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Metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production
Biotechnology for Biofuels ( IF 6.1 ) Pub Date : 2020-03-10 , DOI: 10.1186/s13068-020-01680-5
Evert K Holwerda 1, 2 , Daniel G Olson 1, 2 , Natalie M Ruppertsberger 1 , David M Stevenson 2, 3 , Sean J L Murphy 1 , Marybeth I Maloney 1, 2 , Anthony A Lanahan 1, 2 , Daniel Amador-Noguez 2, 3 , Lee R Lynd 1, 2
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Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving adhE that occurred spontaneously in some of the engineered strains. Contrary to lactate dehydrogenase, deleting phosphotransacetylase (pta, acetate) negatively affected steady-state biomass concentration and caused increased extracellular levels of free amino acids and pyruvate, while no increase in ethanol was detected. Adaptive laboratory evolution (ALE) improved growth and shifted elevated levels of amino acids and pyruvate towards ethanol, but not for all strain lineages. Three out of four end-strains produced ethanol at higher yield, and one did not. The occurrence of a mutation in the adhE gene, expanding its nicotinamide-cofactor compatibility, enabled two end-strains to produce more ethanol. A disruption in the hfsB hydrogenase is likely the reason why a third end-strain was able to make more ethanol. RNAseq analysis showed that the distribution of fermentation products was generally not regulated at the transcript level. At 120 g/L cellulose loadings, deletions of spo0A, ldh and pta and adaptive evolution did not negatively influence cellulose solubilization and utilization capabilities. Strains with a disruption in hfsB or a mutation in adhE produced more ethanol, isobutanol and 2,3-butanediol under these conditions and the highest isobutanol and ethanol titers reached were 5.1 and 29.9 g/L, respectively. Modifications in the organic acid fermentative pathways in Clostridium thermocellum caused an increase in extracellular pyruvate and free amino acids. Adaptive laboratory evolution led to improved growth, and an increase in ethanol yield and production due a mutation in adhE or a disruption in hfsB. Strains with deletions in ldh and pta pathways and subjected to ALE demonstrated undiminished cellulolytic capabilities when cultured on high cellulose loadings.

中文翻译:

热纤梭菌对旨在提高乙醇产量的遗传干预的代谢和进化反应

旨在通过改变热纤梭菌的中央发酵代谢来增加乙醇的工程努力取得了不同程度的成功。在这里,我们的目标是通过多方面的方法来了解这种变异,包括基因组和转录组分析,结合恒化器培养和高固体纤维素发酵。检查了总共包含 16 个菌株的三个菌株谱系。参与导致有机酸和/或孢子形成的途径的基因被敲除的两个菌株谱系在适应性实验室进化(ALE)后产生了四个末端菌株。第三种菌株谱系概括了一些工程菌株中自发发生的涉及 adhE 的突变。与乳酸脱氢酶相反,磷酸转乙酰酶 (pta, 乙酸盐)对稳态生物质浓度产生负面影响,并导致细胞外游离氨基酸和丙酮酸水平增加,而未检测到乙醇增加。适应性实验室进化 (ALE) 改善了生长并将氨基酸和丙酮酸水平升高转向乙醇,但并非适用于所有菌株谱系。四分之三的末端菌株以更高的产率生产乙醇,而一个没有。adhE 基因突变的发生,扩大了其烟酰胺辅因子的相容性,使两个末端菌株能够产生更多的乙醇。hfsB 氢化酶的中断可能是第三个末端菌株能够制造更多乙醇的原因。RNAseq 分析表明,发酵产物的分布通常不受转录水平的调节。在 120 g/L 纤维素装载量下,spo0A 缺失,ldh 和 pta 以及适应性进化对纤维素的溶解和利用能力没有负面影响。在这些条件下,hfsB 中断或 adhE 突变的菌株产生更多的乙醇、异丁醇和 2,3-丁二醇,达到的最高异丁醇和乙醇滴度分别为 5.1 和 29.9 g/L。热纤梭菌中有机酸发酵途径的改变导致细胞外丙酮酸和游离氨基酸的增加。由于adhE突变或hfsB中断,适应性实验室进化导致生长改善,乙醇产量和产量增加。在 ldh 和 pta 途径中缺失并经受 ALE 的菌株在高纤维素负载下培养时表现出不减弱的纤维素分解能力。
更新日期:2020-04-22
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