A pyruvate carbon flux tugging strategy for increasing 2,3-butanediol production and reducing ethanol subgeneration in the yeast Saccharomyces cerevisiae

Ishii, Jun; Morita, Kenichi; Ida, Kengo; Kato, Hiroko; Kinoshita, S.; Hataya, Shoko; Shimizu, Hiroshi; Kondo, Akihiko; Mizutani, Fumio

doi:10.1186/s13068-018-1176-y

Cited by 32 publications

(38 citation statements)

References 57 publications

(77 reference statements)

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“…The 23BD is biologically synthesized from two molecules of pyruvate by four-step reactions catalyzed by acetolactate synthase (ALS), acetolactate decarboxylase (ALDC), and butanediol dehydrogenase (BDH). In this study, a plasmid constructed in the previous study (pATP426-als Lp Op -aldc Ll Op -BDH1, als Lp Op , acdc Ll Op , BDH1 : codon-optimized ALS and ALDC genes derived from Lactobacillus plantarum and Lactococcus lactis and the BDH genes derived from S. cerevisiae [6]) was introduced in S. cerevisiae BY4742 (SCM043 strain) for cytosolic 23BD production.…”

Section: Resultsmentioning

confidence: 99%

“…In S. cerevisiae cells, a large amount of pyruvate is consumed by the synthesis of cell components, ethanol production, and respiration in the mitochondrion. Several studies reported that the knockout or control of ethanol biosynthesis pathway genes in S. cerevisiae improves the production of the pyruvate-derived chemicals [3, 4, 6, 7]. In Escherichia coli , decreasing the citrate synthase expression by CRISPER/Cas9 system increased the n -butanol yield [8].…”

Section: Introductionmentioning

confidence: 99%

“…1). This chemical is generally preferred for yeast metabolic engineering [4, 6, 16, 17] and was used in this study due to its easy handling in metabolic engineering and low cell toxicity [18, 19].

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Section: Introductionmentioning

confidence: 99%

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Repression of mitochondrial metabolism for cytosolic pyruvate-derived chemical production in Saccharomyces cerevisiae

et al. 2019

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Background Saccharomyces cerevisiae is a suitable host for the industrial production of pyruvate-derived chemicals such as ethanol and 2,3-butanediol (23BD). For the improvement of the productivity of these chemicals, it is essential to suppress the unnecessary pyruvate consumption in S. cerevisiae to redirect the metabolic flux toward the target chemical production. In this study, mitochondrial pyruvate transporter gene (MPC1) or the essential gene for mitophagy (ATG32) was knocked-out to repress the mitochondrial metabolism and improve the production of pyruvate-derived chemical in S. cerevisiae. Results The growth rates of both aforementioned strains were 1.6-fold higher than that of the control strain. 13C-metabolic flux analysis revealed that both strains presented similar flux distributions and successfully decreased the tricarboxylic acid cycle fluxes by 50% compared to the control strain. Nevertheless, the intracellular metabolite pool sizes were completely different, suggesting distinct metabolic effects of gene knockouts in both strains. This difference was also observed in the test-tube culture for 23BD production. Knockout of ATG32 revealed a 23.6-fold increase in 23BD titer (557.0 ± 20.6 mg/L) compared to the control strain (23.5 ± 12.8 mg/L), whereas the knockout of MPC1 revealed only 14.3-fold increase (336.4 ± 113.5 mg/L). Further investigation using the anaerobic high-density fermentation test revealed that the MPC1 knockout was more effective for ethanol production than the 23BD production. Conclusion These results suggest that the engineering of the mitochondrial transporters and membrane dynamics were effective in controlling the mitochondrial metabolism to improve the productivities of chemicals in yeast cytosol.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Repression of mitochondrial metabolism for cytosolic pyruvate-derived chemical production in Saccharomyces cerevisiae

et al. 2019

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“…In addition, the concentration of 2,3-BDO (178 g/L) by the engineered polyploid S. cerevisiae strain (YG01_SDBN) is one of the highest titer reported for microbial production of 2,3-BDO. While microbial production of 2,3-BDO has been studied in various species of microorganism and various ways [37], attempts in an industrial polyploid S. cerevisiae are novel [38]. Besides, the results of cassava hydrolysate fermentation using the engineered polyploid S. cerevisiae are not inferior even compared with those of bacteria including K. oxytoca [39].…”

Section: Discussionmentioning

confidence: 99%

Production of 2,3-butanediol from glucose and cassava hydrolysates by metabolically engineered industrial polyploid Saccharomyces cerevisiae

Lee

Seo

2019

Biotechnol Biofuels

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Background 2,3-Butanediol (2,3-BDO) is a valuable chemical for industrial applications. Bacteria can produce 2,3-BDO with a high productivity, though most of their classification as pathogens makes them undesirable for the industrial-scale production. Though Saccharomyces cerevisiae (GRAS microorganism) was engineered to produce 2,3-BDO efficiently in the previous studies, their 2,3-BDO productivity, yield, and titer were still uncompetitive compared to those of bacteria production. Thus, we propose an industrial polyploid S. cerevisiae as a host for efficient production of 2,3-BDO with high growth rate, rapid sugar consumption rate, and resistance to harsh conditions. Genetic manipulation tools for polyploid yeast had been limited; therefore, we engineered an industrial polyploid S. cerevisiae strain based on the CRISPR-Cas9 genome-editing system to produce 2,3-BDO instead of ethanol. Results Endogenous genes coding for pyruvate decarboxylase and alcohol dehydrogenase were partially disrupted to prevent declined growth rate and C 2 -compound limitation. A bacterial 2,3-BDO-producing pathway was also introduced in engineered polyploid S. cerevisiae . A fatal redox imbalance was controlled through the heterologous NADH oxidase from Lactococcus lactis during the 2,3-BDO production. The resulting strain (YG01_SDBN) still retained the beneficial traits as polyploid strains for the large-scale fermentation. The combination of partially disrupted PDC (pyruvate decarboxylase) and ADH (alcohol dehydrogenase) did not cause the severe growth defects typically found in all pdc- or adh-deficient yeast. The YG01_SDBN strain produced 178 g/L of 2,3-BDO from glucose with an impressive productivity (2.64 g/L h). When a cassava hydrolysate was used as a sole carbon source, this strain produced 132 g/L of 2,3-BDO with a productivity of 1.92 g/L h. Conclusions The microbial production of 2,3-BDO has been limited to bacteria and haploid laboratorial S. cerevisiae strains. This study suggests that an industrial polyploid S. cerevisiae (YG01_SDBN) can produce high concentration of 2,3-BDO with various advantages. Integration of metabolic engineering of the industrial yeast at the gene level with optimization of fed-batch fermentation at the process scale resulted in a remarkable achievement of 2,3-BDO production at 178 g/L of 2,3-BDO concentration and 2.64 g/L h of productivity. Furthermore, this strain could make a bioconversion of a cassava hydrolysate to 2,3-BDO with economic and environmental benefits. The engineered industrial polyploid strain could be applicable to production of biofuels and biochemicals in large-scale fermentations particularly when using modified CRISPR-Cas9 tools.

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“…In recent study, S. cerevisiae was shown to produce 81.0 g/l 2,3-BD in a fed-batch fermentation strategy using glucose as a carbon source. For reaching such high concentration, pyruvate carbon flux tugging strategy was used to reduce dominant ethanol production, thus increased 2,3-BD production [51]. In another study, a high 2,3-BD production was achieved by modulating the xylose metabolic pathway in engineered S. cerevisiae .…”

Section: Optimization Of Different Parameters For the Enhanced 23- Bmentioning

confidence: 99%

The current strategies and parameters for the enhanced microbial production of 2,3-butanediol

Hakizimana

Matabaro

Lee

2020

Biotechnology Reports

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A pyruvate carbon flux tugging strategy for increasing 2,3-butanediol production and reducing ethanol subgeneration in the yeast Saccharomyces cerevisiae

Cited by 32 publications

References 57 publications

Repression of mitochondrial metabolism for cytosolic pyruvate-derived chemical production in Saccharomyces cerevisiae

Repression of mitochondrial metabolism for cytosolic pyruvate-derived chemical production in Saccharomyces cerevisiae

Production of 2,3-butanediol from glucose and cassava hydrolysates by metabolically engineered industrial polyploid Saccharomyces cerevisiae

The current strategies and parameters for the enhanced microbial production of 2,3-butanediol

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