普通生酮基古龙酸菌2-酮基-L-古龙酸合成途径在氧化葡萄糖酸杆菌中的整合表达与强化  

Integration and Enhancement of 2-Keto-L-Gulonic Acid Synthesis Pathway in Gluconobactero xydans from Ketogulonicigenium vulgare

作　　者：陈吉铭 堵国成[1,2] 陈坚[1,2] 周景文[1,2] CHEN Jiming DU Guocheng CHEN Ji ZHOU Jingwen(Key Laboratory of Industrial Biotechnology, Ministry of Education , Jiangnan University,Wuxi 214122, China School of Biotechnology. Jiangnan University, Wuxi 214122, China)

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与生物技术学报》2016年第6期611-616,共6页Journal of Food Science and Biotechnology

基　　金：国家863计划项目(2012AA022103)

摘　　要：目前工业生产VC的方法为二步发酵法,但该法涉及两步3种微生物,存在操作工序繁琐、需要消耗大量原材料及能源等缺点。一步发酵法被认为能够降低生产成本并对VC的工业化生产起到革命性的改变。为了克服重组质粒易丢失及需要添加抗生素等不利因素,将普通生酮基古龙酸菌(K.vulgare WSH-001)中的山梨糖脱氢酶基因(sdh)和山梨酮脱氢酶基因(sndh)整合到氧化葡萄糖酸杆菌(G.oxydans WSH-003)基因组,得到一步发酵生产菌株G.oxydans-ss。将其在1 L的罐中发酵,72 h时2-KLG产量达到最高,为37.6 g/L。为了进一步提高2-KLG的产量,还采用了辅因子工程。吡咯喹啉醌(Pyrroloquinoline quinone,PQQ)是SDH和SNDH的辅酶,通过在G.oxydans-ss中过量表达来自K.vulgare WSH-001的pqq ABCDE基因,得到重组菌G.oxydans-ss/pqq ABCDE。将其在1 L的罐中发酵,84 h时2-KLG产量达到最高,为44.5 g/L。以上结果对VC的一步发酵起到了一定的促进作用,使一步发酵法生产VC的工业化生产有望实现。Vitamin C （VC）,as a kind of vitamins and antioxidants,is widely used in pharmaceutical, food, beverage, and feed industries.2-keto-L-gulonic acid （2-KLG） is the precursor of vitamin C. Recently, the two-step fermentation is used as the main route for vitamin C production. However,this route involves three microorganisms and requires an additional second sterilization process,which significantly increases the cost of both raw materials and energy requirement. Therefore, a one-step fermentation process is considered to be more cost-effective and revolutionary in VC industry. In order to overcome the disadvantages ofplasmid overexpression, while the plasmid may be lost and need to add antibiotics in medium,the recombinant strain G. oxydans-ss was constructed by integrating sorbose dehydrogenase gene （sdh） and sorbosone dehydrogenase gene （sndh） from K.vulgare WSH-001 into the genome of G.oxydans WSH-003. This recombinant strain produced 37.6 g/L of 2-KLG after 72 h in 1 L fermentor. The overexpression of pyrroloquinolinequinone （PQQ）, a cofactor of SDH and SNDH, has increased the yield of 2-KLG up to 44.5 g/L after 84 h. These results indicated the significant progress in vitamin C industry and show the possibility of development of one-step fermentation for 2-KLG production in future.

关 键 词：维生素C 山梨糖脱氢酶 山梨酮脱氢酶 吡咯喹啉醌 

分 类 号：TQ920.6[轻工技术与工程—发酵工程] Q78[生物学—分子生物学]