Adenine addition restores cell viability and butanol production in Clostridium saccharoperbutylacetonicum N1-4 (ATCC13564) cultivated at 37°C
Adenine addition restores cell viability and butanol production in Clostridium saccharoperbutylacetonicum N1-4 (ATCC13564) cultivated at 37°C
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添加腺嘌呤可恢复 37°C 培养的糖过丁基丙酮梭菌 N1-4 (ATCC13564) 的细胞活力和丁醇产量
DOI：
10.1128/aem.02960-16
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发表时间：
2017
影响因子：
4.4
通讯作者：
Shunichi Nakayama
中科院分区：
文献类型：
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作者：
Keiji Kiyoshi;Sohei Kawashima;Kosuke Nobuki;Toshimori Kadokura;Atsumi Nakazato;Ken-ichiro Suzuki;Shunichi Nakayama
我们已经开发了丁醇生产巩固的生物加工纤维素底物通过共培养的纤维素分解梭菌和丁醇生产梭菌saccharoperbutylacetonicum菌株N1-4。然而，菌株N1-4（其具有30°C的最佳生长温度）的丁醇发酵对37°C的较高培养温度敏感;这种有害影响的性质仍不清楚。在30°C和37°C下培养的菌株N1-4的细胞内代谢物的比较揭示了在较高温度下生长期间多种初级代谢物（特别是包括核酸和辅因子）的水平降低。在培养基中补充250毫克/升腺嘌呤可促进两种细胞的生长（在600 nm处的光密度从4.3增加到10.2）和丁醇产量（从3.9克/升增加到9.6克/升），与没有补充腺嘌呤获得的那些相比，使得补充的37°C培养物表现出接近在30°C下在没有腺嘌呤补充的情况下观察到的那些的生长和丁醇产生。这些改进的性质是基于细胞活力的维持。我们进一步表明，在37°C下生长期间，腺嘌呤补充通过维持ATP水平和抑制孢子形成来增强细胞活力。这项工作代表了腺嘌呤相关代谢对梭菌丁醇生产的重要性的第一次证明（据我们所知），提出了一种基于代谢物水平增强靶向途径的新方法。重要代谢物组学分析显示，与30°C相比，在37°C下生长期间，C.糖多丁基丙酮菌菌株N1-4。我们发现，腺嘌呤补充恢复了菌株N1-4在37°C下的细胞生长和丁醇生产。腺嘌呤补充的影响反映了维持细胞活力的ATP水平的维持和孢子形成的抑制。因此，我们的代谢组学分析鉴定了维持细胞活力所需的耗尽代谢物。我们的策略，预计将适用于广泛的生物体，允许识别的限制代谢途径，这可以作为一个新的目标分子育种。这项工作的另一个新发现是腺嘌呤补充剂抑制梭菌孢子的形成。丁醇梭菌芽孢形成与代谢组学状态的关系是今后研究的重点。
We have developed butanol-producing consolidated bioprocessing from cellulosic substrates through coculture of cellulolytic clostridia and butanol-producing Clostridium saccharoperbutylacetonicum strain N1-4. However, the butanol fermentation by strain N1-4 (which has an optimal growth temperature of 30°C) is sensitive to the higher cultivation temperature of 37°C; the nature of this deleterious effect remains unclear. Comparison of the intracellular metabolites of strain N1-4 cultivated at 30°C and 37°C revealed decreased levels of multiple primary metabolites (notably including nucleic acids and cofactors) during growth at the higher temperature. Supplementation of the culture medium with 250 mg/liter adenine enhanced both cell growth (with the optical density at 600 nm increasing from 4.3 to 10.2) and butanol production (increasing from 3.9 g/liter to 9.6 g/liter) at 37°C, compared to those obtained without adenine supplementation, such that the supplemented 37°C culture exhibited growth and butanol production approaching those observed at 30°C in the absence of adenine supplementation. These improved properties were based on the maintenance of cell viability. We further showed that adenine supplementation enhanced cell viability during growth at 37°C by maintaining ATP levels and inhibiting spore formation. This work represents the first demonstration (to our knowledge) of the importance of adenine-related metabolism for clostridial butanol production, suggesting a new means of enhancing target pathways based on metabolite levels.IMPORTANCEMetabolomic analysis revealed decreased levels of multiple primary metabolites during growth at 37°C, compared to 30°C, in C. saccharoperbutylacetonicum strain N1-4. We found that adenine supplementation restored the cell growth and butanol production of strain N1-4 at 37°C. The effects of adenine supplementation reflected the maintenance of cell viability originating from the maintenance of ATP levels and the inhibition of spore formation. Thus, our metabolomic analysis identified the depleted metabolites that were required to maintain cell viability. Our strategy, which is expected to be applicable to a wide range of organisms, permits the identification of the limiting metabolic pathway, which can serve as a new target for molecular breeding. The other novel finding of this work is that adenine supplementation inhibits clostridial spore formation. The mechanism linking spore formation and metabolomic status in butanol-producing clostridia is expected to be the focus of further research.