number after transformation via repeated selection in media containing increasing antibiotic concentrations has been reported [22]. Lopes et al. reported that intracellular protein production is highly correlated with the copy number of the gene encoding the protein [23]. Yamada et al. first reported the production of D-lactic acid from methanol by integrating multiple copies of a gene cassette linking the AOX1 promoter to the D-lactate dehydrogenase (D-LDH) gene derived from Leuconostoc mesenteroides at the rDNA locus [24]. However, the maximum reported titer of D-lactic acid from methanol is 3.48 g/L, which is lower than that obtained with sugar as the carbon source [25, 26].

Gene expression can be improved by optimizing the origin of the expressed gene and combination of the promoter, terminator, and secretion signal sequences. Zhong et al. [27] and Watcharawipas et al. [28] investigated the expression of D-LDH gene in yeast Saccharomyces cerevisiae and reported that the origin of D-LDH gene affects D-lactic acid production. Furthermore, previous studies reported that the combination of promoters, terminators, and secretory signal sequences in S. cerevisiae and K. phaffii significantly affects lipase expression [29, 30].

In this study, we investigated the combination of five D-LDH genes of different origins and eight promoters in K. phaffii to achieve efficient D-lactic acid production from methanol. The five D-LDH genes were LdDLDH (Lactobacillus delbrueckii) [31], LmDLDH (L. mesenteroides) [24], LlDLDH (L. lactis) [28], LpDLDH (L. pseudomesenteroides) [28], and SlDLDH (Sporolactobacillus laevolacticus) [32]. These D-LDH genes are also expressed in S. cerevisiae or Escherichia coli [28, 33, 34]. The eight promoters consist of four methanol-inducible promoters (AOX1 promoter [pAOX1], CAT1 promoter [pCAT1], mutant CAT1 promoter [pCATm] [35], and FLD1 promoter [pFLD1] [36]) and four constitutive promoters (GAP1 promoter [pGAP1], ADH3 promoter [pADH3] [37], mutant GAP1 promoter [pGAP1m] [38], and TEF1-α promoter [pTEF1] [39]). Using these combinations, we identified the D-LDH gene and promoter combination suitable for expression in K. phaffii. Finally, the engineered yeast was constructed by integrating a gene cassette containing the identified gene and promoter into the rDNA locus of K. phaffii, followed by post-transformational gene amplification, and D-lactic acid production from methanol was evaluated.

Methods

Strains and media

E. coli strain NEB5α (New England Biolabs Japan, Tokyo, Japan) was used as a host for recombinant DNA manipulation. Genetically modified E. coli cells were cultured in the Luria–Bertani medium (20 g/L Luria–Bertani broth powder [Nacalai Tesque, Kyoto, Japan]) supplemented with 100 μg/mL ampicillin sodium salt (Nacalai Tesque).

K. phaffii GS115 (Thermo Fisher Scientific, Yokohama, Japan) was used as the host for D-lactic acid production. K. phaffii cells were cultured in the yeast/peptone/glucose (YPD) medium (10 g/L yeast extract [Formedium, Norfolk, UK], 20 g/L peptone [Formedium], and 20 g/L glucose [Nacalai Tesque]), yeast/peptone/methanol (YPM) medium (10 g/L yeast extract, 20 g/L peptone, and 30 g/L methanol [Nacalai Tesque]), yeast/peptone/dextrose/sorbitol (YPDS) medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, and 1 mol/L sorbitol [Nacalai Tesque]), RDB medium (182 g/L sorbitol, 10 g/L glucose, 13.4 g/L yeast nitrogen base without amino acid [Formedium], and 4 × 10–4 g/L biotin [Nacalai Tesque]), MD medium (13.4 g/L yeast nitrogen base without amino acids, 20 g/L glucose, and 4 × 10–4 g/L biotin), or MM medium (13.4 g/L yeast nitrogen base without amino acids, 30 g/L methanol, and 4 × 10–4 g/L biotin). As required, 20 g/L agar (Nacalai Tesque) and a predetermined amount of zeocin (InvivoGen, San Diego, CA, USA) were added.

Yeast cultivation

Ninety-six deep-well culture was performed using 1 mL of YPM medium and Deep Well Maximizer (Taitec, Nagoya, Japan) at 30 °C and 1200 rpm. Cultivation was initiated by culturing the cells in a well containing 1 mL of YPD medium at 30 °C and 1200 rpm for 24 h, harvesting and washing the cells, and suspending them in 1 mL of fresh medium.

Test tube cultures were performed using 5 mL of the medium in test tubes (165 mm height and 16 mm diameter) and reciprocating shaker (Taitec) at 30 °C and 150 rpm. Cultures were initiated by inoculating the precultures grown in test tubes containing the MD medium at 30 °C and 150 rpm for 72 h (initial OD600: 3.0).

Flask cultures were performed using a rotary shaker (Taitec) at 30 °C and 200 rpm, with 250-mL flasks equipped with a gas-permeable seal (EXCEL scientific, Victorville, CA, USA) containing 50 mL of medium. Cultures were initiated by inoculating (initial OD600: 11.0) the precultures grown in 250 mL flasks containing the YPD medium for 24 h at 200 rpm and 30 °C.

Plasmid construction and yeast transformation

All plasmids and primers used in this study are listed in Table 1 and Table S1, respectively. pPPE_AOX_XDLDH (X, Ld, Ll, Lp, or Sl), the plasmid used for D-LDH expression, was constructed as described below. Plasmid pPPE [30] was linearized using the restriction enzymes, EcoRI and NotI. Fragments of D-LDH gene from L. mesenteroides linked to pAOX1 were obtained via polymerase chain reaction (PCR) amplification using plasmid pRDZ_AO_DLDH [24] as the template and pAOX_pPPE(F)_ASS and LmeDLDH_pPPE(R)_ASS as the primers. The resulting gene fragments were ligated using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs Japan) to construct the pPPE_AOX_LmDLDH plasmid.

Table 1.

List of plasmids used in this study

Plasmid name Relevant features
pPPE_AOX_Ld Episomal plasmid for expression of LdDLDH (derived from Lactobacillus delbrueckii) by AOX1 promoter
pPPE_AOX_Ll Episomal plasmid for expression of LlDLDH (derived from Leuconostoc lactis) by AOX1 promoter
pPPE_AOX_Lm Episomal plasmid for expression of LmDLDH (derived from Leuconostoc mesenteroides) by AOX1 promoter
pPPE_AOX_Lp Episomal plasmid for expression of LpDLDH (derived from Leuconostoc pseudomesenteroides) by AOX1 promoter
pPPE_AOX_Sl Episomal plasmid for expression of SlDLDH (derived from Sporolactobacillus laevolacticus) by AOX1 promoter
pPPE_ADH_Ll Episomal plasmid for expression of LlDLDH by ADH3 promoter
pPPE_CAT_Ll Episomal plasmid for expression of LlDLDH by CAT1 promoter
pPPE_CATm_Ll Episomal plasmid for expression of LlDLDH by mutant CAT1 promoter
pPPE_FLD_Ll Episomal plasmid for expression of LlDLDH by FLD1 promoter
pPPE_GAP_Ll Episomal plasmid for expression of LlDLDH by GAP1 promoter
pPPE_GAPm_Ll Episomal plasmid for expression of LlDLDH by mutant GAP1 promoter
pPPE_TEF_Ll Episomal plasmid for expression of LlDLDH by TEF1 promoter
pRDZ_CAT_Ll Multicopy integrative plasmid for expression of LlDLDH by CAT1 promoter
pRDZ_FLD_Ll Multicopy integrative plasmid for expression of LlDLDH by FLD1 promoter
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Genes encoding LdDLDH, LlDLDH, LpDLDH, and SlDLDH were PCR-amplified using synthetic genes (Twist Bioscience, South San Francisco, CA, USA) as templates and LdDLDH(F)_ASS and LdDLDH(R)_ASS, LlDLDH(F)_ASS and LlDLDH(R)_ASS, LpDLDH(F)_ASS and LpDLDH(R)_ASS, and SlDLDH2Mu(F)_ASS and SlDLDH2Mu(R)_ASS, respectively, as primers. pPPE_AOX_LmDLDH was linearized using the restriction enzymes, BamHI and NotI. The resulting gene fragments were used to construct the pPPE_AOX_LdDLDH, pPPE_AOX_LlDLDH, pPPE_AOX_LpDLDH, and pPPE_AOX_ SlDLDH plasmids, respectively, by ligating the gene fragments using the NEBuilder HiFi DNA Assembly Master Mix.

pPPE_X_LlDLDH (X; ADH, CAT, CATm, FLD, GAP, GAPm, TEF), the plasmid used for promoter comparison, was constructed as described below. PCR amplification was performed using pPPE_AOX_LlDLDH as the template and pPPE_Ll(F)_ASS and pPPE_Ll(R)_ASS as the primers. The genes encoding pADH3, pCAT1, pFLD1, pGAP1, and pTEF1 were PCR-amplified using K. phaffii genomic DNA as the template and ADH3(F)_ASS and ADH3(R)_ASS, CAT1(F)_ASS and CAT1(R)_ASS, FLD1(F)_ASS and FLD1(R)_ASS, GAP1(F)_ASS and GAP1(R)_ASS, and TEF1(F)_ASS and TEF1(R)_ASS, respectively, as the primers. The genes encoding pCAT1m and pGAP1m were PCR-amplified using CAT1(F)_ASS and CAT1(R)_ASS and GAP1(F)_ASS and GAP1(R)_ASS with synthetic genes (Twist Bioscience) as the templates. The gene fragments were ligated using the NEBuilder HiFi DNA Assembly Master Mix to construct the pPPE_X_LlDLDH plasmid.

Multicopy integration plasmids pRDZ_CAT_LlDLDH and pRDZ_FLD_LlDLDH for D-LDH expression were constructed as described below. PCR amplification was performed using prDZ_AO_DLDH as the template and pRDZ_CAT(F)_ASS and pRDZ_CAT(R)_ASS and pRDZ_FLD(F)_ASS and pRDZ_FLD(R)_ASS, respectively, as the primers. Gene fragments containing pCAT1 and pFLD1 were PCR-amplified using pPPE_CAT_LlDLDH and pPPE_FLD_LlDLDH, respectively, as the templates and pRDZ_CAT_Ll(F)_ASS and pRDZ_CAT_Ll(R)_ASS and pRDZ_FLD_Ll(F)_ASS and pRDZ_FLD_Ll(R)_ASS, respectively, as the primers. The gene fragments were ligated using the NEBuilder HiFi DNA Assembly Master Mix to construct the pRDZ_CAT_LlDLDH and pRDZ_FLD_LlDLDH plasmids. The resulting plasmids were linearized using restriction enzyme AscI prior to transformation.

The resulting plasmids were transformed into K. phaffii GS115 using a previously described electroporation method [40]. Transformants were selected on the RDB medium containing 20 g/L agar or YPDS medium containing 20 g/L agar and 0.1 g/L zeocin.

Analyses of growth and metabolite levels

OD600 of each culture was determined by measuring the absorbance at 600 nm using a spectrophotometer (Shimadzu, Kyoto, Japan).

D-Lactic acid concentration was calculated based on NADH concentration determined from the absorbance at 340 nm using a partially modified enzymatic reaction method with D-LDH described by Rosenberg et al. [41]. Briefly, the culture broth was centrifuged at 10,000 × g for 2 min at 4 °C and diluted appropriately with water. Then, 140 μL of glycine-semicarbazide buffer (15 g/L glycine (Nacalai Tesque) and 22 g/L semicarbazide hydrochloride (Nacalai Tesque), adjusted to pH 8.7 using NaOH), 30 μL of 30 mM NAD+ (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 5 μL of 2 mg/mL D-LDH (Toyobo, Osaka, Japan), and 10 μL of diluted supernatant were mixed and incubated at 30 °C for 3 h. After the reaction, absorbance of the sample was measured at 340 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, US). D-Lactic acid concentration was calculated using a calibration curve plotted using D-lactic acid as the standard.

Methanol concentration was determined using a colorimetric method [42] using 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (AHMT) (Nacalai Tesque). The culture broth was centrifuged at 10,000 × g for 2 min at 4 °C, and the resulting culture supernatant was diluted appropriately. Then, 48 µL of the diluted culture supernatant was mixed with 2 µL of 200 mM potassium phosphate buffer (pH 8.0) containing 0.25 U alcohol oxidase (A2404; Sigma-Aldrich Japan, Tokyo, Japan) and incubated at 25 °C for 80 min. Subsequently, 50 µL of 5 mol/L potassium hydroxide and 50 µL of AHMT solution (5 g/L AHMT [Tokyo Chemical Industry, Tokyo, Japan] and 50 g/L hydrochloric acid [Nacalai Tesque]) were added to the reaction solution and incubated at 25 °C for 20 min. The reaction solution was then mixed with 250 µL of potassium periodate solution (7.5 g/L potassium periodate [Nacalai Tesque] and 0.2 mol/L potassium hydroxide [Nacalai Tesque]), and absorbance at 550 nm was measured. Methanol concentration was calculated based on the calibration curve plotted using methanol as the standard.

D-LDH activity in yeast cells was measured following the method described by Tokuhiro et al. [43]. Yeast cells were cultured in test tubes containing 5 mL of MM medium for 48 h, after which the culture medium was centrifuged at 10,000 × g and 4 °C for 1 min to collect the cells. The collected cells were washed twice with water and resuspended in 300 µL of 100 mM potassium phosphate buffer (pH 7.0). The cell suspension was mixed with 500 µL of zirconia beads (particle size: 0.45 mm) and passed through a bead crusher (Bio Medical Science, Tokyo, Japan) for 900 s. The cell lysate was centrifuged at 10,000 × g and 4 °C for 5 min, and the supernatant was collected. Then, 5 µL of the supernatant was mixed with 180 µL of 100 mM glycine NaOH buffer (pH 10.0), 10 µL of 0.5 M D-lactic acid, and 10 µL of 20 mM NAD+ solution, and the change in absorbance at 340 nm and 30 °C was measured. One unit of D-LDH activity was defined as the amount of enzyme releasing 1 µmol of NADH per minute at 30 °C.

Results

Effect of D-LDH gene type on D-lactic acid production

To express five D-LDH genes (LdDLDH, LlDLDH, LmDLDH, LpDLDH, and SlDLDH) under the control of pAOX1, pPPE_AOX_XDLDH plasmids were constructed by connecting pAOX1 to each D-LDH gene. These plasmids were transformed into K. phaffii GS115, resulting in strains designated as GS115_AOX_X (X; Ld, Ll, Lm, Lp, or Sl; Table 2). Figure 1 shows the D-lactic acid concentration in the culture after cultivating the GS115_AOX_X transformants in MM medium for 288 h. Compared with GS115_AOX_Lm (83 mg/L), which expressed the conventional LmDLDH gene, GS115_AOX_Ld (59 mg/L) and GS115_AOX_Sl (35 mg/L) exhibited lower D-lactic acid concentrations. In contrast, GS115_AOX_Ll (186 mg/L) and GS115_AOX_Lp (155 mg/L) exhibited high D-lactic acid concentrations (2.25- and 1.88-times higher than those of GS115_AOX_Lm, respectively). These results suggest that, among the five types of D-LDH examined, LlDLDH is the most suitable D-LDH for expression in K. phaffii.

Table 2.

List of yeast strains used in this study

Strain name Relevant features
GS115 Komagataella phaffii host strain
GS115_AOX_Ld GS115 harboring pPPE_AOX_Ld
GS115_AOX_Lm GS115 harboring pPPE_AOX_Lm
GS115_AOX_Ll GS115 harboring pPPE_AOX_Ll
GS115_AOX_Lp GS115 harboring pPPE_AOX_Lp
GS115_AOX_Sl GS115 harboring pPPE_AOX_Sl
GS115_ADH_Ll GS115 harboring pPPE_ADH_Ll
GS115_CAT_Ll GS115 harboring pPPE_CAT_Ll
GS115_CATm_Ll GS115 harboring pPPE_CATm_Ll
GS115_FLD_Ll GS115 harboring pPPE_FLD_Ll
GS115_GAP_Ll GS115 harboring pPPE_GAP_Ll
GS115_GAPm_Ll GS115 harboring pPPE_GAPm_Ll
GS115_TEF_Ll GS115 harboring pPPE_TEF_Ll
GS115_CL/Z1/*a GS115 transformed with pRDZ_CAT_Ll and separated using 0.1 g/L zeocin
GS115_CL/Z2/*a GS115 transformed with pRDZ_CAT_Ll and gene amplified using 0.5 g/L zeocin
GS115_CL/Z3/*a) GS115 transformed with pRDZ_CAT_Ll and gene amplified using 1.0 g/L zeocin
GS115_CL/Z4/*a) GS115 transformed with pRDZ_CAT_Ll and gene amplified using 2.0 g/L zeocin
GS115_FL/Z1/*a GS115 transformed with pRDZ_FLD_Ll and separated using 0.1 g/L zeocin
GS115_FL/Z2/*a) GS115 transformed with pRDZ_FLD_Ll and gene amplified using 0.5 g/L zeocin
GS115_FL/Z3/*a) GS115 transformed with pRDZ_FLD_Ll and gene amplified using 1.0 g/L zeocin
GS115_FL/Z4/*a) GS115 transformed with pRDZ_FLD_Ll and gene amplified using 2.0 g/L zeocin
GS115_CFL/Z1/*a) GS115 simultaneously transformed with pRDZ_CAT_Ll and pRDZ_FLD_Ll and separated using 0.1 g/L zeocin
GS115_CFL/Z2/*a) GS115 simultaneously transformed with pRDZ_CAT_Ll and pRDZ_FLD_Ll and gene amplified using 0.5 g/L zeocin
GS115_CFL/Z3/*a) GS115 simultaneously transformed with pRDZ_CAT_Ll and pRDZ_FLD_Ll and gene amplified using 1.0 g/L zeocin
GS115_CFL/Z4/*a) GS115 simultaneously transformed with pRDZ_CAT_Ll and pRDZ_FLD_Ll and gene amplified using 2.0 g/L zeocin
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a)*;1–90

Fig. 1.

Fig. 1

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Comparison of D-lactic acid production by engineered yeasts expressing D-lactate dehydrogenase (D-LDH) from five different microorganisms under the control of pAOX1

Effect of D-LDH-expressing promoter on D-lactic acid production

To select a suitable promoter to express LlDLDH, which is optimal for D-LDH gene expression in K. phaffii, pPPE_X_LlDLDH plasmids were constructed by linking LlDLDH to eight different promoters: four methanol-inducible type (pAOX1, pCAT1, pCATm, and pFLD1) and four constitutive type (pADH3, pGAP1, pGAP1m, and pTEF1) promoters. These plasmids were transformed into K. phaffii GS115, resulting in strains designated as GS115_X_Ll (X: AOX, CAT, CATm, FLD, ADH, GAP, GAPm, and TEF; Table 2). Figure 2A shows the time course of D-lactic acid concentration in the GS115_X_Ll transformants cultured in MM medium. D-Lactic acid production was confirmed in five strains: GS115_AOX_Ll, GS115_CAT_Ll, GS115_GAP_Ll, GS115_FLD_Ll, and GS115_TEF_Ll. In contrast, GS115_ADH_Ll, GS115_CATm_Ll, and GS115_GAPm_Ll produced small amounts of D-lactic acid. Among the five strains producing D-lactic acid, GS115_GAP_Ll produced 69 mg/L D-lactic acid for up to 48 h of cultivation, after which the concentration remained constant. GS115_TEF_Ll produced increased D-lactic acid concentration over time, but the concentration remained at 48 mg/L after 288 h, which was lower than that produced by GS115_AOX_Ll (186 mg/L), which expressed LlDLDH using conventional pAOX1. GS115_CAT1_Ll and GS115_FLD_Ll produced increased D-lactic acid concentrations for up to 48 h, reaching 185 and 182 mg/L, respectively. These values were 6.35- and 6.23-times higher than those produced by GS115_AOX_Ll (29 mg/L) at the same time point. After 288 h of cultivation, D-lactic acid levels by GS115_CAT1_Ll and GS115_FLD_Ll reached 212 and 220 mg/L, respectively, which were 1.24- and 1.29-times higher than those produced by GS115_AOX_Ll.

Fig. 2.

Fig. 2

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A Time course of D-lactic acid production by engineered yeast expressing LlDLDH under the control of eight different promoters. B D-LDH activity in the culture supernatant after 48 h of culture

Figure 2B shows the intracellular D-LDH activity of the GS115_X_Ll strains after 48 h of cultivation. D-LDH activities of GS115_CAT_Ll and GS115_FLD_Ll were 993 and 1026 U/mL-culture, respectively, which were higher than those of GS115_AOX_Ll (639 U/mL-culture). In general, a positive correlation was observed between D-LDH activity and D-lactic acid production in the top three strains with high D-LDH activity. In the three strains showing low D-lactic acid production (GS115_ADH_Ll, GS115_CATm_Ll, and GS115_GAPm_Ll), D-LDH activity was lower than that in GS115_AOX_Ll. These results indicate that pCAT1 and pFLD1 are suitable for the expression of LlDLDH in K. phaffii.

Construction of a strain with D-LDH gene integrated into the rDNA locus and selection of a D-lactic acid-producing strain

Examination of the types of D-LDH genes and promoters revealed that the use of LlDLDH as the D-LDH gene and pCAT1 or pFLD1 as the promoter was suitable for D-lactic acid production in K. phaffii. Therefore, multicopy integrative plasmids pRDZ_CAT_LlDLDH (containing LlDLDH and pCAT1) and pRDZ_FLD_LlDLDH (containing LlDLDH and pFLD1) targeting the rDNA locus were constructed (Table 1) and transformed into K. phaffii GS115. Transformants were separated on YPDS containing 0.1 g/L zeocin, and 90 colonies were randomly isolated. The transformants were named GS115_CL/Z1/* (transformed with pRDZ_CAT_LlDLDH), GS115_FL/Z1/* (transformed with pRDZ_FLD_LlDLDH), and GS115_CFL/Z1/* (transformed with identical amounts of pRDZ_CAT_LlDLDH and pRDZ_FLD_LlDLDH) (*1–90; Table 2). Figure 3 shows the concentration of D-lactic acid in the culture after 48 h of cultivation in a 96-well plate for each of the 90 strains. Among the transformants obtained using pRDZ_CAT_LlDLDH, the highest D-lactic acid-producing strains were GS115_CL/Z1/38 (2.83 g/L), GS115_CL/Z1/83 (2.89 g/L), GS115_CL/Z1/63 (2.96 g/L), and GS115_CL/Z1/70 (3.17 g/L). Similarly, among the transformants obtained using pRDZ_FLD_LlDLDH, the highest producing strains were GS115_FL/Z1/21 (4.00 g/L), GS115_FL/Z1/11 (4.02 g/L), GS115_FL/Z1/63 (4.30 g/L), and GS115_FL/Z1/33 (4.34 g/L). For the transformants constructed using a mixture of both plasmids, the highest producing strains were GS115_CFL/Z1/51 (3.03 g/L), GS115_CFL/Z1/44 (3.44 g/L), GS115_CFL/Z1/62 (3.71 g/L), and GS115_CFL/Z1/03 (4.07 g/L).

Fig. 3.

Fig. 3

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D-Lactic acid production by engineered yeast constructed via multicopy integration after 48 h of deep-well culture. GS115_CL/Z1/* possessed LlDLDH linked to pCAT1, GS115_FL/Z1/* possessed LlDLDH linked to pFLD1, and GS115_FL/Z1/* possessed LlDLDH linked to pCAT1 and pFLD1

Post-transformational gene amplification and selection of strains with high D-lactic acid production

Among the 90 strains obtained via transformation with the pCAT-containing plasmid, pRDZ_CAT_LlDLDH, in the presence of 0.1 g/L zeocin, the top four strains with the highest D-lactic acid production (GS115_CL/Z1/38, GS115_CL/Z1/63, GS115_CL/Z1/83, and GS115_CL/Z1/70; Fig. 3) were separately cultured in test tubes containing YPD medium and 0.1 g/L zeocin for 24 h. Approximately 150 cells were collected from each culture and mixed, and colonies were isolated on YPD agar medium with a zeocin concentration of 0.5 g/L. Ninety randomly isolated colonies were cultured in 96-deep-well plate containing YPM, and the top four strains with the highest D-lactic acid production were selected. The same procedure was performed in a stepwise manner on YPD medium containing 1.0 and 2.0 g/L zeocin. The strains that were subjected to gene amplification in YPD medium containing 0.5, 1.0, and 2.0 g/L zeocin were designated as GS115_CL/Z2/*, GS115_CL/Z3/*, and GS115_CL/Z4/* (*;1–90), respectively (Table 2). The same procedure was performed using the pFLD-containing plasmid pRDZ_FLD_LlDLDH or the pCAT-containing plasmid pRDZ_CAT_LlDLDH and pFLD-containing plasmid pRDZ_FLD_LlDLDH simultaneously and the strains were named as GS115_CL/Z2/*, GS115_CL/Z3/*, GS115_CL/Z4/* (*;1–90) or GS115_CFL/Z2/*, GS115_CFL/Z3/*, and GS115_CFL/Z4/* (*;1–90), respectively (Table 2).

D-Lactic acid concentrations of the strains obtained via the post-transformational gene amplification procedure described above and those of the strains before gene amplification (GS115_CL/Z1/*, GS115_FL/Z1/*, and GS115_CFL/Z1/*) cultured for 24 h in 96-deep-well plates are shown in Fig. 4A, B, and C. The values of the strains with the highest D-lactic acid production among the 90 selected strains at each zeocin concentration are shown in Fig. 4D.

Fig. 4.

Fig. 4

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D-lactic acid production by engineered yeast constructed via multicopy integration and post-transformational gene amplification after 48 h of deep-well culture. A Engineered strains possessing LlDLDH linked to pCAT1. B Engineered strains possessing LlDLDH linked to pFLD1. C Engineered strains possessing LlDLDH linked to both pCAT1 and pFLD1. D Comparison of strains showing the highest D-lactic acid production under each condition. Strains containing Z1 in their names were isolated in the presence of 0.1 g/L zeocin, and strains with Z2, Z3, and Z4 in their names were gene amplified in the presence of 0.5, 1.0, and 2.0 g/L zeocin, respectively

As shown in Fig. 4A, B, and D, when transformed with plasmids carrying pCAT1 or pFLD1, gene amplification at a zeocin concentration of 0.5 g/L improved D-lactic acid production. The strains showing the highest D-lactic acid production were CL/Z2/71 (11.12 g/L) and FL/Z2/38 (9.62 g/L). In contrast, when gene amplification was performed at increased zeocin concentrations of 1.0 and 2.0 g/L, D-lactic acid production was decreased in both transformants.

As shown in Fig. 4C and D, when two types of plasmids carrying pCAT1 or pFLD1 were transformed simultaneously, D-lactic acid production was improved via gene amplification with increasing zeocin concentrations from 0.1 to 0.5 and 1.0 g/L, respectively. The strains showing the highest D-lactic acid production in the presence of 0.5 and 1.0 g/L zeocin were CFL/Z2/57 (6.80 g/L) and CFL/Z3/04 (10.34 g/L), respectively. However, when gene amplification was performed in the presence of 2.0 g/L zeocin, D-lactic acid production was decreased.

Time course of D-lactic acid production by post-transformational gene-amplified strains in a flask culture

To evaluate the culture behavior in flasks of strains GS115_CL/Z2/71 (transformed by plasmid carrying pCAT1 and gene amplified in the presence of 0.5 g/L zeocin), GS115_FL/Z2/38 (transformed by plasmid carrying pFLD1 and gene amplified in the presence of 0.5 g/L zeocin), and GS115_CFL/Z3/04 (transformed by plasmids carrying pCAT1 and pFLD1 and gene amplified in the presence of 1.0 g/L zeocin), which exhibited high D-lactic acid production in 96-deep-well plates (Fig. 4), they were cultured in flasks containing the YPM medium. The methanol concentrations, D-lactic acid concentrations, and OD600 values of the strains are shown in Fig. 5A, B, and C, respectively. As shown in Fig. 5A, no significant difference in methanol consumption was observed among the three strains, and all three strains completely consumed methanol after 192 h of cultivation. Figure 5B shows that GS115_CL/Z2/71 and GS115_FL/Z2/38 strains continued to produce D-lactic acid until 96 h of cultivation, with GS115_CL/Z2/71 and GS115_FL/Z3/38 strains showing maximum D-lactic acid production of 3.48 and 3.61 g/L, respectively, after 96 h of cultivation. GS115_CFL/Z3/04 strain continued to produce D-lactic acid up to 120 h of cultivation, after which it produced 5.09 g/L of D-lactic acid. Figure 5C shows that cell proliferation was observed in strains GS115_CL/Z2/71 and GS115_FL/Z2/38 until 144 h of cultivation, after which cell density decreased slightly in both strains. In contrast, GS115_CFL/Z3/04 strain showed lower growth after 48 h of culture than the other two strains but continued to grow until 192 h of culture. After 192 h of cultivation, OD600 values of all three strains were similar.

Fig. 5.

Fig. 5

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Time course of A methanol concentrations, B D-lactic acid concentrations, and C OD600 values of strains constructed via post-transformational gene amplification in a flask culture

Discussion

In this study, we investigated the type of D-LDH gene expressed and optimal combination of promoters to improve D-lactic acid production from methanol in K. phaffii. The highest D-lactic acid production was achieved by integrating the LlDLDH gene from L. lactis linked to pCAT1 and pFLD1 into the rDNA locus of the K. phaffii genome and performing post-transformational gene amplification. The results indicate that the combination of expressed genes and promoters, use of multiple promoters, and multicopy integration of genes are effective for the production of D-lactic acid from methanol by engineered K. phaffii.

In this study, two types of D-LDH genes, LlDLDH and LpDLDH, were expressed in K. phaffii, and they achieved higher D-lactic acid production than conventional LmDLDH [24]; the highest D-lactic acid production was achieved with LlDLDH (Fig. 1). Watcharawipas et al. also expressed these three D-LDH genes in S. cerevisiae and reported that the highest D-lactic acid production is achieved with LpDLDH, followed by LlDLDH and LmDLDH [28]. In contrast, in a study on D-LDH expression in O. polymorpha, almost no D-lactic acid was produced with LmDLDH [44]. This indicates that the effective D-LDH gene varies depending on the host microorganism. Therefore, type of D-LDH gene expressed in each host should be considered for high D-lactic acid production.

In this study, high D-lactic acid production was achieved when pCAT1 or pFLD1 was used to express D-LDH (Fig. 2A). Of the eight promoters used in this study, two promoters that improved D-lactic acid production over conventional pAOX1, pCAT1, and pFLD1 are known to be strong methanol-inducible promoters, along with pAOX1 [45]. Vogl et al. reported that pFLD1 is comparable to pAOX1 and that pCAT1 reaches expression levels up to 1.8-fold higher than those of pAOX1 in K. phaffii [46]. When pCAT1 or pFLD1 was used, the D-lactic acid concentration nearly reached a plateau at 48 h (Fig. 2A). This is likely due to a decrease in the pH of the medium or a lack of nitrogen sources in the medium. Similarly, Wu et al. reported that the pH decrease due to lactic acid accumulation reduced lactic acid productivity from methanol [47]. In addition, the nitrogen source in the minimal medium was limited [48], and the D-lactic acid production likely reached a plateau due to competition between the nitrogen source for cell growth and D-lactic acid production. In terms of cell proliferation, optimal methanol concentration for culturing K. phaffii is approximately 4.0 g/L [49]. In addition, methanol concentrations < 10 g/L have been used to produce heterologous proteins and useful compounds using methanol-inducible promoters in K. phaffii [35, 50–54]. In contrast, methanol concentration used in this study (30 g/L) was higher than that used in other studies. Only a little is known about the expression intensity of methanol-inducible promoters at methanol concentrations > 10 g/L. However, similar to the galactose-inducible promoter, pGAL1, in S. cerevisiae [55], the level of gene expression driven by the methanol-inducible promoter in K. phaffii may be dependent on the methanol concentration in the medium. pCAT1 and pFLD1 may have higher expression levels than pAOX1 in the presence of high concentrations of methanol. Therefore, exploration of useful promoters at high methanol concentrations is important for the efficient production of various useful chemicals from methanol.

When LlDLDH was expressed in K. phaffii using two mutant promoters, pCAT1m and pGAP1m, almost no D-lactic acid production was observed with either promoter (Fig. 2A). pCAT1m and pGAP1m are mutants of pCAT1 and pGAP1, respectively, with a partial modification of the promoter sequence to improve GFP fluorescence [35, 38]. Gene expression levels vary depending on the gene and promoter combination [29, 30]. Therefore, mutant promoters effective for the expression of GFP were not suitable for the expression of LlDLDH, leading to low D-lactic acid production. In the future, identification of suitable promoters for target gene expression will be important for the production of chemicals from methanol.

GS115_CFL/Z3/04 strain, constructed by integrating the LlDLDH gene linked to two promoters (pCAT1 and pFLD1) and amplifying it after transformation, produced a maximum of 5.18 g/L of D-lactic acid after 168 h of cultivation (Fig. 5B). There have been several reports of lactic acid production by methylotrophic yeasts using methanol as a carbon source. Bachleitner et al. achieved L-lactic acid production of 17 g/L using methanol and glycerol as co-substrates [56]. On the other hand, lactic acid production using methanol as the sole carbon source has been reported in three studies, one for D-lactic acid production [24] and two for L-lactic acid production [44, 47]. The final lactic acid production levels in these studies were 3.48 g/L [24], 3.8 g/L [44], and 4.2 g/L [47], respectively. Therefore, to the best of our knowledge, this study achieved the highest lactic acid production when methanol was used as the sole carbon source.

Previous studies have reported that the combined use of the two promoters, pAOX1 and pGAP1, enhances the production of heterologous proteins in K. phaffii [57]. This is possibly because, even under conditions of repression of one promoter, gene expression is maintained at a high level by the action of the other promoter, thereby increasing the production of the target protein. Strengths of the promoters of some methanol metabolism-related genes in K. phaffii are reduced by amino acids in the culture medium [58], and the repression conditions of pCAT1 and pFLD1 used in this study were different [35, 59]. Combination of the two promoters with different repression conditions, pCAT1 and pFLD1, in this study enabled D-LDH to be constantly expressed at a high level and to continue to stably produce D-lactic acid over a long period, thereby achieving the high D-lactic acid production. Therefore, simultaneous expression of key metabolic enzymes using multiple promoters is a useful strategy for chemical production in K. phaffii.

Conclusions

In this study, LlDLDH gene from L. lactis was linked to pCAT1 and pFLD1, integrated into the rDNA locus of K. phaffii, and subjected to post-transformational gene amplification to construct an engineered yeast strain, GS115_CFL/Z3/04, capable of producing D-lactic acid from methanol. To the best of our knowledge, the amount of D-lactic acid produced by this engineered yeast is the highest reported to date when methanol is used as the sole carbon source. In lactic acid production from methanol, strategies such as overexpression of transcriptional activators [56] and inhibition of the reverse reaction from lactic acid to pyruvate [47] have been employed to increase lactic acid production. Integrating these genetic modification strategies with the strategy in this study likely further increases the production of D-lactic acid. On the other hand, it has been reported that the methanol assimilation ability of K. phaffii decreases when it is cultured for a long period of time using methanol as the sole carbon source [60]. Addressing this issue is also important for efficient production of D-lactic acid from methanol. This study demonstrated the effectiveness of combining different enzyme genes and promoters using multiple promoters with different induction and repression conditions, integrating the genes into the rDNA locus, and amplifying the genes after transformation in K. phaffii. This study outlines a method to engineer other K. phaffii strains capable of producing various useful chemicals in the future.

Supplementary Information

Supplementary Material 1. (21KB, docx)

Acknowledgements

We would like to thank Editage for English language editing.

Abbreviations

AHMT

4-Amino-3-hydrazino-5-mercapto-1,2,4-triazole

D-LDH

D-Lactate dehydrogenase

PCR

Polymerase chain reaction

Author contributions

YI performed the experiments and wrote the manuscript. RY conceptualized the study, wrote the manuscript, supervised the study, and acquired funding. TM and HO supervised the study.

Funding

This study was partially supported by the Japan Society for the Promotion of Science (grant number JP22H03803).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Wendisch VF, Brito LF, Lopez MG, Hennig G, Pfeifenschneider J, et al. The flexible feedstock concept in Industrial biotechnology: metabolic engineering of Escherichia coli, Corynebacterium glutamicum, Pseudomonas, Bacillus and yeast strains for access to alternative carbon sources. J Biotechnol. 2016;234:139–47. [DOI] [PubMed] [Google Scholar]
  • 2.Dummer NF, Willock DJ, He Q, Howard MJ, Lewis RJ, et al. Methane oxidation to methanol. Chem Rev. 2023;123:6359–411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Sugiyama H, Miyazaki M, Sasase M, Kitano M, Hosono H. Room-temperature CO2 hydrogenation to methanol over air-stable hcp-PdMo intermetallic catalyst. J Am Chem Soc. 2023;145:9410–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Ohkubo K, Hirose K. Light-driven C–H oxygenation of methane into methanol and formic acid by molecular oxygen using a perfluorinated solvent. Angew Chem Int Ed. 2017;57:2126–9. [DOI] [PubMed] [Google Scholar]
  • 5.Zhang W, Song M, Yang Q, Dai Z, Zhang S, et al. Current advance in bioconversion of methanol to chemicals. Biotechnol Biofuels. 2018;11:260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Schrader J, Schilling M, Holtmann D, Sell D, Filho MV, et al. Methanol-based industrial biotechnology: current status and future perspectives of methylotrophic bacteria. Trends Biotechnol. 2008;27:107–15. [DOI] [PubMed] [Google Scholar]
  • 7.Hartner FS, Glieder A. Regulation of methanol utilisation pathway genes in yeasts. Microb Cell Fact. 2006;5:39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Gan Y, Meng X, Gao C, Song W, Liu L, Chen X. Metabolic engineering strategies for microbial utilization of methanol. Eng Microbiol. 2023;3:3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Zhu WL, Cui JY, Cui LY, Liang WF, Yang S, et al. Bioconversion of methanol to value-added mevalonate by engineered Methylobacterium extorquens AM1 containing an optimized mevalonate pathway. Appl Microbiol Biotechnol. 2016;100:2171–82. [DOI] [PubMed] [Google Scholar]
  • 10.Zhai X, Gao J, Li Y, Grininger M, Zhou YJ. Peroxisomal metabolic coupling improves fatty alcohol production from sole methanol in yeast. Proc Natl Acad Sci. 2023. 10.1073/pnas.2220816120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Gupta B, Revagade N, Hilborn J. Poly(lactic acid) fiber: an overview. Prog Polym Sci. 2007;32:455–82. [Google Scholar]
  • 12.Palm MD, Woodhall KE, Butterwick KJ, Goldman MP. Cosmetic use of poly-l-lactic acid: a retrospective study of 130 patients. Dermatol Surg. 2010;36:161–70. [DOI] [PubMed] [Google Scholar]
  • 13.Wee YJ, Kim JN, Ryu HW. Biotechnological production of lactic acid and its recent applications. Food Technol Biotechnol. 2006;44:163–72. [Google Scholar]
  • 14.Santoro M, Shah SR, Walker JL, Mikos AG. Poly(lactic acid) nanofibrous scaffolds for tissue engineering. Adv Drug Deliv Rev. 2016;107:206–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Tsuji H. Poly(lactide) stereocomplexes: formation, structure, properties, degradation, and applications. Macromol Biosci. 2005;5:569–97. [DOI] [PubMed] [Google Scholar]
  • 16.Abdel-Rahman MA, Sonomoto K. Opportunities to overcome the current limitations and challenges for efficient microbial production of optically pure lactic acid. J Biotechnol. 2016;236:176–92. [DOI] [PubMed] [Google Scholar]
  • 17.Ahmad M, Hirz M, Pichler H, Schwab H. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol. 2014;98:5301–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Guo F, Dai Z, Peng W, Zhang S, Zhou J, et al. Metabolic engineering of Pichia pastoris for malic acid production from methanol. Biotechnol Bioeng. 2021;118:357–71. [DOI] [PubMed] [Google Scholar]
  • 19.Zhang X, Wang D, Duan Y, Zheng X, Lin Y, Liang S. Production of lycopene by metabolically engineered Pichia pastoris. Biosci Biotechnol Biochem. 2020;84:463–70. [DOI] [PubMed] [Google Scholar]
  • 20.Sarwar A, Lee EY. Methanol-based biomanufacturing of fuels and chemicals using native and synthetic methylotrophs. Synth Syst Biotechnol. 2023;8(3):396–415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Steinborn G, Boer E, Scholz A, Tag K, Kunze G, Gellissen G. Application of a wide-range yeast vector (CoMed™) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts. Microb Cell Fact. 2006;5:33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Marx H, Mecklenbrauker A, Gasser B, Sauer M, Mattanovich D. Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus. FEMS Yeast Res. 2009;9:1260–70. [DOI] [PubMed] [Google Scholar]
  • 23.Lopes TS, Klootwijk J, Veenstra AE, Van der Aar PC, van Heerikhuizen H, et al. High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression. Gene. 1989;79:199–206. [DOI] [PubMed] [Google Scholar]
  • 24.Yamada R, Ogura K, Kimoto Y, Ogino H. Toward the construction of a technology platform for chemicals production from methanol: D-lactic acid production from methanol by an engineered yeast Pichia pastoris. World J Microbiol Biotechnol. 2019;35:37. [DOI] [PubMed] [Google Scholar]
  • 25.Mitsui R, Yamada R, Matsumoto T, Yoshihara S, Tokumoto H, Ogino H. Construction of lactic acid-tolerant Saccharomyces cerevisiae by using CRISPR-Cas-mediated genome evolution for efficient D-lactic acid production. Appl Microbiol Biotechnol. 2020;104:9147–58. [DOI] [PubMed] [Google Scholar]
  • 26.Huang S, Xue Y, Yu B, Wang L, Zhou C, Ma Y. A review of the recent developments in the bioproduction of polylactic acid and its precursors optically pure lactic acids. Molecules. 2021. 10.3390/molecules26216446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Zhong W, Yang M, Mu T, Wu T, Hao X, et al. Systematically redesigning and optimizing the expression of D-lactate dehydrogenase efficiently produces high-optical-purity D-lactic acid in Saccharomyces cerevisiae. Biochem Eng J. 2019;144:217–26. [Google Scholar]
  • 28.Watcharawipas A, Sae-tang K, Sansatchanon K, Sudying P, Boonchoo P, et al. Systematic engineering of Saccharomyces cerevisiae for D-lactic acid production with near theoretical yield. FEMS Yeast Res. 2021;21:4. [DOI] [PubMed] [Google Scholar]
  • 29.Kajiwara S, Yamada R, Ogino H. Secretory overexpression of Bacillus thermocatenulatus lipase in Saccharomyces cerevisiae using combinatorial library strategy. Biotechnol J. 2018;13:1700409. [DOI] [PubMed] [Google Scholar]
  • 30.Yamada R, Kimoto Y, Ogino H. Combinatorial library strategy for strong overexpression of the lipase from Geobacillus thermocatenulatus on the cell surface of yeast Pichia pastoris. Biochem Eng J. 2016;113:7–11. [Google Scholar]
  • 31.Kadam SR, Patil SS, Bastawde KB, Khire JM, Gokhale DV. Strain improvement of Lactobacillus delbrueckii NCIM 2365 for lactic acid production. Process Biochem. 2006;41(1):120–6. [DOI] [PubMed] [Google Scholar]
  • 32.Mimitsuka T, Na K, Morita K, Sawai H, Minegishi S, et al. A membrane-integrated fermentation reactor system: Its effects in reducing the amount of sub-raw materials for D-lactic acid continuous fermentation by Sporolactobacillus laevolacticus. Biosci Biotechnol Biochem. 2012;76:67–72. [DOI] [PubMed] [Google Scholar]
  • 33.Williams SA, Hadges RA, Strilke TL, Show R, Kunkee RE. Cloning the gene for the malolactic fermentation of wine from Lactobacillus delbrueckii in Escherichia coli and yeasts. Appl Environ Microbiol. 1984;47:288–93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Nakano K, Sawada S, Yamada R, Mimitsuka T, Ogino H. Enhancement of the catalytic activity of d-lactate dehydrogenase from Sporolactobacillus laevolacticus by site-directed mutagenesis. Biochem Bioeng J. 2018;133(15):214–8. [Google Scholar]
  • 35.Nong L, Zhang Y, Duan Y, Hu S, Lin Y, Liang S. Engineering the regulatory site of the catalase promoter for improved heterologous protein production in Pichia pastoris. Biotechnol Lett. 2020;42:2703–9. [DOI] [PubMed] [Google Scholar]
  • 36.Wang J, Zhang T, Li Y, Li L, Wang Y, et al. High-level expression of Thermomyces dupontii thermo-alkaline lipase in Pichia pastoris under the control of different promoters. 3 Biotech. 2019;9:33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Karaoglan M, Karaoglan FE, Inan M. Functional analysis of alcohol dehydrogenase (ADH) genes in Pichia pastoris. Biotechnol Lett. 2016;38:463–9. [DOI] [PubMed] [Google Scholar]
  • 38.Qin X, Qian J, Yao G, Zhuang Y, Zhang S, Chu J. GAP promoter library for fine-tuning of gene expression in Pichia pastoris. Appl Environ Microbiol. 2011;77:11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Ahn J, Hong J, Lee H, Park M, Lee E, et al. Translation elongation factor 1-α gene from Pichia pastoris: molecular cloning, sequence, and use of its promoter. Appl Microbiol Biotechnol. 2007;74:601–8. [DOI] [PubMed] [Google Scholar]
  • 40.Wu S, Letchworth GJ. High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques. 2004;36:152–4. [DOI] [PubMed] [Google Scholar]
  • 41.Rosenberg JC, Rush BF. An enzymatic-spectrophotometric determination of pyruvic and lactic acid in blood: methodologic aspects. Clin Chem. 1966;12:299–307. [PubMed] [Google Scholar]
  • 42.Avigad G. A simple spectrophotometric determination of formaldehyde and other aldehydes: application to periodate-oxidized glycol systems. Anal Biochem. 1983;134:499–504. [DOI] [PubMed] [Google Scholar]
  • 43.Tokuhiro K, Ishida N, Nagamori E, Saitoh S, Ohnishi T, et al. Double mutation of the PDC1 and ADH1 genes improves lactate production in the yeast Saccharomyces cerevisiae expressing the bovine lactate dehydrogenase gene. Appl Microbiol Biotechnol. 2009;82:883–90. [DOI] [PubMed] [Google Scholar]
  • 44.Wefelmeier K, Schmitz S, Haut AM, Otten J, Julich T, Blank LM. Engineering the methylotrophic yeast Ogataea polymorpha for lactate production from methanol. Front Bioeng Biotechnol. 2023;11:1223726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Schwarzhans JP, Luttermann T, Geier M, Kalinowski J, Friehs K. Towards systems metabolic engineering in Pichia pastoris. Biotechnol Adv. 2017;35:681–710. [DOI] [PubMed] [Google Scholar]
  • 46.Vogl T, Kickenweiz T, Pitzer J, Sturmberger L, Weninger A, et al. Engineered bidirectional promoters enable rapid multi-gene co-expression optimization. Nat Commun. 2018;9:3589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Wu X, Ren Y, Chen S, Cai P, Zhou Y. Production of L-lactic acid from methanol by engineered yeast Pichia pastoris. Bioresour Technol. 2025;415: 131730. [DOI] [PubMed] [Google Scholar]
  • 48.Corbacho I, Teixido F, Velazquez R, Hernandez LM, Olivero I. Standard YPD, even supplemented with extra nutrients, does not always compensate growth defects of Saccharomyces cerevisiae auxotrophic strains. Antonie Van Leeuwenhoek. 2010;99:591–600. [DOI] [PubMed] [Google Scholar]
  • 49.Kupcsulik B, Sevella B. Effect of methanol concentration on the recombinant Pichia pastoris MUTS fermentation. Period Polytech Chem Eng. 2004;48:73–87. [Google Scholar]
  • 50.Krainer FW, Dietzsch C, Hajek T, Herwig C, Spadiut O, Glieder A. Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway. Microb Cell Fact. 2012;11:1–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Cos O, Serrano A, Montesions JL, Ferrer P, Cregg JM, Valero F. Combined effect of the methanol utilization (Mut) phenotype and gene dosage on recombinant protein production in Pichia pastoris fed-batch cultures. J Biotechnol. 2005;116:321–35. [DOI] [PubMed] [Google Scholar]
  • 52.Zhang T, Ge C, Deng L, Tan T, Wang F. C4-dicarboxylic acid production by overexpressing the reductive TCA pathway. FEMS Microbiol Lett. 2015;362:9. [DOI] [PubMed] [Google Scholar]
  • 53.Cai P, Duan X, Wu X, Gao L, Ye M, Zhou YJ. Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichia pastoris. Nucl Acid Res. 2021;49:7791–805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Wu X, Cai P, Gao L, Li Y, Yao L, Zhou YJ. Efficient bioproduction of 3-hydroxypropionic acid from methanol by a synthetic yeast cell factory. ACS Sustain Chem Eng. 2023;11:6445–53. [Google Scholar]
  • 55.Li J, Wang S, VanDusen WJ, Schultz LD, George HA, et al. Green fluorescent protein in Saccharomyces cerevisiae: real-time studies of the GAL1 promoter. Biotechnol Bioeng. 2020;70:187–96. [DOI] [PubMed] [Google Scholar]
  • 56.Bachleitner S, Severinsen MM, Lutz G, Mattanovich D. Overexpression of the transcriptional activators Mxr1 and Mit1 enhances lactic acid production on methanol in Komagataella phaffii. Metab Eng. 2024;85:133–44. [DOI] [PubMed] [Google Scholar]
  • 57.Parashar D, Satyanarayana T. Enhancing the production of recombinant acidic α-amylase and phytase in Pichia pastoris under dual promoters [constitutive (GAP) and inducible (AOX)] in mixed fed batch high cell density cultivation. Process Biochem. 2016;51:1315–22. [Google Scholar]
  • 58.Velastegui E, Theron C, Berrios J, Fickers P. Downregulation by organic nitrogen of AOX1 promoter used for controlled expression of foreign genes in the yeast Pichia pastoris. Yeast. 2019;36:297–304. [DOI] [PubMed] [Google Scholar]
  • 59.Wang XF, Shen XG, Sun YC, Zhao HY, Xu L, et al. Production of Yarrowia lipolytica lipase LIP2 in Pichia pastoris using the nitrogen source-regulated FLD1 promoter. J Chem Technol Biotechnol. 2011;87:553–8. [Google Scholar]
  • 60.Wang C, Jiang W, Yu C, Xia J. Transcriptional downregulation of methanol metabolism key genes during yeast death in engineered Pichia pastoris. Biotechnol J. 2024;19(10): e202400328. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

Supplementary Material 1. (21KB, docx)

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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