Yeast has been widely used for large-scale production of terpenoids. In yeast, modifications of terpenoid biosynthetic pathways have been intensively studied. tHMG1 (encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase of yeast) and UPC2-1 (the G888D mutant of UPC2 encoding a transcription factor) were integrated into yeast chromosome, and ERG9 (the squalene synthase gene of yeast) was knocked down to yield the chassis strain DH02. A F96C mutation in ERG20 (farnesyl diphosphate synthase of yeast) was conducted to obtain mERG20 which can function as a geranylgeranyl diphosphate synthase (GGPS). Then, three fused genes, including BTS1 (the yeast innate GGPS)-ERG20, ERG20-mERG20 and mERG20-ERG20, were constructed, and expressed either by the pESC-based plasmids in DH02, or by being integrated into DH02 chromosome. The highest geranylgeraniol (GGOH) content was observed in the extracts of DH12 integrated with ERG20-mERG20, corresponding to 3.2 and 2.3 folds of those of the strains integrated with BTS1 and mERG20, respectively. Finally, three genes encoding nor-copalyl diphosphate synthase (nor-CPS), ent-CPS and syn-CPS were integrated into the chromosome of DH12, respectively, to construct yeasts for producing corresponding copalyl diphosphates (CPPs). Thus, a yeast-based platform was built for characterizing all types of diterpene synthases using GGPP or various CPPs as their substrates.
Keywords: Fused enzymes; Geranylgeranyl diphosphate; Metabolic engineering; Yeast.
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