Systematic engineering of Saccharomyces cerevisiae for D-lactic acid production with near theoretical yield

2021 ◽  
Vol 21 (4) ◽  
Author(s):  
Akaraphol Watcharawipas ◽  
Kittapong Sae-tang ◽  
Kitisak Sansatchanon ◽  
Pipat Sudying ◽  
Kriengsak Boonchoo ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lactic Acid ◽  
Lactate Dehydrogenase ◽  
Single Gene ◽  
Lactic Acid Production ◽  
Gene Deletions ◽  
Theoretical Yield ◽  
Pathway Gene ◽  
Fed Batch Fermentation ◽  
Leuconostoc Pseudomesenteroides

Abstract D-lactic acid is a chiral three-carbon organic acid that can improve the thermostability of polylactic acid. Here, we systematically engineered Saccharomyces cerevisiae to produce D-lactic acid from glucose, a renewable carbon source, at near theoretical yield. Specifically, we screened D-lactate dehydrogenase (DLDH) variants from lactic acid bacteria in three different genera and identified the Leuconostoc pseudomesenteroides variant (LpDLDH) as having the highest activity in yeast. We then screened single-gene deletions to minimize the production of the side products ethanol and glycerol as well as prevent the conversion of D-lactic acid back to pyruvate. Based on the results of the DLDH screening and the single-gene deletions, we created a strain called ASc-d789M which overexpresses LpDLDH and contains deletions in glycerol pathway genes GPD1 and GPD2 and lactate dehydrogenase gene DLD1, as well as downregulation of ethanol pathway gene ADH1 using the L-methionine repressible promoter to minimize impact on growth. ASc-d789M produces D-lactic acid at a titer of 17.09 g/L in shake-flasks (yield of 0.89 g/g glucose consumed or 89% of the theoretical yield). Fed-batch fermentation resulted in D-lactic acid titer of 40.03 g/L (yield of 0.81 g/g glucose consumed). Altogether, our work represents progress towards efficient microbial production of D-lactic acid.

scholarly journals Efficient Homolactic Fermentation byKluyveromyces lactis Strains Defective in Pyruvate Utilization and Transformed with the HeterologousLDH Gene

2001 ◽  
Vol 67 (12) ◽  
pp. 5621-5625 ◽  
Author(s):  
Michele M. Bianchi ◽  
Luca Brambilla ◽  
Francesca Protani ◽  
Chi-Li Liu ◽  
Jefferson Lievense ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactate Dehydrogenase ◽  
Fermentation Broth ◽  
Kluyveromyces Lactis ◽  
Large Fraction ◽  
Lactic Acid Production ◽  
Pyruvate Decarboxylase ◽  
High Yield ◽  
Theoretical Yield ◽  
Ldh Activity

ABSTRACT A high yield of lactic acid per gram of glucose consumed and the absence of additional metabolites in the fermentation broth are two important goals of lactic acid production by microrganisms. Both purposes have been previously approached by using aKluyveromyces lactis yeast strain lacking the single pyruvate decarboxylase gene (KlPDC1) and transformed with the heterologous lactate dehydrogenase gene (LDH). The LDH gene was placed under the control theKlPDC1 promoter, which has allowed very high levels of lactate dehydrogenase (LDH) activity, due to the absence of autoregulation by KlPdc1p. The maximal yield obtained was 0.58 g g−1, suggesting that a large fraction of the glucose consumed was not converted into pyruvate. In a different attempt to redirect pyruvate flux toward homolactic fermentation, we usedK. lactis LDH transformant strains deleted of the pyruvate dehydrogenase (PDH) E1α subunit gene. A great process improvement was obtained by the use of producing strains lacking both PDH and pyruvate decarboxylase activities, which showed yield levels of as high as 0.85 g g−1 (maximum theoretical yield, 1 g g−1), and with high LDH activity.

scholarly journals Improvement of Lactic Acid Production in Saccharomyces cerevisiae by Cell Sorting for High Intracellular pH

2006 ◽  
Vol 72 (8) ◽  
pp. 5492-5499 ◽  
Author(s):  
Minoska Valli ◽  
Michael Sauer ◽  
Paola Branduardi ◽  
Nicole Borth ◽  
Danilo Porro ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lactic Acid ◽  
Intracellular Ph ◽  
Cell Sorting ◽  
Acid Production ◽  
Lactic Acid Production ◽  
Low Ph ◽  
The Mean ◽  
High Level ◽  
Lactate Dehydrogenases

ABSTRACT Yeast strains expressing heterologous l-lactate dehydrogenases can produce lactic acid. Although these microorganisms are tolerant of acidic environments, it is known that at low pH, lactic acid exerts a high level of stress on the cells. In the present study we analyzed intracellular pH (pHi) and viability by staining with cSNARF-4F and ethidium bromide, respectively, of two lactic-acid-producing strains of Saccharomyces cerevisiae, CEN.PK m850 and CEN.PK RWB876. The results showed that the strain producing more lactic acid, CEN.PK m850, has a higher pHi. During batch culture, we observed in both strains a reduction of the mean pHi and the appearance of a subpopulation of cells with low pHi. Simultaneous analysis of pHi and viability proved that the cells with low pHi were dead. Based on the observation that the better lactic-acid-producing strain had a higher pHi and that the cells with low pHi were dead, we hypothesized that we might find better lactic acid producers by screening for cells within the highest pHi range. The screening was performed on UV-mutagenized populations through three consecutive rounds of cell sorting in which only the viable cells within the highest pHi range were selected. The results showed that lactic acid production was significantly improved in the majority of the mutants obtained compared to the parental strains. The best lactic-acid-producing strain was identified within the screening of CEN.PK m850 mutants.

scholarly journals Synthesis and initial screening of lactate dehydrogenase inhibitor activity of 1,3-benzodioxole derivatives

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Dicky Annas ◽  
Se-Yun Cheon ◽  
Mohammad Yusuf ◽  
Sung-Jin Bae ◽  
Ki-Tae Ha ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactate Dehydrogenase ◽  
Cancer Cells ◽  
Aerobic Glycolysis ◽  
Dominant Role ◽  
Lactic Acid Production ◽  
Human Pancreatic Cancer ◽  
Drug Candidate ◽  
Acid Fermentation ◽  
High Level

AbstractCancer is one of the main causes of mortality in the world. Many cancer cells produce ATP through high-level lactic acid fermentation catalyzed by lactate dehydrogenase (LDH), which converts pyruvic acid to lactic acid. LDH plays a dominant role in the Warburg effect, wherein aerobic glycolysis is favored over oxidative phosphorylation. Due to the high lactic acid production level in cancer cells, LDH-targeting could be a potential cancer treatment strategy. A few approaches, such as drug treatment, reportedly inhibited LDH activity. In this study, we describe new 1,3-benzodioxole derivatives that might be potential small molecule candidates for LDHA inhibition. The synthesis was carried out by trans-esterification between aryl ester and alcohol groups from piperonyl alcohol. Compounds 2 and 10 exhibited a selective LDHA IC50 value of 13.63 µM and 47.2 µM, respectively. Whereas only compound 10 showed significant cytotoxicity in several lines of cancer cells, especially in human pancreatic cancer PANC-1 cells. These synthesized compounds possess 2 aromatic rings and –CF3 moiety, which expectedly contributes to LDHA inhibition. The presented products have the potential to become a promising LDHA inhibitor drug candidate.

scholarly journals Xylose utilization stimulates mitochondrial production of isobutanol and 2-methyl-1-butanol in Saccharomyces cerevisiae

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Yanfei Zhang ◽  
Stephan Lane ◽  
Jhong-Min Chen ◽  
Sarah K. Hammer ◽  
Jake Luttinger ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biosynthetic Pathway ◽  
Xylose Isomerase ◽  
Biofuel Production ◽  
Mitochondrial Activity ◽  
Xylose Utilization ◽  
Gene Deletions ◽  
Theoretical Yield ◽  
Byproduct Formation ◽  
Branched Chain

Abstract Background Branched-chain higher alcohols (BCHAs), including isobutanol and 2-methyl-1-butanol, are promising advanced biofuels, superior to ethanol due to their higher energy density and better compatibility with existing gasoline infrastructure. Compartmentalizing the isobutanol biosynthetic pathway in yeast mitochondria is an effective way to produce BCHAs from glucose. However, to improve the sustainability of biofuel production, there is great interest in developing strains and processes to utilize lignocellulosic biomass, including its hemicellulose component, which is mostly composed of the pentose xylose. Results In this work, we rewired the xylose isomerase assimilation and mitochondrial isobutanol production pathways in the budding yeast Saccharomyces cerevisiae. We then increased the flux through these pathways by making gene deletions of BAT1, ALD6, and PHO13, to develop a strain (YZy197) that produces as much as 4 g/L of BCHAs (3.10 ± 0.18 g isobutanol/L and 0.91 ± 0.02 g 2-methyl-1-butanol/L) from xylose. This represents approximately a 28-fold improvement on the highest isobutanol titers obtained from xylose previously reported in yeast and the first report of 2-methyl-1-butanol produced from xylose. The yield of total BCHAs is 57.2 ± 5.2 mg/g xylose, corresponding to ~ 14% of the maximum theoretical yield. Respirometry experiments show that xylose increases mitochondrial activity by as much as 7.3-fold compared to glucose. Conclusions The enhanced levels of mitochondrial BCHA production achieved, even without disrupting ethanol byproduct formation, arise mostly from xylose activation of mitochondrial activity and are correlated with slow rates of sugar consumption.

Lactic acid production from xylose by engineered Saccharomyces cerevisiae without PDC or ADH deletion

2015 ◽  
Vol 99 (19) ◽  
pp. 8023-8033 ◽  
Author(s):  
Timothy L. Turner ◽  
Guo-Chang Zhang ◽  
Soo Rin Kim ◽  
Vijay Subramaniam ◽  
David Steffen ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lactic Acid ◽  
Acid Production ◽  
Lactic Acid Production ◽  
Engineered Saccharomyces Cerevisiae

d-Lactic acid production by metabolically engineered Saccharomyces cerevisiae

2006 ◽  
Vol 101 (2) ◽  
pp. 172-177 ◽  
Author(s):  
Nobuhiro Ishida ◽  
Tomiko Suzuki ◽  
Kenro Tokuhiro ◽  
Eiji Nagamori ◽  
Toru Onishi ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lactic Acid ◽  
Acid Production ◽  
Lactic Acid Production ◽  
Engineered Saccharomyces Cerevisiae

scholarly journals Production of biopolymer precursors beta-alanine and L-lactic acid from CO2 with metabolically versatile Rhodococcus opacus DSM 43205

2020 ◽  
Author(s):  
Laura Salusjärvi ◽  
Leo Ojala ◽  
Gopal Peddinti ◽  
Michael Lienemann ◽  
Paula Jouhten ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Metabolic Engineering ◽  
Lactate Dehydrogenase ◽  
Acid Production ◽  
Lactic Acid Production ◽  
Water Electrolysis ◽  
Rhodococcus Opacus ◽  
Beta Alanine ◽  
Polymer Precursors ◽  
Autotrophic Bacteria

AbstractHydrogen oxidizing autotrophic bacteria are promising hosts for CO2 conversion into chemicals. In this work, we engineered the metabolically versatile lithoautotrophic bacterium Rhodococcus opacus strain DSM 43205 for synthesis of polymer precursors. Aspartate decarboxylase (panD) or lactate dehydrogenase (ldh) were expressed for beta-alanine or L-lactic acid production, respectively. The heterotrophic cultivations on glucose produced 25 mg L-1 beta-alanine and 742 mg L-1 L-lactic acid, while autotrophic cultivations with CO2, H2 and O2 resulted in the production of 1.8 mg L-1 beta-alanine and 146 mg L-1 L-lactic acid. Beta-alanine was also produced at 345 µg L-1 from CO2 in electrobioreactors, where H2 and O2 were provided by water electrolysis. This work demonstrates that R. opacus DSM 43205 can be readily engineered to produce chemicals from CO2 and provides base for its further metabolic engineering.

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