ME: Metabolic Engineering of Succinate Production in Actinobacillus Succinogenes

丁二酸酯具有广泛的工业应用前景，有望成为生产大宗化学品的中间体。丁二酸酯通常由石化产品生产，也可以由葡萄糖发酵生产。开发一种低成本、高琥珀酸产率的发酵工艺将为农产品创造新的用途。琥珀酸的最佳产生菌是琥珀酸放线杆菌，其琥珀酸产率可达100g/L。为了设计一种能够以更高的化学产量生产更多琥珀酸的琥珀酸杆菌菌株，首席调查员(PI)将确定将哪些代谢通量作为基因操作的目标。以产琥珀酸菌代谢网络为基础，建立线性化学计量方程系统(以矩阵形式表示)。在13C-NaHCO3或2-13C-葡萄糖存在下生长的连续培养物收集的实验数据(即发酵平衡、生物量生产和标记在琥珀酸、甲酸盐和醋酸酯中的分布)将被输入到这个化学计量矩阵中，以计算不同途径中的通量。不同代谢分支对通量分配的控制将通过在不同条件下生长的培养来评估。每种生长条件都与优化后的标准条件相差一个参数。参数将包括特定于酶的抑制剂、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(通过电子还原的人造电子载体)。从这些代谢通量分析得出的结论应该对如何改造产琥珀酸杆菌来生产更多的琥珀酸有一定的指导意义。

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.