Use of an EZ-Tn5-based random mutagenesis system to create aZymomonas mobiliswith significant tolerance to heat stress and malnutrition

Abstract

During ethanol production, the fermentation cells are always exposed to stresses like high temperature and low nutritional conditions, which affect their growth and productivity. Stress-tolerant strains with high ethanol yield are highly desirable. Therefore, a recombinant Zymomonas mobilis (Z. mobilis) designated as HYM was constructed by integrating three genes (yfdZ, metB, and Pfu-sHSP) into the genome of Z. mobilis CP4 (CP4) via Tn5 transposon in the present study. The yfdZ and metB genes from E. coli were used to decrease the nutritional requirement. The small heat shock protein gene (Pfu-sHSP) from Pyrococcus furiosus (P. furiosus) was used to increase the heat tolerance. The genomic integration of three genes confers on Z. mobilis the ability to grow in simple chemical defined medium without the addition of amino acid. The HYM not only demonstrated the high tolerance to unfavorable lower nutrition stresses but also the capability of converting glucose to ethanol with high yield at higher temperature. What is more, these genetic characteristics were stable up to 100 generations on nonselective medium. The effects of glucose concentration, fermentation temperature, and initial pH on ethanol production of the mutant strain HYM were optimized using a Box-Behnken design (BBD) experiment. The integration of three genes led to a significant increase in ethanol production by 9 % compared with its original Z. mobilis counterpart. The maximum ethanol production of HYM was as high as 105 g/l.

Food load manipulation ability shapes flight morphology in females of central-place foraging Hymenoptera

“…females per species on average] were caught in the field, in natural populations found in the Parque Natural de la Albufera, Valencia province (South-Eastern Spain), during the spring-summer 2009–2010. Specimens were determined to species level following taxonomic keys [57-59] and with the aid of experts; however, for seven of them we could only reach the genus level and therefore assigned specimens to morphospecies (Table 1). Additional data for seven species were obtained from the literature (Table 1).…”

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

“…A significant collection of classical genetics tools have been explored and are now routine metabolic engineering practices in Z. mobilis , including stable and transferable plasmids, shuttle vectors, promoters, transformation methods such as conjugation and electroporation, reporter genes such as green fluorescent protein (GFP) and ice nucleation activity, and transposon mutagenesis strategies (Skotnicki et al ., ; Carey et al ., ; Browne et al ., ; Conway et al ., ,b; Arfman et al ., ; Delgado et al ., ; Drainas et al ., ; Zhang et al ., , ; Pappas et al ., ; Douka et al ., ; Yang et al ., ,b, ; Dong et al ., , ; Pappas, ; Jia et al ., ; Dunn and Rao, ; Yi et al ., ; Wang et al ., ). These methods have been widely reviewed (Panesar et al ., ; He et al ., ) and are not further described here.…”

“…The possibility to substitute freshwater with seawater in the culture medium could further mitigate the socio‐environmental challenges for the expansion of ethanol production (Swings and De Ley, ; Goncalves et al ., ). In addition to the ongoing efforts to engineer Z. mobilis for fermentation under heat stress conditions without supplementation of amino acids and vitamins (Jia et al ., ; Zhang et al ., ; Wang et al ., ), a recent report demonstrated that Z. mobilis can utilize N 2 as a nitrogen source and thus replace NH 4 or the industrial nitrogen supplement, corn steep liquor. It was also observed that nitrogen fixation did not affect ethanol yield, but rather increased the specific ethanol productivity at lower biomass loadings, which could significantly reduce the cellulosic ethanol production cost by millions of dollars annually (Kremer et al ., ), although the utility of this process at an industrial scale requires further investigation.…”

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).…”