氨基葡萄糖是一种重要的功能单糖,具有广阔的市场需求。目前,氨基葡萄糖主要的工业化生产方式为酸水解虾壳蟹壳,但该方法会造成环境污染等问题,而单一微生物发酵法生产氨基葡萄糖因受到中间代谢产物的影响导致目标产物无法大量积累。该研究通过构建混菌发酵体系,将氨基葡萄糖的合成分为N-乙酰氨基葡萄糖的高效合成与脱乙酰化2个步骤,实现了混菌发酵过程中的劳动分工。分别强化内源性和外源性的脱乙酰酶基因,进一步优化菌种比例和发酵条件,提高N-乙酰氨基葡萄糖在胞内的转化率。所获得混菌体系摇瓶发酵24 h产生11.21 g/L氨基葡萄糖,较初始对照体系提高了约3.1倍。该策略可以为其他因合成酶受代谢产物抑制而无法大量积累的产物合成提供借鉴。
Glucosamine is an important functional monosaccharide, which has large market demand. At present, the main industrial production method of glucosamine is acid hydrolysis using shells of shrimps and crabs, but this method can cause environmental pollution, while the single-microorganism fermentation method cannot accumulate a large amount of glucosamine due to the influence of intermediate metabolite. In this study, the synthesis of glucosamine was divided into two steps: efficient synthesis of N-acetyl glucosamine and its deacetylation via division of labor of Escherichia coli co-cultures. Endogenous and exogenous deacetylase encoding genes were respectively enhanced and compared, and further optimization of inoculation ratio of two strains and fermentation conditions were conducted to improve the conversion efficiency from N-acetyl glucosamine to glucosamine. The final co-culture system produced 11.21 g/L glucosamine at 24 h in shake flask, which was about 3.1 times higher than the initial control system. This strategy can be used as a reference for the synthesis of other products which cannot be highly accumulated due to the feedback inhibition of key enzymes from intermediate metabolites.
[1] NAGAOKA I, IGARASHI M, SAKAMOTO K. Biological activities of glucosamine and its related substances[J]. Advances in Food and Nutrition Research, 2012,65: 337-352.
[2] KAPOOR M, MINEAU F, FAHMI H, et al. Glucosamine sulfate reduces prostaglandin E2 production in osteoarthritic chondrocytes through inhibition of microsomal PGE synthase-1[J]. The Journal of Rheumatology, 2012, 39(3):635-644.
[3] YADAV V, PANILAITIS B, SHI H, et al.N-acetylglucosamine 6-phosphate deacetylase (nagA) is required for N-acetyl glucosamine assimilation in Gluconacetobacter xylinus[J].PLoS One, 2011, 6(6):e18099.
[4] CHEN J K, SHEN C R, YEH C H, et al.N-acetyl glucosamine obtained from chitin by chitin degrading factors in Chitinbacter tainanesis[J].International Journal of Molecular Sciences, 2011, 12(2):1187-1195.
[5] LIU L, LIU Y F, SHIN H D, et al.Microbial production of glucosamine and N-acetylglucosamine:Advances and perspectives[J].Applied Microbiology and Biotechnology, 2013, 97(14):6149-6158.
[6] DENG M D, SEVERSON D K, GRUND A D, et al.Metabolic engineering of Escherichia coli for industrial production of glucosamine and N-acetylglucosamine[J].Metabolic Engineering, 2005, 7(3):201-214.
[7] 董瑞真, 李丕武, 石凤, 等.glmU基因单功能敲除对大肠杆菌生产氨基葡萄糖的影响[J].中国酿造, 2018, 37(4):23-27.
DONG R Z, LI P W, SHI F, et al.Effect of single function knockout of glmU gene on glucosamine production by Escherichia coli[J].China Brewing, 2018, 37(4):23-27.
[8] 董瑞真. 大肠杆菌代谢途径的改造对氨基葡萄糖积累的影响[D].济南:齐鲁工业大学, 2018.
DONG R Z.Effect of modification of Escherichia coli metabolic pathway on accumulation of glucosamine[D].Jinan:Qilu University of Technology, 2018.
[9] 陈欣, 刘龙, 李江华, 等.Red同源重组敲除nagE和manX对大肠杆菌发酵生产氨基葡萄糖的影响[J].生物工程学报, 2012, 28(3):305-319.
CHEN X, LIU L, LI J H, et al.Influence of nagE and manX knockout with red homologous recombination on the microbial production of glucosamine by Escherichia coli[J].Chinese Journal of Biotechnology, 2012, 28(3):305-319.
[10] 周丁, 王倩, 祁庆生.glmS核糖开关研究进展[J].微生物学报, 2017, 57(8):1152-1159.
ZHOU D, WANG Q, QI Q S.Research progress in glmS riboswitch[J].Acta Microbiologica Sinica, 2017, 57(8):1152-1159.
[11] 徐颖宣, 徐尔尼, 冯乃宪, 等.微生物混菌发酵应用研究进展[J].中国酿造, 2008, 27(9):1-4.
XU Y X, XU E N, FENG N X, et al.Research progress on application of microbial mixed fermentation[J].China Brewing, 2008, 27(9):1-4.
[12] 朱可丽, 张海宏, 孙传宝, 等.维生素C二步发酵混菌接种控制[J].微生物学杂志, 1998, 18(2):13-15.
ZHU K L, ZHANG H H, SUN C B, et al.Studies on inoculation in the two-step-fermentation of vitamin c[J].Journal of Microbiology, 1998, 18(2):13-15.
[13] SHONG J, JIMENEZ DIAZ M R, COLLINS C H.Towards synthetic microbial consortia for bioprocessing[J].Current Opinion in Biotechnology, 2012, 23(5):798-802.
[14] TSAI S L, GOYAL G, CHEN W.Surface display of a functional minicellulosome by intracellular complementation using a synthetic yeast consortium and its application to cellulose hydrolysis and ethanol production[J].Applied and Environmental Microbiology, 2010, 76(22):7514-7520.
[15] GARIMA G, SHEN-LONG T, BHAWNA M, et al.Simultaneous cell growth and ethanol production from cellulose by an engineered yeast consortium displaying a functional mini-cellulosome[J].Microbial Cell Factories, 2011, 10:89.
[16] LI Y F, LIN Z Q, HUANG C, et al.Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing[J].Metabolic Engineering, 2015, 31:13-21.
[17] 孙全伟. 大肠杆菌复合碳源利用进行N-乙酰氨基葡萄糖合成研究[D].天津:天津科技大学, 2021.
SUN Q W.Study on synthesis of N- acetylglucosamine by Escherichia coli combined with carbon source[D].Tianjin:Tianjin University of Science & Technology, 2021.
[18] JIANG Z, LYU X Q, LIU Y F, et al.Biocatalytic production of glucosamine from N-acetylglucosamine by diacetylchitobiose deacetylase[J].Journal of Microbiology and Biotechnology, 2018, 28(11):1850-1858.
[19] LV Y M, LABORDA P, HUANG K, et al.Highly efficient and selective biocatalytic production of glucosamine from chitin[J].Green Chemistry, 2017, 19(2):527-535.
[20] 李康. 大肠杆菌氨基葡萄糖代谢途径的改造[D].济南:齐鲁工业大学, 2020.
LI K.Modification of glucosamine metabolic pathway in Escherichia coli[D].Jinan:Qilu University of Technology, 2020.
[21] CHEN R Z, BAILEY J E.Observations of aerobic, growing Escherichia coli metabolism using an on-line nuclear magnetic resonance spectroscopy system[J].Biotechnology and Bioengineering, 1993, 42(2):215-221.
[22] LEE S W, OH M K.Improved production of N-acetylglucosamine in Saccharomyces cerevisiae by reducing glycolytic flux[J].Biotechnology and Bioengineering, 2016, 113(11):2524-2528.
[23] LI Z J, CAI L, QI Q S, et al.Synthesis of rare sugars with L-fuculose-1-phosphate aldolase (FucA) from Thermus thermophilus HB8[J].Bioorganic & Medicinal Chemistry Letters, 2011, 21(17):5084-5087.
