Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production

Microbial Cell Factories

Research

Ruiyang Zou¹  Kang Zhou¹  Heng-Phon Too²  Gregory Stephanopoulos³ 

[1] Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, 4 Engineering Drive 3, Singapore, Singapore;Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, 4 Engineering Drive 3, Singapore, Singapore;Department of Biochemistry Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive Blk MD7 #05-04,, 117597, Singapore, Singapore;Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, 4 Engineering Drive 3, Singapore, Singapore;Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, USA;

关键词: Isoprenoids;    Protein solubility;    Deoxyxylulose phosphate pathway;    Activity analysis;    Metabolic engineering;   

DOI  :  10.1186/1475-2859-11-148

 received in 2012-06-19, accepted in 2012-11-07,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundRecombinant proteins are routinely overexpressed in metabolic engineering. It is well known that some over-expressed heterologous recombinant enzymes are insoluble with little or no enzymatic activity. This study examined the solubility of over-expressed homologous enzymes of the deoxyxylulose phosphate pathway (DXP) and the impact of inclusion body formation on metabolic engineering of microbes.ResultsFour enzymes of this pathway (DXS, ISPG, ISPH and ISPA), but not all, were highly insoluble, regardless of the expression systems used. Insoluble dxs (the committed enzyme of DXP pathway) was found to be inactive. Expressions of fusion tags did not significantly improve the solubility of dxs. However, hypertonic media containing sorbitol, an osmolyte, successfully doubled the solubility of dxs, with the concomitant improvement in microbial production of the metabolite, DXP. Similarly, sorbitol significantly improved the production of soluble and functional ERG12, the committed enzyme in the mevalonate pathway.ConclusionThis study demonstrated the unanticipated findings that some over-expressed homologous enzymes of the DXP pathway were highly insoluble, forming inclusion bodies, which affected metabolite formation. Sorbitol was found to increase both the solubility and function of some of these over-expressed enzymes, a strategy to increase the production of secondary metabolites.

【 授权许可】

Unknown   
© Zhou et al.; licensee BioMed Central Ltd. 2012. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.