高产柠檬酸工业Aspergillus niger菌株转化体系构建及衣康酸发酵

Establishment of transformation system of an industrial Aspergillus niger strain with high citric-acid productivity and itaconic acid fermentation

柠檬酸高产菌株Aspergillus niger YX-1217无法成功建立遗传转化体系,极大地限制了其代谢机制和分子机制的研究。对该菌株进行遗传改造,最终建立了一种农杆菌(Agrobacterium tumefacien)介导的遗传转化体系:农杆菌AGL-1作为介导菌株、A.niger YX-1217孢子密度为1×107m L-1、孢子与农杆菌的体积比为1∶9、乙酰丁香酮浓度为300μmol/L。在最佳转化体系下获得25±1个转化子(以107个分生孢子计)。基于上述遗传转化技术,对A.niger YX-1217产柠檬酸的应用进行拓展,通过引入乌头酸脱羧酶cad A基因构建衣康酸的合成途径,基于搅拌转速三阶段控制利用补料分批发酵生产衣康酸,质量浓度最高达到4. 21 g/L,最大产率为0. 040 g/(L·h),从而延伸原有柠檬酸的代谢通路来实现衣康酸的生产。

Compared with other filamentous fungi,the genetic transformation of Aspergillus niger YX-1217 with high citric-acid productivity is imperfect,which greatly limits the molecular mechanism and the metabonomics development of A.niger YX-1217.The optimum genetic transformation of an industrial A.niger YX-1217 strain mediated by Agrobacterium tumefacien was determined with A.tumefaciens AGL-1 as the mediated strains,the initial A.niger YX-1217 spores concentration of 10 7 mL-1 suspension,acetosyringone(AS)concentration of 300μmol/L,and the volume ratio of the initial A.niger YX-1217 spores to A.tumefaciens of 1∶9.Under such optimal transformation conditions,the transformation frequency was 25 transformants per plate using 10 7 conidia.Based on the genetic transformation,the application of A.niger YX-1217 as a high citric-acid production was expanded.The key gene cadA encoding conitate decarboxylase was overexpressed in the A.niger YX-1217 to increase itaconic acid production.Based on fed-batch fermentation,with three stage control of agitation speed,the highest itaconic acid production could reach 4.21 g/L with a productivity of 0.040 g/(L·h).Results showed the remarkable potential of A.niger YX-1217 to improve itaconic acid production.