Dimethyl sulfoxide enhances both cellulose dissolution ability and biocompatibility of a carboxylate-type liquid zwitterion

金沢大学理工研究域生命理工学系The cellulose dissolution ability of a liquid zwitterion, the most biocompatible cellulose solvent, was improved by adding a co-solvent, dimethylsulfoxide. Moreover, the biocompatibility of the liquid zwitterion was also improved by adding dimethylsulfoxide although it is toxic relative to the liquid zwitterion. This mixture is an efficient and extremely biocompatible cellulose solvent.Embargo Period 12 monthsThis paper has supplementary information

Design of Wall-Destructive but Membrane-Compatible Solvents

金沢大学理工研究域生命理工学系We report an extremely biocompatible solvent for plant cell walls based on a polar liquid zwitterion that dissolves cellulose, the most recalcitrant component of the plant cell walls. The polar liquid zwitterion does not affect the viability and activity of Escherichia coli, even at high concentrations. We demonstrate conversion of cell walls to ethanol via a starch-like process, namely successive dissolution, hydrolysis and fermentation in the same reaction pot.Embargo Period 12 monthsThe Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b08914.This paper has supplementary information

汎用的な細菌の凝集誘導システムの開発とそれを利用した二段階物質生産システムの構築

金沢大学新学術創成研究機構 / 神戸大学発酵阻害物質であるフルフラールのアミノ酸生産菌の増殖と発酵に対する影響を調べた。その結果、増殖は影響を受けるが、時間経過とともにフルフリルアルコールと2-フランカルボン酸に変換されることが分かった。遺伝子欠損株の解析から、フルフラールをフルフリルアルコールに変換する遺伝子を見出した。本遺伝子の精製タンパク質は試験管内でNADPHを補因子にフルフラールをフルフリルアルコールに変換した。また、酸素を代謝スイッチとして好気条件下でアミノ酸のリジンを、嫌気条件下で有機酸であるコハク酸を同一株で生産する代謝改変株を作製した。We firstly investigated cell growth of amino acid producing Corynebacterium glutamicum under furfural stress. The growth was inhibited by furfural, but furfural was degraded into furfuryl alcohol and 2-furoic acid overtime. Through constructing deletion strains of alcohol dehydrogenase genes, we identified the gene responsible for the conversion of furfural into furfuryl alcohol. Purified protein of this gene converted furfural into furfuryl alcohol in vitro using NADPH as a co-factor.We moreover constructed a strain by metabolic engineering producing amino acid lysine under aerobic conditions and organic acid succinic acid under anaerobic conditions using oxygen as a metabolic switch.研究課題/領域番号:26870363, 研究期間(年度):2009-201

酸素に応答する代謝経路のスイッチング機構の解明と新規バイオプロセスへの応用

本研究では酸素を代謝スイッチとして用いることで、コリネ型細菌の解糖系とペントースリン酸経路の切り換えに成功した。解糖系とペントースリン酸経路の切り換えは分岐点であるグルコース-6-リン酸を基質とするグルコース-6-リン酸イソメラーゼ遺伝子のプロモーターを置換することで実現した。本代謝スイッチを用いることで、樹脂原料であるカダベリンとコハク酸を好気条件と嫌気条件それぞれ効率的に生産する菌株を作製した。Controlling carbon flow in the metabolic pathway is critically important for improving production titer and yield in metabolic engineering. When synthesizing target chemicals from glucose, carbon is firstly passed through either glycolysis or pentose phosphate pathway. The split ratio between glycolysis and pentose phosphate pathway largely affects the titer and yield of the target chemicals. Here, we demonstrate that the switching of the carbon flow between glycolysis and pentose phosphate pathway by oxygen level in Corynebacterium glutamicum. By using the metabolic switch, cadaverine and succinate were efficiently produced under aerobic and anaerobic conditions, respectively. This approach is a useful and practical strategy to automatically redirect carbon flow into a desirable metabolic pathway using environmental factor as a metabolic switch.研究課題/領域番号:17K14866, 研究期間(年度):2011-201

Changes in the microbial consortium during dark hydrogen fermentation in a bioelectrochemical system increases methane production during a two-stage process

Abstract Background Bioelectrochemical systems (BESs) are an innovative technology developed to influence conventional anaerobic digestion. We examined the feasibility of applying a BES to dark hydrogen fermentation and its effects on a two-stage fermentation process comprising hydrogen and methane production. The BES used low-cost, low-reactivity carbon sheets as the cathode and anode, and the cathodic potential was controlled at − 1.0 V (vs. Ag/AgCl) with a potentiostat. The operation used 10 g/L glucose as the major carbon source. Results The electric current density was low throughout (0.30–0.88 A/m2 per electrode corresponding to 0.5–1.5 mM/day of hydrogen production) and water electrolysis was prevented. At a hydraulic retention time of 2 days with a substrate pH of 6.5, the BES decreased gas production (hydrogen and carbon dioxide contents: 52.1 and 47.1%, respectively), compared to the non-bioelectrochemical system (NBES), although they had similar gas compositions. In addition, a methane fermenter (MF) was applied after the BES, which increased gas production (methane and carbon dioxide contents: 85.1 and 14.9%, respectively) compared to the case when the MF was applied after the NBES. Meta 16S rRNA sequencing revealed that the BES accelerated the growth of Ruminococcus sp. and Veillonellaceae sp. and decreased Clostridium sp. and Thermoanaerobacterium sp., resulting in increased propionate and ethanol generation and decreased butyrate generation; however, unknowingly, acetate generation was increased in the BES. Conclusions The altered redox potential in the BES likely transformed the structure of the microbial consortium and metabolic pattern to increase methane production and decrease carbon dioxide production in the two-stage process. This study showed the utility of the BES to act on the microbial consortium, resulting in improved gas production from carbohydrate compounds

New Multiple-Deletion Method for the Corynebacterium glutamicum Genome, Using a Mutant lox Sequence

Due to the difficulty of multiple deletions using the Cre/loxP system, a simple, markerless multiple-deletion method based on a Cre/mutant lox system combining a right-element (RE) mutant lox site with a left-element (LE) mutant lox site was employed for large-scale genome rearrangements in Corynebacterium glutamicum. Eight distinct genomic regions that had been identified previously by comparative analysis of C. glutamicum R and C. glutamicum 13032 genomes were targeted for deletion. By homologous recombination, LE and RE mutant lox sites were integrated at each end of a target region. Highly efficient and accurate deletions between the two chromosomal mutant lox sites in the presence of Cre recombinase were realized. A deletion mutant lacking 190 kb of chromosomal regions, encoding a total of 188 open reading frames (ORFs), was obtained. These deletions represent the largest genomic excisions in C. glutamicum reported to date. Despite the loss of numerous predicted ORFs, the mutant exhibited normal growth under standard laboratory conditions. The Cre/loxP system using a pair of mutant lox sites provides a new, efficient genome rearrangement technique for C. glutamicum. It should facilitate the understanding of genome functions of microorganisms