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Heterologous production of levopimaric acid in Saccharomyces cerevisiae.

ABSTRACT: BACKGROUND:Levopimaric acid (LA), a type of diterpene resin acid produced by plants, is a significant industrial intermediate that is mainly produced via phytoextraction. This work aimed to apply synthetic biology to produce LA in yeast strains from a simple carbon source. RESULTS:Levopimaradiene (LP), the precursor of LA, was produced via LP synthase (LPS) expression in yeast. LPS was then modified by N-terminal truncating and site-directed mutagenesis. The strain containing t79LPSMM (79 N-terminal amino acid truncating and M593I/Y700F mutation) produced 6.92 mg/L of LP, which were 23-fold higher than the strain containing LPS. Next, t79LPSMM was expressed in a new metabolically engineered chassis, and the final LP production increased 164-folds to 49.21 mg/L. Three cytochrome P450 reductases (CPRs) were co-expressed with CYP720B1 (the enzyme responsible for LA production from LP) in yeast to evaluate their LA producing abilities, and the CPR from Taxus cuspidata (TcCPR) was found to be the best (achieved 23.13 mg/L of LA production). CYP720B1 and TcCPR genes overexpression in the multi-copy site of the S.cerevisiae genome led to a 1.9-fold increase in LA production to 45.24 mg/L in a shake-flask culture. Finally, LA production was improved to 400.31 mg/L via fed-batch fermentation in a 5-L bioreactor. CONCLUSIONS:This is the first report to produce LA in a yeast cell factory and the highest titer of LA is achieved.

SUBMITTER: Liu T

PROVIDER: S-EPMC6050663 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Heterologous production of levopimaric acid in Saccharomyces cerevisiae.

Liu Ting T Zhang Chuanbo C Lu Wenyu W

Microbial cell factories 20180718 1

<h4>Background</h4>Levopimaric acid (LA), a type of diterpene resin acid produced by plants, is a significant industrial intermediate that is mainly produced via phytoextraction. This work aimed to apply synthetic biology to produce LA in yeast strains from a simple carbon source.<h4>Results</h4>Levopimaradiene (LP), the precursor of LA, was produced via LP synthase (LPS) expression in yeast. LPS was then modified by N-terminal truncating and site-directed mutagenesis. The strain containing t79L ...[more]

PMID: 30021574

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Project description:BACKGROUND:Decarboxylation of ?-ketoisovalerate to isobutyraldehyde is a key reaction in metabolic engineering of Saccharomyces cerevisiae for isobutanol production with published studies relying on overexpression of either the native ARO10 gene or of the Lactococcus lactis kivD decarboxylase gene resulting in low enzymatic activities. Here, we compare relevant properties for isobutanol production of Aro10, KivD and an additional, less studied, L. lactis decarboxylase KdcA. RESULTS:To eliminate interference by native decarboxylases, each 2-oxo acid decarboxylase was overexpressed in a 'decarboxylase-negative' (pdc1? pdc5? pdc6? aro10?) S. cerevisiae background. Kinetic analyses in cell extracts revealed a superior V max/K m ratio of KdcA for ?-ketoisovalerate and a wide range of linear and branched-chain 2-oxo acids. However, KdcA also showed the highest activity with pyruvate which, in engineered strains, can contribute to formation of ethanol as a by-product. Removal of native decarboxylase genes eliminated growth on valine as sole nitrogen source and subsequent complementation of this growth impairment by expression of each decarboxylase indicated that based on the increased growth rate, the in vivo activity of KdcA with ?-ketoisovalerate was higher than that of KivD and Aro10. Moreover, during oxygen-limited incubation in the presence of glucose, strains expressing kdcA or kivD showed a ca. twofold higher in vivo rate of conversion of ?-ketoisovalerate into isobutanol than an ARO10-expressing strain. Finally, cell extracts from cultures grown on different nitrogen sources revealed increased activity of constitutively expressed KdcA after growth on both valine and phenylalanine, while KivD and Aro10 activity was only increased after growth on phenylalanine suggesting a difference in the regulation of these enzymes. CONCLUSIONS:This study illustrates important differences in substrate specificity, enzyme kinetics and functional expression between different decarboxylases in the context of isobutanol production and identifies KdcA as a promising alternative decarboxylase not only for isobutanol production but also for other branched-chain and linear alcohols.

Dataset Information

Heterologous production of levopimaric acid in Saccharomyces cerevisiae.

Publications

Heterologous production of levopimaric acid in Saccharomyces cerevisiae.

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