Department of Microbial Biotechnology, School of Biology, College of Science, University of Tehran, Tehran, Iran.
Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran.
PLoS One. 2020 Oct 9;15(10):e0240330. doi: 10.1371/journal.pone.0240330. eCollection 2020.
Zymomonas mobilis, as an ethanologenic microorganism with many desirable industrial features, faces crucial obstacles in the lignocellulosic ethanol production process. A significant hindrance occurs during the pretreatment procedure that not only produces fermentable sugars but also releases severe toxic compounds. As diverse parts of regulation networks are involved in different aspects of complicated tolerance to inhibitors, we developed ZM4-hfq and ZM4-sigE strains, in which hfq and sigE genes were overexpressed, respectively. ZM4-hfq is a transcription regulator and ZM4-sigE is a transcription factor that are involved in multiple stress responses. In the present work, by overexpressing these two genes, we evaluated their impact on the Z. mobilis tolerance to furfural, acetic acid, and sugarcane bagasse hydrolysates. Both recombinant strains showed increased growth rates and ethanol production levels compared to the parental strain. Under a high concentration of furfural, the growth rate of ZM4-hfq was more inhibited compared to ZM4-sigE. More precisely, fermentation performance of ZM4-hfq revealed that the yield of ethanol production was less than that of ZM4-sigE, because more unused sugar had remained in the medium. In the case of acetic acid, ZM4-sigE was the superior strain and produced four and two-fold more ethanol compared to the parental strain and ZM4-hfq, respectively. Comparison of inhibitor tolerance between single and multiple toxic inhibitors in the fermentation of sugarcane bagasse hydrolysate by ZM4-sigE strain showed similar results. In addition, ethanol production performance was considerably higher in ZM4-sigE as well. Finally, the results of the qPCR analysis suggested that under both furfural and acetic acid treatment experiments, overproduction of both hfq and sigE improves the Z. mobilis tolerance and its ethanol production capability. Overall, our study showed the vital role of the regulatory elements to overcome the obstacles in lignocellulosic biomass-derived ethanol and provide a platform for further improvement by directed evolution or systems metabolic engineering tools.
运动发酵单胞菌作为一种具有许多理想工业特性的产乙醇微生物，在木质纤维素乙醇生产过程中面临着关键的障碍。在预处理过程中会产生可发酵糖，但同时也会释放出严重的有毒化合物，这是一个重大的阻碍。由于调节网络的不同部分涉及到对抑制剂的复杂耐受性的不同方面，我们开发了 ZM4-hfq 和 ZM4-sigE 菌株，分别过表达了 hfq 和 sigE 基因。ZM4-hfq 是一种转录调节因子，ZM4-sigE 是一种转录因子，它们都参与多种应激反应。在本工作中，通过过表达这两个基因，我们评估了它们对运动发酵单胞菌耐受糠醛、乙酸和甘蔗渣水解物的影响。与亲本菌株相比，这两个重组菌株的生长速率和乙醇产率都有所提高。在高浓度糠醛下，ZM4-hfq 的生长速率比 ZM4-sigE 受到更大的抑制。更准确地说，ZM4-hfq 的发酵性能表明，乙醇的产率低于 ZM4-sigE，因为培养基中仍有未使用的糖。在乙酸的情况下，ZM4-sigE 是更好的菌株，与亲本菌株和 ZM4-hfq 相比，分别产生了 4 倍和 2 倍的乙醇。ZM4-sigE 菌株在发酵甘蔗渣水解物时，比较了单一和多种有毒抑制剂的抑制剂耐受性，结果相似。此外，ZM4-sigE 的乙醇产率也显著提高。最后，qPCR 分析的结果表明，在糠醛和乙酸处理实验中，hfq 和 sigE 的过量表达都提高了运动发酵单胞菌的耐受性及其乙醇生产能力。总的来说，我们的研究表明，调控元件在克服木质纤维素生物质衍生乙醇中的障碍方面起着至关重要的作用，并为通过定向进化或系统代谢工程工具进一步提高提供了一个平台。
