CAS Key Laboratory of Bio-Based Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
Microb Cell Fact. 2017 Dec 19;16(1):227. doi: 10.1186/s12934-017-0839-x.
Phloroglucinol is an important chemical which has been successfully produced by engineered Escherichia coli. However, the toxicity of phloroglucinol can enormously inhibit E. coli cell growth and viability, and the productivity is still too low and not economically feasible for industrial applications. Therefore, strain tolerance to toxic metabolites remains a key issue during the production of chemicals using biological processes.
In the present work, we examined the impact of the native GroESL chaperone system with different overexpression levels on phloroglucinol tolerance and production in E. coli. The groESL gene was cloned into an expression vector, of which expression level was regulated by three different promoters (natural, tac and T7 promoter). Strain tolerance was evaluated employing viable cell counts and phloroglucinol production. In comparison with the control strain, all GroESL overexpressing strains showed good characteristics in cell viability and phloroglucinol synthesis. Strain which overexpressed GroESL under tac promoter was found to show the best tolerance in all of those tested, resulting in a 3.19-fold increase in viable cell numbers compared with control strain of agar-plate culture under the condition of 0.7 g/L phloroglucinol, and a 39.5% increase in phloroglucinol production under fed-batch fermentation. This engineered strain finally accumulated phloroglucinol up to 5.3 g/L in the fed-batch cultivation 10 h after induction, and the productivity was 0.53 g/L/h. To date, the highest phloroglucinol production was achieved in this work compared with the previous reports, which is promising to make the bioprocess feasible from the economical point.
The data show that appropriate expression level of GroESL plays a critical role in improving phloroglucinol tolerance and production in E. coli, and maybe involve in controlling some aspects of the stress response system through upregulation of GroESL. GroESL overexpression is therefore a feasible and efficient approach for improvement of E. coli tolerance.
间苯三酚是一种重要的化学物质，已成功通过工程大肠杆菌生产。然而，间苯三酚的毒性会极大地抑制大肠杆菌细胞的生长和活力，并且其生产力仍然太低，从经济角度来看，不适合工业应用。因此，在使用生物过程生产化学品时，菌株对有毒代谢物的耐受性仍然是一个关键问题。
在本工作中，我们研究了不同过表达水平的天然 GroESL 伴侣系统对大肠杆菌中间苯三酚耐受性和生产的影响。groESL 基因被克隆到表达载体中，其表达水平由三个不同的启动子（天然、 tac 和 T7 启动子）调节。通过活菌计数和间苯三酚生产来评估菌株的耐受性。与对照菌株相比，所有 GroESL 过表达菌株在细胞活力和间苯三酚合成方面均表现出良好的特性。在所有测试中，发现过表达 tac 启动子下的 GroESL 的菌株表现出最好的耐受性，与对照菌株相比，在 0.7 g/L 间苯三酚的琼脂平板培养条件下，活菌数增加了 3.19 倍，在补料分批发酵条件下，间苯三酚产量增加了 39.5%。该工程菌株最终在诱导后 10 小时的补料分批培养中积累了 5.3 g/L 的间苯三酚，产率为 0.53 g/L/h。迄今为止，与以前的报告相比，本工作中实现了最高的间苯三酚产量，这从经济角度来看有望使生物过程可行。
数据表明，GroESL 的适当表达水平在提高大肠杆菌中对间苯三酚的耐受性和生产方面起着关键作用，并且可能通过上调 GroESL 来控制应激反应系统的某些方面。因此，GroESL 的过表达是提高大肠杆菌耐受性的一种可行且有效的方法。
