Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production

Abstract

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.

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

“…This finding was consistent with previous reports that enhancement in production of various metabolites requires only dxs and idi but not ispDF to be overexpressed or modulated (Jin and Stephanopoulos 2007;Morrone et al 2010;Sun et al 2014) and that the overexpression of ispDF operon even decreased isoprenoid production . The enzymes ispD and ispF were reported to be more soluble than the enzymes dxs and idi and may have higher activities (Zhou et al 2012); hence, the overexpression of ispDF may be unnecessary when the pathway was optimized, and further study is required to address this issue. The fourth module was required to be expressed at higher levels as the activity of heterologous ADS from Artemisia annua is known to be poor in E. coli Green et al 2009).…”

Decoupling production from growth by magnesium sulfate limitation boosts de novo limonene production

“…Operational enzyme instability thus constitutes an optimization target for improving terpene production with resting as well as growing cells, although the reason for this instability remains to be elucidated. In addition, a resting-cell configuration can also be applied to individually dissect and exploit the effects of perturbations/modifications such as medium additives to enhance expression (Zhou et al, 2012), carbon sources (Yoon et al, 2009), or (pathway) enzyme inhibitors/promoters, for example acetate, on growth and product formation. The values for growing cells refer to data from (Willrodt et al, 2014).…”

Improving d-glucaric acid production from myo-inositol in E. coli by increasing MIOX stability and myo-inositol transport

“…The unpredictable effects of protein fusion tags have therefore limited their usage in metabolic engineering contexts, where proteins must function even while fused: in vivo cleavage of the fusion tag would require expression of a potentially promiscuous protease, and freshly cleaved proteins may simply aggregate into insoluble and inactive inclusion bodies. Although increased product formation due to increased enzyme solubility has been reported, these gains were not due to protein fusion tags (Zhou et al, 2012). In contrast, directed evolution has been used successfully for a number of metabolic engineering problems (Marcheschi et al, 2013).…”