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Open Access Research

Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production

Kang Zhou1, Ruiyang Zou1, Gregory Stephanopoulos13 and Heng-Phon Too12*

Author Affiliations

1 Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, 4 Engineering Drive 3, Singapore, Singapore

2 Department of Biochemistry Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive Blk MD7 #05-04, Singapore 117597, Singapore

3 Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, USA

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Microbial Cell Factories 2012, 11:148  doi:10.1186/1475-2859-11-148

Published: 14 November 2012

Abstract

Background

Recombinant 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.

Results

Four 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.

Conclusion

This 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.

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