摘要
10,11-dehydrocurvularin是一种来源于真菌Aspergillus terreus的一种具有多种生物活性的聚酮化合物。课题组前期的工作中以酿酒酵母(Saccharomyces cerevisiae)为宿主细胞,通过异源表达来源于A.terreus的还原性聚酮合酶(highly-reducing polyketide synthase,hrPKS)与非还原性聚酮合酶(non-reducing PKS,nrPKS)成功实现了10,11-dehydrocurvularin的合成。该研究通过调控质粒拷贝数,研究不同酶通量对多酶系统的最终产物产量的影响,以优化10,11-dehydrocurvularin在酿酒酵母中的产量。该研究以遗传霉素和潮霉素B两种抗生素抗性为选择压,通过截短抗性基因启动子长度,降低其单位拷贝抗性能力,经使用抗生素胁迫即可控制质粒拷贝数。结果表明,在一定抗生素浓度胁迫下,荧光及其质粒拷贝数与截短抗性基因的启动子长度呈负相关。将该系统分别应用至hrPKS和nrPKS,在遗传霉素与潮霉素B质量浓度都为300 mg/L的合成培养基中发酵2 d后,在构建的不同酶通量系统中,有不同程度的产量优化,其中启动子截短长度分别为全长和100 bp时,得到最优组合的产量为0.359 mg/L,为对照组的3.63倍。该研究表明在多酶系统中,通过组合优化酶表达量能够优化目标产物产量,但并非表达量越高效果越好,该研究将有利于今后非天然聚酮化合物的发现及优化生产。
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 hr-PKS 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.
作者
严豪
王志远
庞子萱
林苹鑫
吴疆
李业
白仲虎
YAN Hao;WANG Zhiyuan;PANG Zixuan;LIN Pingxin;WU Jiang;LI Ye;BAI Zhonghu(School of Biotechnology,Jiangnan University,Wuxi 214122,China;National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2021年第14期63-69,共7页
Food and Fermentation Industries
基金
江南大学青年基金(JUSRP12057)。
