Alpha-Terpineol production from an engineered Saccharomyces cerevisiae cell factory

Abstract Background Alpha-Terpineol (α-Terpineol), a C10 monoterpenoid alcohol, is widely used in the cosmetic and pharmaceutical industries. Construction Saccharomyces cerevisiae cell factories for producing monoterpenes offers a promising means to substitute chemical synthesis or phytoextraction. Results α-Terpineol was produced by expressing the truncated α-Terpineol synthase (tVvTS) from Vitis vinifera in S. cerevisiae. The α-Terpineol titer was increased to 0.83 mg/L with overexpression of the rate-limiting genes tHMG1, IDI1 and ERG20F96W-N127W. A GSGSGSGSGS linker was applied to fuse ERG20F96W-N127W with tVvTS, and expressing the fusion protein increased the α-Terpineol production by 2.87-fold to 2.39 mg/L when compared with the parental strain. In addition, we found that farnesyl diphosphate (FPP) accumulation by down-regulation of ERG9 expression and deletion of LPP1 and DPP1 did not improve α-Terpineol production. Therefore, ERG9 was overexpressed and the α-Terpineol titer was further increased to 3.32 mg/L. The best α-Terpineol producing strain LCB08 was then used for batch and fed-batch fermentation in a 5 L bioreactor, and the production of α-Terpineol was ultimately improved to 21.88 mg/L. Conclusions An efficient α-Terpineol production cell factory was constructed by engineering the S. cerevisiae mevalonate pathway, and the metabolic engineering strategies could also be applied to produce other valuable monoterpene compounds in yeast.

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Synthesizing ginsenoside Rh2 in Saccharomyces cerevisiae cell factory at high-efficiency

Cell Discovery ◽

10.1038/s41421-018-0075-5 ◽

2019 ◽

Vol 5 (1) ◽

Cited By ~ 24

Author(s):

Pingping Wang ◽

Wei Wei ◽

Wei Ye ◽

Xiaodong Li ◽

Wenfang Zhao ◽

...

Keyword(s):

Yeast Cell ◽

Batch Fermentation ◽

Cell Factory ◽

Ginsenoside Rh2 ◽

Fed Batch ◽

Microbial Cell Factories ◽

Cell Factories ◽

Fed Batch Fermentation ◽

Shake Flasks ◽

Produce Plant

AbstractSynthetic biology approach has been frequently applied to produce plant rare bioactive compounds in microbial cell factories by fermentation. However, to reach an ideal manufactural efficiency, it is necessary to optimize the microbial cell factories systemically by boosting sufficient carbon flux to the precursor synthesis and tuning the expression level and efficiency of key bioparts related to the synthetic pathway. We previously developed a yeast cell factory to produce ginsenoside Rh2 from glucose. However, the ginsenoside Rh2 yield was too low for commercialization due to the low supply of the ginsenoside aglycone protopanaxadiol (PPD) and poor performance of the key UDP-glycosyltransferase (UGT) (biopart UGTPg45) in the final step of the biosynthetic pathway. In the present study, we constructed a PPD-producing chassis via modular engineering of the mevalonic acid pathway and optimization of P450 expression levels. The new yeast chassis could produce 529.0 mg/L of PPD in shake flasks and 11.02 g/L in 10 L fed-batch fermentation. Based on this high PPD-producing chassis, we established a series of cell factories to produce ginsenoside Rh2, which we optimized by improving the C3–OH glycosylation efficiency. We increased the copy number of UGTPg45, and engineered its promoter to increase expression levels. In addition, we screened for more efficient and compatible UGT bioparts from other plant species and mutants originating from the direct evolution of UGTPg45. Combining all engineered strategies, we built a yeast cell factory with the greatest ginsenoside Rh2 production reported to date, 179.3 mg/L in shake flasks and 2.25 g/L in 10 L fed-batch fermentation. The results set up a successful example for improving yeast cell factories to produce plant rare natural products, especially the glycosylated ones.

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Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.00277-09 ◽

2009 ◽

Vol 75 (17) ◽

pp. 5536-5543 ◽

Cited By ~ 68

Author(s):

Kenro Tokuhiro ◽

Masayoshi Muramatsu ◽

Chikara Ohto ◽

Toshiya Kawaguchi ◽

Shusei Obata ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Metabolic Flux ◽

Mevalonate Pathway ◽

Fusion Gene ◽

Pharmaceutical Products ◽

Test Tube ◽

Ethanol Solution ◽

Multicopy Integration ◽

Engineered Saccharomyces Cerevisiae ◽

Rate Limiting

ABSTRACT (E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter−1) rather than GGOH (0.2 mg liter−1) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter−1 GGOH and 6.5 mg liter−1 squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter−1 GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering.

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Efficient Alcoholic Conversion of Glycerol by Engineered Saccharomyces cerevisiae

10.1101/2021.05.24.445540 ◽

2021 ◽

Author(s):

Takashi Watanabe ◽

Sadat M. R. Khattab

Keyword(s):

Saccharomyces Cerevisiae ◽

Nicotinamide Adenine Dinucleotide ◽

Batch Fermentation ◽

Plant Biomass ◽

Glycerol Oxidation ◽

Fed Batch ◽

Fed Batch Fermentation ◽

Engineered Saccharomyces Cerevisiae ◽

Biomass Decomposition ◽

Engineered Strain

Glycerol is an eco-friendly solvent that enhances plant biomass decomposition via glycerolysis in many pretreatment methods. Nonetheless, the lack of efficient conversion of glycerol by natural Saccharomyces cerevisiae hinders its use in these methods. Here, we have aimed to develop a complete strategy for the generation of efficient glycerol-converting yeast by modifying the oxidation of cytosolic nicotinamide adenine dinucleotide (NADH) by an O2-dependent dynamic shuttle, while abolishing both glycerol phosphorylation and biosynthesis. By following a vigorous glycerol oxidation pathway, the engineered strain increased the conversion efficiency (CE) to up to 0.49 g ethanol/g glycerol (98% of theoretical CE), with production rate > 1 g×L×h, when glycerol was supplemented in a single fed-batch fermentation in a rich medium. Furthermore, the engineered strain fermented a mixture of glycerol and glucose, producing > 86 g/L bioethanol with 92.8% CE. To our knowledge, this is the highest ever reported titer in this field. Notably, this strategy changed conventional yeast from a non-grower on minimal medium containing glycerol to a fermenting strain with productivity of 0.25-0.5 g×L×h and 84-78% CE, which converted 90% of the substrate to products. Our findings may improve the utilization of glycerol in several eco-friendly biorefinery approaches.

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Improving xylitol yield by deletion of endogenous xylitol-assimilating genes: a study of industrial Saccharomyces cerevisiae in fermentation of glucose and xylose

FEMS Yeast Research ◽

10.1093/femsyr/foaa061 ◽

2020 ◽

Vol 20 (8) ◽

Author(s):

Bai-Xue Yang ◽

Cai-Yun Xie ◽

Zi-Yuan Xia ◽

Ya-Jing Wu ◽

Min Gou ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Batch Fermentation ◽

Xylose Reductase ◽

Fed Batch ◽

Fed Batch Fermentation ◽

Engineered Saccharomyces Cerevisiae ◽

Sorbitol Dehydrogenases ◽

Simultaneous Saccharification ◽

Xylitol Yield ◽

Endogenous Enzymes

ABSTRACT Engineered Saccharomyces cerevisiae can reduce xylose to xylitol. However, in S.cerevisiae, there are several endogenous enzymes including xylitol dehydrogenase encoded by XYL2, sorbitol dehydrogenases encoded by SOR1/SOR2 and xylulokinase encoded by XKS1 may lead to the assimilation of xylitol. In this study, to increase xylitol accumulation, these genes were separately deleted through CRISPR/Cas9 system. Their effects on xylitol yield of an industrial S. cerevisiae CK17 overexpressing Candida tropicalis XYL1 (encoding xylose reductase) were investigated. Deletion of SOR1/SOR2 or XKS1 increased the xylitol yield in both batch and fed-batch fermentation with different concentrations of glucose and xylose. The analysis of the transcription level of key genes in the mutants during fed-batch fermentation suggests that SOR1/SOR2 are more crucially responsible for xylitol oxidation than XYL2 under the genetic background of S.cerevisiae CK17. The deletion of XKS1 gene could also weaken SOR1/SOR2 expression, thereby increasing the xylitol accumulation. The XKS1-deleted strain CK17ΔXKS1 produced 46.17 g/L of xylitol and reached a xylitol yield of 0.92 g/g during simultaneous saccharification and fermentation (SSF) of pretreated corn stover slurry. Therefore, the deletion of XKS1 gene provides a promising strategy to meet the industrial demands for xylitol production from lignocellulosic biomass.

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