Metabolic Engineering Of Escherichia Coli And Saccharomyces Cerevisiae For The Production Of Isoprene

Isoprene is the monomeric building block of the most diverse and abundant group of biological organic compounds referred to as isoprenoids. As a feedstock in the synthetic chemistry industry, it has significant potential and is widely used in the synthesis of rubber, medicines, spices and pesticides. Currently, the industrial supply of isoprene is limited by the petrochemical sources. Its green manufacturing remains a major challenge. Escherichia coli and Saccharomyces cerevisiae are attractive microbial cell factories due to their clear genetic background and ease of genetic manipulation. Therefore, in this study, they were used as the host strains for isoprene biosynthesis research.For isoprene biosynthesis of Escherichia coli, firstly, an isoprene biosynthetic pathway was built by introducing the isoprene synthase (ISPS) from Populus alba. By overexpressing genes of MEP pathway, Dxs, Dxr and Idi were identified as the rate-limiting genes and the effect of these genes on the isoprene production was found to follow the order of Idi>Dxs>Dxr. Secondly, we established an expression system by combining incompatible plasmids system with IRES method, achieved the efficient coexpression of the genes (IspS, Dxs, Dxr, Idi) and explored its synergistic effect on isoprene production. Thirdly, metabolic balance was investigated by regulating the gene order. It was found that the gene order with the highest isoprene production yield was consistent with that of the metabolic pathway, indicating that order of genes is a significant concern in metabolic engineering. Finally, protein engineering and metabolic engineering were combined together for directed co-evolution of the rate-limiting enzymes. The isoprene production in the final mutant strain reached 4.18 mg·g-1·h-1, which was improved by 60%.Multigene pathway assembly is a key bottleneck in synthetic biology of S. cerevisiae for high-value complex biochemicals, with high demands of robust, broadly accessible methods and tools. In the engineering of S. cerevisiae engineering for isoprene biosynthesis, firstly, a new reiterative recombination system was developed, in which a ready-to-use marker recyclable integrative toolbox pUMRI was designed and constructed. As proof of principle, we employed this system to assemble a functional β-carotene biosynthesis pathway (～10 kb DNA consisting of four genes), a functional mevalonic acid biosynthesis pathway (～17 kb DNA consisting of eight genes) and a functional lycopene biosynthesis pathway (～22 kb DNA consisting eleven genes). We further demonstrated its application potential in GAL-regulated metabolic engineering for high production of isoprenoids (-16.3 mg/g β-carotene in Y-carotene-01 and ～39.6 mg/g squalene in BY4742-C-04).As the precursor of the MVA pathway, the sufficient supply of acetyl-CoA is also important for the accumulation of isoprenoids. To maximize the utilization efficiency of acetyl-CoA, cytoplasm and mitochondria were co-engineered for isoprene production improvement. A rational push-pull-restrain strategy was proposed and applied to engineer the mevalonic acid pathway (MVA), the acetyl-CoA pathway and the isoprene branch pathway in cytoplasm. The rate-limiting steps involved in these three modules were identified and eliminated. Eventually, isoprene production reached 18.5 mg/L, which was improved by 394 fold. In the metabolic engineering of mitochondria, firstly the strategy for gene subcellular localization was established in which mitochondrial localization signal (MLS) was characterized by fluorescenes with GFP as the reportor. MLS was then introduced into the pURMI toolbox for MVA pathway construction in mitochondria. In order to improve isoprene production and relieve the toxicity of intermediate metabolites accumulation to cell growth, GAL regulation and aerobiotic regulation were applied. As a result, isoprene production was improved to 133 mg/L. The results indicated the effectiveness of mitochondria regulation. Finally, synergistic regulation of cytoplasm and mitochondria was investigated, and the final strain can accumulated 158 mg/L of isoprene.In summary, through development of novel pathway assembly tools and metabolic engineering strategies, the biosynthetic efficiency of isoprene by E. coli and S. cerevisiae were significantly improved, which might provide technical support for future industrial production of bio-based isoprene.