通过消除转运蛋白 ProP 和 ProVWX 来提高大肠杆菌中 L-苏氨酸的产量。
Improving L-threonine production in Escherichia coli by elimination of transporters ProP and ProVWX.
机构信息
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China.
Key Laboratory of Industrial Biotechnology, School of Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.
出版信息
Microb Cell Fact. 2021 Mar 2;20(1):58. doi: 10.1186/s12934-021-01546-x.
BACKGROUND
Betaine, an osmoprotective compatible solute, has been used to improve L-threonine production in engineered Escherichia coli L-threonine producer. Betaine supplementation upregulates the expression of zwf encoding glucose-6-phosphate dehydrogenase, leading to the increase of NADPH, which is beneficial for L-threonine production. In E. coli, betaine can be taken through ProP encoded by proP or ProVWX encoded by proVWX. ProP is a H-osmolyte symporter, whereas ProVWX is an ABC transporter. ProP and ProVWX mediate osmotic stress protection by transporting zwitterionic osmolytes, including glycine betaine. Betaine can also be synthesized in E. coli by enzymes encoded by betABIT. However, the influence of ProP, ProVWX and betABIT on L-threonine production in E. coli has not been investigated.
RESULTS
In this study, the influence of ProP, ProVWX and betABIT on L-threonine production in E. coli has been investigated. Addition of betaine slightly improved the growth of the L-threonine producing E. coli strain TWF001 as well as the L-threonine production. Deletion of betABIT retarded the growth of TWF001 and slightly decreased the L-threonine production. However, deletion of proP or/and proVWX significantly increased the L-threonine production. When proP was deleted, the L-threonine production increased 33.3%; when proVWX was deleted, the L-threonine production increased 40.0%. When both proP and proVWX were deleted, the resulting strain TSW003 produced 23.5 g/l L-threonine after 36 h flask cultivation. The genes betABIT, proC, fadR, crr and ptsG were individually deleted from TSW003, and it was found that further absence of either crr (TWS008) or ptsG (TWS009) improved L-threonine production. TSW008 produced 24.9 g/l L-threonine after 36 h flask cultivation with a yield of 0.62 g/g glucose and a productivity of 0.69 g/l/h. TSW009 produced 26 g/l L-threonine after 48 h flask cultivation with a yield of 0.65 g/g glucose and a productivity of 0.54 g/l/h, which is 116% increase compared to the control TWF001.
CONCLUSIONS
In this study, L-threonine-producing E. coli strains TSW008 and TSW009 with high L-threonine productivity were developed by regulating the intracellular osmotic pressure. This strategy could be used to improve the production of other products in microorganisms.
背景
甜菜碱作为一种渗透保护剂兼容溶质，已被用于提高工程大肠杆菌 L-苏氨酸生产菌的 L-苏氨酸产量。甜菜碱的补充上调了编码葡萄糖-6-磷酸脱氢酶的 zwf 的表达，导致 NADPH 的增加，这有利于 L-苏氨酸的生产。在大肠杆菌中，甜菜碱可以通过 proP 编码的 ProP 或 proVWX 编码的 ProVWX 摄取。ProP 是一种 H-渗透物协同转运体，而 ProVWX 是一种 ABC 转运体。ProP 和 ProVWX 通过转运两性离子渗透物（包括甘氨酸甜菜碱）来介导渗透胁迫保护。大肠杆菌还可以通过 betABIT 编码的酶合成甜菜碱。然而，ProP、ProVWX 和 betABIT 对大肠杆菌中 L-苏氨酸生产的影响尚未得到研究。
结果
在本研究中，研究了 ProP、ProVWX 和 betABIT 对大肠杆菌中 L-苏氨酸生产的影响。添加甜菜碱可轻微改善 L-苏氨酸生产大肠杆菌菌株 TWF001 的生长和 L-苏氨酸的生产。betABIT 的缺失会减缓 TWF001 的生长并略微降低 L-苏氨酸的产量。然而，proP 或/和 proVWX 的缺失会显著增加 L-苏氨酸的产量。当 proP 缺失时，L-苏氨酸的产量增加了 33.3%；当 proVWX 缺失时，L-苏氨酸的产量增加了 40.0%。当 proP 和 proVWX 都缺失时，所得菌株 TSW003 在 36 小时摇瓶培养后产生 23.5 g/L L-苏氨酸。从 TSW003 中分别缺失 betABIT、proC、fadR、crr 和 ptsG 基因，发现进一步缺失 crr（TWS008）或 ptsG（TWS009）均可提高 L-苏氨酸的产量。TSW008 在 36 小时摇瓶培养后产生 24.9 g/L L-苏氨酸，产率为 0.62 g/g 葡萄糖，生产率为 0.69 g/L/h。TSW009 在 48 小时摇瓶培养后产生 26 g/L L-苏氨酸，产率为 0.65 g/g 葡萄糖，生产率为 0.54 g/L/h，与对照 TWF001 相比提高了 116%。
结论
在这项研究中，通过调节细胞内渗透压，开发了具有高 L-苏氨酸生产能力的 L-苏氨酸生产大肠杆菌菌株 TSW008 和 TSW009。该策略可用于提高微生物中其他产物的产量。
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