Engineered monoculture and co-culture of methylotrophic yeast for de novo production of monacolin J and lovastatin from methanol

Liu, Yiqi; Tu, Xiaohu; Xu, Qin; Bai, Chenxiao; Kong, Chuixing; Liu, Qi; Yu, Jiahui; Peng, Qiangqiang; Zhou, Xiangshan; Zhang, Yuanxing; Cai, Menghao

doi:10.1016/j.ymben.2017.12.009

Cited by 120 publications

(108 citation statements)

References 44 publications

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“…As shown in Fig. 5a, P. p/LV_V#9-TapA produced 32.9 ± 1.6 mg/L lovastatin, which was 69 and 520% higher than the productivity of P. p/LV_V#9 and of the single-copy strain (Liu et al 2018), respectively. The pH values in the medium of lovastatin-producing strains were nearly the same at the end of fermentation (Additional file 1: Figure S5).…”

Section: Improvement In Lovastatin Production By Coexpression Of Tapamentioning

confidence: 87%

“…Plasmids pZ_BCGN, pK_sAR, and pG_sDF were constructed as previously described (Liu et al 2018). pZ_ BCGN was linearized by digestion with BlnI and was transferred into P. pastoris GS115 by electroporation in accordance with the manufacturer's instructions (Invitrogen).…”

Section: Construction Of Plasmids and Strainsmentioning

confidence: 99%

“…The latter was inoculated into the MGY medium until it reached OD 600 of 6 and then served as the second seed culture; 300 mL of it was aseptically transferred into the bioreactor. For bioprocess control, a batch phase involving the fermentation basal salt medium containing 40 g/L glycerol (PFPG, Invitrogen; Liu et al 2018) was first conducted and maintained for approximately 18 h. A glycerol-fed batch phase was next carried out with a batch using 50% (w/v) glycerol for approximately 12 h, followed by a period of 0.5 h without any feeding, to allow the glycerol to be fully consumed. The methanol feed was implemented via a gradual increase in the methanol feeding rate from 2.4 to 24 g/h during the first 7.5 h, to allow the cells to adapt to methanol catabolism; after that, a constant rate of 24 g/h was maintained until the end of the experiment.…”

Section: Bioreactor Fermentationmentioning

confidence: 99%

“…Both strains were cultured in the methanol medium (YNM), and a strain of P. p/LV_SC with all genes at single-copy levels (Liu et al 2018) was cultured as a control. As shown in Fig.…”

Section: Improvement In Lovastatin Production By Coexpression Of Tapamentioning

confidence: 99%

“…Previously, we assembled the biosynthetic pathway of lovastatin in the methylotrophic yeast P. pastoris (Fig. 1) and successfully produced lovastatin from methanol (Liu et al 2018). Nonetheless, gene dosages in the lovastatinproducing strains were all maintained at single-copy levels, and a titer of only 250.8 mg/L lovastatin was obtained via an optimal coculture strategy (Liu et al 2018); this titer is still much lower than that of a native lovastatinproducing strain (Singh and Pandey 2013;Huang et al 2017).…”

Section: Introductionmentioning

confidence: 99%

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Improved methanol-derived lovastatin production through enhancement of the biosynthetic pathway and intracellular lovastatin efflux in methylotrophic yeast

Liu

Bai

et al. 2018

Bioresour. Bioprocess.

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Background: Methanol has attracted interest as a substrate for improvement of product titers and yields of bioprocesses, because methanol has a more reduced state than sugars do and can be renewably synthesized from abundant natural gas supplies. Our aim was to engineer methylotrophic Pichia pastoris to convert methanol into value-added lovastatin more productively. Results:A strengthened biosynthetic pathway of lovastatin was constructed through the assembly of three modules with increasing module-specific antibiotic stress in the methylotrophic yeast P. pastoris. The resulting strain (P. p/LV_V#9) produced 287.5 ± 2.0 mg/L lovastatin in a 5-L bioreactor from methanol. The production was further improved by identification and overexpression of a statin pump protein, TapA, a membrane protein capable of lovastatin efflux out of the cell. A TapA-overexpressing strain, P. p/LV_V#9-TapA, produced 419.0 ± 9.5 mg/L lovastatin from methanol: 46% more than P. p/LV_V#9 did, and 520% more relative to the strain (P. p/LV_SC) with single-copy genes. Conclusions:A methylotrophic yeast strain producing 419.0 ± 9.5 mg/L lovastatin was constructed by optimization of biosynthetic gene dosages and coexpression of a statin pump protein; these results proved that P. pastoris is a promising chassis organism for natural-product biosynthesis. A membrane protein, TapA was found to perform the function of exporting intracellular lovastatin and enhanced lovastatin production.

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Section: Improvement In Lovastatin Production By Coexpression Of Tapamentioning

confidence: 87%

Section: Construction Of Plasmids and Strainsmentioning

confidence: 99%

Section: Bioreactor Fermentationmentioning

confidence: 99%

“…Both strains were cultured in the methanol medium (YNM), and a strain of P. p/LV_SC with all genes at single-copy levels (Liu et al 2018) was cultured as a control. As shown in Fig.…”

Section: Improvement In Lovastatin Production By Coexpression Of Tapamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Improved methanol-derived lovastatin production through enhancement of the biosynthetic pathway and intracellular lovastatin efflux in methylotrophic yeast

Liu

Bai

et al. 2018

Bioresour. Bioprocess.

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The “Three Cs” of Novel Antibiotic Discovery and Production through Synthetic Biology: Biosynthetic Gene Clusters, Heterologous Chassis, and Synthetic Microbial Consortia

2018

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Antimicrobial resistance is a rapidly growing problem worldwide, with bacteria showing resistance to last‐resort antibiotic treatments such as carbapenems and colistin becoming more abundant. Synthetic biology may be able to contribute to solving this growing antimicrobial resistance crisis by facilitating the engineering of diversified collections of novel antibiotics, targeting multidrug resistant bacteria in new ways. This review discusses recent advances in the use of synthetic biology methodologies to discover and produce novel antimicrobials; in particular, the work being carried out on biosynthetic gene clusters, heterologous chassis, and synthetic microbial consortia, the “three Cs” of the title.

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Bioretrosynthesis of Functionalized N‐Heterocycles from Glucose via One‐Pot Tandem Collaborations of Designed Microbes

Feng

Zhang

et al. 2020

Advanced Science

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The design of multistrain systems has markedly expanded the prospects of using long biosynthetic pathways to produce natural compounds. However, the cooperative use of artificially engineered microbes to synthesize xenobiotic chemicals from renewable carbohydrates is still in its infancy. Here, a microbial system is developed for the production of high‐added‐value N‐heterocycles directly from glucose. Based on a retrosynthetic analysis, eleven genes are selected, systematically modulated, and overexpressed in three Escherichia coli strains to construct an artificial pathway to produce 5‐methyl‐2‐pyrazinecarboxylic acid, a key intermediate in the production of the important pharmaceuticals Glipizide and Acipimox. Via one‐pot tandem collaborations, the designed microbes remarkably realize high‐level production of 5‐methyl‐2‐pyrazinecarboxylic acid (6.2 ± 0.1 g L−1) and its precursor 2,5‐dimethylpyrazine (7.9 ± 0.7 g L−1). This study is the first application of cooperative microbes for the total biosynthesis of functionalized N‐heterocycles and provides new insight into integrating bioretrosynthetic principles with synthetic biology to perform complex syntheses.

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Engineered monoculture and co-culture of methylotrophic yeast for de novo production of monacolin J and lovastatin from methanol

Cited by 120 publications

References 44 publications

Improved methanol-derived lovastatin production through enhancement of the biosynthetic pathway and intracellular lovastatin efflux in methylotrophic yeast

Improved methanol-derived lovastatin production through enhancement of the biosynthetic pathway and intracellular lovastatin efflux in methylotrophic yeast

The “Three Cs” of Novel Antibiotic Discovery and Production through Synthetic Biology: Biosynthetic Gene Clusters, Heterologous Chassis, and Synthetic Microbial Consortia

Bioretrosynthesis of Functionalized N‐Heterocycles from Glucose via One‐Pot Tandem Collaborations of Designed Microbes

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