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-?-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-?-1,3-glucanase can hydrolyze curdlan to curdlan oligosaccharides. To improve the production efficiency of curdlan oligosaccharides, a coupled fermentation system of Agrobacterium sp.?Pichia pastoris is constructed, in which the Agrobacterium sp. metabolite curdlan can be directly used to produce curdlan oligosaccharides by endo-β-1,3-glucanase (secreted by Pichia pastoris). 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 Pichia pastoris GS115 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 reached 51.24, 49.64, and 46.99 U/mL, respectively. On this basis, the Pichia pastoris engineered 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-?-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 Pichia pastoris was 1:2, and fermentation time in shaker flasks was 79 h, respectively, the maximum production of curdlan oligosaccharides (degree of polymerization between 17 and 22) reached 4.278 g/L.
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