ME: Metabolic Engineering of Succinate Production in Actinobacillus Succinogenes

琥珀酸具有许多工业应用，未来可能用作生产大宗化学品的中间商品化学原料。 琥珀酸通常由石化产品生产，也可以通过葡萄糖发酵生产。 开发具有高琥珀酸产量的低成本发酵工艺将为农产品创造新用途。 最好的琥珀酸生产者，Actinobacillus succinogenes，可生产高达 100 g/l 的琥珀酸。 为了设计出能够以更高的化学产率生产更多琥珀酸的 A. succinogenes 菌株，首席研究员 (PI) 将确定哪些代谢通量是基因操作的目标。 将基于 A. succinogenes 代谢网络建立线性化学计量方程系统（表示为矩阵）。 从在 13 C-NaHCO3 或 2- 13 C-葡萄糖存在下生长的连续培养物中收集的实验数据（即，发酵平衡、生物量生产以及琥珀酸盐、甲酸盐和乙酸盐中的标签分布）将被输入到该化学计量矩阵中，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生长的培养物来评估。 每个生长条件都与优化的标准条件存在一个参数的差异。 参数将包括酶特异性抑制剂、不同的碳源、新酶活性（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）。 从这些代谢流分析中得出的结论应该能够为如何改造 A. succinogenes 来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.