Sander Kyle B, Chung Daehwan, Klingeman Dawn M, Giannone Richard J, Rodriguez Miguel, Whitham Jason, Hettich Robert L, Davison Brian H, Westpheling Janet, Brown Steven D
1BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN USA.
2Bredesen Center for Interdisciplinary Graduate Research and Education, University of Tennessee, Knoxville, TN USA.
Biotechnol Biofuels. 2020 Mar 13;13:50. doi: 10.1186/s13068-020-01690-3. eCollection 2020.
, a promising biocatalyst being developed for use in consolidated bioprocessing of lignocellulosic materials to ethanol, grows poorly and has reduced conversion at elevated medium osmolarities. Increasing tolerance to elevated fermentation osmolarities is desired to enable performance necessary of a consolidated bioprocessing (CBP) biocatalyst.
Two strains of showing growth phenotypes in elevated osmolarity conditions were identified. The first strain, ORCB001, carried a deletion of the FapR fatty acid biosynthesis and malonyl-CoA metabolism repressor and had a severe growth defect when grown in high-osmolarity conditions-introduced as the addition of either ethanol, NaCl, glycerol, or glucose to growth media. The second strain, ORCB002, displayed a growth rate over three times higher than its genetic parent when grown in high-osmolarity medium. Unexpectedly, a genetic complement ORCB002 exhibited improved growth, failing to revert the observed phenotype, and suggesting that mutations other than the deleted transcription factor (the gene) are responsible for the growth phenotype observed in ORCB002. Genome resequencing identified several other genomic alterations (three deleted regions, three substitution mutations, one silent mutation, and one frameshift mutation), which may be responsible for the observed increase in osmolarity tolerance in the /-deficient strain, including a substitution mutation in , a gene previously implicated in osmoresistance in bacteria. Differential expression analysis and transcription factor binding site inference indicates that FapR negatively regulates malonyl-CoA and fatty acid biosynthesis, as it does in many other bacteria. FruR/Cra regulates neighboring fructose metabolism genes, as well as other genes in global manner.
Two systems able to effect tolerance to elevated osmolarities in are identified. The first is fatty acid biosynthesis. The other is likely the result of one or more unintended, secondary mutations present in another transcription factor deletion strain. Though the locus/loci and mechanism(s) responsible remain unknown, candidate mutations are identified, including a mutation in the chaperone coding sequence. These results illustrate both the promise of targeted regulatory manipulation for osmotolerance (in the case of ) and the challenges (in the case of ).
作为一种有前景的生物催化剂，正被开发用于木质纤维素材料的整合生物加工以生产乙醇，但在高渗透压培养基中生长不良且转化率降低。提高对升高的发酵渗透压的耐受性对于实现整合生物加工（CBP）生物催化剂所需的性能至关重要。
鉴定出两株在高渗透压条件下表现出生长表型的菌株。第一株菌株ORCB001缺失FapR脂肪酸生物合成和丙二酰辅酶A代谢阻遏物，当在高渗透压条件下生长时（通过向生长培养基中添加乙醇、NaCl、甘油或葡萄糖引入）存在严重的生长缺陷。第二株菌株ORCB002在高渗透压培养基中生长时，其生长速率比其基因亲本高出三倍以上。出乎意料的是，基因互补的ORCB002表现出改善的生长，未能恢复观察到的表型，这表明除了缺失的转录因子（基因）之外的其他突变是导致ORCB002中观察到的生长表型的原因。基因组重测序鉴定出其他几个基因组改变（三个缺失区域、三个替换突变、一个沉默突变和一个移码突变），这些改变可能是导致/缺陷菌株中观察到的渗透压耐受性增加的原因，包括中的一个替换突变，该基因先前在细菌的渗透压抗性中起作用。差异表达分析和转录因子结合位点推断表明，FapR如在许多其他细菌中一样，负向调节丙二酰辅酶A和脂肪酸生物合成。FruR/Cra调节邻近的果糖代谢基因以及其他全局基因。
鉴定出两个能够影响对升高的渗透压耐受性的系统。第一个是脂肪酸生物合成。另一个可能是另一个转录因子缺失菌株中存在的一个或多个意外的二次突变的结果。尽管负责的基因座和机制仍然未知，但已鉴定出候选突变，包括伴侣蛋白编码序列中的一个突变。这些结果既说明了靶向调节操纵对渗透压耐受性的前景（在的情况下），也说明了挑战（在的情况下）。
