Grousseau Estelle, Lu Jingnan, Gorret Nathalie, Guillouet Stéphane E, Sinskey Anthony J
Université de Toulouse, INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077, Toulouse, France.
Appl Microbiol Biotechnol. 2014 May;98(9):4277-90. doi: 10.1007/s00253-014-5591-0. Epub 2014 Mar 7.
Alleviating our society's dependence on petroleum-based chemicals has been highly emphasized due to fossil fuel shortages and increasing greenhouse gas emissions. Isopropanol is a molecule of high potential to replace some petroleum-based chemicals, which can be produced through biological platforms from renewable waste carbon streams such as carbohydrates, fatty acids, or CO2. In this study, for the first time, the heterologous expression of engineered isopropanol pathways were evaluated in a Cupriavidus necator strain Re2133, which was incapable of producing poly-3-hydroxybutyrate [P(3HB)]. These synthetic production pathways were rationally designed through codon optimization, gene placement, and gene dosage in order to efficiently divert carbon flow from P(3HB) precursors toward isopropanol. Among the constructed pathways, Re2133/pEG7c overexpressing native C. necator genes encoding a β-ketothiolase, a CoA-transferase, and codon-optimized Clostridium genes encoding an acetoacetate decarboxylase and an alcohol dehydrogenase produced up to 3.44 g l(-1) isopropanol in batch culture, from fructose as a sole carbon source, with only 0.82 g l(-1) of biomass. The intrinsic performance of this strain (maximum specific production rate 0.093 g g(-1) h(-1), yield 0.32 Cmole Cmole(-1)) corresponded to more than 60 % of the respective theoretical performance. Moreover, the overall isopropanol production yield (0.24 Cmole Cmole(-1)) and the overall specific productivity (0.044 g g(-1) h(-1)) were higher than the values reported in the literature to date for heterologously engineered isopropanol production strains in batch culture. Strain Re2133/pEG7c presents good potential for scale-up production of isopropanol from various substrates in high cell density cultures.
由于化石燃料短缺和温室气体排放增加，减轻我们社会对石油基化学品的依赖已受到高度重视。异丙醇是一种具有高潜力替代某些石油基化学品的分子，它可通过生物平台由可再生废碳流（如碳水化合物、脂肪酸或二氧化碳）生产。在本研究中，首次在不能产生聚-3-羟基丁酸酯[P(3HB)]的拜氏固氮菌菌株Re2133中评估了工程化异丙醇途径的异源表达。这些合成生产途径通过密码子优化、基因定位和基因剂量进行合理设计，以便有效地将碳流从P(3HB)前体转向异丙醇。在所构建的途径中，过表达编码β-酮硫解酶、辅酶A转移酶的天然拜氏固氮菌基因以及编码乙酰乙酸脱羧酶和醇脱氢酶的密码子优化的梭菌基因的Re2133/pEG7c，在分批培养中以果糖作为唯一碳源时，可产生高达3.44 g l(-1)的异丙醇，而生物量仅为0.82 g l(-1)。该菌株的内在性能（最大比生产率0.093 g g(-1) h(-1)，产率0.32 Cmol Cmol(-1)）相当于各自理论性能的60%以上。此外，异丙醇的总产率（0.24 Cmol Cmol(-1)）和总比生产率（0.044 g g(-1) h(-1)）高于迄今文献报道的分批培养中异源工程化异丙醇生产菌株的值。菌株Re2133/pEG7c在高细胞密度培养中从各种底物大规模生产异丙醇方面具有良好潜力。
