【作者】 张颖；

【导师】 徐美娟；

【作者基本信息】 江南大学 ， 发酵工程， 2025， 硕士

【摘要】 L-苯丙氨酸作为一种重要的芳香族氨基酸,在食品与医药领域具有广泛的应用价值。目前,L-苯丙氨酸主要通过微生物发酵法生产,但其生产效率受限于代谢流分配不均及关键基因表达调控机制不明确等问题。为提高L-苯丙氨酸的产量和生产效率,本研究以大肠杆菌（Escherichia coli）W3110为底盘菌株,通过代谢工程策略对其生物合成路径进行系统性优化,同时结合培养基优化和发酵罐参数优化,进一步提升L-苯丙氨酸的产量和生产效率。主要研究内容如下:（1）构建高效合成L-苯丙氨酸的底盘菌株。本研究以野生型E.coli W3110为底盘菌株,通过敲除乳糖操纵子阻遏基因lac I和反馈抑制调节因子基因tyr R,基因aro G是关键限速酶DAHP（3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase,DAHP合成酶）的合成基因之一,本研究过表达了经过突变的限速酶基因aro G^fbr,解除了L-苯丙氨酸对基因aro G的反馈抑制。随后,敲除全局调控因子基因csr A和丙酮酸氧化酶基因pox B,提高了菌体量并扩大L-苯丙氨酸合成途径的碳流量,得到的菌株M4最终摇瓶产量为6.1 g/L。（2）进一步优化代谢路径。整合了经过点突变的关键限速酶基因phe A^fbr、解除了L-苯丙氨酸结合引起该酶的反馈抑制,另外整合了基因ilv E、aro B、aro D、aro K和aro C,最终保留了有正向效果的基因phe A（预苯酸脱氢酶Prephenate dehydrogenase）,aro D（莽草酸脱氢酶Shikimate dehydrogenase）、aro K（分支酸激酶Chorismate kinase）,此时摇瓶产量上升到11.8 g/L,显著提高了菌体生长和L-苯丙氨酸的合成效率。同时加强L-苯丙氨酸的分泌,整合了ydd G基因、敲除了aro P基因,保留了有正向效果的ydd G基因,得到菌株M13,其摇瓶产量达到13.5 g/L。（3）建立基因型与表型的最终联系。通过RT-PCR的手段,量化了代谢改造后菌株M13与L-苯丙氨酸生产相关基因的表达情况。相对表达水平的分析表明,过表达后的基因表达明显上调;而未被改动的基因tyr A、tyr B被促进了表达;基因tkt A、pyk F和pps A在没有改造的情况下表达下降。（4）进行发酵优化。优化摇瓶培养基成分与发酵罐发酵参数,通过溶氧水平和补料策略,进一步优化了发酵条件,最终优化后摇瓶培养基中葡萄糖底糖添加量为15 g/L;酵母粉、蛋白胨和（NH₄）₂SO₄的最佳添加量分别为4.5 g/L、1.5 g/L和2.5 g/L;磷酸二氢钾（KH₂PO₄）、维生素B₂及谷氨酸的优化添加量分为3 g/L、5 mg/L,1.2 g/L。发酵罐中25-35%为最佳溶氧控制范围,残糖浓度控制在5 g/L以下为最佳补料策略。在5 L发酵罐中,M13菌株经过60 h发酵,L-苯丙氨酸积累量达到62.7 g/L,糖酸转化率为22.3%。该结果为突破L-苯丙氨酸生物合成提供了数据支持,更为其他芳香族氨基酸及其衍生物的高产菌株开发提供方法论参考。更多还原

【Abstract】 L-Phenylalanine,as a crucial aromatic amino acid,holds extensive application value in the food and pharmaceutical industries.Currently,microbial fermentation serves as the primary production method for L-phenylalanine,yet its efficiency remains constrained by issues such as imbalanced metabolic flux distribution and unclear regulatory mechanisms of key gene expression.To enhance the production yield and efficiency of L-phenylalanine,this study employed Escherichia coli W3110 as the chassis strain.Through metabolic engineering strategies,we systematically optimized its biosynthetic pathway,while further improving L-phenylalanine production by combining culture medium optimization and fermentation parameter adjustments.The main research contents are as follows:（1）Construction of a High-Efficiency Chassis Strain for L-Phenylalanine Synthesis.In this study,wild-type Escherichia coli W3110 was used as the chassis strain.By knocking out the lactose operon repressor gene lac I and the feedback inhibition regulator gene tyr R,a genetic basis for enhanced production was established.The aro G gene encodes DAHP synthase（3-deoxy-D-arabino-heptulosonate 7-phosphate synthase）,one of the key rate-limiting enzymes in the pathway.Overexpression of the mutated rate-limiting enzyme gene aro G^fbr relieved feedback inhibition of aro G by L-phenylalanine.Subsequent knockout of the global regulator gene csr A and pyruvate oxidase gene pox B increased biomass and redirected carbon flux toward L-Phe biosynthesis.The final engineered strain,M4,achieved a shake-flask yield of 6.1 g/L.（2）Further optimization of the metabolic pathway:The key rate-limiting enzyme gene phe A^fbr was integrated to abolish feedback inhibition caused by L-Phe binding.Additionally,genes ilv E,aro B,aro D,aro K,and aro C were integrated.Subsequent evaluation retained genes with positive effects:phe A（prephenate dehydrogenase）,aro D（shikimate dehydrogenase）,and aro K（chorismate kinase）.This increased the shake-flask titer to 11.8 g/L,significantly improving cell growth and L-Phe synthesis efficiency.To enhance L-Phe secretion,the ydd G transporter gene was integrated,and the aro P gene was deleted.Retaining the functional ydd G gene yielded strain M13,achieving a shake-flask titer of 13.5 g/L.（3）Establishing the final link between genotype and phenotype.The expression levels of L-phenylalanine pathway-specific genes in the metabolically engineered strain M13 were quantified using RT-PCR.Relative expression analysis showed significant upregulation of overexpressed genes.Native genes tyr A and tyr B were transcriptionally promoted without direct modification,while tkt A,pyk F,and pps A expression decreased despite no genetic alterations.（4）Fermentation Optimization.To further enhance production,the composition of the shake-flask medium and the fermentation parameters for the bioreactor were optimized.This involved controlling dissolved oxygen（DO）levels and implementing a feeding strategy to refine the fermentation conditions.Optimal shake-flask conditions included:15 g/L initial glucose,4.5 g/L yeast extract,1.5 g/L peptone,2.5 g/L（NH₄）₂SO₄,3 g/L KH₂PO₄,5 mg/L vitamin B₂,and 1.2 g/L glutamate.In a 5-L bioreactor,dissolved oxygen was maintained at 25-35%,with residual glucose controlled below 5 g/L via fed-batch strategy.In a 5 L fermenter,the M13 strain accumulated 62.7 g/L of L-phenylalanine after 60 hours of fermentation,achieving a sugar-to-acid conversion rate of 22.3%.This study provides critical data support for advancing the biosynthesis of L-phenylalanine and offers methodological references for developing high-yielding strains of other aromatic amino acids and their derivatives.更多还原