代谢改造克雷伯氏菌合成D-1,2,4-丁三醇

Production of D-1,2,4-butanetriol by engineering Klebsiella pneumoniae

【背景】D-1,2,4-丁三醇(D-1,2,4-butanetriol,BT)是一种重要的四碳多元醇,应用范围广,以木糖为底物的四步生化反应是目前最高效的BT生物合成路线。但大肠杆菌宿主存在严重的碳代谢抑制,限制了工程菌在木糖葡萄糖混合糖下的生长和BT合成。然而克雷伯氏菌具有生长速度更快、葡萄糖木糖混合糖利用效果好等优点。【目的】在碳代谢抑制效应较弱的克雷伯氏菌中构建以木糖为底物的BT合成途径,以提高混合糖下BT合成能力。【方法】将来源于Clostridium crescenti的木糖脱氢酶基因xdh和来源于Lactococcus lactis的2-酮异戊酸脱羧酶基因kivD及来源于Escherichia coli W3110的木糖酸脱水酶基因yjhG克隆至KlebsiellapneumoniaeZG25,得到重组菌K.pneumoniae ZG25-BT,对重组菌进行培养条件和培养基优化,进一步敲除xylA以提高BT产量。【结果】在37°C、200 r/min、接种量1%、诱导时间2 h、添加10.0 g/L CaCO3控制pH条件下,敲除xylA的重组菌在1.5倍LB培养基中以30.0 g/L木糖和10.0 g/L葡萄糖为底物,BT的产量达到4.52 g/L,摩尔转化率为0.21mol/mol,收率为15%,较优化前分别提高150%、62%和67%。【结论】实现了BT在K.pneumoniaeZG25中的发酵生产,同时通过培养条件和培养基的优化及xylA的敲除提高了BT合成能力,为进一步实验奠定了基础。

[Background] D-1,2,4-butanetriol(BT) is an important four-carbon polyol with a wide range of applications in industries. The four-step biochemical reaction using xylose as a substrate is currently the most efficient BT biosynthetic route. However, the Escherichia coli host has serious carbon catabolite repression which limits the growth and BT synthesis of xylose and glucose sugar mixture. Klebsiella pneumoniae has the advantages of faster growth rate, better utilization of xylose and glucose sugar mixture. [Objective] Establishing BT synthesis pathway using xylose as a substrate in K. pneumonia, which has a weak carbon catabolite repression, to improve BT production of sugar mixture. [Methods] The heterologous pathway of BT biosynthesis was constructed in K. pneumoniae ZG25 by co-overexpressing the xylose dehydrogenase gene(xdh) from Clostridium crescenti, 2-ketoisovalerate decarboxylase gene(kivD) from Lactococcus lactis, and xylose dehydratase gene(yjhG) from E. coli W3110, obtaining recombinant strain K. pneumoniae ZG25-BT. After optimization of the medium and culture conditions and deletion of xylA, significant increase in BT production was achieved. [Results] The optimal procedure for BT production in recombinant K. pneumoniae ZG25-BT strain without the xylA was achieved in 1.5-fold LB medium under the conditions of 30.0 g/L xylose, 10.0 g/L glucose, 37 °C, 200 r/min rotation speed, adding 10.0 g/L CaCO3 to control pH and 1% inoculation amount followed by 2 h of induction with the addition of IPTG, which made BT titer, molar conversion and yield up to 4.52 g/L, 0.21 mol/mol and 15%, respectively, representing a 150%, 62% and 67% increase compared with that of unoptimizable condition, respectively. [Conclusion] A fermentative process of BT production in K. pneumoniae ZG25 is achieved. Optimization of the culture conditions and medium and the knockout of xylA are applied to improve BT production. Furthermore, this work provides a useful host for the improved BT production through metabolic engineering.