Metabolic engineering of Escherichia coli for high-specificity production of isoprenol and prenol as next generation of biofuels

Abstract

BackgroundThe isopentenols, including isoprenol and prenol, are excellent alternative fuels. However, they are not compounds largely accumulated in natural organism. The need for the next generation of biofuels with better physical and chemical properties impels us to develop biosynthetic routes for the production of isoprenol and prenol from renewable sugar. In this study, we use the heterogenous mevalonate-dependent (MVA) isoprenoid pathway for the synthesis of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) intermediates, and then convert IPP and DMAPP to isoprenol and prenol, respectively.ResultsA mevalonate titer of 1.7 g/L was obtained by constructing an efficient MVA upper pathway in engineered E. coli. Different phosphatases and pyrophosphatases were investigated for their abilities in hydrolyzing the IPP and DMAPP. Consequently, ADP-ribose pyrophosphatase was found to be an efficient IPP and DMAPP hydrolase. Moreover, ADP-ribose pyrophosphatase from Bacillus subtilis (BsNudF) exhibited a equivalent substrate specificity towards IPP and DMAPP, while ADP-ribose pyrophosphatase from E. coli (EcNudF) presented a high substrate preference for DMAPP. Without the expression of any phosphatases or pyrophosphatases, a background level of isopentenols was synthesized. When the endogenous pyrophosphatase genes (EcNudF and yggV) that were capable of enhancing the hydrolyzation of the IPP and DMAPP were knocked out, the background level of isopentenols was still obtained. Maybe the synthesized IPP and DMAPP were hydrolyzed by some unknown hydrolases of E. coli. Finally, 1.3 g/L single isoprenol was obtained by blocking the conversion of IPP to DMAPP and employing the BsNudF, and 0.2 g/L ~80% prenol was produced by employing the EcNudF. A maximal yield of 12% was achieved in both isoprenol and prenol producing strains.ConclusionsTo the best of our knowledge, this is the first successful report on high-specificity production of isoprenol and prenol by microbial fermentation. Over 1.3 g/L isoprenol achieved in shake-flask experiments represents a quite encouraging titer of higher alcohols. In addition, the substrate specificities of ADP-ribose pyrophosphatases were determined and successfully applied for the high-specificity synthesis of isoprenol and prenol. Altogether, this work presents a promising strategy for high-specificity production of two excellent biofuels, isoprenol and prenol.

Utilization of alkaline phosphatase PhoA in the bioproduction of geraniol by metabolically engineered Escherichia coli

“…The theoretical molecular mass of DPP1, LPP1, NudF and PhoA were known as 32 kDa, 30 kDa, 23 kDa and 52 kDa, which were determined from their amino acids. 14 The results of SDS-PAGE show that all phosphatases were expressed and matched their theoretical molecular mass respectively (Fig. 1).…”

“…Four different phosphatase genes, alkaline phosphatase (PhoA) gene from E. coli, ADP-ribose pyrophosphatase (NudF) gene, bifunctional diacylglycerol diphosphate phosphatase (DPP1) gene and lipid phosphate phosphatase (LPP1) gene were cloned into pCOLADuet-1 creating pYY11, pYY12, pYY13 and pYY16 respectively in our previous study, 14 and LWG2, LWG3, LWG4, LWG5 were formed by transferred these plasmid into E. coli BL21 (DE3) separately.…”

Metabolic Engineering for Advanced Biofuels Production and Recent Advances Toward Commercialization

“…Product purity and yield of isopentenol could be improved when the pathway was overexpressed in the absence of idi, which isomerizes IPP to DMAPP. [37] Further optimization of the MVA pathway enzyme expression based on multiple "-omics" data analysis, coupled with ribosome binding site optimization for nudB, pushed the yield of isopentenol significantly to 2.2 g L À1 from 1% glucose, 70% of its theoretical yield. [38,39] Efforts to tailor microbial isopentenol for industrial implementation were leveraged by creating a more energetically efficient "IPP-bypass" pathway, [40] and subsequently, the isopentenol titer was further improved by engineering a key enzyme, a phosphomevalonate decarboxylase (pmd), which enables bypass of one phosphorylation step in the original MVA pathway.…”

“…[43] Efforts in S. cerevisiae were also underway, positing the IPP-DMAPP isomerase (idi1) is a rate-limiting enzyme for geraniol production by limiting the supply of precursors for GPP synthesis. [37] Overexpression of idi1 in strains containing a geraniol synthase has pushed titers to its highest level reported in S. cerevisiae at 293 mg L À1 . [44] Substantial advances have been reported for biologically producing several potential jet fuel candidates.…”

Overexpression of key enzymes of the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway for improving squalene production in Escherichia coli

“…E. coli BL21(DE3), BL21 Star(DE3), OverExpress C43(DE3) and Tuner(DE3) (Shanghai Weidi Biotechnology Co., Ltd) were used to produce squalene. Recombinant strains were cultured in fermentation medium (Zheng et al, 2013) for squalene production.…”