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.
Feifei LI Shuxia JIN Li ZHU Xiaobei ZHAN Yue ZHAO Liping LIU Minjie GAO
. Direct production of curdlan oligosaccharides by coupled fermentation system of Agrobacterium sp.-Pichia pastoris[J]. The Chinese Journal of Process Engineering, 2019
, 19(4)
: 801
-808
.
DOI: 10.12034/j.issn.1009-606X.218319
[1] 张洪涛，詹晓北，郑志永，等. 热凝胶的高产策略及功能研究进展 [J]. 生物加工过程, 2009, 7(5):8-12.
Zhang HT, Zhan XB, Zheng ZY. et al. Advances in curdlan of production by Alcaligenes faecalis and function of curdlan [J]. Chinese Journal of Bioprocess Engineering, 2009, 7(5):8-12.
[2] Jamois F, Ferrieres V, Guegan JP, et al. Glucan-like synthetic oligosaccharides: iterative synthesis of linear oligo-beta-(1,3)-glucans and immunostimulatory effects [J]. Glycobiology, 2005, 15(4):393-407.
[3] Miyanishi N, Iwamoto Y, Watanabe E, et al. Induction of TNF-alpha production from human peripheral blood monocytes with beta-1,3-glucan oligomer prepared from laminarin with beta-1,3-glucanase from Bacillus clausii NM-1 [J]. J Biosci Bioeng, 2003, 95(2):192-195.
[4] Fu YB, Yin H, Wang WX, Wang MY, et al. beta-1,3-Glucan with different degree of polymerization induced different defense responses in tobacco [J]. Carbohydr Polym, 2011, 86(2):774-782.
[5] 李晶. 热凝胶制备β-1,3-葡聚寡糖及其诱导抗真菌功能的研究 [D]. 江南大学; 2014.
[6] 徐敏，李晶，郑志永，等. 哈茨木霉产水解热凝胶的内切β-1,3-葡聚糖酶的分离纯化 [J]. 食品工业科技, 2014, 35(12):157-161.
Xu M, Li J, Zheng ZY, et al. Purification of an endo-β-1，3-glucanase to degrade curdlan from Trichoderma harzianum [J]. Science and Technology of Food Industry, 2014, 35(12):157-161.
[7] Liang Y, Zhu L, Gao M, et al. Effective production of biologically active water-soluble beta-1,3-glucan by a coupled system of Agrobacterium sp. and Trichoderma harzianum [J]. Prep Biochem Biotechnol, 2018, 48(5):446-456.
[8] 李珊. 哈茨木霉发酵生产内切β-1,3-葡聚糖酶的工艺研究 [D]. 江南大学; 2014.
[9] Zhang H, Wang X. Modular co-culture engineering, a new approach for metabolic engineering [J]. Metab Eng, 2016, 37:114-121.
[10] Zhou K, Qiao K, Edgar S, et al. Distributing a metabolic pathway among a microbial consortium enhances production of natural products [J]. Nat Biotechnol, 2015, 33(4):377-383.
[11] J.Minty J, Singer ME, A.Scholz S, et al. Design and characterization of synthetic fungal-bacterial consortia for direct production of isobutanol from cellulosic biomass [J]. Proc Natl Acad Sci USA, 2013, 110(36):14592-14597.
[12] Saini M, Chen MH, Chung-Jen C, et al. Potential production platform of n-butanol in Escherichia coli [J]. Metab Eng, 2015, 27:76-82.
[13] Saini M, Chiang CJ, Li SY, et al. Production of biobutanol from cellulose hydrolysate by the Escherichia coli co-culture system [J]. FEMS Microbiol Lett, 2016, 363(4):fnw008.
[14] Zhang H, Pereira B, Li Z, Stephanopoulos G. Engineering Escherichia coli coculture systems for the production of biochemical products [J]. Proc Natl Acad Sci USA, 2015, 112(27):8266-8271.
[15] Zhang H, Stephanopoulos G. Co-culture engineering for microbial biosynthesis of 3-amino-benzoic acid in Escherichia coli [J]. Biotechnol. J., 2016, 11(7):981-987.
[16] Wang EX, Ding MZ, Ma Q, et al. Reorganization of a synthetic microbial consortium for one-step vitamin C fermentation [J]. Microb Cell Fact, 2016, 15:21.
[17] Daly R, Hearn MT. Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production [J]. J Mol Recognit, 2005, 18(2):119-138.
[18] Ahmad M, Hirz M, Pichler H, et al. Protein in Pichia pastoris: recent achievements and perspectives for heterologous protein production [J]. Appl Microbiol Biotechnol, 2014, 98(12):5301-5317.
[19] 郭美锦，朱泰承，张明，等. 重组毕赤酵母甲醇利用表型与基因拷贝数对外源基因表达的影响 [J]. 中国生物工程杂志, 2007, 27(7),7-11.
Guo MJ, Zhu TC, Zhang M, et al. Influences of Methanol Utilization Phenotype and Gene Dosage on Heterologous Protein Expression in Recombinant Pichia pastoris [J]. Journal of Chinese Biotechnology, 2007, 27(7),7-11.
[20] Ellis SB, Brust PF, Koutz PJ, et al. Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris [J]. Mol Cell Biol, 1985, 5(5):1111-1121.
[21] Waterham HR, Digan ME, Koutz PJ, et al. Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene, 1997, 186(1):37-44.
[22] Shen SG, Sulter G, Jeffries TW, et al. A strong nitrogen source-regulated promoter for controlled of foreign genes in the yeast Pichia pastoris [J]. Gene, 1998, 216(1):93-102.
