Production of pyruvate in Saccharomyces cerevisiae through adaptive evolution and rational cofactor metabolic engineering

doi:10.1016/j.bej.2012.06.006

Biochemical Engineering Journal

Volume 67, 15 August 2012, Pages 126-131

https://doi.org/10.1016/j.bej.2012.06.006 Get rights and content

Abstract

Pyruvate-decarboxylase (Pdc)-negative Saccharomyces cerevisiae has been proven as a suitable metabolic engineering platform to produce organic acids. S. cerevisiae BY5419 Pdc⁻ strain cannot grow in batch cultures on synthetic medium with glucose as the sole carbon source, yet grows well on synthetic medium with ethanol or acetate. In this study, by combining adaptive evolution and cofactor engineering, we obtained a series of engineered yeasts that can produce pyruvate using glucose as sole carbon source. Differential expression of noxE, encoding a water-forming NADH oxidase from Lactococcus lactis, and udhA, encoding a soluble pyridine nucleotide transhydrogenase from Escherichia coli, was investigated. Of all the constructed recombinant strains, G2U1-A₀ was able to produce 75.1 g l⁻¹ pyruvate, increased 21% compared with the original strain A₀. The production yield of this strain reached 0.63 g of pyruvate g of glucose⁻¹. This study demonstrates that the fine regulation of intracellular NADH/NAD⁺ ratio is critical for cell metabolism and pyruvate production. Combining the adaptive evolution and fine regulation of intracellular NADH/NAD⁺ ratio provides a new strategy for improving the Pdc⁻ strain engineering platform.

Highlights

► Combining adaptive evolution and cofactor engineering obtained engineered yeasts. ► G2U1-A₀ was able to produce 75.1 g l⁻¹ pyruvate. ► Differential expression of noxE and udhA was investigated. ► Fine regulation of NADH/NAD⁺ ratio is critical to pyruvate production. ► A new strategy for improvement of the Pdc⁻ strain engineering platform is provided.

Introduction

Saccharomyces cerevisiae is extensively known for its molecular genetics, physiology and genomics, allowing excellent accessibility for genetic modification and making it an excellent candidate as a biocatalyst for metabolic engineering [1]. The collection of compounds that are produced using S. cerevisiae has expanded to include organic acids and even secondary metabolites [2].

Pyruvate, a product of glycolysis, is located at an important branching point in the metabolism of carbohydrates in S. cerevisiae. Apart from its significance in metabolism, pyruvate serves as an effective starting material for the synthesis of many drugs and agrochemicals and is presently in the food industry as a fat burning supplement. It is also a valuable substrate for the enzymatic production of amino acids such as l-tryptophan, l-tyrosine, and l-dihydroxyphenylalanine (l-DOPA) [3]. Even under fully aerobic conditions, high glycolytic fluxes in wild-type S. cerevisiae strains are intrinsically linked to alcoholic fermentation [4]. Therefore, to avoid reduced product yields as a result of ethanol co-production, the metabolic engineering strategy for high-yield production of pyruvate and other organic acids with S. cerevisiae must focus on the reduction or elimination of ethanol formation. One approach for eliminating ethanol formation was to block pyruvate decarboxylase activity [5]. Pyruvate decarboxylase is critical for switching sugar catabolism from respiratory to fermentative. Pyruvate decarboxylase negative S. cerevisiae (Pdc⁻), in which three structural genes (PDC1, PDC5 and PDC6) [6] that encode active pyruvate decarboxylase isoenzymes were knocked out, could excrete significant amounts of pyruvate into the medium when limited amount of glucose was supplied. However, this strain was unable to grow in batch culture under normal growth conditions, and the growth of this strain in synthetic media with limited glucose required the supply of C₂ compound (acetate or ethanol) [7]. Over-expression of relative enzymes producing acetaldehyde could not completely restore the growth of Pdc⁻ strains [8]. The requirement for C₂-compounds and high glucose sensitivity presented obstacles in the use of Pdc⁻ strains for production of pyruvate and other organic acids. Glucose sensitivity has been observed in Pdc⁻ strain constructed S. cerevisiae with genetically different backgrounds [7], [8]. However, its molecular basis remains unknown.

Adaptive evolution, which does not require a priori knowledge on the molecular basis of cellular tolerance, provides a global regulation strategy using irrational metabolic engineering, which is especially useful for metabolite resistance or sugar utilization [9]. During evolution, microbial populations or single-celled microorganisms are driven to adapt to environmental changes. The response to environmental changes includes physiological and genetic changes, which ranges from alternations of DNA/RNA sequences to major variation of genome structure [9], [10]. This evolutionary process could be replicated in the laboratory or in industrial environments for improving strain phenotype or biotechnological processes, thus is named evolutionary engineering [11].

Maintenance of redox balance is extremely important for metabolism and cell growth. The redox couples NADH/NAD⁺ and NADPH/NADP⁺ cannot pass the mitochondrial membrane [12]. Thus, the surplus of cytosolic NADH has to be re-oxidized in the compartment where they are produced. Usually, the maintenance of cytosolic redox balance is limited in the presence of oxygen at high glucose concentration, in which glycerol formation and external NADH dehydrogenase take effect [13]. Production of pyruvate from glucose generates two mole NADH per mole glucose in the cytoplasm through the catalyzation of glyceraldehyde phosphate dehydrogenase. The surplus of NADH in the cytoplasm may inhibit the glucose consumption and cell growth if it is not immediately consumed during the subsequent metabolism. Since the emergence of metabolic engineering, it has achieved many successful applications in optimization of cellular processes by manipulating the throughput of certain pathways [14], [15]. In this view, cofactor engineering by alternation of intracellular NADH pool or NADH/NAD⁺ ratio might be helpful to alleviate redox unbalance burden in vivo. NoxE, encoding a water-forming NADH oxidase from Lactococcus lactis [16], could potently decrease the intracellular NADH concentration and NADH/NAD⁻ ratio, causing a large redistribution of metabolic flux. Another candidate gene, UdhA, encoding a soluble pyridine nucleotide transhydrogenase from Escherichia coli, could catalyze the reversible transfer between NADH and NADPH [17].

In this study, we first performed the adaptive evolution of the Pdc⁻ yeast BY5419, aiming at metabolic utilization of glucose as sole carbon sources in batch culture. Then, noxE and udhA were expressed in the evolved strain at different levels using various promoter strengths and plasmid copy number to investigate the regulation of NADH/NAD⁺ on cell metabolism and pyruvate production. Pyruvate decarboxylase-negative (Pdc⁻) strain has been proven as a suitable metabolic engineering platform to produce organic acids. Our present study provides a new strategy to improve this strain as an engineering platform.

Section snippets

Strains and cultivation conditions

E. coli DH5α was used for molecular manipulation, and was cultivated in LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) at 37 °C. S. cerevisiae BY5419 (pdc1Δ::LEU2 pdc5Δ::URA3 pdc6Δ::TRP1 leu2-3,112 ura3-52 trp1-92 GAL) was obtained from the Japanese Yeast Genetic Resource Center (NBRP), and was cultivated in YPE-3% medium (1% yeast extract, 2% tryptone, 3% ethanol). YPD medium (1% yeast extract, 2% tryptone, 2% glucose) or SD minimal medium (0.67% yeast nitrogen base without amino acids,

Adapted evolution of Pdc⁻ strain BY5419

The Pdc⁻ S. cerevisiae BY5419 has two growth defects: (i) growth on synthetic medium in glucose-limited chemostat requires the addition of small amounts of ethanol or acetate; and (ii) even in the presence of a C₂ compound, the strain cannot grow in batch cultures on synthetic medium with high a concentration of glucose [8]. This defect phenotype greatly limits the application of the strain for industrial pyruvate production. Therefore, we performed the adapted evolution of BY5419 in batch

Discussion

Although S. cerevisiae does not naturally produce organic acid (e.g. pyruvate) in large quantities, its robustness, pH tolerance, simple nutrient requirements compared with bacteria and long history as an industrial workhorse make it an excellent candidate biocatalyst for such process. The absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc⁻) strains of S. cerevisiae an interesting platform for further metabolic engineering of central metabolism. However, Pdc⁻ S.

Conclusion

In this study, by combining adaptive evolution and cofactor engineering, we obtained a series of yeasts that can produce pyruvate using glucose as a sole carbon source. The best strain G2U1-A₀ produced 75.1 g l⁻¹ pyruvate, which increased 21% compared with the original strain A₀. Expression of udhA and noxE in the evolved strain A₀ improved cell pyruvate production and/or growth. Expression of udhA at a high level is beneficial for cell growth, but results in increased glycerol accumulation.

Acknowledgments

This work was financially supported by National key Basic Research Program of China (973 Program) 2012CB725202, Shandong Science and Technology Development Plan (2010GSF10202, 2011GSF12120), grants from the National Natural Science Foundation of China (30870022, 31170097), and Independent Innovation Foundation of Shandong University, IIFSDU (2012ZD029). We would like to thank Razel Blaza for critically reading the manuscript.

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Regular articleProduction of pyruvate in Saccharomyces cerevisiae through adaptive evolution and rational cofactor metabolic engineering

Abstract

Highlights

Introduction

Section snippets

Strains and cultivation conditions

Adapted evolution of Pdc− strain BY5419

Discussion

Conclusion

Acknowledgments

FEMS Microbiol. Lett.

Curr. Opin. Microbiol.

Curr. Opin. Biotechnol.

Gene

FEMS Microbiol. Rev.

Metab. Eng.

Metab. Eng.

Metab. Eng.

Progress in metabolic engineering of Saccharomyces cerevisiae

Microbiol. Mol. Biol. Rev.

Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export

Appl. Environ. Microbiol.

Biotechnological production of pyruvic acid

Appl. Microbiol. Biotechnol.

Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae

Appl. Environ. Microbiol.

Pyruvate decarboxylase: an indispensable enzyme for growth of Saccharomyces cerevisiae on glucose

Yeast

Characterization of PDC6, a third structural gene for pyruvate decarboxylase in Saccharomyces cerevisiae

J. Bacteriol.

Overproduction of threonine aldolase circumvents the biosynthetic role of pyruvate decarboxylase in glucose-limited chemostat cultures of Saccharomyces cerevisiae

Appl. Environ. Microbiol.

Adaptive evolution of a lactose-consuming Saccharomyces cerevisiae recombinant

Appl. Environ. Microbiol.

Evolutionary engineering of industrially important microbial phenotypes

Adv. Biochem. Eng. Biotechnol.

Pathways of hydrogen in mitochondria of Saccharomyces carlsbergensis

Eur. J. Biochem.

Regular article
Production of pyruvate in Saccharomyces cerevisiae through adaptive evolution and rational cofactor metabolic engineering

Adapted evolution of Pdc⁻ strain BY5419