基于钝齿棒杆菌argGH启动子改造的L-瓜氨酸高效合成

Enhancement of L-Citrulline Production by Promoter Modification ofargGHinCorynebacerium crenatum

DOI：10.3969/j.issn.1673-1689.2019.03.012

中文关键词: 钝齿棒杆菌 L-瓜氨酸 L-精氨酸 启动子 代谢工程

英文关键词: Corynebacterium crenatum,L-citrulline,L-arginine,promoter,metabolic engineering

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中文摘要:

L-瓜氨酸是尿素循环的重要中间体，在argGH编码酶的催化下易分解为L-精氨酸，在微生物体内难以大量积累。作者通过启动子替换手段，以弱启动子P-dapAB6替换调控argGH表达的启动子P-argG，构建了弱化L-瓜氨酸分解代谢途径的重组菌株Corynebacterium crenatumH-7-PdapAB6：argGH。重组菌株的转录水平和酶活力结果显示，重组菌株分解途径中argG和argH的基因表达水平下调，精胺琥珀酸合成酶ASS（argG编码）和精胺琥珀酸裂解酶ASL（argH编码）酶活分别降低91.80%和55.35%。摇瓶发酵结果表明，L-瓜氨酸的产量、糖酸转化率和生产强度分别为33.85 g/L、0.25 g/g和0.34 g/（L·h），较原始菌株分别提高了4.91、5.00和4.86倍。通过启动子替换策略，构建了L-瓜氨酸分解代谢途径弱化的工程菌株，初步实现了L-瓜氨酸的高效合成。

英文摘要:

L-citrulline isan importantintermediate metabolite inurea cycle. The enzymes encoded by geneargGHcatalyze thereaction of L-citrullineto L-arginineresults in difficulty of L-citrulline accumulation via microbial metabolism. In this study，promoter P-argG wasreplaced by a weak promoter P-dapAB6to down-regulateargGHexpression. The recombinant strain Corynebacterium crenatum H-7-PdapAB6：argGHshowed lowerargGHexpression level，with ASS（argininosuccinate synthase，encoding byargG） and ASL（argininosuccinase，encoding byargH） activities reducing by 91.80% and 55.35%，respectively，compared with the parent strain. Via fermentation in shake flasks，the L-citrulline titer，yield and productivity ofCorynebacterium crenatumH-7-PdapAB6：argGH were 33.85 g/L，0.25 g/g and 0.34 g/（L·h），respectively，which were 4.91，5.00 and 4.86-fold of those obtained from the parentstrain. These results indicatedthat，by promoter engineeringapproach，the preliminary construction of L-citrulline biosynthesis pathway was achieved.

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