土壤杆菌-毕赤酵母耦合培养直接生产热凝胶低聚糖

Direct production of curdlan oligosaccharides by coupled fermentation system of Agrobacterium sp.-Pichiapastoris

为提高热凝胶低聚糖生产效率,构建了土壤杆菌-毕赤酵母耦合培养体系,其中土壤杆菌代谢产物热凝胶可被毕赤酵母分泌的内切-β-1,3-葡聚糖酶利用直接生产热凝胶低聚糖。用基于不同启动子(AOX1,GAP,FLD)调控的毕赤酵母重组菌株分泌表达内切-β-1,3-葡聚糖酶BGN13.1a,验证其均能有效水解热凝胶得到热凝胶低聚糖。在此基础上,选取GAP启动子调控的毕赤酵母工程菌与土壤杆菌耦合培养,通过设计两种物种之间的共生关系实现稳定共培养并生产聚合度为17~22的热凝胶低聚糖,产量为4.278 g/L。

Curdlan oligosaccharides are widely used in biomedicine and food.Agrobacterium sp.ATCC 31749 can produce curdlan under nitrogen-deficient conditions,and endo-β-1,3-glucanasecan hydrolyzecurdlan to curdlanoligosaccharides.To improve the production efficiency of curdlanoligosaccharides,a coupled fermentation system of Agrobacterium sp.-Pichiapastorisis constructed,in which the Agrobacterium sp.metabolite curdlan can be directly used to produce curdlan oligosaccharides by endo-β-1,3-glucanase(secreted by Pichiapastoris).This microbial consortium omits the steps of extraction,purification,and drying of curdlan and endo-β-1,3-glucanase,and it has considerable potential for use in the industrial production of curdlan oligosaccharides.The commercially available β-1,3-glucanase is a complex enzyme(it contains a variety of exoglucanases and endoglucanases),and the specific endoglucanase is difficult to obtain.In is work,to avoid the effects of exo-β-1,3-glucanase,the endo-β-1,3-glucanase(BGN13.1)was expressed in PichiapastorisGS115 by different promoters(AOX1,GAP and FLD),and they were all verified to be effective in hydrolyzing curdlan to curdlan oligosaccharides;the endo-β-1,3-glucanase enzyme activities reached51.24,49.64,and 46.99 U/mL,respectively.On this basis,the Pichiapastorisengineered by the GAP promoter was selected for co-culture with Agrobacterium sp.to avoid the methanol-induced process,and soybean sprout extract was the optimal source of nitrogen for both curdlan and endo-β-1,3-glucanase production.In the end,the coupled fermentation system was divided into two stages to produce the best culture effect:Agrobacterium sp.cell growth stage(pH=7.0),and curdlan oligosaccharides production stage(pH=5.6).Potassium phosphate buffer was added to the medium to keep the pH of the fermentation broth within a constant range.When the initial inoculation ratio of Agrobacterium sp.to Pichiapastoriswas 1:2,and fermentation time in shaker flaskswas 79 h,respectively,the maximum production of curdlan oligosaccharides(degree of polymerization between 17 and 22)reached 4.278 g/L.