Whitham Jason M, Moon Ji-Won, Rodriguez Miguel, Engle Nancy L, Klingeman Dawn M, Rydzak Thomas, Abel Malaney M, Tschaplinski Timothy J, Guss Adam M, Brown Steven D
1Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN USA.
2BioEnergy Science Center, National Laboratory, Oak Ridge, TN USA.
Biotechnol Biofuels. 2018 Apr 5;11:98. doi: 10.1186/s13068-018-1095-y. eCollection 2018.
is a model fermentative anaerobic thermophile being studied and engineered for consolidated bioprocessing of lignocellulosic feedstocks into fuels and chemicals. Engineering efforts have resulted in significant improvements in ethanol yields and titers although further advances are required to make the bacterium industry-ready. For instance, fermentations at lower pH could enable co-culturing with microbes that have lower pH optima, augment productivity, and reduce buffering cost. is typically grown at neutral pH, and little is known about its pH limits or pH homeostasis mechanisms. To better understand pH homeostasis we grew strain LL1210 ( DSM1313 -), currently the highest ethanol producing strain of , at different pH values in chemostat culture and applied systems biology tools.
LL1210 was found to be growth-limited below pH 6.24 at a dilution rate of 0.1 h. FF-ATPase gene expression was upregulated while many ATP-utilizing enzymes and pathways were downregulated at pH 6.24. These included most flagella biosynthesis genes, genes for chemotaxis, and other motility-related genes (> 50) as well as sulfate transport and reduction, nitrate transport and nitrogen fixation, and fatty acid biosynthesis genes. Clustering and enrichment of differentially expressed genes at pH values 6.48, pH 6.24 and pH 6.12 (washout conditions) compared to pH 6.98 showed inverse differential expression patterns between the FF-ATPase and genes for other ATP-utilizing enzymes. At and below pH 6.24, amino acids including glutamate and valine; long-chain fatty acids, their iso-counterparts and glycerol conjugates; glycolysis intermediates 3-phosphoglycerate, glucose 6-phosphate, and glucose accumulated intracellularly. Glutamate was 267 times more abundant in cells at pH 6.24 compared to pH 6.98, and intercellular concentration reached 1.8 μmol/g pellet at pH 5.80 (stopped flow).
LL1210 can grow under slightly acidic conditions, similar to limits reported for other strains. This foundational study provides a detailed characterization of a relatively acid-intolerant bacterium and provides genetic targets for strain improvement. Future studies should examine adding gene functions used by more acid-tolerant bacteria for improved pH homeostasis at acidic pH values.
是一种用于研究和工程改造的发酵性厌氧嗜热菌，用于将木质纤维素原料整合生物加工为燃料和化学品。尽管还需要进一步改进以使该细菌能够投入工业应用，但工程改造已使乙醇产量和滴度有了显著提高。例如，在较低pH值下进行发酵可以实现与最适pH值较低的微生物共培养，提高生产力，并降低缓冲成本。通常在中性pH值下生长，对其pH值限制或pH值稳态机制了解甚少。为了更好地理解pH值稳态，我们在恒化器培养中以不同pH值培养菌株LL1210（DSM1313 -），该菌株目前是乙醇产量最高的菌株，并应用了系统生物学工具。
发现在稀释率为0.1 h时，LL1210在pH 6.24以下生长受限。在pH 6.24时，FF - ATP酶基因表达上调，而许多利用ATP的酶和途径下调。这些包括大多数鞭毛生物合成基因、趋化性基因和其他与运动相关的基因（超过50个），以及硫酸盐转运和还原、硝酸盐转运和固氮以及脂肪酸生物合成基因。与pH 6.98相比，在pH 6.48、pH 6.24和pH 6.12（冲出条件）下差异表达基因的聚类和富集显示，FF - ATP酶与其他利用ATP的酶的基因之间存在相反的差异表达模式。在pH 6.24及以下，包括谷氨酸和缬氨酸在内的氨基酸；长链脂肪酸、其异构体对应物和甘油共轭物；糖酵解中间体3 - 磷酸甘油酸、6 - 磷酸葡萄糖和葡萄糖在细胞内积累。与pH 6.98相比，pH 6.24时细胞内谷氨酸含量高267倍，在pH 5.80（停流）时细胞间浓度达到1.8 μmol/g沉淀。
LL1210可以在微酸性条件下生长，类似于其他菌株报道的限制。这项基础研究详细描述了一种相对不耐酸的细菌，并为菌株改良提供了遗传靶点。未来的研究应研究添加更耐酸细菌使用的基因功能，以改善酸性pH值下的pH值稳态。
