10,11-dehydrocurvularin is a polyketide initially isolated from Aspergillus terreus, which shows potent multiple biological activities. In previous work of our group, we successfully cloned and expressed the corresponding highly-reducing PKS (hrPKS) gene and non-reducing PKS (nrPKS) gene in Saccharomyces cerevisiae strain BJ5464-NpgA, which enabled heterologous biosynthesis of 10,11-dehydrocurvularin in this model eukaryotic system. To enhance the production of 10,11-dehydrocurvularin, we optimized the expression levels of hrPKS and nrPKS by regulating the plasmid copy numbers. As for the dominant selection markers (KanR and HgyR), promoter truncation from 5' end of the marker genes was positively correlated with the increase of plasmid copy numbers, when 300 mg/L of geneticin and/or hygromycin B were applied. We hence built up the combination of hrPKS and nrPKS with different copy numbers by placing hrPKS and nrPKS genes onto KanR truncated plasmids and HgyR truncated plasmids, respectively. After two days of fermentation in the synthetic media containing both geneticin and hygromycin B, the optimal combination with max truncation of KanR promoter and 100 bp truncation of HygR promoter resulted in the highest titer of 10,11-dehydrocurvularin at 0.359 mg/L, which was 3.63-fold compared to that of the control with full-length promoters for both KanR and HygR. These results showed that in a multi-enzyme system, the combined optimization of enzyme expression could improve the yield of the target product, but not that the higher the expression, the better the effect. This study could serve as the basis for identification and optimization of unnatural polyketides in future.
YAN Hao
,
WANG Zhiyuan
,
PANG Zixuan
,
LIN Pingxin
,
WU Jiang
,
LI Ye
,
BAI Zhonghu
. Optimized heterologous production of 10,11-dehydrocurvularin in Saccharomyces cerevisiae by tuning plasmid copy number[J]. Food and Fermentation Industries, 2021
, 47(14)
: 63
-69
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DOI: 10.13995/j.cnki.11-1802/ts.027402
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