Effect of puuC overexpression and nitrate addition on glycerol metabolism and anaerobic 3-hydroxypropionic acid production in recombinant Klebsiella pneumoniae ΔglpKΔdhaT
Effect of puuC overexpression and nitrate addition on glycerol metabolism and anaerobic 3-hydroxypropionic acid production in recombinant Klebsiella pneumoniae ΔglpKΔdhaT
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DOI：
10.1016/j.ymben.2012.09.004
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发表时间：
2013-01-01
影响因子：
8.4
通讯作者：
Park, Sunghoon
中科院分区：
文献类型：
--
作者：
Ashok, Somasundar;Raj, Subramanian Mohan;Park, Sunghoon
3-羟基丙酸（3-HP）是一种重要的工业平台化学品，在许多重要的商业化学品的生产过程中用作前体。近年来，K. pneumoniae过表达克雷伯氏菌的NAD（+）依赖性γ-谷氨酰-γ-氨基丁醛脱氢酶（PuuC）。pneumoniae DSM 2026显示在不添加昂贵的辅酶B-12的情况下从甘油生产3-HP。然而，K.肺炎伴随着NADH的产生，这总是导致1，3-丙二醇（1，3-PDO）和乳酸的大量积累。在这项研究中，我们研究了硝酸盐作为电子受体的潜在用途，既可以再生NAD（+），又可以防止在从甘油厌氧生产3-HP过程中形成副产物。添加硝酸盐可提高NAD（+）的再生率，但降低了甘油向3-HP转化的速率。为了将更多的甘油转化为3-HP，一种新的重组菌株K。通过破坏编码甘油激酶的glpK基因和编码1，3-丙二醇氧化还原酶的dhaT基因开发了Δ glpK Δ dhaT（puuC）。该菌株显示出改善的细胞NAD（+）浓度和朝向3-HP生产的高碳通量。通过在硝酸盐存在下的厌氧培养，该重组菌株在摇瓶中产生超过40 +/- 3 mM 3-HP，其中基于甘油的产率超过50%，并且在补料分批生物反应器中产生250 +/- 10 mM 3-HP，其中基于甘油的产率约为30%。(C)2012 Elsevier Inc. All rights reserved.
3-Hydroxypropionic acid (3-HP), an industrially important platform chemical, is used as a precursor during the production of many commercially important chemicals. Recently, recombinant strains of K. pneumoniae overexpressing an NAD(+)-dependent gamma-glutamyl-gamma-aminobutyraldehyde dehydrogenase (PuuC) enzyme of K. pneumoniae DSM 2026 were shown to produce 3-HP from glycerol without the addition coenzyme B-12, which is expensive. However, 3-HP production in K. pneumoniae is accompanied with NADH generation, and this always results in large accumulation of 1,3-propanediol (1,3-PDO) and lactic acid. In this study, we investigated the potential use of nitrate as an electron acceptor both to regenerate NAD(+) and to prevent the formation of byproducts during anaerobic production of 3-HP from glycerol. Nitrate addition could improve NAD(+) regeneration, but decreased glycerol flux towards 3-HP production. To divert more glycerol towards 3-HP, a novel recombinant strain K. pneumoniae Delta glpK Delta dhaT (puuC) was developed by disrupting the glpK gene, which encodes glycerol kinase, and the dhaT gene, which encodes 1,3-propanediol oxidoreductase. This strain showed improved cellular NAD(+) concentrations and a high carbon flux towards 3-HP production. Through anaerobic cultivation in the presence of nitrate, this recombinant strain produced more than 40 +/- 3 mM 3-HP with more than 50% yield on glycerol in shake flasks and 250 +/- 10 mM 3-HP with approximately 30% yield on glycerol in a fed-batch bioreactor. (C) 2012 Elsevier Inc. All rights reserved.