Department of Microbiology, North Carolina State University, Raleigh, NC 27695-7615, USA.
Department of Biological and Agricultural Engineering, North Carolina State University, Raleigh, NC 27695-7615, USA.
Microbiology (Reading). 2013 Dec;159(Pt 12):2558-2570. doi: 10.1099/mic.0.069534-0. Epub 2013 Sep 25.
Production of butanol by solventogenic clostridia is controlled through metabolic regulation of the carbon flow and limited by its toxic effects. To overcome cell sensitivity to solvents, stress-directed evolution methodology was used three decades ago on Clostridium beijerinckii NCIMB 8052 that spawned the SA-1 strain. Here, we evaluated SA-1 solventogenic capabilities when growing on a previously validated medium containing, as carbon- and energy-limiting substrates, sucrose and the products of its hydrolysis d-glucose and d-fructose and only d-fructose. Comparative small-scale batch fermentations with controlled pH (pH 6.5) showed that SA-1 is a solvent hyper-producing strain capable of generating up to 16.1 g l(-1) of butanol and 26.3 g l(-1) of total solvents, 62.3 % and 63 % more than NCIMB 8052, respectively. This corresponds to butanol and solvent yields of 0.3 and 0.49 g g(-1), respectively (63 % and 65 % increase compared with NCIMB 8052). SA-1 showed a deficiency in d-fructose transport as suggested by its 7 h generation time compared with 1 h for NCIMB 8052. To potentially correlate physiological behaviour with genetic mutations, the whole genome of SA-1 was sequenced using the Illumina GA IIx platform. PCR and Sanger sequencing were performed to analyse the putative variations. As a result, four errors were confirmed and validated in the reference genome of NCIMB 8052 and a total of 10 genetic polymorphisms in SA-1. The genetic polymorphisms included eight single nucleotide variants, one small deletion and one large insertion that it is an additional copy of the insertion sequence ISCb1. Two of the genetic polymorphisms, the serine threonine phosphatase cbs_4400 and the solute binding protein cbs_0769, may possibly explain some of the observed physiological behaviour, such as rerouting of the metabolic carbon flow, deregulation of the d-fructose phosphotransferase transport system and delayed sporulation.
溶剂梭菌生产丁醇受碳流代谢调控，其产量受到其毒性的限制。为了克服细胞对溶剂的敏感性，三十年前就采用了定向进化方法对凝结芽孢杆菌 NCIMB 8052 进行了研究，由此产生了 SA-1 菌株。在这里，我们评估了 SA-1 在以前经过验证的培养基上生长时的溶剂生成能力，该培养基含有蔗糖及其水解产物 d-葡萄糖和 d-果糖作为碳源和能量限制底物，且仅含 d-果糖。在 pH （6.5）可控的小型分批发酵实验中，结果表明 SA-1 是一株高产溶剂的菌株，最高可生成 16.1 g/L 的丁醇和 26.3 g/L 的总溶剂，分别比 NCIMB 8052 高出 62.3%和 63%。这相当于丁醇和溶剂的产率分别为 0.3 和 0.49 g/g（与 NCIMB 8052 相比，分别提高了 63%和 65%）。与 NCIMB 8052 相比，SA-1 的 d-果糖转运能力存在缺陷，这表明其代时为 7 h，而 NCIMB 8052 为 1 h。为了潜在地将生理行为与基因突变相关联，我们使用 Illumina GA IIx 平台对 SA-1 的全基因组进行了测序。通过 PCR 和 Sanger 测序对可能的变异进行了分析。结果，在 NCIMB 8052 的参考基因组中确认并验证了四个错误，并在 SA-1 中发现了总共 10 个遗传多态性。遗传多态性包括 8 个单核苷酸变异、1 个小缺失和 1 个大插入，这是插入序列 ISCb1 的额外副本。其中两个遗传多态性，丝氨酸-苏氨酸磷酸酶 cbs_4400 和溶质结合蛋白 cbs_0769，可能解释了一些观察到的生理行为，例如代谢碳流的改道、d-果糖磷酸转移酶运输系统的去调控以及延迟孢子形成。
