Enhancement of D-lactic acid production by the methylotrophic yeast, Komagataella phaffii, via optimization of D-LDH gene and promoter and post-transformational gene amplification

Enhancement of D-lactic acid production by the methylotrophic yeast, Komagataella phaffii, via optimization of D-LDH gene and promoter and post-transformational gene amplification | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhancement of D-lactic acid production by the methylotrophic yeast, Komagataella phaffii, via optimization of D-LDH gene and promoter and post-transformational gene amplification Yoshifumi Inoue, Ryosuke Yamada, Takuya Matsumoto, Hiroyasu Ogino This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5101349/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Dec, 2024 Read the published version in Biotechnology for Biofuels and Bioproducts → Version 1 posted 8 You are reading this latest preprint version 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, including both D- and L- lactic acids. 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. D-lactic acid Komagataella phaffii Methanol Metabolic engineering Yeast Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 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 CO 2 and methane, has increased ( 3 – 5 ). 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 ( 6 ). 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 ( 7 – 9 ). These methylotrophic microorganisms can be used to produce useful compounds, such as mevalonic acid ( 10 ) and fatty alcohols ( 11 ), 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 ( 12 – 15 ). 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 (16). Consequently, the demand for both L- and D-lactic acids is increasing ( 17 ), 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 ( 18 ). Recently, research on the development of recombinant K. phaffii capable of producing useful chemicals, such as malic acid ( 19 ) and lycopene ( 20 ), from methanol is attracting attention. 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 ), LmDLDH ( L. mesenteroides ), LlDLDH ( L. lactis ), LpDLDH ( L. pseudomesenteroides ), and SlDLDH ( Sporolactobacillus laevolacticus ). These D-LDH genes are also expressed in S. cerevisiae ( 28 , 31 , 32 ). The eight promoters consist of four methanol-inducible promoters ( AOX1 promoter [pAOX1], CAT1 promoter [pCAT1], mutant CAT1 promoter [pCATm] ( 33 ), and FLD1 promoter [pFLD1] ( 34 )) and four constitutive promoters ( GAP1 promoter [pGAP1], A DH3 promoter [pADH3] ( 35 ), mutant GAP1 promoter [pGAP1m] ( 36 ), and TEF1-α promoter [pTEF1] ( 37 )). 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 Escherichia 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 1,200 rpm. Cultivation was initiated by culturing the cells in a well containing 1 mL of YPD medium at 30°C and 1,200 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 OD 600 : 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 OD 600 : 11.0) the pre-cultures 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 LdDLDH (derived from Leuconostoc mesenteroides ) by AOX1 promoter pPPE_AOX_Lp Episomal plasmid for expression of LdDLDH (derived from Leuconostoc pseudomesenteroides) by AOX1 promoter pPPE_AOX_Sl Episomal plasmid for expression of LdDLDH (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 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 ( 38 ). 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 OD 600 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. ( 39 ). 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 ( 40 ) 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. ( 40 ). 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 a) *;1–90 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 2 A 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. Figure 2 B 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). 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. 4 A, 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. 4 D. As shown in Fig. 4 A, 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. 4 C 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 OD 600 values of the strains are shown in Figs. 5 A, B, and C, respectively. As shown in Fig. 5 A, 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 5 B 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 5 C 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, OD 600 values of all three strains were similar. 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 ( 42 ). 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. 2 A). 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 ( 43 ). 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 ( 44 ). In terms of cell proliferation, optimal methanol concentration for culturing K. phaffii is approximately 4.0 g/L ( 45 ). In addition, methanol concentrations < 10 g/L have been used to produce heterologous proteins and useful compounds using methanol-inducible promoters in K. phaffii ( 33 , 46 – 50 ). 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 ( 51 ), 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. 2 A). pCAT1m and pGAP1m are mutants of pCAT1 and pGAP1, respectively, with a partial modification of the promoter sequence to improve GFP fluorescence ( 33 , 36 ). 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. 5 B). In a previous study, D-lactic acid production of 3.48 g/L was achieved using engineered K. phaffii ( 23 ). In addition, production of 3.8 g/L L-lactic acid from methanol was achieved using O. polymorpha ( 42 ). Therefore, to the best of our knowledge, this study achieved the highest reported lactic acid production from methanol for both D- and L-lactic acids. Previous studies have reported that the combined use of the two promoters, pAOX1 and pGAP1, enhances the production of heterologous proteins in K. phaffii ( 52 ). 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 ( 53 ), and the repression conditions of pCAT1 and pFLD1 used in this study were different ( 33 , 54 ). 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 from methanol produced by this engineered yeast (5.18 g/L) is the highest reported value to date. 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. Abbreviations AHMT 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole D-LDH D-lactate dehydrogenase PCR polymerase chain reaction Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding This study was partially supported by the Japan Society for the Promotion of Science (grant number JP22H03803). Author Contribution 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. Acknowledgements We would like to thank Editage for English language editing. Data Availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. 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Supplementary Files MetLacsupplementaryinformation.docx Cite Share Download PDF Status: Published Journal Publication published 22 Dec, 2024 Read the published version in Biotechnology for Biofuels and Bioproducts → Version 1 posted Editorial decision: Revision requested 07 Dec, 2024 Reviews received at journal 27 Oct, 2024 Reviewers agreed at journal 15 Oct, 2024 Reviewers agreed at journal 07 Oct, 2024 Reviewers invited by journal 07 Oct, 2024 Editor assigned by journal 18 Sep, 2024 Submission checks completed at journal 18 Sep, 2024 First submitted to journal 17 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5101349","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":370857887,"identity":"9bd93467-cefc-4788-9b4f-842ff8382fdd","order_by":0,"name":"Yoshifumi Inoue","email":"","orcid":"","institution":"Osaka Metropolitan University","correspondingAuthor":false,"prefix":"","firstName":"Yoshifumi","middleName":"","lastName":"Inoue","suffix":""},{"id":370857888,"identity":"c2af82d4-e654-43c1-a1c2-0160ef2d1e7d","order_by":1,"name":"Ryosuke Yamada","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYNACNgY5GJOZaC3GpGtJbCDaSeYzkp9u+FFml94vdsaA4UcNA7s5IS0yN9LMbvacS86dOTvHgLHnGAOzJSH7JCQSzG7wtjHnbridY8DA28DAbHCAoJb0bzf/ttWnGwC1MP4lTkuO2W3etsMJIC3MxNnC86bstsy544YzZ6cVHJY5JkGEX9jTt918U1Ytzy+dvPHhmxqbZIIhxiCQgGADnSSRbEBQC/8BVL4dYS2jYBSMglEw0gAA3N878kNBJCwAAAAASUVORK5CYII=","orcid":"","institution":"Osaka Metropolitan University","correspondingAuthor":true,"prefix":"","firstName":"Ryosuke","middleName":"","lastName":"Yamada","suffix":""},{"id":370857889,"identity":"12e50ce9-14ee-4e36-823b-bd76ee9bdb20","order_by":2,"name":"Takuya Matsumoto","email":"","orcid":"","institution":"Osaka Metropolitan University","correspondingAuthor":false,"prefix":"","firstName":"Takuya","middleName":"","lastName":"Matsumoto","suffix":""},{"id":370857890,"identity":"89628653-6344-48fc-8a04-3c164c4b1b44","order_by":3,"name":"Hiroyasu Ogino","email":"","orcid":"","institution":"Osaka Metropolitan University","correspondingAuthor":false,"prefix":"","firstName":"Hiroyasu","middleName":"","lastName":"Ogino","suffix":""}],"badges":[],"createdAt":"2024-09-17 07:30:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5101349/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5101349/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13068-024-02596-0","type":"published","date":"2024-12-22T15:57:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69108178,"identity":"bae242a1-53f5-4ed7-a7d6-00169e3ce416","added_by":"auto","created_at":"2024-11-15 17:58:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":80664,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of D-lactate production by engineered yeasts expressing D-lactate dehydrogenase (D-LDH) from five different microorganisms under the control of pAOX1.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5101349/v1/b1e20e5d119a0ab3d939d09d.png"},{"id":69108182,"identity":"97f67249-e39c-45c4-a383-7fea3ddc6e31","added_by":"auto","created_at":"2024-11-15 17:58:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":205018,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Time course of D-lactate 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.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5101349/v1/f126dad152dd3ed72b2990fc.png"},{"id":69108180,"identity":"59bf83ea-a077-4ee0-b3b5-8b0f86ce16f4","added_by":"auto","created_at":"2024-11-15 17:58:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":94622,"visible":true,"origin":"","legend":"\u003cp\u003eD-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.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5101349/v1/d21cd7889f7f6fc2a05966e2.png"},{"id":69108181,"identity":"2bbaa8b6-1ee8-43b7-9eea-bba44e4b752c","added_by":"auto","created_at":"2024-11-15 17:58:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":219343,"visible":true,"origin":"","legend":"\u003cp\u003eD-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.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5101349/v1/2f1a447600fab5e4f2c631ce.png"},{"id":69108183,"identity":"ce7dcbb1-5d90-49fa-82cb-f5448b022ce7","added_by":"auto","created_at":"2024-11-15 17:58:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":120504,"visible":true,"origin":"","legend":"\u003cp\u003eTime course of (A) methanol concentrations, (B) D-lactic acid concentrations, and (C) OD\u003csub\u003e600\u003c/sub\u003e values of strains constructed via post-transformational gene amplification in a flask culture.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5101349/v1/35c64927f9e95ee10768f492.png"},{"id":72201748,"identity":"e0b0d02c-3984-4663-a937-24821b1c0640","added_by":"auto","created_at":"2024-12-23 16:10:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1163993,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5101349/v1/57040a93-35a1-40ee-bfa4-07c87ef7c38c.pdf"},{"id":69108179,"identity":"e7ca6adc-b8a8-442d-b149-f61ba9aae721","added_by":"auto","created_at":"2024-11-15 17:58:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":21467,"visible":true,"origin":"","legend":"","description":"","filename":"MetLacsupplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5101349/v1/4afad709341b304c0ef24a48.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancement of D-lactic acid production by the methylotrophic yeast, Komagataella phaffii, via optimization of D-LDH gene and promoter and post-transformational gene amplification","fulltext":[{"header":"Background","content":"\u003cp\u003eCurrently, technologies producing various useful chemicals from alternative carbon resources to replace petroleum are in demand (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Methanol has attracted considerable attention as an alternative carbon source as it is a liquid at room temperature, easy to transport, and stable (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). In recent years, development of new production technologies with low environmental impact, such as the production of methanol from CO\u003csub\u003e2\u003c/sub\u003e and methane, has increased (\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003eChemical 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 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Many microorganisms can utilize methanol, including bacteria, such as \u003cem\u003eMethylobacterium extorquens\u003c/em\u003e and \u003cem\u003eBacillus ethanolicus\u003c/em\u003e, and yeasts, such as \u003cem\u003eKomagataella phaffii\u003c/em\u003e (formerly known as \u003cem\u003ePichia pastoris\u003c/em\u003e), \u003cem\u003eCandida boidinii\u003c/em\u003e, and \u003cem\u003eOgataea polymorpha\u003c/em\u003e. These microorganisms metabolize methanol via the serine, ribulose phosphate, and xylose phosphate pathways (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). These methylotrophic microorganisms can be used to produce useful compounds, such as mevalonic acid (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) and fatty alcohols (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), from methanol.\u003c/p\u003e \u003cp\u003eLactic acid is used as a chemical in the food and pharmaceutical industries and as a raw material for polylactic acid, a biodegradable plastic (\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). 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 (16). Consequently, the demand for both L- and D-lactic acids is increasing (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), 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.\u003c/p\u003e \u003cp\u003e \u003cem\u003eK. phaffii\u003c/em\u003e, a type of methanol-utilizing yeast, is safe and used in well-established genetic engineering techniques as a host for protein production (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Recently, research on the development of recombinant \u003cem\u003eK. phaffii\u003c/em\u003e capable of producing useful chemicals, such as malic acid (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) and lycopene (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), from methanol is attracting attention. In \u003cem\u003eK. phaffii\u003c/em\u003e, integration of a multicopy plasmid into the non-transcribed spacer of the ribosomal DNA (rDNA) locus leads to high mitotic stability and gene expression (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). 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 (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Lopes et al. reported that intracellular protein production is highly correlated with the copy number of the gene encoding the protein (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Yamada et al. first reported the production of D-lactic acid from methanol by integrating multiple copies of a gene cassette linking the \u003cem\u003eAOX1\u003c/em\u003e promoter to the D-lactate dehydrogenase (\u003cem\u003eD-LDH\u003c/em\u003e) gene derived from \u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e at the rDNA locus (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). 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 (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGene 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. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) and Watcharawipas et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) investigated the expression of \u003cem\u003eD-LDH\u003c/em\u003e gene in yeast \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and reported that the origin of \u003cem\u003eD-LDH\u003c/em\u003e gene affects D-lactic acid production. Furthermore, previous studies reported that the combination of promoters, terminators, and secretory signal sequences in \u003cem\u003eS. cerevisiae\u003c/em\u003e and \u003cem\u003eK. phaffii\u003c/em\u003e significantly affects lipase expression (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we investigated the combination of five \u003cem\u003eD-LDH\u003c/em\u003e genes of different origins and eight promoters in \u003cem\u003eK. phaffii\u003c/em\u003e to achieve efficient D-lactic acid production from methanol. The five \u003cem\u003eD-LDH\u003c/em\u003e genes were \u003cem\u003eLdDLDH\u003c/em\u003e (\u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e), \u003cem\u003eLmDLDH\u003c/em\u003e (\u003cem\u003eL. mesenteroides\u003c/em\u003e), \u003cem\u003eLlDLDH\u003c/em\u003e (\u003cem\u003eL. lactis\u003c/em\u003e), \u003cem\u003eLpDLDH\u003c/em\u003e (\u003cem\u003eL. pseudomesenteroides\u003c/em\u003e), and \u003cem\u003eSlDLDH\u003c/em\u003e (\u003cem\u003eSporolactobacillus laevolacticus\u003c/em\u003e). These \u003cem\u003eD-LDH\u003c/em\u003e genes are also expressed in \u003cem\u003eS. cerevisiae\u003c/em\u003e (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). The eight promoters consist of four methanol-inducible promoters (\u003cem\u003eAOX1\u003c/em\u003e promoter [pAOX1], \u003cem\u003eCAT1\u003c/em\u003e promoter [pCAT1], mutant \u003cem\u003eCAT1\u003c/em\u003e promoter [pCATm] (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), and \u003cem\u003eFLD1\u003c/em\u003e promoter [pFLD1] (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e)) and four constitutive promoters (\u003cem\u003eGAP1\u003c/em\u003e promoter [pGAP1], A\u003cem\u003eDH3\u003c/em\u003e promoter [pADH3] (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), mutant \u003cem\u003eGAP1\u003c/em\u003e promoter [pGAP1m] (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), and \u003cem\u003eTEF1-α\u003c/em\u003e promoter [pTEF1] (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e)). Using these combinations, we identified the \u003cem\u003eD-LDH\u003c/em\u003e gene and promoter combination suitable for expression in \u003cem\u003eK. phaffii\u003c/em\u003e. Finally, the engineered yeast was constructed by integrating a gene cassette containing the identified gene and promoter into the rDNA locus of \u003cem\u003eK. phaffii\u003c/em\u003e, followed by post-transformational gene amplification, and D-lactic acid production from methanol was evaluated.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains and media\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e strain NEB5α (New England Biolabs Japan, Tokyo, Japan) was used as a host for recombinant DNA manipulation. Genetically modified \u003cem\u003eE. coli\u003c/em\u003e cells were cultured in the Luria\u0026ndash;Bertani medium (20 g/L Luria\u0026ndash;Bertani broth powder [Nacalai Tesque, Kyoto, Japan]) supplemented with 100 \u0026micro;g/mL ampicillin sodium salt (Nacalai Tesque).\u003c/p\u003e \u003cp\u003e \u003cem\u003eK. phaffii\u003c/em\u003e GS115 (Thermo Fisher Scientific, Yokohama, Japan) was used as the host for D-lactic acid production. \u003cem\u003eK. phaffii\u003c/em\u003e 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 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e g/L biotin [Nacalai Tesque]), MD medium (13.4 g/L yeast nitrogen base without amino acids, 20 g/L glucose, and 4 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e g/L biotin), or MM medium (13.4 g/L yeast nitrogen base without amino acids, 30 g/L methanol, and 4 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e g/L biotin). As required, 20 g/L agar (Nacalai Tesque) and a predetermined amount of zeocin (InvivoGen, San Diego, CA, USA) were added.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eYeast cultivation\u003c/h2\u003e \u003cp\u003eNinety-six-deep well culture was performed using 1 mL of YPM medium and Deep Well Maximizer (Taitec, Nagoya, Japan) at 30\u0026deg;C and 1,200 rpm. Cultivation was initiated by culturing the cells in a well containing 1 mL of YPD medium at 30\u0026deg;C and 1,200 rpm for 24 h, harvesting and washing the cells, and suspending them in 1 mL of fresh medium.\u003c/p\u003e \u003cp\u003eTest 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\u0026deg;C and 150 rpm. Cultures were initiated by inoculating the precultures grown in test tubes containing the MD medium at 30\u0026deg;C and 150 rpm for 72 h (initial OD\u003csub\u003e600\u003c/sub\u003e: 3.0).\u003c/p\u003e \u003cp\u003eFlask cultures were performed using a rotary shaker (Taitec) at 30\u0026deg;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 OD\u003csub\u003e600\u003c/sub\u003e: 11.0) the pre-cultures grown in 250-mL flasks containing the YPD medium for 24 h at 200 rpm and 30\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid construction and yeast transformation\u003c/h2\u003e \u003cp\u003eAll plasmids and primers used in this study are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, respectively. pPPE_AOX_XDLDH (X, Ld, Ll, Lp, or Sl), the plasmid used for \u003cem\u003eD-LDH\u003c/em\u003e expression, was constructed as described below. Plasmid pPPE (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) was linearized using the restriction enzymes, EcoRI and NotI. Fragments of \u003cem\u003eD-LDH\u003c/em\u003e gene from \u003cem\u003eL. mesenteroides\u003c/em\u003e linked to pAOX1 were obtained via polymerase chain reaction (PCR) amplification using plasmid pRDZ_AO_DLDH (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) 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.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of plasmids used in this study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlasmid name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRelevant features\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epPPE_AOX_Ld\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEpisomal plasmid for expression of LdDLDH (derived from \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e) by \u003cem\u003eAOX1\u003c/em\u003e promoter\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epPPE_AOX_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEpisomal plasmid for expression of LlDLDH (derived from \u003cem\u003eLeuconostoc lactis\u003c/em\u003e) by \u003cem\u003eAOX1\u003c/em\u003e promoter\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epPPE_AOX_Lm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEpisomal plasmid for expression of LdDLDH (derived from \u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e) by \u003cem\u003eAOX1\u003c/em\u003e promoter\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epPPE_AOX_Lp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEpisomal plasmid for expression of LdDLDH (derived from Leuconostoc pseudomesenteroides) by \u003cem\u003eAOX1\u003c/em\u003e promoter\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epPPE_AOX_Sl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEpisomal plasmid for expression of LdDLDH (derived from \u003cem\u003eSporolactobacillus laevolacticus\u003c/em\u003e) by \u003cem\u003eAOX1\u003c/em\u003e promoter\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epPPE_ADH_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEpisomal plasmid for expression of LlDLDH by \u003cem\u003eADH3\u003c/em\u003e promoter\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epPPE_CAT_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEpisomal plasmid for expression of LlDLDH by \u003cem\u003eCAT1\u003c/em\u003e promoter\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epPPE_CATm_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEpisomal plasmid for expression of LlDLDH by mutant \u003cem\u003eCAT1\u003c/em\u003e promoter\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epPPE_FLD_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEpisomal plasmid for expression of LlDLDH by \u003cem\u003eFLD1\u003c/em\u003e promoter\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epPPE_GAP_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEpisomal plasmid for expression of LlDLDH by \u003cem\u003eGAP1\u003c/em\u003e promoter\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epPPE_GAPm_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEpisomal plasmid for expression of LlDLDH by mutant \u003cem\u003eGAP1\u003c/em\u003e promoter\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epPPE_TEF_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEpisomal plasmid for expression of LlDLDH by \u003cem\u003eTEF1\u003c/em\u003e promoter\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epRDZ_CAT_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMulticopy integrative plasmid for expression of LlDLDH by \u003cem\u003eCAT1\u003c/em\u003e promoter\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epRDZ_FLD_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMulticopy integrative plasmid for expression of LlDLDH by \u003cem\u003eFLD1\u003c/em\u003e promoter\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGenes 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.\u003c/p\u003e \u003cp\u003epPPE_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 \u003cem\u003eK. phaffii\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003eMulticopy integration plasmids pRDZ_CAT_LlDLDH and pRDZ_FLD_LlDLDH for \u003cem\u003eD-LDH\u003c/em\u003e 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 \u003cem\u003eAscI\u003c/em\u003e prior to transformation.\u003c/p\u003e \u003cp\u003eThe resulting plasmids were transformed into \u003cem\u003eK. phaffii\u003c/em\u003e GS115 using a previously described electroporation method (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnalyses of growth and metabolite levels\u003c/h2\u003e \u003cp\u003eOD\u003csub\u003e600\u003c/sub\u003e of each culture was determined by measuring the absorbance at 600 nm using a spectrophotometer (Shimadzu, Kyoto, Japan).\u003c/p\u003e \u003cp\u003eD-lactic acid concentration was calculated based on NADH concentration determined from the absorbance at 340 nm using a partially modified enzymatic reaction method with \u003cem\u003eD-LDH\u003c/em\u003e described by Rosenberg et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Briefly, the culture broth was centrifuged at 10,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 2 min at 4\u0026deg;C and diluted appropriately with water. Then, 140 \u0026micro;L 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 \u0026micro;L of 30 mM NAD\u003csup\u003e+\u003c/sup\u003e (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 5 \u0026micro;L of 2 mg/mL D-LDH (Toyobo, Osaka, Japan), and 10 \u0026micro;L of diluted supernatant were mixed and incubated at 30\u0026deg;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.\u003c/p\u003e \u003cp\u003eMethanol concentration was determined using a colorimetric method (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) using 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (AHMT) (Nacalai Tesque). The culture broth was centrifuged at 10,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 2 min at 4\u0026deg;C, and the resulting culture supernatant was diluted appropriately. Then, 48 \u0026micro;L of the diluted culture supernatant was mixed with 2 \u0026micro;L 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\u0026deg;C for 80 min. Subsequently, 50 \u0026micro;L of 5 mol/L potassium hydroxide and 50 \u0026micro;L 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\u0026deg;C for 20 min. The reaction solution was then mixed with 250 \u0026micro;L 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.\u003c/p\u003e \u003cp\u003e \u003cem\u003eD-LDH\u003c/em\u003e activity in yeast cells was measured following the method described by Tokuhiro et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). 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 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C for 1 min to collect the cells. The collected cells were washed twice with water and resuspended in 300 \u0026micro;L of 100 mM potassium phosphate buffer (pH 7.0). The cell suspension was mixed with 500 \u0026micro;L 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 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C for 5 min, and the supernatant was collected. Then, 5 \u0026micro;L of the supernatant was mixed with 180 \u0026micro;L of 100 mM glycine NaOH buffer (pH 10.0), 10 \u0026micro;L of 0.5 M D-lactic acid, and 10 \u0026micro;L of 20 mM NAD\u003csup\u003e+\u003c/sup\u003e solution, and the change in absorbance at 340 nm and 30\u0026deg;C was measured. One unit of \u003cem\u003eD-LDH\u003c/em\u003e activity was defined as the amount of enzyme releasing 1 \u0026micro;mol of NADH per minute at 30\u0026deg;C.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffect of\u003c/b\u003e \u003cb\u003eD-LDH\u003c/b\u003e \u003cb\u003egene type on D-lactic acid production\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo express five \u003cem\u003eD-LDH\u003c/em\u003e genes \u003cem\u003e(LdDLDH\u003c/em\u003e, \u003cem\u003eLlDLDH\u003c/em\u003e, \u003cem\u003eLmDLDH\u003c/em\u003e, \u003cem\u003eLpDLDH\u003c/em\u003e, and \u003cem\u003eSlDLDH\u003c/em\u003e) under the control of pAOX1, pPPE_AOX_XDLDH plasmids were constructed by connecting pAOX1 to each \u003cem\u003eD-LDH\u003c/em\u003e gene. These plasmids were transformed into \u003cem\u003eK. phaffii\u003c/em\u003e GS115, resulting in strains designated as GS115_AOX_X (X; Ld, Ll, Lm, Lp, or Sl; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e 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 \u003cem\u003eLmDLDH\u003c/em\u003e 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 \u003cem\u003eD-LDH\u003c/em\u003e examined, \u003cem\u003eLlDLDH\u003c/em\u003e is the most suitable \u003cem\u003eD-LDH\u003c/em\u003e for expression in \u003cem\u003eK. phaffii\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of yeast strains used in this study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRelevant features\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eKomagataella phaffii\u003c/em\u003e host strain\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_AOX_Ld\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 harboring pPPE_AOX_Ld\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_AOX_Lm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 harboring pPPE_AOX_Lm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_AOX_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 harboring pPPE_AOX_Ll\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_AOX_Lp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 harboring pPPE_AOX_Lp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_AOX_Sl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 harboring pPPE_AOX_Sl\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_ADH_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 harboring pPPE_ADH_Ll\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_CAT_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 harboring pPPE_CAT_Ll\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_CATm_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 harboring pPPE_CATm_Ll\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_FLD_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 harboring pPPE_FLD_Ll\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_GAP_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 harboring pPPE_GAP_Ll\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_GAPm_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 harboring pPPE_GAPm_Ll\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_TEF_Ll\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 harboring pPPE_TEF_Ll\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_CL/Z1/*\u003csup\u003ea)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 transformed with pRDZ_CAT_Ll and separated using 0.1 g/L zeocin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_CL/Z2/* \u003csup\u003ea)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 transformed with pRDZ_CAT_Ll and gene amplified using 0.5 g/L zeocin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_CL/Z3/* \u003csup\u003ea)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 transformed with pRDZ_CAT_Ll and gene amplified using 1.0 g/L zeocin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_CL/Z4/* \u003csup\u003ea)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 transformed with pRDZ_CAT_Ll and gene amplified using 2.0 g/L zeocin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_FL/Z1/* \u003csup\u003ea)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 transformed with pRDZ_FLD_Ll and separated using 0.1 g/L zeocin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_FL/Z2/* \u003csup\u003ea)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 transformed with pRDZ_FLD_Ll and gene amplified using 0.5 g/L zeocin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_FL/Z3/* \u003csup\u003ea)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 transformed with pRDZ_FLD_Ll and gene amplified using 1.0 g/L zeocin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_FL/Z4/* \u003csup\u003ea)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 transformed with pRDZ_FLD_Ll and gene amplified using 2.0 g/L zeocin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_CFL/Z1/* \u003csup\u003ea)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 simultaneously transformed with pRDZ_CAT_Ll and pRDZ_FLD_Ll and separated using 0.1 g/L zeocin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_CFL/Z2/* \u003csup\u003ea)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 simultaneously transformed with pRDZ_CAT_Ll and pRDZ_FLD_Ll and gene amplified using 0.5 g/L zeocin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_CFL/Z3/* \u003csup\u003ea)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 simultaneously transformed with pRDZ_CAT_Ll and pRDZ_FLD_Ll and gene amplified using 1.0 g/L zeocin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGS115_CFL/Z4/* \u003csup\u003ea)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGS115 simultaneously transformed with pRDZ_CAT_Ll and pRDZ_FLD_Ll and gene amplified using 2.0 g/L zeocin\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003ea) *;1\u0026ndash;90\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of\u003c/b\u003e \u003cb\u003eD-LDH\u003c/b\u003e\u003cb\u003e-expressing promoter on D-lactic acid production\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo select a suitable promoter to express LlDLDH, which is optimal for \u003cem\u003eD-LDH\u003c/em\u003e gene expression in \u003cem\u003eK. phaffii\u003c/em\u003e, 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 \u003cem\u003eK. phaffii\u003c/em\u003e GS115, resulting in strains designated as GS115_X_Ll (X: AOX, CAT, CATm, FLD, ADH, GAP, GAPm, and TEF; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB shows the intracellular \u003cem\u003eD-LDH\u003c/em\u003e activity of the GS115_X_Ll strains after 48 h of cultivation. \u003cem\u003eD-LDH\u003c/em\u003e 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 \u003cem\u003eD-LDH\u003c/em\u003e activity and D-lactic acid production in the top three strains with high \u003cem\u003eD-LDH\u003c/em\u003e activity. In the three strains showing low D-lactic acid production (GS115_ADH_Ll, GS115_CATm_Ll, and GS115_GAPm_Ll), \u003cem\u003eD-LDH\u003c/em\u003e activity was lower than that in GS115_AOX_Ll. These results indicate that pCAT1 and pFLD1 are suitable for the expression of LlDLDH in \u003cem\u003eK. phaffii\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConstruction of a strain with\u003c/b\u003e \u003cb\u003eD-LDH\u003c/b\u003e \u003cb\u003egene integrated into the rDNA locus and selection of a D-lactic acid producing strain\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExamination of the types of \u003cem\u003eD-LDH\u003c/em\u003e genes and promoters revealed that the use of \u003cem\u003eLlDLDH\u003c/em\u003e as the \u003cem\u003eD-LDH\u003c/em\u003e gene and pCAT1 or pFLD1 as the promoter was suitable for D-lactic acid production in \u003cem\u003eK. phaffii\u003c/em\u003e. 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\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and transformed into \u003cem\u003eK. phaffii\u003c/em\u003e 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\u0026ndash;90; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e 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).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003ePost-transformational gene amplification and selection of strains with high D-lactic acid production\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eAmong 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) 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\u0026ndash;90), respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). 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\u0026ndash;90) or GS115_CFL/Z2/*, GS115_CFL/Z3/*, and GS115_CFL/Z4/* (*;1\u0026ndash;90), respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eD-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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, 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.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eTime course of D-lactic acid production by post-transformational gene-amplified strains in a flask culture\u003c/h2\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), they were cultured in flasks containing the YPM medium. The methanol concentrations, D-lactic acid concentrations, and OD\u003csub\u003e600\u003c/sub\u003e values of the strains are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, and C, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, 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\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB 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\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC 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, OD\u003csub\u003e600\u003c/sub\u003e values of all three strains were similar.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the type of \u003cem\u003eD-LDH\u003c/em\u003e gene expressed and optimal combination of promoters to improve D-lactic acid production from methanol in \u003cem\u003eK. phaffii\u003c/em\u003e. The highest D-lactic acid production was achieved by integrating the \u003cem\u003eLlDLDH\u003c/em\u003e gene from \u003cem\u003eL. lactis\u003c/em\u003e linked to pCAT1 and pFLD1 into the rDNA locus of the \u003cem\u003eK. phaffii\u003c/em\u003e 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 \u003cem\u003eK. phaffii\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn this study, two types of \u003cem\u003eD-LDH\u003c/em\u003e genes, \u003cem\u003eLlDLDH\u003c/em\u003e and \u003cem\u003eLpDLDH\u003c/em\u003e, were expressed in \u003cem\u003eK. phaffii\u003c/em\u003e, and they achieved higher D-lactic acid production than conventional \u003cem\u003eLmDLDH\u003c/em\u003e (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e); the highest D-lactic acid production was achieved with \u003cem\u003eLlDLDH\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Watcharawipas et al. also expressed these three \u003cem\u003eD-LDH\u003c/em\u003e genes in \u003cem\u003eS. cerevisiae\u003c/em\u003e and reported that the highest D-lactic acid production is achieved with \u003cem\u003eLpDLDH\u003c/em\u003e, followed by \u003cem\u003eLlDLDH\u003c/em\u003e and \u003cem\u003eLmDLDH\u003c/em\u003e (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In contrast, in a study on \u003cem\u003eD-LDH\u003c/em\u003e expression in \u003cem\u003eO. polymorpha\u003c/em\u003e, almost no D-lactic acid was produced with \u003cem\u003eLmDLDH\u003c/em\u003e (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). This indicates that the effective \u003cem\u003eD-LDH\u003c/em\u003e gene varies depending on the host microorganism. Therefore, type of \u003cem\u003eD-LDH\u003c/em\u003e gene expressed in each host should be considered for high D-lactic acid production.\u003c/p\u003e \u003cp\u003eIn this study, high D-lactic acid production was achieved when pCAT1 or pFLD1 was used to express \u003cem\u003eD-LDH\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). 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 (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). 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 \u003cem\u003eK. phaffii\u003c/em\u003e (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). In terms of cell proliferation, optimal methanol concentration for culturing \u003cem\u003eK. phaffii\u003c/em\u003e is approximately 4.0 g/L (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). In addition, methanol concentrations\u0026thinsp;\u0026lt;\u0026thinsp;10 g/L have been used to produce heterologous proteins and useful compounds using methanol-inducible promoters in \u003cem\u003eK. phaffii\u003c/em\u003e (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan additionalcitationids=\"CR47 CR48 CR49\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). 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\u0026thinsp;\u0026gt;\u0026thinsp;10 g/L. However, similar to the galactose-inducible promoter, pGAL1, in \u003cem\u003eS. cerevisiae\u003c/em\u003e (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e), the level of gene expression driven by the methanol-inducible promoter in \u003cem\u003eK. phaffii\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003eWhen LlDLDH was expressed in \u003cem\u003eK. phaffii\u003c/em\u003e using two mutant promoters, pCAT1m and pGAP1m, almost no D- lactic acid production was observed with either promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). pCAT1m and pGAP1m are mutants of pCAT1 and pGAP1, respectively, with a partial modification of the promoter sequence to improve GFP fluorescence (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Gene expression levels vary depending on the gene and promoter combination (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003eGS115_CFL/Z3/04 strain, constructed by integrating the \u003cem\u003eLlDLDH\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In a previous study, D-lactic acid production of 3.48 g/L was achieved using engineered \u003cem\u003eK. phaffii\u003c/em\u003e (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). In addition, production of 3.8 g/L L-lactic acid from methanol was achieved using \u003cem\u003eO. polymorpha\u003c/em\u003e (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Therefore, to the best of our knowledge, this study achieved the highest reported lactic acid production from methanol for both D- and L-lactic acids. Previous studies have reported that the combined use of the two promoters, pAOX1 and pGAP1, enhances the production of heterologous proteins in \u003cem\u003eK. phaffii\u003c/em\u003e (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). 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 \u003cem\u003eK. phaffii\u003c/em\u003e are reduced by amino acids in the culture medium (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e), and the repression conditions of pCAT1 and pFLD1 used in this study were different (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Combination of the two promoters with different repression conditions, pCAT1 and pFLD1, in this study enabled \u003cem\u003eD-LDH\u003c/em\u003e 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 \u003cem\u003eK. phaffii\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, \u003cem\u003eLlDLDH\u003c/em\u003e gene from \u003cem\u003eL. lactis\u003c/em\u003e was linked to pCAT1 and pFLD1, integrated into the rDNA locus of \u003cem\u003eK. phaffii\u003c/em\u003e, 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 from methanol produced by this engineered yeast (5.18 g/L) is the highest reported value to date. 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 \u003cem\u003eK. phaffii\u003c/em\u003e. This study outlines a method to engineer other \u003cem\u003eK. phaffii\u003c/em\u003e strains capable of producing various useful chemicals in the future.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAHMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e4-amino-3-hydrazino-5-mercapto-1,2,4-triazole\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eD-LDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eD-lactate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was partially supported by the Japan Society for the Promotion of Science (grant number JP22H03803).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYI 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.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe would like to thank Editage for English language editing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWendisch VF, Brito LF, Lopez MG, Hennig G, Pfeifenschneider J, et al. The flexible feedstock concept in Industrial Biotechnology: Metabolic engineering of \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e and yeast strains for access to alternative carbon sources. 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Enhancing the production of recombinant acidic α-amylase and phytase in \u003cem\u003ePichia pastoris\u003c/em\u003e under dual promoters [constitutive (\u003cem\u003eGAP\u003c/em\u003e) and inducible (\u003cem\u003eAOX\u003c/em\u003e)] in mixed fed batch high cell density cultivation. Process Biochem. 2016;51:1315\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVelastegui E, Theron C, Berrios J, Fickers P. Downregulation by organic nitrogen of \u003cem\u003eAOX1\u003c/em\u003e promoter used for controlled expression of foreign genes in the yeast \u003cem\u003ePichia pastoris\u003c/em\u003e. Yeast. 2019;36:297\u0026ndash;304.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang XF, Shen XG, Sun YC, Zhao HY, Xu L, et al. Production of \u003cem\u003eYarrowia lipolytica\u003c/em\u003e lipase LIP2 in \u003cem\u003ePichia pastoris\u003c/em\u003e using the nitrogen source-regulated \u003cem\u003eFLD1\u003c/em\u003e promoter. J Chem Technol Biotechnol. 2011;87:553\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"D-lactic acid, Komagataella phaffii, Methanol, Metabolic engineering, Yeast","lastPublishedDoi":"10.21203/rs.3.rs-5101349/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5101349/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCurrently, efficient technologies producing useful chemicals from alternative carbon resources, such as methanol, to replace petroleum are in demand. The methanol-utilizing yeast, \u003cem\u003eKomagataella phaffii\u003c/em\u003e, 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 (\u003cem\u003eD-LDH\u003c/em\u003e) genes and promoters in \u003cem\u003eK. phaffii\u003c/em\u003e. The yeast strain was constructed by integrating a gene cassette containing the identified gene and promoter into the rDNA locus of \u003cem\u003eK. phaffii\u003c/em\u003e, followed by post-transformational gene amplification. Subsequently, D-lactic acid production from methanol was evaluated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAmong the five \u003cem\u003eD-LDH\u003c/em\u003e genes and eight promoters tested, the combination of LlDLDH derived from \u003cem\u003eLeuconostoc lactis\u003c/em\u003e and \u003cem\u003eCAT1\u003c/em\u003e and \u003cem\u003eFLD1\u003c/em\u003e promoters was suitable for expression in \u003cem\u003eK. phaffii\u003c/em\u003e. GS115_CFL/Z3/04, the best-engineered strain constructed via integration of LlDLDH linked to \u003cem\u003eCAT1\u003c/em\u003e and \u003cem\u003eFLD1\u003c/em\u003e 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, including both D- and L- lactic acids.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis 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 \u003cem\u003eK. phaffii\u003c/em\u003e. Using our established method, other \u003cem\u003eK. phaffii\u003c/em\u003e strains can be engineered to produce various useful chemicals in the future.\u003c/p\u003e","manuscriptTitle":"Enhancement of D-lactic acid production by the methylotrophic yeast, Komagataella phaffii, via optimization of D-LDH gene and promoter and post-transformational gene amplification","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-15 17:58:28","doi":"10.21203/rs.3.rs-5101349/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-07T23:23:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-27T12:45:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148372592265752832488307353955795252425","date":"2024-10-15T06:15:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4430152818722663090726229394929396396","date":"2024-10-08T00:57:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-07T16:28:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-18T11:16:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-18T11:16:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology for Biofuels and Bioproducts","date":"2024-09-17T07:28:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8347075f-9c55-495a-98ee-81915772ba4b","owner":[],"postedDate":"November 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-23T16:02:06+00:00","versionOfRecord":{"articleIdentity":"rs-5101349","link":"https://doi.org/10.1186/s13068-024-02596-0","journal":{"identity":"biotechnology-for-biofuels-and-bioproducts","isVorOnly":false,"title":"Biotechnology for Biofuels and Bioproducts"},"publishedOn":"2024-12-22 15:57:38","publishedOnDateReadable":"December 22nd, 2024"},"versionCreatedAt":"2024-11-15 17:58:28","video":"","vorDoi":"10.1186/s13068-024-02596-0","vorDoiUrl":"https://doi.org/10.1186/s13068-024-02596-0","workflowStages":[]},"version":"v1","identity":"rs-5101349","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5101349","identity":"rs-5101349","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}