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Biosynthesis of natural flavanones in Saccharomyces cerevisiae.

ABSTRACT: A four-step flavanone biosynthetic pathway was constructed and introduced into Saccharomyces cerevisiae. The recombinant yeast strain was fed with phenylpropanoid acids and produced the flavanones naringenin and pinocembrin 62 and 22 times more efficiently compared to previously reported recombinant prokaryotic strains. Microbial biosynthesis of the flavanone eriodictyol was also achieved.

SUBMITTER: Yan Y

PROVIDER: S-EPMC1214632 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature

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Biosynthesis of natural flavanones in Saccharomyces cerevisiae.

Yan Yajun Y Kohli Abhijeet A Koffas Mattheos A G MA

Applied and environmental microbiology 20050901 9

A four-step flavanone biosynthetic pathway was constructed and introduced into Saccharomyces cerevisiae. The recombinant yeast strain was fed with phenylpropanoid acids and produced the flavanones naringenin and pinocembrin 62 and 22 times more efficiently compared to previously reported recombinant prokaryotic strains. Microbial biosynthesis of the flavanone eriodictyol was also achieved. ...[more]

PMID: 16151160

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Project description:BackgroundDue to excellent performance in antitumor, antioxidation, antihypertension, antiatherosclerotic and antidepressant activities, crocetin, naturally exists in Crocus sativus L., has great potential applications in medical and food fields. Microbial production of crocetin has received increasing concern in recent years. However, only a patent from EVOVA Inc. and a report from Lou et al. have illustrated the feasibility of microbial biosynthesis of crocetin, but there was no specific titer data reported so far. Saccharomyces cerevisiae is generally regarded as food safety and productive host, and manipulation of key enzymes is critical to balance metabolic flux, consequently improve output. Therefore, to promote crocetin production in S. cerevisiae, all the key enzymes, such as CrtZ, CCD and ALD should be engineered combinatorially.ResultsBy introduction of heterologous CrtZ and CCD in existing β-carotene producing strain, crocetin biosynthesis was achieved successfully in S. cerevisiae. Compared to culturing at 30 °C, the crocetin production was improved to 223 μg/L at 20 °C. Moreover, an optimal CrtZ/CCD combination and a titer of 351 μg/L crocetin were obtained by combinatorial screening of CrtZs from nine species and four CCDs from Crocus. Then through screening of heterologous ALDs from Bixa orellana (Bix_ALD) and Synechocystis sp. PCC6803 (Syn_ALD) as well as endogenous ALD6, the crocetin titer was further enhanced by 1.8-folds after incorporating Syn_ALD. Finally a highest reported titer of 1219 μg/L at shake flask level was achieved by overexpression of CCD2 and Syn_ALD. Eventually, through fed-batch fermentation, the production of crocetin in 5-L bioreactor reached to 6278 μg/L, which is the highest crocetin titer reported in eukaryotic cell.ConclusionsSaccharomyces cerevisiae was engineered to achieve crocetin production in this study. Through combinatorial manipulation of three key enzymes CrtZ, CCD and ALD in terms of screening enzymes sources and regulating protein expression level (reaction temperature and copy number), crocetin titer was stepwise improved by 129.4-fold (from 9.42 to 1219 μg/L) as compared to the starting strain. The highest crocetin titer (6278 μg/L) reported in microbes was achieved in 5-L bioreactors. This study provides a good insight into key enzyme manipulation involved in serial reactions for microbial overproduction of desired compounds with complex structure.

Dataset Information

Biosynthesis of natural flavanones in Saccharomyces cerevisiae.

Publications

Biosynthesis of natural flavanones in Saccharomyces cerevisiae.

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