Systematically redesigning and optimizing the expression of D-lactate dehydrogenase efficiently produces high-optical-purity D-lactic acid in Saccharomyces cerevisiae

Zhong, Wanxie; Yang, Maohua; Mu, Tingzhen; Wu, Fan; Hao, Xuemi; Chen, Ruonan; Sharshar, Moustafa Mohamed; Thygesen, Anders; Wang, Qinhong; Xing, Jianmin

doi:10.1016/j.bej.2018.09.013

Cited by 14 publications

(21 citation statements)

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“…The strains and plasmids used in this study are shown in Table 1 and supporting information Table S1. The engineered strains used for d ‐LA production were based on the YIP‐pTCSt‐301 and YIP‐J‐C‐D‐A1strains 20 . All of the strains were engineered through the CRISPR‐Cas9 method 22,23 .…”

Section: Methodsmentioning

confidence: 99%

“…An LB medium supplemented with 100 mg L –1 ampicillin was used to culture the Escherichia coli strains. Yeast cells and wild‐type I. orientalis were cultivated in a YPD medium (20 g L –1 peptone, 20 g L –1 glucose, 10 g L –1 yeast extract) or synthetic dropout (SD) medium (6.7 g L –1 yeast nitrogen base without amino acids, 20 g L –1 glucose and 1 g L –1 amino acids dropout mixture lacking His, Try, Leu and Ura) 20 . The pH values of the SD‐based medium were adjusted to 2.2 or 4.2 by addition of H 2 SO 4 or NaOH.…”

Section: Methodsmentioning

confidence: 99%

“…PCR fusion was used to amplify the DNA fragments 24 . The Xma I, Sac I, Bam HI, Spe I and Not I restriction endonucleases, and T4 DNA ligase were used to construct plasmids 20 . All of the promoters and terminators [P TEF1 , P TEF1V1 , UAS CLB(3x) ‐P TEF1 , P synth1v3 , T TEF1 and T synth25 ] used in this study were referenced in previous studies 20 .…”

Section: Methodsmentioning

confidence: 99%

“…The plasmids not containing upstream activating sequences were constructed by T4 DNA Ligase using Xma I and Sac I restriction endonucleases. (d) Diagram of the gene editing method by CRISPR‐Cas9 19, 20 …”

Section: Methodsmentioning

confidence: 99%

“…The hot acidic phenol RNA isolation method was used for the extraction of total RNA from yeast strains 27 . A relative amount of messenger RNA (mRNA) was detected via qRT‐PCR 20 . ACT1 was used as a reference gene for expression analysis 28 .…”

Section: Methodsmentioning

confidence: 99%

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Improvement of D‐lactic acid production at low pH through expressing acid‐resistant gene IoGAS1 in engineered Saccharomyces cerevisiae

Zhong

Yang

Hao

et al. 2020

J of Chemical Tech & Biotech

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BACKGROUND Blending d‐lactic acid (d‐LA) with l‐lactic acid can significantly improve the thermostability of polylactic (PLA). Although microbial production of d‐LA under acidic conditions is beneficial for the reduction of production costs, the yield is low due to the acidic toxicity of the source strains. Herein, an Issatchenkia orientalis glycosylphosphatidylinositol‐anchored protein IoGas1, which is required for resistance to low pH and salt stress, was expressed in the YIP‐J‐C‐D‐A1 yeast strain. This strain was integrated with Escherichia coli d‐lactate dehydrogenase gene and several attenuated key pathway genes, including pyruvate decarboxylases (PDC1, PDC6), JEN1 (a monocarboxylate transporter), d‐lactate dehydrogenase1 (DLD1), l‐lactate cytochrome‐c oxidoreductase (CYB2) and alcohol dehydrogenase 1(ADH1). RESULTS The results revealed that the production of d‐LA by the modified strain YIP‐I‐J‐C‐D‐A1 was remarkably improved and reached 85.3 g L–1 d‐LA, with a yield of 0.71 g g–1 and a productivity of 1.20 g L h–1 in batch‐fed fermentation. The d‐LA production of the YIP‐I‐J‐C‐D‐A1 strain (CGMCC2.5785) was further improved by attenuating the ethanol and glycerol pathways. The resulting strain YIP‐A15G12 (CGMCC2.5803) produced 92.0 g L–1 d‐LA with a yield of 0.70 g g–1 and a productivity of 1.21 g L h–1 in batch‐fed fermentation at a final pH of 3.58. CONCLUSION Taken together, the expression of the acid‐resistant gene IoGAS1 in a modified yeast strain can significantly improve the efficiency of producing d‐LA at low pH, which may prove beneficial for the industrial production of the biodegradable material, PLA.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Improvement of D‐lactic acid production at low pH through expressing acid‐resistant gene IoGAS1 in engineered Saccharomyces cerevisiae

Zhong

Yang

Hao

et al. 2020

J of Chemical Tech & Biotech

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Improvement of d‐lactic acid productivity by introducing Escherichia coli acetyl‐CoA synthesis pathway in engineered Saccharomyces cerevisiae

Zhong

Yang

Hao

et al. 2021

J of Chemical Tech & Biotech

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BACKGROUND d‐Lactic acid (d‐LA) is gaining increased attention as it can be applied in the food, pharmaceutical, cosmetic, polylactic acid and textile industries. Acid‐tolerant Saccharomyces cerevisiae is often engineered to produce organic acids. For the pyruvate decarboxylases (PDCs) or alcohol dehydrogenase 1 (ADH1) disrupted strains, the productivity of d‐LA significantly decreases due to the accumulation of acetaldehyde and acetic acid. To overcome the problem, Escherichia coli acetylating acetaldehyde dehydrogenase (A‐ALD) enzyme genes (MHPF and EUTE) (EC 1.2.1.10) were integrated into the d‐LA producing strain YIP‐A15G12 strain (CGMCC2.5803). The function of these genes is to directly convert acetaldehyde into acetyl‐CoA without energy consumption. This strain was integrated with Escherichia coli d‐lactate dehydrogenase and Issatchenkia orientalis glycosylphosphatidylinositol‐anchored protein IoGas1gene and several attenuated key pathway genes, including PDC1, PDC6, ADH1, ADH5, JEN1 (a monocarboxylate transporter), d‐lactate dehydrogenase 1 (DLD1), l‐lactate cytochrome c oxidoreductase (CYB2) and glycerol‐3‐phosphate dehydrogenases (GPD1, GPD2). RESULTS The strain expressions of the A‐ALD enzymes effectively complemented the attenuated acetaldehyde dehydrogenase (ALD) acetyl‐CoA synthetase (ACS) pathway and significantly improved the levels of acetyl‐CoA and the productivity of d‐LA. Finally, the resulting strainYIP‐m‐ald6 (CGMCC2.5826) produced 86.9 g L–1 d‐LA with a yield of 0.77 g g–1 glucose and a volumetric productivity of 1.77 g L–1 h–1 at pH 3.58 under fed‐batch fermentation conditions. CONCLUSION Introducing the E. coli acetyl‐CoA synthesis pathway into the yeast strain can significantly enhance the growth of the strain and improve the d‐LA productivity, as a benefit for d‐LA production in industry. © 2021 Society of Chemical Industry (SCI).

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Comparative responses of flocculating and nonflocculating yeasts to cell density and chemical stress in lactic acid fermentation

Pangestu,

Kahar,

Ogino

et al. 2023

Yeast

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While flocculation has demonstrated its efficacy in enhancing yeast robustness and ethanol production, its potential application for lactic acid fermentation remains largely unexplored. Our study examined the differences between flocculating and nonflocculating Saccharomyces cerevisiae strains in terms of their metabolic dynamics when incorporating an exogenous lactic acid pathway, across varying cell densities and in the presence of lignocellulose‐derived byproducts. Comparative gene expression profiles revealed that cultivating a nonflocculant strain at higher cell density yielded a substantial upregulation of genes associated with glycolysis, energy metabolism, and other key pathways, resulting in elevated levels of fermentation products. Meanwhile, the flocculating strain displayed an inherent ability to sustain high glycolytic activity regardless of the cell density. Moreover, our investigation revealed a significant reduction in glycolytic activity under chemical stress, potentially attributable to diminished ATP supply during the energy investment phase. Conversely, the formation of flocs in the flocculating strain conferred protection against toxic chemicals present in the medium, fostering more stable lactic acid production levels. Additionally, the distinct flocculation traits observed between the two examined strains may be attributed to variations in the nucleotide sequences of the flocculin genes and their regulators. This study uncovers the potential of flocculation for enhanced lactic acid production in yeast, offering insights into metabolic mechanisms and potential gene targets for strain improvement.

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Systematically redesigning and optimizing the expression of D-lactate dehydrogenase efficiently produces high-optical-purity D-lactic acid in Saccharomyces cerevisiae

Cited by 14 publications

References 50 publications

Improvement of D‐lactic acid production at low pH through expressing acid‐resistant gene IoGAS1 in engineered Saccharomyces cerevisiae

Improvement of D‐lactic acid production at low pH through expressing acid‐resistant gene IoGAS1 in engineered Saccharomyces cerevisiae

Improvement of d‐lactic acid productivity by introducing Escherichia coli acetyl‐CoA synthesis pathway in engineered Saccharomyces cerevisiae

Comparative responses of flocculating and nonflocculating yeasts to cell density and chemical stress in lactic acid fermentation

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