Metabolic engineering of Cupriavidus necator H16 for the production of high value chemicals

基于微生物将一氧化碳（CO）、二氧化碳（CO2）和甲烷（CH4）转化为化学品和燃料的气体发酵技术有可能取代现有的基于化石燃料的技术，并为运输用低碳燃料、绿色化学品或其他可进一步加工成生物聚合物和其他高价值化学品的有价值单体的生产提供理想的替代方案。这些过程通过将钢铁制造、炼油、煤炭和天然气/页岩气产生的废气转化为有价值的产品，将大大有助于减少温室气体（GHG）的排放。Cupriavidus necator H16（以前称为Ralstonia eutropha）是一种革兰氏阴性，非孢子形成，兼性化能石自养细菌，能够在有氧条件下在有机基质或H2和CO2上生长。其以二氧化碳为唯一碳源的生长能力使其成为从废气中可持续生产高价值平台化学品的有吸引力的底盘生物。这个项目的目的是代谢工程铜杆菌necator H16产生一个有价值的平台化学品，3-羟基丙酸或3HP。该中间产物可转化为丙烯酸、可生物降解聚酯、高吸水性聚合物和丙烯酸等高价值工业产品。它可以通过甘油、乳酸、丙二酰辅酶a或β -丙氨酸中间体通过至少七种不同的生物合成途径合成（Kumar, 2013）。提出的三种途径在热力学上是有利的，其中最有利的途径是通过β -丙氨酸进行的，将在本博士期间在C. necator中进行3HP生产测试。从β -丙氨酸生产3-HP的合成途径先前已在大肠杆菌和酵母中描述过。在酵母中，中间产物β -丙氨酸通过β -丙氨酸-丙酮酸转氨酶（BAPAT）或y-氨基丁酸转氨酶（GABT）转化为丙二醛半醛，并通过3-羟基丙酸脱氢酶（HPDH）或3-羟基异丁酸脱氢酶（HIBADH）进一步还原为3-HP。所有四个候选基因的同源物已经在C. necator H16基因组中被鉴定出来，并将进行酶鉴定。此外，还将构建和测试几种合成操纵子，以有效地生产β -丙氨酸并将β -丙氨酸转化为3HP

Gas fermentation technology based on microbial conversion of carbon monoxide (CO), carbon dioxide (CO2), and methane (CH4) into chemicals and fuels has the potential to replace the existing fossil fuel based technologies and could provide a desirable alternative for the production of low carbon fuels for transportation, green chemicals or other valuable monomers that can be further processed into biopolymers and other high value chemicals. These processes would greatly contribute to reduction of Green House Gas (GHG) emissions by converting waste gasses from steel manufacturing, oil refining, coal and natural/shale gas into valuable products. Cupriavidus necator H16 (formerly known as Ralstonia eutropha) is a Gram-negative, non-spore forming, facultatively chemolithoautotrophic bacterium able to grow on organic substrates or H2 and CO2 under aerobic conditions. Its ability to grow on CO2 as sole carbon source makes it an attractive chassis organism for the sustainable production of high value platform chemicals from waste gasses. The aim of this project is to metabolically engineer Cupriavidus necator H16 to produce a valuable platform chemical, 3-hydroxypropanoic acid or 3HP. This intermediate product can be converted into acrylic acid, biodegradable polyesters, superabsorbent polymers and acrylic acids among other highly valued industrial products. It can be synthesised via glycerol, lactate, malonyl-CoA or beta-alanine intermediates via at least seven different biosynthetic pathways (Kumar, 2013). Three of the proposed pathways are thermodynamically favourable, and the most favourable pathway, proceeding via beta-alanine will be tested during this PhD for 3HP production in C. necator.The synthetic pathway for 3-HP production from beta-alanine has been previously described in E. coli and yeast. In yeast, the intermediate beta-alanine was converted into malonic semialdehyde either by the action of beta-alanine-pyruvate aminotransferase (BAPAT) or y-amino butyrate transaminase (GABT), and further reduced into 3-HP by the action of either 3-hydroxypropionate dehydrogenase (HPDH) or 3-hydroxyisobutyrate dehydrogenase (HIBADH). Homologues of all four candidate genes have been identified in the genome of C. necator H16 and will be subjected to enzyme characterisation. In addition, several synthetic operons will be built and tested for efficient production of beta-alanine and conversion of beta-alanine to 3HP