A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris

In methylotrophic yeasts, glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required for the metabolism of methanol as a carbon source and certain alkylated amines such as methylamine as nitrogen sources. We describe the isolation and characterization of the FLD1 gene from the yeast Pichia pastoris. The gene contains a single short intron with typical yeast-splicing signals near its 5′ end, the first intron to be demonstrated in this yeast. The predicted FLD1 product (Fld1p) is a protein of 379 amino acids (approx. 40 kDa) with 71% identity to the FLD protein sequence from the n-alkane-assimilating yeast Candida maltosa and 61–65% identity with dehydrogenase class III enzymes from humans and other higher eukaryotes. Using β-lactamase as a reporter, we show that the FLD1 promoter (P_FLD1) is strongly and independently induced by either methanol as sole carbon source (with ammonium sulfate as nitrogen source) or methylamine as sole nitrogen source (with glucose as carbon source). Furthermore, with either methanol or methylamine induction, levels of β-lactamase produced under control of P_FLD1 are comparable to those obtained with the commonly used alcohol oxidase I gene promoter (P_AOX1). Thus, P_FLD1 is an attractive alternative to P_AOX1 for expression of foreign genes in P. pastoris, allowing the investigator a choice of carbon (methanol) or nitrogen source (methylamine) regulation with the same expression strain.     

Engineering of carboligase activity reaction in Candida glabrata for acetoin production

Utilization of Candida glabrata overproducing pyruvate is a promising strategy for high-level acetoin production. Based on the known regulatory and metabolic information, acetaldehyde and thiamine were fed to identify the key nodes of carboligase activity reaction (CAR) pathway and provide a direction for engineering C. glabrata. Accordingly, alcohol dehydrogenase, acetaldehyde dehydrogenase, pyruvate decarboxylase, and butanediol dehydrogenase were selected to be manipulated for strengthening the CAR pathway. Following the rational metabolic engineering, the engineered strain exhibited increased acetoin biosynthesis (2.24 g/L). In addition, through in silico simulation and redox balance analysis, NADH was identified as the key factor restricting higher acetoin production. Correspondingly, after introduction of NADH oxidase, the final acetoin production was further increased to 7.33 g/L. By combining the rational metabolic engineering and cofactor engineering, the acetoin-producing C. glabrata was improved stepwise, opening a novel pathway for rational development of microorganisms for bioproduction.     

Artificial consortium that produces riboflavin regulates distribution of acetoin and 2,3‐butanediol by Paenibacillus polymyxa CJX518

The introduction of an NADH/NAD⁺ regeneration system can regulate the distribution between acetoin and 2,3‐butanediol. NADH regeneration can also enhance butanol production in coculture fermentation. In this work, a novel artificial consortium of Paenibacillus polymyxa CJX518 and recombinant Escherichia coli LS02T that produces riboflavin (VB₂) was used to regulate the NADH/NAD⁺ ratio and, consequently, the distribution of acetoin and 2,3‐butanediol by P. polymyxa. Compared with a pure culture of P. polymyxa, the level of acetoin was increased 76.7% in the P. polymyxa and recombinant E. coli coculture. Meanwhile, the maximum production and yield of acetoin in an artificial consortium with fed‐batch fermentation were 57.2 g/L and 0.4 g/g glucose, respectively. Additionally, the VB₂ production of recombinant E. coli could maintain a relatively low NADH/NAD⁺ ratio by changing NADH dehydrogenase activity. It was also found that 2,3‐butanediol dehydrogenase activity was enhanced and improved acetoin production by the addition of exogenous VB₂ or by being in the artificial consortium that produces VB₂. These results illustrate that the coculture of P. polymyxa and recombinant E. coli has enormous potential to improve acetoin production. It was also a novel strategy to regulate the NADH/NAD⁺ ratio to improve the acetoin production of P. polymyxa.     

Metabolic Engineering of Bacillus subtilis for Ethanol Production: Lactate Dehydrogenase Plays a Key Role in Fermentative Metabolism

Wild-type Bacillus subtilis ferments 20 g/liter glucose in 48 h, producing lactate and butanediol, but not ethanol or acetate. To construct an ethanologenic B. subtilis strain, homologous recombination was used to disrupt the native lactate dehydrogenase (LDH) gene (ldh) by chromosomal insertion of the Zymomonas mobilis pyruvate decarboxylase gene (pdc) and alcohol dehydrogenase II gene (adhB) under the control of the ldh native promoter. The values of the intracellular PDC and ADHII enzymatic activities of the engineered B. subtilis BS35 strain were similar to those found in an ethanologenic Escherichia coli strain. BS35 produced ethanol and butanediol; however, the cell growth and glucose consumption rates were reduced by 70 and 65%, respectively, in comparison to those in the progenitor strain. To eliminate butanediol production, the acetolactate synthase gene (alsS) was inactivated. In the BS36 strain (BS35 ΔalsS), ethanol production was enhanced, with a high yield (89% of the theoretical); however, the cell growth and glucose consumption rates remained low. Interestingly, kinetic characterization of LDH from B. subtilis showed that it is able to oxidize NADH and NADPH. The expression of the transhydrogenase encoded by udhA from E. coli allowed a partial recovery of the cell growth rate and an early onset of ethanol production. Beyond pyruvate-to-lactate conversion and NADH oxidation, an additional key physiological role of LDH for glucose consumption under fermentative conditions is suggested. Long-term cultivation showed that 8.9 g/liter of ethanol can be obtained using strain BS37 (BS35 ΔalsS udhA⁺). As far as we know, this is the highest ethanol titer and yield reported with a B. subtilis strain.     

An “energy-auxotroph” Escherichia coli provides an in vivo platform for assessing NADH regeneration systems

An efficient in vivo regeneration of the primary cellular resources NADH and ATP is vital for optimizing the production of value-added chemicals and enabling the activity of synthetic pathways. Currently, such regeneration routes are tested and characterized mainly in vitro before being introduced into the cell. However, in vitro measurements could be misleading as they do not reflect enzyme activity under physiological conditions. Here, we construct an in vivo platform to test and compare NADH regeneration systems. By deleting dihydrolipoyl dehydrogenase in Escherichia coli, we abolish the activity of pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. When cultivated on acetate, the resulting strain is auxotrophic to NADH and ATP: acetate can be assimilated via the glyoxylate shunt but cannot be oxidized to provide the cell with reducing power and energy. This strain can, therefore, serve to select for and test different NADH regeneration routes. We exemplify this by comparing several NAD-dependent formate dehydrogenases and methanol dehydrogenases. We identify the most efficient enzyme variants under in vivo conditions and pinpoint optimal feedstock concentrations that maximize NADH biosynthesis while avoiding cellular toxicity. Our strain thus provides a useful platform for comparing and optimizing enzymatic systems for cofactor regeneration under physiological conditions.     

Efficient secretion of inulin fructotransferase in Pichia pastoris using the formaldehyde dehydrogenase 1 promoter

Inulin fructotransferase (IFTase) has received considerable attention due to its ability to catalyse inulin hydrolysis to difructose anhydride (DFA III), a natural low-calorie functional sweetener. In the present study, for the first time, we describe the expression of IFTase in Pichia pastoris under the control of the formaldehyde dehydrogenase 1 promoter (PFLD1). Using this system, we achieved efficient secretion with four substrate fed-batch strategies in a 3-L fermenter. The co-feeding induction strategy with methylamine hydrochloride and methanol achieved the maximum extracellular IFTase activity of 62.72 U mL^?1, which was 3.2-fold higher than that obtained with the wild-type strain. In addition to methanol, carbon sources such as glucose and glycerol could also be utilised by PFLD1-controlled P. pastoris for IFTase production using methylamine hydrochloride induction. However, we found that glycerol and glucose should be strictly controlled at low concentrations of 0.5–1.5 % (v/v) and 1–1.5 % (w/v), respectively. The use of glycerol and glucose demonstrated that P. pastoris was also attractive for IFTase production via methanol-free cultivation strategies. This study may provide the basis for the industrial use of this recombinant IFTase for the production of DFA III.     

Expression of NAD<Superscript>+</Superscript>-dependent formate dehydrogenase in <Emphasis Type="Italic">Enterobacter aerogenes</Emphasis> and its involvement in anaerobic metabolism and H<Subscript>2</Subscript> production

An expression system for NAD⁺-dependent formate dehydrogenase gene (fdh1), from Candida boidinii, was constructed and cloned into Enterobacter aerogenes IAM1183. With the fdh1 expression, the total H₂ yield was attributed to a decrease in activity of the lactate pathway and an increase of the formate pathway flux due to the NADH regeneration. Analysis of the redox state balance and ethanol-to-acetate ratio in the fdhl-expressed strain showed that increased reducing power arose from the reconstruction of NADH regeneration pathway from formate thereby contributing to the improved H₂ production.     

Intracellular expression of Vitreoscilla hemoglobin improves S-adenosylmethionine production in a recombinant Pichia pastoris

To develop an efficient way to produce S-adenosylmethionine (SAM), methionine adenosyltransferase gene (mat) from Streptomyces spectabilis and Vitreoscilla hemoglobin gene (vgb) were coexpressed intracellularly in Pichia pastoris, both under control of methanol-inducible promoter. Expression of mat in P. pastoris resulted in about 27 times higher specific activity of methionine adenosyltransferase (SMAT) and about 19 times higher SAM production relative to their respective control, suggesting that overexpression of mat could be used as an efficient method for constructing SAM-accumulating strain. Under induction concentration of 0.8 and 2.4% methanol, coexpression of vgb improved, though to different extent, cell growth, SAM production, and respiratory rate. However, the effects of VHb on SAM content (specific yield of SAM production) and SMAT seemed to be methanol concentration-dependent. When cells were induced with 0.8% methanol, no significant effects of VHb expression on SAM content and specific SMAT could be detected. When the cells were induced with 2.4% methanol, vgb expression increased SAM content significantly and depressed SMAT remarkably. We suggested that under our experimental scheme, the presence of VHb might improve ATP synthesis rate and thus improve cell growth and SAM production in the recombinant P. pastoris.     

Metabolic engineering of Bacillus subtilis for ethanol production: lactate dehydrogenase plays a key role in fermentative metabolism

Wild-type Bacillus subtilis ferments 20 g/liter glucose in 48 h, producing lactate and butanediol, but not ethanol or acetate. To construct an ethanologenic B. subtilis strain, homologous recombination was used to disrupt the native lactate dehydrogenase (LDH) gene (ldh) by chromosomal insertion of the Zymomonas mobilis pyruvate decarboxylase gene (pdc) and alcohol dehydrogenase II gene (adhB) under the control of the ldh native promoter. The values of the intracellular PDC and ADHII enzymatic activities of the engineered B. subtilis BS35 strain were similar to those found in an ethanologenic Escherichia coli strain. BS35 produced ethanol and butanediol; however, the cell growth and glucose consumption rates were reduced by 70 and 65%, respectively, in comparison to those in the progenitor strain. To eliminate butanediol production, the acetolactate synthase gene (alsS) was inactivated. In the BS36 strain (BS35 delta alsS), ethanol production was enhanced, with a high yield (89% of the theoretical); however, the cell growth and glucose consumption rates remained low. Interestingly, kinetic characterization of LDH from B. subtilis showed that it is able to oxidize NADH and NADPH. The expression of the transhydrogenase encoded by udhA from E. coli allowed a partial recovery of the cell growth rate and an early onset of ethanol production. Beyond pyruvate-to-lactate conversion and NADH oxidation, an additional key physiological role of LDH for glucose consumption under fermentative conditions is suggested. Long-term cultivation showed that 8.9 g/liter of ethanol can be obtained using strain BS37 (BS35 delta alsS udhA+). As far as we know, this is the highest ethanol titer and yield reported with a B. subtilis strain.     

Expression of functional single-chain variable domain fragment (scFv) antibody against Metolcarb in Pichia pastoris

A Pichia pastoris system was used to express a single-chain variable fragment (scFv) antibody targeted against Metolcarb. The specific scFv gene was amplified from the phage-display scFv library and then subcloned into the expression vector pPICZα C. The resulting plasmid, pPICZα C-scFv, was linearized and transformed into P. pastoris strain X-33. A transformant named X-33-Pp-SMW-12-6, which showed strong expression of antibodies, was isolated, and the culture conditions, including methanol induction concentrations, inoculum densities, and pH, were optimized. Under optimal conditions, P. pastoris cultures yielded much higher levels of the scFv product than the Escherichia coli expression system. Immunochemical characterization of the scFv antibodies produced in P. pastoris indicated that the affinity and specificity of scFv against Metolcarb are comparable to those of scFv antibodies produced in E. coli. Recoveries of Metolcarb demonstrated that the P. pastoris-derived scFv antibodies can be used to determine the content of Metolcarb residue in environmental and agricultural samples using a competitive inhibition enzyme-linked immunosorbent assay. For our purposes, expression in Pichia proved to be an efficient and economical method for the large-scale production of functional scFv antibodies against Metolcarb for downstream applications.     

Expression of multidrug transporter P-glycoprotein in Pichia pastoris affects the host's methanol metabolism

Pichia pastoris KM71H (Mut^S) is an efficient producer of hard-to-express proteins such as the membrane protein P-glycoprotein (Pgp), an ATP-powered efflux pump which is expressed properly, but at very low concentration, using the conventional induction strategy. Evaluation of different induction strategies indicated that it was possible to increase Pgp expression by inducing the culture with 20% media containing 2.5% methanol. By quantifying methanol, formaldehyde, hydrogen peroxide and formate, and by measuring alcohol oxidase, catalase, formaldehyde dehydrogenase, formate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenases, it was possible to correlate Pgp expression to the induction strategy. Inducing the culture by adding methanol with fresh media was associated with decreases in formaldehyde and hydrogen peroxide, and increases in formaldehyde dehydrogenase, formate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenases. At these conditions, Pgp expression was 1400-fold higher, an indication that Pgp expression is affected by increases in formaldehyde and hydrogen peroxide. It is possible that Pgp is responsible for this behaviour, since the increased metabolite concentrations and decreased enzymatic activities were not observed when parental Pichia was subjected to the same growth conditions. This report adds information on methanol metabolism during expression of Pgp from P. pastoris Mut^S strain and suggests an expression procedure for hard-to-express proteins from P. pastoris.     

Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichia pastoris

The industrial yeast Pichia pastoris has been harnessed extensively for production of proteins, and it is attracting attention as a chassis cell factory for production of chemicals. However, the lack of synthetic biology tools makes it challenging in rewiring P. pastoris metabolism. We here extensively engineered the recombination machinery by establishing a CRISPR-Cas9 based genome editing platform, which improved the homologous recombination (HR) efficiency by more than 54 times, in particular, enhanced the simultaneously assembly of multiple fragments by 13.5 times. We also found that the key HR-relating gene RAD52 of P. pastoris was largely repressed in compared to that of Saccharomyces cerevisiae. This gene editing system enabled efficient seamless gene disruption, genome integration and multiple gene assembly with positive rates of 68–90%. With this efficient genome editing platform, we characterized 46 potential genome integration sites and 18 promoters at different growth conditions. This library of neutral sites and promoters enabled two-factorial regulation of gene expression and metabolic pathways and resulted in a 30-fold range of fatty alcohol production (12.6–380 mg/l). The expanding genetic toolbox will facilitate extensive rewiring of P. pastoris for chemical production, and also shed light on engineering of other non-conventional yeasts.     

Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by Pichia pastoris

The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G₄S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation. This immunotoxin was produced extracellularly in toxin-sensitive Pichia pastoris JW102 (Mut⁺) under control of the AOX1 promoter. There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for P. pastoris and the limited capacity of P. pastoris to secrete the immunotoxin. The immunotoxin toxicity resulted in a decrease in the methanol consumption rate, cessation of cell growth, and low immunotoxin productivity after the first 22 h of methanol induction. Continuous cell growth and continuous immunotoxin secretion after the first 22 h of methanol induction were obtained by adding glycerol to the methanol feed by using a 4:1 methanol-glycerol mixed feed as an energy source and by continuously adding a yeast extract solution during methanol induction. The secretory capacity was increased from 22.5 to 37 mg/liter by lowering the induction temperature. A low temperature reduced the methanol consumption rate and protease activity in the supernatant but not cell growth. The effects of adding glycerol and yeast extract to the methanol feed were synergistic. Adding yeast extract primarily enhanced methanol utilization and cell growth, while adding glycerol primarily enhanced immunotoxin production. The synergy was further enhanced by decreasing the induction temperature from 23 to 15°C, which resulted in a robust process with a yield of 37 mg/liter, which was sevenfold greater than the yield previously reported for a toxin-resistant CHO cell expression system. This methodology should be applicable to other toxin-related recombinant proteins in toxin-sensitive P. pastoris.     

Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter⁻¹ acetate during fermentation of 114 g liter⁻¹ glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter⁻¹, this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter⁻¹ and raised the ethanol yield to 7% above the wild-type level.     

谷氨酸脱氢酶与醇脱氢酶的共表达及其在制备(R)-2-氯-1-苯乙醇中的应用

【背景】醇脱氢酶AdhS能催化不对称还原反应制备(R)-2-氯-1-苯乙醇,但由于自身再生辅酶NADH的能力不足,需要辅酶再生酶协助其再生NADH。谷氨酸脱氢酶能以谷氨酸为底物,再生辅酶NAD(P)H,具有辅酶再生酶的潜力。【目的】克隆表达谷氨酸脱氢酶基因gdhA,构建谷氨酸脱氢酶GdhA与醇脱氢酶AdhS的大肠杆菌共表达体系,提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。【方法】从枯草芽孢杆菌(Bacillus subtilis) 168中克隆基因gdhA,并在大肠杆菌(Escherichia coli) BL21(DE3)中表达,分析辅酶再生活力;再与醇脱氢酶AdhS共表达,优化表达条件;分析不同辅酶再生方案对制备(R)-2-氯-1-苯乙醇的转化效率的影响。【结果】谷氨酸脱氢酶GdhA再生NADH的比活力为694 U/g。经GdhA与AdhS的共表达及表达条件优化后,制备(R)-2-氯-1-苯乙醇的转化效率达465 U/L。经比较,GdhA协助再生辅酶NADH,可使AdhS制备(R)-2-氯-1-苯乙醇的转化效率提高到约3倍。【结论】谷氨酸脱氢酶GdhA为NADH高效再生酶,与醇脱氢酶AdhS共表达可显著提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。     

Enhanced protein production by sorbitol co-feeding with methanol in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis> strains

Pichia pastoris strains carrying 1, 6, 12, and 18 copies of the porcine insulin precursor (PIP) gene, were employed to investigate the effects of sorbitol co-feeding with methanol on the physiology of the strains. Multicopy clones of the methylotrophic yeast were generated to vary the PIP gene dosage and recombinant proteins. Elevated gene dosage increased levels of the recombinant PIP protein when methanol served as the sole carbon and energy source i.e., an increase of 1.9% for a strain carrying 1 copy, 42.6% for a strain carrying 6 copies, 34.7% for a strain carrying 12 copies and 80.9% for a strain carrying 18 copies, respectively (using sorbitol co-feeding with methanol during the induction phase). However, it had no significant influence on a lower gene dosage strain (1 copy), but this approach affirmed enhancement in cell growth and PIP production for higher gene dosage strain (6, 12, and 18 copies) via using sorbitol co-feeding with methanol. Additionally, the co-feeding strategy could hold vital importance for recombinant protein production by a multi-copy P. pastoris system.     

Reduced Oxidative Pentose Phosphate Pathway Flux in Recombinant Xylose-Utilizing Saccharomyces cerevisiae Strains Improves the Ethanol Yield from Xylose

In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD⁺. In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g⁻¹) and the lowest xylitol yield (0.05 g g⁻¹) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.     

Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris: A review

Pichia pastoris has been recognized as one of the most industrially important hosts for heterologous protein production. Despite its high protein productivity, the optimization of P. pastoris cultivation is still imperative due to strain- and product-specific challenges such as promoter strength, methanol utilization type and oxygen demand. To address the issues, strategies involving genetic and process engineering have been employed. Optimization of codon usage and gene dosage, as well as engineering of promoters, protein secretion pathways and methanol metabolic pathways have proved beneficial to innate protein expression levels. Large-scale production of proteins via high cell density fermentation additionally relies on the optimization of process parameters including methanol feed rate, induction temperature and specific growth rate. Recent progress related to the enhanced production of proteins in P. pastoris via various genetic engineering and cultivation strategies are reviewed. Insight into the regulation of the P. pastoris alcohol oxidase 1 (AOX1) promoter and the development of methanol-free systems are highlighted. Novel cultivation strategies such as mixed substrate feeding are discussed. Recent advances regarding substrate and product monitoring techniques are also summarized. Application of P. pastoris to the production of biodiesel and other value-added products via metabolic engineering are also reviewed. P. pastoris is becoming an indispensable platform through the use of these combined engineering strategies.