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

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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Alpha-Terpineol production from an engineered Saccharomyces cerevisiae cell factory

Microbial Cell Factories ◽

10.1186/s12934-019-1211-0 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 3

Author(s):

Chuanbo Zhang ◽

Man Li ◽

Guang-Rong Zhao ◽

Wenyu Lu

Keyword(s):

Saccharomyces Cerevisiae ◽

Mevalonate Pathway ◽

Cell Factory ◽

Fed Batch ◽

Cell Factories ◽

Fed Batch Fermentation ◽

Pharmaceutical Industries ◽

Engineered Saccharomyces Cerevisiae ◽

Rate Limiting ◽

Production Cell

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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De Novo Biosynthesis of C-Arabinosylated Flavones by Utilization of Indica Rice C-Glycosyltransferases

10.21203/rs.3.rs-365369/v1 ◽

2021 ◽

Author(s):

Zhuo Chen ◽

Yuwei Sun ◽

Guangyi Wang ◽

Ying Zhang ◽

Qian Zhang ◽

...

Keyword(s):

De Novo ◽

Indica Rice ◽

Fed Batch ◽

Rice Varieties ◽

E Coli ◽

De Novo Biosynthesis ◽

Microbial Cell Factories ◽

Cell Factories ◽

Fed Batch Fermentation ◽

Heterologous Pathway

Abstract Flavone C-arabinosides/xylosides are plant-originated glycoconjugates with various bioactivities. However, the potential utility of these molecules is hindered by their low abundance in nature. Engineering biosynthesis pathway in heterologous bacterial chassis provides a sustainable source of these C-glycosides. We previously reported bifunctional C-glucosyl/C-arabinosyltransferases in Oryza sativa japonica and O. sativa indica, which influence the C-glycoside spectrum in different rice varieties. In this study, we proved the C-arabinosyltransferring activity of rice C-glycosyltransferases (CGTs) on the mono-C-glucoside substrate nothofagin, followed by taking advantage of specific CGTs and introducing heterologous UDP-pentose supply, to realize the production of eight different C-arabinosides/xylosides in recombinant E. coli. Fed-batch fermentation and precursor supplement maximized the titer of rice-originated C-arabinosides to 20~110 mg/L in an E. coli chassis. The optimized final titer of schaftoside and apigenin di-C-arabinoside reached 19.87 and 113.16 mg/L respectively. We demonstrate here the success of de novo bio-production of C-arabinosylated and C-xylosylated flavones by heterologous pathway reconstitution. These results lay a foundation for further optimal manufacture of complex flavonoid compounds in microbial cell factories.

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Enhancement of β-Alanine Biosynthesis in Escherichia coli Based on Multivariate Modular Metabolic Engineering

Biology ◽

10.3390/biology10101017 ◽

2021 ◽

Vol 10 (10) ◽

pp. 1017

Author(s):

Jian Xu ◽

Li Zhou ◽

Zhemin Zhou

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

Industrial Production ◽

Metabolic Flux ◽

Coli Strain ◽

Biosynthesis Pathway ◽

Fed Batch ◽

Microbial Cell Factories ◽

Cell Factories ◽

Fed Batch Fermentation

β-alanine is widely used as an intermediate in industrial production. However, the low production of microbial cell factories limits its further application. Here, to improve the biosynthesis production of β-alanine in Escherichia coli, multivariate modular metabolic engineering was recruited to manipulate the β-alanine biosynthesis pathway through keeping the balance of metabolic flux among the whole metabolic network. The β-alanine biosynthesis pathway was separated into three modules: the β-alanine biosynthesis module, TCA module, and glycolysis module. Global regulation was performed throughout the entire β-alanine biosynthesis pathway rationally and systematically by optimizing metabolic flux, overcoming metabolic bottlenecks and weakening branch pathways. As a result, metabolic flux was channeled in the direction of β-alanine biosynthesis without huge metabolic burden, and 37.9 g/L β-alanine was generated by engineered Escherichia coli strain B0016-07 in fed-batch fermentation. This study was meaningful to the synthetic biology of β-alanine industrial production.

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De novo biosynthesis of C-arabinosylated flavones by utilization of indica rice C-glycosyltransferases

Bioresources and Bioprocessing ◽

10.1186/s40643-021-00404-3 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Zhuo Chen ◽

Yuwei Sun ◽

Guangyi Wang ◽

Ying Zhang ◽

Qian Zhang ◽

...

Keyword(s):

De Novo ◽

Indica Rice ◽

Fed Batch ◽

Rice Varieties ◽

E Coli ◽

De Novo Biosynthesis ◽

Microbial Cell Factories ◽

Cell Factories ◽

Fed Batch Fermentation ◽

Heterologous Pathway

AbstractFlavone C-arabinosides/xylosides are plant-originated glycoconjugates with various bioactivities. However, the potential utility of these molecules is hindered by their low abundance in nature. Engineering biosynthesis pathway in heterologous bacterial chassis provides a sustainable source of these C-glycosides. We previously reported bifunctional C-glucosyl/C-arabinosyltransferases in Oryza sativa japonica and O. sativa indica, which influence the C-glycoside spectrum in different rice varieties. In this study, we proved the C-arabinosyl-transferring activity of rice C-glycosyltransferases (CGTs) on the mono-C-glucoside substrate nothofagin, followed by taking advantage of specific CGTs and introducing heterologous UDP-pentose supply, to realize the production of eight different C-arabinosides/xylosides in recombinant E. coli. Fed-batch fermentation and precursor supplement maximized the titer of rice-originated C-arabinosides to 20–110 mg/L in an E. coli chassis. The optimized final titer of schaftoside and apigenin di-C-arabinoside reached 19.87 and 113.16 mg/L, respectively. We demonstrate here the success of de novo bio-production of C-arabinosylated and C-xylosylated flavones by heterologous pathway reconstitution. These results lay a foundation for further optimal manufacture of complex flavonoid compounds in microbial cell factories.

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