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Abstract
BackgroundLevopimaric 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.ResultsLevopimaradiene (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.ConclusionsThis is the first report to produce LA in a yeast cell factory and the highest titer of LA is achieved.
Highlights
Levopimaric acid (LA), a type of diterpene resin acid produced by plants, is a significant industrial intermediate that is mainly produced via phytoextraction
LP, the precursor of LA, was produced via a precursor pathway metabolically engineered E.coli; the levopimaradiene synthase (LPS) from Ginkgo biloba and geranylgeranyl diphosphate synthase (GGPPS) from Taxus canadensis were designed by protein engineering, and the combination of these two strategies increased levopimaradiene production about 2600-fold to 700 mg/L in a benchscale bioreactor [11]
LPS modification to improve levopimaradiene productivity According to Ohto [15], geranylgeraniol (GGOH) accumulated when HMG1 and the ERG20-BTS1 fusion protein were co-expressed
Summary
Levopimaric acid (LA), a type of diterpene resin acid produced by plants, is a significant industrial intermediate that is mainly produced via phytoextraction. Diterpenes, types of compounds with 20 carbon atoms in their skeleton, are important plants metabolites that are used to defend insects or pathogens [3]. LA, an important diterpene resin acid in conifers, is a significant industrial intermediate; its Diels–Alder reaction products are widely used in coatings, printing inks, plasticizers and adhesives [7, 8]. These high value-added terpenoids rarely accumulate in their native host and are difficult to chemically synthesize due to their complicated chemical structure. Cytochrome P450 monooxygenase (P450 s) from plants often loses function
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