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

Jung-Hun Kim,Hui-Jung Jang,Eui-Sung Choi,Chonglong Wang,Seon-Won Kim,Ju-Eon Park,Myeong-Seok Cha,Seon-Yeong Jo

doi:10.1186/s12934-016-0612-6

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.

Highlights

Isoprene, 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
Zhao et al [15] constructed an isoprene synthesis pathway in Escherichia coli based on the endogenous methylerythritol phosphate (MEP) pathway by overexpression of the native 1-deoxy-d-xylulose-5-phosphate (DXP) synthase gene and the foreign DXP reductoisomerase gene from Bacillus subtilis combined with introduction of the ispS gene from Populus nigra, which resulted in isoprene production of 314 mg/L
The isoprene synthases of Populus alba and Pueraria montana have been used in most microbial engineering processes for isoprene production

Summary

Results

Escherichia coli MG1655 harboring Populus trichocarpa isoprene synthase (PtispS) and the exog‐ enous 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 produc‐ tion 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

Conclusions

Background

Results and discussion

Conclusion

Methods