Yuan Jianfeng, Wu Mianbin, Lin Jianping, Yang Lirong
Key Laboratory of Biomass Chemical Engineering of the Ministry of Education,College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China.
BMC Biotechnol. 2016 May 17;16(1):42. doi: 10.1186/s12896-016-0272-y.
L-(+)-tartaric acid (L-TA) is an important organic acid, which is produced from the cream of tartar or stereospecific hydrolysis of the cis-epoxysuccinate. The former method is limited by the availability of raw material and the latter is dependent on the petrochemical material. Thus, new processes for the economical preparation of L-TA from carbohydrate or renewable resource would be much more attractive. Production of 5-keto-D-gluconate (5-KGA) from glucose by Gluconobacter oxydans is the first step to produce L-TA. The aim of this work is to enhance 5-KGA accumulation using combinatorial metabolic engineering strategies in G. oxydans. The sldAB gene, encoding sorbitol dehydrogenase, was overexpressed in an industrial strain G. oxydans ZJU2 under a carefully selected promoter, P0169. To enhance the efficiency of the oxidation by sldAB, the coenzyme pyrroloquinoline quinone (PQQ) and respiratory chain were engineered. Besides, the role in sldAB overexpression, coenzyme and respiratory chain engineering and their subsequent effects on 5-KGA production were investigated.
An efficient, stable recombinant strain was constructed, whereas the 5-KGA production could be enhanced. By self-overexpressing the sldAB gene in G. oxydans ZJU2 under the constitutive promoter P0169, the resulting strain, G. oxydans ZJU3, produced 122.48 ± 0.41 g/L of 5-KGA. Furthermore, through the coenzyme and respiratory chain engineering, the titer and productivity of 5-KGA reached 144.52 ± 2.94 g/L and 2.26 g/(L · h), respectively, in a 15 L fermenter. It could be further improved the 5-KGA titer by 12.10 % through the fed-batch fermentation under the pH shift and dissolved oxygen tension (DOT) control condition, obtained 162 ± 2.12 g/L with the productivity of 2.53 g/(L · h) within 64 h.
The 5-KGA production could be significantly enhanced with the combinatorial metabolic engineering strategy in Gluconobacter strain, including sldAB overexpression, coenzyme and respiratory chain engineering. Fed-batch fermentation could further enlarge the positive effect and increase the 5-KGA production. All of these demonstrated that the robust recombinant strain can efficiently produce 5-KGA in larger scale to fulfill the industrial production of L-TA from 5-KGA.
L-(+)-酒石酸（L-TA）是一种重要的有机酸，可由酒石制得或通过顺式环氧琥珀酸的立体选择性水解获得。前一种方法受限于原材料的可得性，而后一种方法依赖于石化原料。因此，从碳水化合物或可再生资源经济制备L-TA的新工艺将更具吸引力。氧化葡萄糖杆菌将葡萄糖转化为5-酮基-D-葡萄糖酸（5-KGA）是生产L-TA的第一步。本研究旨在利用组合代谢工程策略提高氧化葡萄糖杆菌中5-KGA的积累量。编码山梨醇脱氢酶的sldAB基因在精心挑选的启动子P0169控制下，在工业菌株氧化葡萄糖杆菌ZJU2中过表达。为提高sldAB介导的氧化效率，对辅酶吡咯喹啉醌（PQQ）和呼吸链进行了改造。此外，还研究了sldAB过表达、辅酶和呼吸链工程在5-KGA生产中的作用及其后续影响。
构建了一株高效、稳定的重组菌株，其5-KGA产量得到提高。通过在组成型启动子P0169控制下，使氧化葡萄糖杆菌ZJU2中的sldAB基因自过表达，得到的菌株氧化葡萄糖杆菌ZJU3可产生122.48±0.41 g/L的5-KGA。此外，通过辅酶和呼吸链工程，在15 L发酵罐中，5-KGA的产量和产率分别达到144.52±2.94 g/L和2.26 g/(L·h)。在pH值变化和溶解氧张力（DOT）控制条件下进行补料分批发酵，5-KGA产量可进一步提高12.10%，在64 h内达到162±2.12 g/L，产率为2.53 g/(L·h)。
利用组合代谢工程策略，包括sldAB过表达、辅酶和呼吸链工程，可显著提高氧化葡萄糖杆菌菌株中5-KGA的产量。补料分批发酵可进一步扩大其积极作用，提高5-KGA产量。所有这些都表明，该强大的重组菌株能够大规模高效生产5-KGA，以实现从5-KGA工业生产L-TA。
