ME: Metabolic Engineering of Succinate Production in Actinobacillus Succinogenes

琥珀酸盐具有许多工业应用，并且其在未来可能用作生产大宗化学品的中间商品化学原料。 除了从石油化工产品中生产之外，琥珀酸也可以通过葡萄糖发酵生产。 开发一种低成本、高琥珀酸产量的发酵工艺将为农产品创造一个新的用途。 最好的琥珀酸生产者，琥珀酸放线杆菌，产生高达100克/升的琥珀酸。 设计一个A为了使产琥珀酸菌菌株能够以更高的化学产率产生更多的琥珀酸，主要研究者（PI）将确定哪些代谢通量是遗传操作的目标。 基于A.琥珀酸代谢网络。 实验数据（即，发酵平衡、生物量生产和标记物在琥珀酸、甲酸和乙酸中的分布）将加入到该化学计量矩阵中以计算不同途径中的通量。 将通过在各种条件下生长培养物来评价不同代谢分支对通量分布的控制。 每种生长条件与优化的标准条件相差一个参数。 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（通过电还原人工电子载体的中介）。 从这些代谢通量分析得出的结论应该对如何设计A。产琥珀酸菌以产生更多的琥珀酸。

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.