Engineering the acyltransferase domain of epothilone polyketide synthase to alter the substrate specificity

Microbial Cell Factories

Huimin Wang¹  Yan Shi¹  Junheng Liang¹  Qianwen Yue¹  Xiaoming Ding¹  Guoping Zhao²  Xiaoying Bian³  Youming Zhang³  Yue-zhong Li³  Long Li⁴  Guosong Chen⁴ 

[1] Collaborative Innovation Center for Genetics and Development, State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, Department of Microbiology, School of Life Sciences, Fudan University, 200438, Shanghai, People’s Republic of China;Collaborative Innovation Center for Genetics and Development, State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, Department of Microbiology, School of Life Sciences, Fudan University, 200438, Shanghai, People’s Republic of China;CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032, Shanghai, People’s Republic of China;State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University-Helmholtz Institute of Biotechnology, Shandong University, Qingdao, Shandong, People’s Republic of China;The State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, 200433, Shanghai, People’s Republic of China;

关键词: Polyketide synthase;    AT;    Epothilone;    Domain swap;    Substrate specificity;   

DOI  :  10.1186/s12934-021-01578-3

来源: Springer

【 摘 要 】

BackgroundPolyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal.ResultsThe substitution of module 4 drastically diminished the activity of epothilone PKS. However, with careful design of the KS-AT linker and the post-AT linker, replacing EPOAT4 with EPOAT2, EPOAT6, EPOAT7 or EPOAT8 (specifically incorporating methylmalonyl-CoA (MMCoA)) significantly increased the ratio of epothilone D (4) to epothilone C (3) (the highest ratio of 4:3 = 4.6:1), whereas the ratio of 4:3 in the parental strain Schlegelella brevitalea 104-1 was 1.4:1. We also obtained three strains by swapping EPOAT4 with EPOAT3, EPOAT5, or EPOAT9, which specifically incorporate malonyl-CoA (MCoA). These strains produced only epothilone C, and the yield was increased by a factor of 1.8 compared to that of parental strain 104-1. Furthermore, mutations of five residues in the AT domain identified Ser310 as the critical factor for MMCoA recognition in EPOAT4. Then, the mutation of His308 to valine or tyrosine combined with the mutation of Phe310 to serine further altered the product ratios. At the same time, we successfully deleted the inactive module 9 DH and ER domains and fused the ΨKR domain with the KR domain through an ~ 25-residue linker to generate a productive and simplified epothilone PKS.ConclusionsThese results suggested that the substitution and deletion of catalytic domains effectively produces desirable compounds and that selection of the linkers between domains is crucial for maintaining intact PKS catalytic activity.

【 授权许可】

CC BY