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. 2018 Jan 31;9(1):448.
doi: 10.1038/s41467-018-02883-z.

Engineering yeast for the production of breviscapine by genomic analysis and synthetic biology approaches

Xiaonan Liu  1   2 , Jian Cheng  1 , Guanghui Zhang  3 , Wentao Ding  1 , Lijin Duan  1 , Jing Yang  3   4 , Ling Kui  2   5 , Xiaozhi Cheng  1 , Jiangxing Ruan  1 , Wei Fan  3 , Junwen Chen  3 , Guangqiang Long  3 , Yan Zhao  3 , Jing Cai  6 , Wen Wang  5   7 , Yanhe Ma  1 , Yang Dong  3   4 , Shengchao Yang  8   9 , Huifeng Jiang  10
Affiliations

Engineering yeast for the production of breviscapine by genomic analysis and synthetic biology approaches

Xiaonan Liu et al. Nat Commun. .

Abstract

The flavonoid extract from Erigeron breviscapus, breviscapine, has increasingly been used to treat cardio- and cerebrovascular diseases in China for more than 30 years, and plant supply of E. breviscapus is becoming insufficient to satisfy the growing market demand. Here we report an alternative strategy for the supply of breviscapine by building a yeast cell factory using synthetic biology. We identify two key enzymes in the biosynthetic pathway (flavonoid-7-O-glucuronosyltransferase and flavone-6-hydroxylase) from E. breviscapus genome and engineer yeast to produce breviscapine from glucose. After metabolic engineering and optimization of fed-batch fermentation, scutellarin and apigenin-7-O-glucuronide, two major active ingredients of breviscapine, reach to 108 and 185 mg l-1, respectively. Our study not only introduces an alternative source of these valuable compounds, but also provides an example of integrating genomics and synthetic biology knowledge for metabolic engineering of natural compounds.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Proposed pathway for breviscapine biosynthesis in E. breviscapus and construction of the apigenin-7-O-glucuronide producing platform strain. a Proposed pathway for breviscapine biosynthesis in E. breviscapus. Genes that have been reported are shown in blue and those which were identified in this study are shown in red. Abbreviations: apigenin-7-O-G, apigenin-7-O-glucuronide; 4CL, 4-coumaroyl-CoA ligase; C4H, cinnamate 4-hydroxylase; CHI, chalcone isomerase; CHS, chalcone synthase; FSII, flavone synthase II; F6H, flavone-6-hydroxylase; F7GAT, flavonoid-7-O-glucuronosyltransferase; PAL, phenylalanine ammonia lyase; UDPGDH, UDP-glucose dehydrogenase. b Construction of the apigenin-producing strain SC1. c HPLC analysis of the apigenin-7-O-glucuronide standard, the fermented products of the apigenin-7-O-glucuronide producing strain (SC1-Y33-EbF7GAT-EbUDPGDH) and the negative control strain (SC1-Y33). Y33 is the abbreviation of plasmid YCPlac33. d LC-MS analysis of apigenin-7-O-glucuronide in the fermented products of SC1-Y33-EbF7GAT-EbUDPGDH
Fig. 2
Fig. 2
Analysis of scutellarin extracted from 13 species and the classification and expression of P450 genes. a The phylogenetic relationship among 13 species. The neighbor-joining tree was constructed based on 369 one-to-one orthologous genes in all studied species. All nodes received bootstrap support values from 100 replicates. b LC-MS analysis of flavonoids extracted from thirteen plants at 335 nm with molecular masses from 462 to 464 by extracted ion chromatographic (EIC) analysis. The molecular mass of scutellarin was highlighted. c The distribution of P450 genes from 13 plants in 411 P450 subfamilies. Each row indicates the plant species and each column indicates the P450 subfamily. The color scale from 0 to 8 indicates the number of P450 genes in a subfamily. d The correlation of gene expression in both the cultivated types and wild-type species. The average of gene expression in six cultivated types (Y axis) and in five wild types (X axis) is shown. Gray dots: all genes in E. breviscapus, green asterisks: the non-Asteraceae specific P450 genes, red asterisks: the Asteraceae-specific p450 genes, blue pentagrams: key enzymes in the breviscapine biosynthesis pathway
Fig. 3
Fig. 3
P450 enzymes screening and functional identification. a Overview of the P450 gene screening method. The detailed method is described in Methods. b HPLC analysis of the apigenin-7-O-glucuronide standard, scutellarin standard, the fermented product of the scutellarin-producing strain (SC1-Y22-EbF6H-EbCPR&Y33-EbF7GAT-EbUDPGDH), and the negative control strain (SC1-Y22&Y33). Y22 and Y33 represent the plasmids Ycplac22 and YcplacY33. c MS analysis of scutellarin in the fermented product of the scutellarin-producing strain. d HPLC analysis of microsomal enzyme assays (containing EbF6H and EbCPR) incubated with apigenin in vitro. The microsomal fraction containing only EbCPR was used as negative control. e HPLC analysis of microsomal enzyme assays incubated with apigenin-7-O-glucuronide in vitro. The microsomal fraction containing only EbCPR was used as the negative control
Fig. 4
Fig. 4
Metabolic engineering and fed-batch fermentation. a Metabolic engineering to improve the production of malonyl-CoA in the cytosol. Overexpressed genes are shown in orange and deleted genes are shown in red. Abbreviations: ACSSEL641P, acetyl-CoA synthase variant from Salmonella enterica; ADH2, endogenous alcohol dehydrogenase; ALD6, endogenous aldehyde dehydrogenase from S. cerevisiae; CIT2, citrate synthase; MLS1, malate synthase. b Evaluation of the engineered yeast strains SC1-FU-FC (SC1-Y22-EbF6H-EbCPR and Y33-EbF7GAT-EbUDPGDH), ΔM-FU-FC (SC1-FU-FC-Δmls1), ΔMC-FU-FC (SC1-FU-FC-Δmls1-Δcit2), and ΔM-FU-FC-AAA (ΔMC-FU-FC-ACS-ALD6-ADH2) (more details in Supplementary Table 4) for apigenin-7-O-glucuronide and scutellarin production in shake flask fermentations. c The production of scutellarin and apigenin-7-O-glucuronide by strain ΔM-FU-FC-AAA in fed-batch fermentation. Three replicates were performed for each analysis and the error bars represented the SD
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References

    1. Lan, M. Austroyunnanese Mater. Med. 2, 300-302 (1977).
    1. Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China. China Medical Science and Technology Presspart 1, 138, 379, 848 (2010).
    1. Prince MJ, et al. The burden of disease in older people and implications for health policy and practice. Lancet. 2015;385:549–562. doi: 10.1016/S0140-6736(14)61347-7. - DOI - PubMed
    1. Ma Y, Li H, Guan S. Enhancement of the oral bioavailability of breviscapine by nanoemulsions drug delivery system. Drug. Dev. Ind. Pharm. 2015;41:177–182. doi: 10.3109/03639045.2014.947510. - DOI - PubMed
    1. Jia JH, et al. Breviscapine, a traditional Chinese medicine, alleviates myocardial ischaemia reperfusion injury in diabetic rats. Acta Cardiol. 2008;63:757. doi: 10.2143/AC.63.6.2033394. - DOI - PubMed

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