Introduction of a bacterial acetyl-CoA synthesis pathway improves lactic acid production in Saccharomyces cerevisiae

Song, Ji-Yoon; Park, Joon-Song; Kang, Chang Duk; Cho, Hwa-Young; Yang, Daejong; Lee, Seung–Hyun; Cho, Kwang Myung

doi:10.1016/j.ymben.2015.09.006

Cited by 56 publications

(52 citation statements)

References 45 publications

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“…Strain JHY703 was generated by additional deletion of the STD1 gene in strain JHY701 by using the same method. Lactic acid-producing strains SP2006 and SP1130, possessing lactate dehydrogenase (LDH) genes originating from Pelodiscus sinensis subspecies japonicus (PsjLDH) or Bos taurus (BtLDH), were described previously (29). In CEN.PK2-1C, open reading frames (ORFs) of ADH1, PDC1, GPD1, and CYB2 genes were replaced with either the BtLDH gene or the PsjLDH gene controlled by the CCW12 promoter, resulting in strain SP2006 (Table 1) (29).…”

Section: Methodsmentioning

confidence: 99%

“…Lactic acid-producing strains SP2006 and SP1130, possessing lactate dehydrogenase (LDH) genes originating from Pelodiscus sinensis subspecies japonicus (PsjLDH) or Bos taurus (BtLDH), were described previously (29). In CEN.PK2-1C, open reading frames (ORFs) of ADH1, PDC1, GPD1, and CYB2 genes were replaced with either the BtLDH gene or the PsjLDH gene controlled by the CCW12 promoter, resulting in strain SP2006 (Table 1) (29). To generate strain SP1130 (Table 1), P TDH3 -controlled Escherichia coli mhpF and eutE genes, encoding acetylating acetaldehyde dehydrogenase (A-ALD), were introduced into the chromosome of SP2006, and ALD6 was additionally deleted (29).…”

Section: Methodsmentioning

confidence: 99%

“…In CEN.PK2-1C, open reading frames (ORFs) of ADH1, PDC1, GPD1, and CYB2 genes were replaced with either the BtLDH gene or the PsjLDH gene controlled by the CCW12 promoter, resulting in strain SP2006 (Table 1) (29). To generate strain SP1130 (Table 1), P TDH3 -controlled Escherichia coli mhpF and eutE genes, encoding acetylating acetaldehyde dehydrogenase (A-ALD), were introduced into the chromosome of SP2006, and ALD6 was additionally deleted (29). To generate strains SP2018 and SP1141 (overexpressing GCR1), the P TDH3 -GCR1/HA-URA3-HA cassette was PCR amplified using primers i_GCR1 F and i_GCR1 R from plasmid pUC57-URA3-GCR1 and integrated into the chromosomes of SP2006 and SP1130, respectively, by replacing his3⌬1.…”

Section: Methodsmentioning

confidence: 99%

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Improvement of Glucose Uptake Rate and Production of Target Chemicals by Overexpressing Hexose Transporters and Transcriptional Activator Gcr1 in Saccharomyces cerevisiae

Kim

Song

Hahn

2015

Appl Environ Microbiol

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Metabolic engineering to increase the glucose uptake rate might be beneficial to improve microbial production of various fuels and chemicals. In this study, we enhanced the glucose uptake rate in Saccharomyces cerevisiae by overexpressing hexose transporters (HXTs). Among the 5 tested HXTs (Hxt1, Hxt2, Hxt3, Hxt4, and Hxt7), overexpression of high-affinity transporter Hxt7 was the most effective in increasing the glucose uptake rate, followed by moderate-affinity transporters Hxt2 and Hxt4. Deletion of STD1 and MTH1, encoding corepressors of HXT genes, exerted differential effects on the glucose uptake rate, depending on the culture conditions. In addition, improved cell growth and glucose uptake rates could be achieved by overexpression of GCR1, which led to increased transcription levels of HXT1 and ribosomal protein genes. All genetic modifications enhancing the glucose uptake rate also increased the ethanol production rate in wild-type S. cerevisiae. Furthermore, the growth-promoting effect of GCR1 overexpression was successfully applied to lactic acid production in an engineered lactic acid-producing strain, resulting in a significant improvement of productivity and titers of lactic acid production under acidic fermentation conditions. Saccharomyces cerevisiae has been used as an important cell factory for production of fuels and chemicals (1, 2). Although yeast can utilize a wide range of carbon sources, glucose is the most widely preferred carbon source for yeast fermentation (3). Glucose uptake in S. cerevisiae is elaborately regulated to adapt cellular metabolism in response to ever-changing environmental conditions (3, 4). However, to achieve high titers and productivity of target chemicals using metabolically engineered yeast cells, it may be beneficial to inactivate, circumvent, or modify some of such natural regulatory mechanisms.The first regulatory step of glucose uptake is the facilitated diffusion of glucose through hexose transporters (HXTs) in the plasma membrane (4, 5). S. cerevisiae contains 18 HXT family members of hexose transporters, Hxt1 to Hxt17 and Gal2, among which Hxt1 to Hxt7 function as major glucose transporters (6). Expression of the HXTs, all of which exhibit different levels of glucose affinity, is differentially regulated depending on extracellular glucose concentrations (7-9). Hxt1 (K m ϭ ϳ100 mM) and Hxt3 (K m ϭ ϳ30 to 60 mM) are classified as low-affinity glucose transporters. HXT1 is highly induced in the presence of high (Ͼ1%) concentrations of glucose, whereas HXT3 is induced by both low and high levels of glucose. When the glucose concentrations are lowered to around 0.1%, HXT2 and HXT4, encoding moderate-affinity glucose transporters (K m ϭ ϳ5 to 10 mM), are expressed (10). HXT6 and HXT7, encoding highly homologous glucose transporters with very high glucose affinity (K m ϭ ϳ1 mM), are expressed just before the depletion of glucose in the medium (11). Expression of HXT5, encoding a moderate-affinity transporter (K m ϭ ϳ10 mM), is not regulated by extracellular glucose ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Improvement of Glucose Uptake Rate and Production of Target Chemicals by Overexpressing Hexose Transporters and Transcriptional Activator Gcr1 in Saccharomyces cerevisiae

Kim

Song

Hahn

2015

Appl Environ Microbiol

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“…Further, two successive laboratory evolution experiments for glycine prototrophy and faster growth were performed with this strain. Finally, overexpression of isocitrate lyase, Icl1p, in the evolved strain, resulted Deletion of PDC1 and expression of multiple copies of L-LDH from bovine [30] Inhibition of L-LDH consumption by deletion of DLD1 and JEN1, elimination of ethanol and glycerol production by deleting PDC1, ADH1, GPD1 and GPD2, and improvement of lactic acid tolerance by adaptive evolution and overexpression of HAA1 [31] Overexpression of HXT1 and HXT7 hexose transporters [32] Repression of ethanol production by deleting PDC1 and ADH1 and enhanced acetylCoA supply by the introduction of the genes encoding acetylating acetaldehyde dehydrogenase enzyme from Escherichia coli [33] Enhancement of lactic acid transport by expressing JEN1 and ADY1 [34] Expression of ESBP6, a novel target isolated by screening a multi-copy yeast genomic DNA library [35] in a succinic acid yield of 0.07 mol/mol glucose under aerobic conditions without glycine addition. Metabolic proiling analysis of a succinic acid-producing recombinant S. cerevisiae hinted a metabolic engineering strategy involving expression of a malic acid transporter from Schizosaccharomyces pombe (MAE1) to export succinic acid out of cells [16].…”

Section: Production Of Bulk Chemicalsmentioning

confidence: 99%

“…Therefore, deletions of PDH encoding pyruvate dehydrogenase, ADH encoding alcohol dehydrogenase and GPD1 encoding glycerol-3-phosphate dehydrogenase were reported to improve lactic acid production in LDH-expressing yeast strains [30,31]. Another approach was the improvement of cell growth either by an increased glucose uptake via overexpression of hexose transporters (HXT1 and HXT7) or an enhanced acetyl-CoA supply through implementing an acetyl-CoA synthesis pathway from E. coli in lactic acid-producing S. cerevisiae [32,33]. In addition, elimination of NADH-consuming reactions through deletions of NDE1 and NDE2 encoding mitochondrial external NADH dehydrogenases was shown to improve lactic acid production due to increased cofactor availability.…”

Section: Production Of Bulk Chemicalsmentioning

confidence: 99%

Advances in Metabolic Engineering of Saccharomyces cerevisiae for the Production of Industrially and Clinically Important Chemicals

Turanlı-Yıldız¹,

Hacısalihoğlu²,

Çakar³

2017

Old Yeasts - New Questions

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Sustainable production of chemicals is of increasing importance, due to depletion of petroleum and environmental concerns. In addition to its importance in basic research as a simple, eukaryotic model organism, Saccharomyces cerevisiae has long been exploited in industry because of its physiological properties. And today, the development in genetic engineering toolbox and genome-scale metabolic models of S. cerevisiae has extended its application range to new products and bioprocesses. In addition, evolutionary engineering strategies have been useful in improving cellular properties of S. cerevisiae, such as tolerance to product toxicity and inhibitors. In this chapter, recent metabolic and evolutionary engineering studies that involve S. cerevisiae for the production of bulk chemicals and ine chemicals including lavours and pharmaceuticals are reviewed. It was shown that metabolic engineering particularly allowed the improvement of pharmaceuticals production, which will enable economic and large-scale production of many valuable pharmaceuticals. It is clear that S. cerevisiae will continue to be an important host for future metabolic engineering and metabolic pathway engineering applications to produce a variety of industrially and clinically important chemicals.

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Improvement of d‐Lactic Acid Production in Saccharomyces cerevisiae Under Acidic Conditions by Evolutionary and Rational Metabolic Engineering

Baek

Kwon

Bae

et al. 2017

Biotechnology Journal

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Microbial lactic acid (LA) production under acidic fermentation conditions is favorable to reduce the production cost, but circumventing LA toxicity is a major challenge. A d-LA-producing Saccharomyces cerevisiae strain JHY5610 is generated by expressing d-lactate dehydrogenase gene (Lm. ldhA) from Leuconostoc mesenteroides, while deleting genes involved in ethanol production (ADH1, ADH2, ADH3, ADH4, and ADH5), glycerol production (GPD1 and GPD2), and degradation of d-LA (DLD1). Adaptive laboratory evolution of JHY5610 lead to a strain JHY5710 having higher LA tolerance and d-LA-production capability. Genome sequencing of JHY5710 reveal that SUR1 mutation increases LA tolerance and d-LA-production, whereas a loss-of-function mutation of ERF2 only contributes to increasing d-LA production. Introduction of both SUR1 and erf2Δ mutations into JHY5610 largely mimic the d-LA-production capability of JHY5710, suggesting that these two mutations, which could modulate sphingolipid production and protein palmitoylation, are mainly responsible for the improved d-LA production in JHY5710. JHY5710 is further improved by deleting PDC1 encoding pyruvate decarboxylase and additional integration of Lm. ldhA gene. The resulting strain JHY5730 produce up to 82.6 g L of d-LA with a yield of 0.83 g g glucose and a productivity of 1.50 g/(L · h) in fed-batch fermentation at pH 3.5.

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Introduction of a bacterial acetyl-CoA synthesis pathway improves lactic acid production in Saccharomyces cerevisiae

Cited by 56 publications

References 45 publications

Improvement of Glucose Uptake Rate and Production of Target Chemicals by Overexpressing Hexose Transporters and Transcriptional Activator Gcr1 in Saccharomyces cerevisiae

Improvement of Glucose Uptake Rate and Production of Target Chemicals by Overexpressing Hexose Transporters and Transcriptional Activator Gcr1 in Saccharomyces cerevisiae

Advances in Metabolic Engineering of Saccharomyces cerevisiae for the Production of Industrially and Clinically Important Chemicals

Improvement of d‐Lactic Acid Production in Saccharomyces cerevisiae Under Acidic Conditions by Evolutionary and Rational Metabolic Engineering

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