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Efficient synthesis of d-phenyllactic acid by a whole-cell biocatalyst co-expressing glucose dehydrogenase and a novel d-lactate dehydrogenase from Lactobacillus rossiae.

ABSTRACT: d-Phenyllactic acid is a versatile natural organic acid, which is used as an antiseptic agent, monomer of the biodegradable material poly-phenyllactic acid and in the synthesis chiral intermediate of pharmaceuticals. In this report, the novel NADH-dependent d-lactate dehydrogenase LrLDH was identified by screening a shotgun genome of Lactobacillus rossiae. To improve cofactor regeneration, the Exiguobacterium sibiricum glucose dehydrogenase EsGDH was overexpressed together with LrLDH in E. coli BL21(DE3)-pCDFDuet-1-gdh-ldh. The total enzyme activity in the fermentation broth of E. coli BL 21(DE3)-pCDFDuet-1-gdh-ldh peaked at 2359.0 U l^-1 when induced by 10 g l^-1 lactose at 28 °C and 150 rpm for 14 h. The biocatalytic reduction of sodium phenylpyruvate to d-phenyllactic acid was successfully carried out using whole cells of the engineered E. coli. Under the optimized biocatalysis conditions, 50 g l^-1 sodium phenylpyruvate was completely converted to d-phenyllactic acid with a space-time yield and enantiomeric excess of 262.8 g l^-1 day^-1 and >?99.5%, respectively. To our best knowledge, it is the highest productivity reported to date, with great potential for the mass production of d-phenyllactic acid.

SUBMITTER: Luo X

PROVIDER: S-EPMC6904706 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Efficient synthesis of d-phenyllactic acid by a whole-cell biocatalyst co-expressing glucose dehydrogenase and a novel d-lactate dehydrogenase from Lactobacillus rossiae.

Luo Xi X Zhang Yingying Y Yin Longfei L Zheng Weilong W Fu Yongqian Y

3 Biotech 20191210 1

d-Phenyllactic acid is a versatile natural organic acid, which is used as an antiseptic agent, monomer of the biodegradable material poly-phenyllactic acid and in the synthesis chiral intermediate of pharmaceuticals. In this report, the novel NADH-dependent d-lactate dehydrogenase LrLDH was identified by screening a shotgun genome of Lactobacillus rossiae. To improve cofactor regeneration, the Exiguobacterium sibiricum glucose dehydrogenase EsGDH was overexpressed tog ...[more]

PMID: 31879578

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Project description:BACKGROUND/AIM:Nearly all mammalian tumors of diverse tissues are believed to be dependent on fermentative glycolysis, marked by elevated production of lactic acid and expression of glycolytic enzymes, most notably lactic acid dehydrogenase (LDH). Therefore, there has been significant interest in developing chemotherapy drugs that selectively target various isoforms of the LDH enzyme. However, considerable questions remain as to the consequences of biological ablation of LDH or upstream targeting of the glycolytic pathway. MATERIALS AND METHODS:In this study, we explore the biochemical and whole transcriptomic effects of CRISPR-Cas9 gene knockout (KO) of lactate dehydrogenases A and B [LDHA/B double KO (DKO)] and glucose-6-phosphate isomerase (GPI KO) in the human colon cancer cell line LS174T, using Affymetrix 2.1 ST arrays. RESULTS:The metabolic biochemical profiles corroborate that relative to wild type (WT), LDHA/B DKO produced no lactic acid, (GPI KO) produced minimal lactic acid and both KOs displayed higher mitochondrial respiration, and minimal use of glucose with no loss of cell viability. These findings show a high biochemical energy efficiency as measured by ATP in glycolysis-null cells. Next, transcriptomic analysis conducted on 48,226 mRNA transcripts reflect 273 differentially expressed genes (DEGS) in the GPI KO clone set, 193 DEGS in the LDHA/B DKO clone set with 47 DEGs common to both KO clones. Glycolytic-null cells reflect up-regulation in gene transcripts typically associated with nutrient deprivation / fasting and possible use of fats for energy: thioredoxin interacting protein (TXNIP), mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), PPAR? coactivator 1? (PGC-1?), and acetyl-CoA acyltransferase 2 (ACAA2). Other changes in non-ergometric transcripts in both KOs show losses in "stemness", WNT signaling pathway, chemo/radiation resistance, retinoic acid synthesis, drug detoxification, androgen/estrogen activation, and extracellular matrix reprogramming genes. CONCLUSION:These findings demonstrate that: 1) The "Warburg effect" is dispensable, 2) loss of the LDHAB gene is not only inconsequential to viability but fosters greater mitochondrial energy, and 3) drugs that target LDHA/B are likely to be ineffective without a plausible combination second drug target.

Dataset Information

Efficient synthesis of d-phenyllactic acid by a whole-cell biocatalyst co-expressing glucose dehydrogenase and a novel d-lactate dehydrogenase from Lactobacillus rossiae.

Publications

Efficient synthesis of d-phenyllactic acid by a whole-cell biocatalyst co-expressing glucose dehydrogenase and a novel d-lactate dehydrogenase from <i>Lactobacillus rossiae</i>.

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