Adaptive laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors

Abstract

Furfural and acetic acid from lignocellulosic hydrolysates are the prevalent inhibitors to Zymomonas mobilis during cellulosic ethanol production. Developing a strain tolerant to furfural or acetic acid inhibitors is difficul by using rational engineering strategies due to poor understanding of their underlying molecular mechanisms. In this study, strategy of adaptive laboratory evolution (ALE) was used for development of a furfural and acetic acid-tolerant strain. After three round evolution, four evolved mutants (ZMA7-2, ZMA7-3, ZMF3-2, and ZMF3-3) that showed higher growth capacity were successfully obtained via ALE method. Based on the results of profiling of cell growth, glucose utilization, ethanol yield, and activity of key enzymes, two desired strains, ZMA7-2 and ZMF3-3, were achieved, which showed higher tolerance under 7 g/l acetic acid and 3 g/l furfural stress condition. Especially, it is the first report of Z. mobilis strain that could tolerate higher furfural. The best strain, Z. mobilis ZMF3-3, has showed 94.84% theoretical ethanol yield under 3-g/l furfural stress condition, and the theoretical ethanol yield of ZM4 is only 9.89%. Our study also demonstrated that ALE method might also be used as a powerful metabolic engineering tool for metabolic engineering in Z. mobilis. Furthermore, the two best strains could be used as novel host for further metabolic engineering in cellulosic ethanol or future biorefinery. Importantly, the two strains may also be used as novel-tolerant model organisms for the genetic mechanism on the "omics" level, which will provide some useful information for inverse metabolic engineering.

Zymomonas mobilis as a model system for production of biofuels and biochemicals

“…Several recent successful examples include the enhancement of Z. mobilis stability towards known hydrolysate inhibitors (e.g. acetate, furfural) or the complex hydrolysate itself using lab‐directed evolution (Mohagheghi et al ., ; Shui et al ., ). Recent examples to successfully increase inhibitor tolerance through reverse genetics include the identification of Hfq using microarray studies and the demonstration of its role in conveying tolerance to multiple hydrolysate inhibitors, such as acetate, vanillin, furfural and HMF (Yang et al ., , ).…”

Progress and perspective on lignocellulosic hydrolysate inhibitor tolerance improvement in Zymomonas mobilis

“…As a model bioethanol producer, Zymomonas mobilis has attracted considerable attention over the past decades due to its excellent industrial characteristics, such as the unique Entner-Doudoroff (ED) pathway under anaerobic conditions resulting in low cell mass formation, high specific rate of sugar uptake, high ethanol yield, notable ethanol tolerance, and the generally regarded as safe (GRAS) status (Panesar et al 2006;Rogers et al 2007). Furthermore, the availability of multiple genome sequences for 12 Zymomonas strains with small genome size around 2 Mb (Seo et al 2005;Yang et al 2009a;Zhao et al 2012), multiple genome-scale metabolic models (Kalnenieks et al 2014;Pentjuss et al 2013;Widiastuti et al 2011), and versatile genetic engineering strategies (Jia et al 2013;Shui et al 2015;Tan et al 2016) also accelerates the research progress in Z. mobilis. Z. mobilis has also been engineered for the production of sorbitol, gluconic acid, levan, 2,3-butanediol, isobutanol, and other biochemicals, which is proposed as an ideal microbial chassis for future synthetic biology and biorefinery (He et al 2014;Yang et al 2016).…”

Biochar-mediated enhanced ethanol fermentation (BMEEF) in Zymomonas mobilis under furfural and acetic acid stress

“…The BMEEF is an efficient and convenient method to promote the production of ethanol by Z. mobilis wildtype strain ZM4 under the high concentration of acetic acid. Compared with the reported acetic acid-tolerant Z. mobilis strains [4,10,30], biochar addition remarkably shortened the fermentation time and enhanced ethanol productivity. For example, a mutant ZMA7-2 (tolerant to 7.0 g/L acetic acid) was obtained via three rounds of adaptive laboratory evolution (ALE) [4], which consumed 96% glucose within 48 h. Besides, a flocculent mutant ZM401 and mutants ZMAQ8-1 and ZMAC8-9 with high tolerance to acetic acid were obtained by nitrosoguanidine (NTG) and ARTP mutagenesis, respectively [10,30].…”

“…"An" and "Fn" indicate Z. mobilis ZM4 fermented in the presence of n g/L acetic acid and n g/L furfural, respectively. "AnC" and "FnC" indicate Z. mobilis ZM4 co-cultured with 3.5‰ biochar fermented in the presence of n g/L acetic acid and n g/L furfural, respectively 80% glucose within 48 h [4]. In addition, mutant ZM4-MF2 was obtained by error-prone PCR of the global transcription sigma factor RpoD in Z. mobilis ZM4, which consumed 92.8% glucose and produced 9.8 g/L ethanol within 54 h in the presence of 3.0 g/L furfural under 20.0 g/L glucose [31].…”

“…However, the two most toxic inhibitors, i.e., furfural and acetic acid, were generated during this process, and these inhibitors would adversely affect the cellular growth, metabolism, and ethanol fermentation efficiency of ethanologenic bacteria [2,3]. The minimum inhibitory tolerant levels vary from microbes but are generally below 1.5 g/L furfural and 3.0 g/L acetic acid, respectively [4,5]. Furthermore, furfural can form synergistic inhibition with acetic acid [6].…”