Caldicellulosiruptor bescii, 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 C. bescii 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 fruR/cra 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 fruR/cra-deficient strain, including a substitution mutation in dnaK, 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 C. bescii 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 dnaK chaperone coding sequence. These results illustrate both the promise of targeted regulatory manipulation for osmotolerance (in the case of fapR) and the challenges (in the case of fruR/cra).

中文翻译：

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用于工程化贝西卡迪纤维素渗透压耐受性的基因靶标

Caldicellulosiruptor bescii 是一种很有前途的生物催化剂，正在开发用于将木质纤维素材料综合生物加工成乙醇，在升高的中等渗透压下生长不良并且转化率降低。希望增加对升高的发酵渗透压的耐受性，以实现综合生物加工 (CBP) 生物催化剂所需的性能。鉴定了两种在高渗透压条件下表现出生长表型的贝西梭菌菌株。第一个菌株 ORCB001 缺失了 FapR 脂肪酸生物合成和丙二酰辅酶 A 代谢抑制因子，并且在高渗透压条件下生长时具有严重的生长缺陷——作为添加乙醇、氯化钠、甘油或葡萄糖引入生长培养基。第二个菌株，ORCB002，在高渗透压培养基中生长时，其生长速度是其遗传亲本的三倍以上。出乎意料的是，遗传补体 ORCB002 表现出改善的生长，未能恢复观察到的表型，并表明除缺失的转录因子（fruR/cra 基因）之外的突变是导致 ORCB002 中观察到的生长表型的原因。基因组重测序确定了其他几个基因组改变（三个缺失区域、三个替代突变、一个沉默突变和一个移码突变），这可能是观察到 fruR/cra 缺陷菌株的渗透压耐受性增加的原因，包括一个替代突变在 dnaK 中，一种先前与细菌的渗透压抗性有关的基因。差异表达分析和转录因子结合位点推断表明，FapR 负调控丙二酰辅酶A 和脂肪酸生物合成，就像在许多其他细菌中一样。FruR/Cra 以全局方式调节邻近的果糖代谢基因以及其他基因。确定了两种能够对贝西梭菌中升高的渗透压产生耐受性的系统。首先是脂肪酸的生物合成。另一种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/基因座和机制仍然未知，但确定了候选突变，包括 dnaK 伴侣编码序列中的突变。