【作者】 刘辉；

【导师】 陈宁；

【作者基本信息】 天津科技大学 ， 工程硕士（全日制）（专业学位）， 2016， 硕士

【摘要】 L-色氨酸是一种重要的芳香族氨基酸,广泛应用于饲料和医药方面。本研究依据代谢工程理论,以L-色氨酸工程菌TRTH为出发菌株,构建L-色氨酸高产菌。tktA基因编码的转酮醇酶在磷酸戊糖途径中生成4-磷酸赤藓糖起主要作用。在本实验中,将两种启动子控制的tktA基因整合至假基因位点ybfG处,获得菌株TRTH（△ybfG::Ptrc 1-tktA）和 TRTH（△ybfG::Ptrc2-tktA）。通过30L发酵罐进行发酵实验,结果表明,菌株TRTH（△ybfG::Ptrc1-tktA）L-色氨酸产量36.2 g/L,相比较于菌株TRTH（32.2 g/L）产量提高12.4%。而菌株TRTH（△ybfG::Ptrc2-tktA）L-色氨酸产量 12.3 g/L,相比较于菌株TRTH下降61.8%。发酵结果表明适当增强tktA基因表达量能够增加L-色氨酸前体物E4P的合成,减少耗糖量提高L-色氨酸产量。将将解除了反馈抑制的基因trpE^fbrD整合至假基因位点处构建菌株TRTH(△ybfG::Ptrc-trpE^fbrD)。发酵结果显示 TRTH(△ybfG::Ptrc-trpE^fbrD)L-色氨酸产量为29.3 g/L,比出发菌TRTH降低9.3%。这表明L-色氨酸工程菌质粒本身所带有的L-色氨酸合成相关基因表达量已足够。以增加NADPH为目的,将gapC基因替换大肠杆菌自身基因gapA构建了菌株TRTH（△gapA::PgapA-gapC）。发酵 L-色氨酸最终产量为 37.5 g/L,相较于 TRTH（32.2 g/L）,增加15.4%。说明通过增加NADPH的供应量对提高L-色氨酸产量和减少乙酸有正向效果。菌株TRTH生产L-色氨酸时,发酵周期较长,容易染菌,若将培养基pH降低可减少生产过程中染菌的机率。构建了耐酸性菌株TRTH（△araBAD::Para-arcA）和TRTH（AaraBAD::Para-gadBC）。将菌株TRTH（AaraBAD::Para-arcA）分别在pH5.5、6.0及双阶段情况下发酵,L-色氨酸产量均提高。菌株TRTH（△araBAD::Para-gadBC）在pH6.0情况下发酵,最终L-色氨酸产量为27.45 g/L下降18.86%。发酵结果表明谷氨酸依赖性系统的强化并没有显著提高TRTH的耐酸能力。更多还原

【Abstract】 Trytophan is an important aromatic acid,which is widely used in feeding animals and pharmaceutical industries.In this paper,based on metabolic engineering theory,with E.coli TRTH as starting strain,we attempted to construct L-tryptophan high-producing strain.Gene tktA encoding transketolase erythrose to produce 4-phosphate plays a major role in the pentose phosphate pathway.In this experiment,we overexpressed tktA gene to increase the precursor E4P.The tktA gene under different promoters were integrated at the locus of ybfG gene to obtain strains TRTH（△ybfG::Ptrc1-tktA）and TRTH（△ybfG::Ptrc2-tkt A）.Fermentation experiments by 30 L fermentor showed that TRTH（△ybfG::Ptrc1-tktA）produced 36.2 g/L L-tryptophan,and L-tryptophan of TRTH was 32.2g/L,12.4%increase in L-tryptophan was obtained.TRTH（△ybfG::Ptrc2-tktA）produced 12.3 g/L L-tryptophan,compared to strain TRTH（32.2 g/L）decreased 61.8%.Overexpression of tktA gene has a positive effect on the production of L-tryptophan.We integrated trpE^fbrD gene to the site of ybfG to obtain strain TRTH(△ybfG::Ptrc-trpE^fbrD).Fermentation results showed that L-tryptophan production was 29.3 g/L,9.3%decrease compared with the original strain.Expression of genes in the plasmid of TRTH is sufficient,additional expression is not necessary.To increase NADPH concentration,we used gapC gene to replace gene gapA,strain TRTH（△gapA::PgapA-gapC）was constructed.Fermentation results showed that L-tryptophan production was 37.5 g/L,compared to TRTH（32.2 g/L）increased by 15.4%,illustrating that increase of NADPH supply has a positive effect on increasing L-tryptophan and reducing acetic acid.A longer fermentation period could increase the risk of other bacteria contamination.If maintaining a lower pH of the medium could help to reduce possibility of bacteria contamination in the production process.We constructed the acid resistance strain TRTH（△araBAD::Para-arcA）and（△araBAD::Para-gadBC）.The strain TRTH（△araBAD::Para-arcA）was fermented at pH5.5,6.0 and two-stage pH respectively,L-tryptophan production increased.TRTH（△araBAD::Para-gadBC）was fermented at pH6.0,L-tryptophan production was 27.45 g/L,decreased 18.86%.Strengthen glutamate-dependent acid resistance system did not significantly improve acid resistace ability.更多还原