Isoprene production by Escherichia coli through the exogenous mevalonate pathway with reduced formation of fermentation byproducts

Abstract

BackgroundIsoprene, a volatile C5 hydrocarbon, is an important platform chemical used in the manufacturing of synthetic rubber for tires and various other applications, such as elastomers and adhesives.ResultsIn this study, Escherichia coli MG1655 harboring Populus trichocarpa isoprene synthase (PtispS) and the exogenous mevalonate (MVA) pathway produced 80 mg/L isoprene. Codon optimization and optimal expression of the ispS gene via adjustment of the RBS strength and inducer concentration increased isoprene production to 199 and 337 mg/L, respectively. To augment expression of MVA pathway genes, the MVA pathway was cloned on a high-copy plasmid (pBR322 origin) with a strong promoter (Ptrc), which resulted in an additional increase in isoprene production up to 956 mg/L. To reduce the formation of byproducts derived from acetyl-CoA (an initial substrate of the MVA pathway), nine relevant genes were deleted to generate the E. coli AceCo strain (E. coli MG1655 ΔackA-pta, poxB, ldhA, dld, adhE, pps, and atoDA). The AceCo strain harboring the ispS gene and MVA pathway showed enhanced isoprene production of 1832 mg/L in flask culture with reduced accumulation of byproducts.ConclusionsWe achieved a 23-fold increase in isoprene production by codon optimization of PtispS, augmentation of the MVA pathway, and deletion of genes involved in byproduct formation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0612-6) contains supplementary material, which is available to authorized users.

Metabolic engineering of E. coli for improving mevalonate production to promote NADPH regeneration and enhance acetyl‐CoA supply

“…Blocking pathways that catabolize acetyl‐CoA is an effective approach for obtaining acetyl‐CoA derived compounds (Dong et al, 2017; Wu & Eiteman, 2016). Acetate formation is another pathway to reduce the supply of acetyl‐CoA (Kim et al, 2016; Parimi, Durie, Wu, Niyas, & Eiteman, 2017; Saini, Wang, Chiang, & Chao, 2014). Acetyl‐CoA is metabolized by phosphate acetyltransferase, which is encoded by pta , and the resultant product (acetyl‐phosphate) is converted to acetate by acetate kinase ( ackA ) Pyruvate dehydrogenase ( poxB ) produces acetate from pyruvate, a precursor of acetyl‐CoA.…”

Engineered Escherichia coli strains as platforms for biological production of isoprene

“…The production of isoprene was further improved in E. coli MG1655 and BL21 using 5‐L fed‐batch cultivation and a two‐vector system (pTS‐sPt‐MVA and pS‐NA). This improvement is considered to be derived from the increased supply of IPP and DMAPP from pS‐NA through augmentation of the MVA pathway, resulting in high levels of isoprenoid compounds such as isoprene and lycopene [16,17]. Based on these results, we further tested a 300‐L scale‐up fermentation process with the two‐vector system using E. coli BL21 for 72‐h cultivation to improve isoprene production.…”

“…[16] suggested that the whole MVA pathway of pS‐NA could provide a sufficient amount of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Therefore, the strains were additionally transfected with the plasmid pTS‐sPt‐MVA derived from pTrc99K, encoding isoprene synthase from Populus trichocarpa and the MVA pathway operon from pS‐NA [17].…”

Metabolic engineering for the production of isoprene and isopentenol by Escherichia coli

“…In different studies, the measured k cat values of IspS from kudzu were different, and variable isoprene production was obtained (Table 1 ). In a recent study, more IspSs from poplar were identified, and an approximately 3-fold isoprene production increase was detected (Table 1 ) (Kim et al 2016 ). Novel IspS enzymes from other plants ( Ipomoea batatas , Mangifera indica , Elaeocarpus photiniifolius , and Mucuna pruriens ) have been identified through genome mining, and high isoprene production has been detected (Table 1 ) (Ilmen et al 2015 ).…”