玉米浆发酵产生物丁醇的氨基酸代谢动力学模拟

为了深入挖掘利用丙酮丁醇梭菌产生物丁醇过程中氨基酸代谢的动态过程，探究利用廉价氮源玉米浆中的氨基酸用于丙酮丁醇梭菌产生物丁醇的生产策略，寻找生产丁醇的高效率廉价氮源来降低发酵生产成本。该研究首先利用高通量测序技术对玉米浆中微生物多样性进行分析；同时基于丙酮丁醇梭菌（Clostridium acetobutylicum）生产丙酮-丁醇-乙醇（Acetone-Butanol-Ethanol，ABE）碳代谢动态模型的基础上，构建氨基酸代谢模型，以此模拟15种氨基酸在利用木糖为碳源发酵生产ABE中的氨基酸代谢过程，并对氨基酸的代谢与丙酮丁醇梭菌的生物量以及ABE的合成相关性关系进行冗余分析；通过模型预测实际生产中利用玉米浆发酵时氨基酸的消耗过程。结果表明，梭状芽胞杆菌属（Clostridium）占细菌总数的68.76%，是玉米浆中的优势菌群；最佳参数校正后构建了氨基酸代谢模型，模拟值与试验值有较好拟合度；11种氨基酸（苯丙氨酸、苏氨酸、异亮氨酸、亮氨酸、蛋氨酸、缬氨酸、酪氨酸、甘氨酸、丝氨酸、精氨酸、天冬酰胺）在培养过程中迅速消耗用于细胞生长和溶剂生成，3种氨基酸（脯氨酸、组织胺、天冬氨酸）保持稳定状态，同时发酵过程中谷氨酰胺积累；冗余分析表明其中5种氨基酸对发酵产物及生物量影响具有相关性（P<0.05），相关性排序从大到小依次为丝氨酸、甘氨酸、亮氨酸、缬氨酸、天冬酰胺；模拟预测玉米浆中缬氨酸、甘氨酸、丝氨酸在发酵过程中基本被消耗，推测其为发酵后期的营养限制性因子。该结论可证实玉米浆可作为丙酮丁醇梭菌发酵丁醇的优势氮源，为丙酮丁醇梭菌的氨基酸代谢调控及下一步利用并优化玉米浆作为氮源生产生物丁醇提供一定的理论参考和数据支撑。

Kinetic simulation of amino acid metabolism of butanol produced from the fermentation of corn steep liquor

Abstract: This study aims to investigate the dynamic process of amino acid metabolism in the Clostridium acetobutylicum fermentation of biobutanol. In a production strategy, a type of cheap nitrogen source, such as amino acid in the corn steep liquor (CSL), can be used to produce butanol from Clostridium acetobutylicum. The efficient and cheap nitrogen source is seeking for butanol production to reduce the production cost of fermentation. In this study, a high-throughput sequencing technology was used to analyze the microbial diversity in CSL, based on the dynamic model of carbon metabolism for ABE (acetone-butanol-ethanol) produce by Clostridium acetobutylicum. A model of amino acid metabolism was constructed, using the metabolism network of amino acid, chemical reaction flux dynamics, and mass balance measurement. 15 amino acids were selected to simulate the metabolism process in ABE fermentation, using xylose as a carbon source. A redundancy analysis was conducted to establish the correlation between amino acid metabolism, the biomass of Clostridium acetobutylicum, and the synthesis of ABE. The predict model was achieved for the consumption process of amino acid in the CSL fermentation in an actual production. The results showed that the Clostridium spp. accounted for 68.76% of the total number of bacteria, indicating the dominant bacterial group in the CSL. The prediction results showed that the gene function of original bacteria was mainly focused on the transformation of carbon and nitrogen sources in the CSL. The optimal parameter correction demonstrated that the simulated values were in good agreement with the experimental ones, indicating that the proposed model can accurately reveal the dynamic metabolic process of amino acid under the utilization by C. acetobutylicum. Both experimental and simulation results indicated that 11 amino acids (phenylalanine, threonine, isoleucine, leucine, methionine, valine, tyrosine, glycine, serine, arginine, asparagine) were rapidly consumed for cell growth and solvent generation during the cultivation process, whereas 3 amino acids (proline, histamine, aspartic acid) remained stable, particularly that the glutamine accumulation was detected during fermentation. In addition, the metabolism of sugar and amino acid can be beneficial to the cell growth in the early stage, but not to the butanol synthesis. Correlation analysis results showed that five types of amino acids (serine, asparagine, glycine, leucine, and valine) were closely related to the fermentation products and biomass accumulation (P<0.05). The correlation sequence was serine, glycine, leucine, valine, asparagine in order from the largest to the smallest. A high consumption rate was obtained for valine, glycine, and serine during the fermentation process, indicating a nutrient limiting factor in the later stage of fermentation. A fully consumption was found in the predicted values of valine, glycine, and serine in the fermentation of CSL, indicating that the C. acetobutylicum had the highest utilization rate of three amino acids. Therefore, amino acids can be expected to add in the late period of fermentation, to avoid the deficiency of nutrient limiting factors. The CSL can also serve as the dominant nitrogen source of butanol fermentation by Clostridium spp. The findings can provide a sound theoretical reference and data support for the amino acid metabolism of Clostridium acetobutylicum, and for the subsequent utilization and optimization of CSL as a promising nitrogen source to produce biobutanol.