ME: Metabolic Engineering of Succinate Production in Actinobacillus Succinogenes

琥珀酸盐具有广泛的工业用途，可作为大宗化工产品的中间原料。琥珀酸盐通常由石油化工产品生产，也可由葡萄糖发酵生产。开发一种成本低、琥珀酸盐产量高的发酵工艺将为农产品创造新的用途。琥珀酸最好的生产者是琥珀酸放线杆菌，其琥珀酸产量可达100克/升。为了设计一种能够以更高的化学产量生产更多琥珀酸盐的琥珀酸杆菌菌株，首席研究员（pi）将确定基因操作的代谢通量目标。一个线性化学计量方程系统（表示为矩阵）将建立基于琥珀酸根代谢网络。从13 C-NaHCO3或2- 13 c -葡萄糖连续培养中收集的实验数据（即发酵平衡、生物量生产和琥珀酸盐、甲酸盐和乙酸盐中的标签分布）将被输入该化学计量矩阵，以计算不同途径中的通量。不同代谢分支对通量分布的控制将通过在各种条件下的培养来评估。每个生长条件与优化后的标准条件只相差一个参数。参数将包括酶特异性抑制剂、不同的碳源、新的酶活性（通过引入重组基因）和增加的还原能力（通过电还原的人工电子载体的中介）。从这些代谢通量分析中得出的结论应该为如何设计琥珀酸a产生更多的琥珀酸提供见解。

Succinate has many industrial applications and it maybe used in the future as an intermediary commodity chemical feedstock for producing bulk chemicals. Routinely produced from petrochemicals, succinate can also be produced by fermentation from glucose. Developing a low-cost fermentation process with high succinate yields would create a new use for agricultural products. The best succinate producer, Actinobacillus succinogenes, produces up to 100 g/l of succinate. To engineer an A. succinogenes strain able to produce even more succinate at a higher chemical yield, the Principal Investigators (PIs) will determine which metabolic fluxes to target for genetic manipulation. A system of linear stoichiometric equations (expressed as a matrix) will be built, based on A. succinogenes metabolic network. Experimental data (i.e., fermentation balance, biomass production, and label distribution in succinate, formate, and acetate) collected from continuous cultures grown in the presence of 13 C-NaHCO3 or 2- 13 C-glucose will be fed into this stoichiometric matrix to calculate the fluxes in the different pathways. Control exerted on flux distribution by different metabolic branches will be evaluated by growing cultures in a variety of conditions. Each growth condition will differ from the optimized, standard conditions by a single parameter. Parameters will include enzyme-specific inhibitors, different carbon sources, new enzyme activities (by introduction of recombinant genes), and added reducing power (by the intermediary of an electrically-reduced artificial electron carrier). The conclusions drawn from these metabolic flux analyses should give insights on how to engineer A. succinogenes to produce more succinate.