Abstract
Isoprene is a useful phytochemical with high commercial values in many industrial applications including synthetic rubber, elastomers, isoprenoid medicines, and fossil fuel. Currently, isoprene is on large scale produced from petrochemical sources. An efficient biological process for isoprene production utilizing renewable feedstocks would be an important direction of research due to the fossil raw material depletion and air pollution. In this study, we introduced the mevalonate (MVA) pathway genes/acetoacetyl-coenzyme A thiolase (mvaE) and MVA synthase (mvaS) from Enterococcus faecalis (E. faecalis); MVA kinase (mvk) derived from Methanosarcina mazei (M. mazei); and phosphomevalonate kinase (pmk), diphosphomevalonate decarboxylase (mvaD), and isopentenyl diphosphate isomerase (idi) from Streptococcus pneumoniae (S. pneumoniae) to accelerate dimethylallyl diphosphate (DMAPP) accumulation in Escherichia coli (E. coli). Together with a codon-optimized isoprene synthase (ispS) from Populus alba (P. alba), E. coli strain succeeded in formation of isoprene. We then manipulated the heterologous MVA pathway for high-level production of isoprene, by controlling the gene expression levels of the MVA pathway genes. We engineered four E. coli strains which showed different gene expression levels and different isoprene productivities, and we also characterized them with quantitative real-time PCR and metabolite analysis. To further improve the isoprene titers and release the toxicity to cells, we developed the extraction fermentation by adding dodecane in cultures. Finally, strain BL2T7P1TrcP harboring balanced gene expression system produced 587 ± 47 mg/L isoprene, with a 5.2-fold titer improvement in comparison with strain BL7CT7P. This work indicated that a balanced metabolic flux played a significant role to improve the isoprene production via MVA pathway.
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This work was supported by the State key laboratory of organic–inorganic composites, the National Basic Research Program of China (973 program) (2013CB733600), the National Nature Science Foundation of China (21390202, 21476017).
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C.-L. Liu and T.-W. Tan designed the study. C.-L. Liu and H.-R. Bi performed the experiments. C.-L. Liu, B. Hu, and L.-H. Fan are involved in the manuscript writing and editing. All the authors read and approved the manuscript.
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Liu, CL., Bi, HR., Bai, Z. et al. Engineering and manipulation of a mevalonate pathway in Escherichia coli for isoprene production. Appl Microbiol Biotechnol 103, 239–250 (2019). https://doi.org/10.1007/s00253-018-9472-9
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DOI: https://doi.org/10.1007/s00253-018-9472-9