由于几丁寡糖难以分离单分子,从而难以对同一聚合度的壳寡糖的功能活性进行研究,因此如何大规模的获得同一聚合度的寡糖是几丁二糖的功能活性研究以及大规模普及应用的关键。该研究旨在利用价格较为低廉的N-乙酰氨基葡萄糖作为底物,ATP作为辅因子参与,在聚磷酸化酶(polyphosphate kinase, PPK)、N-乙酰己糖胺激酶(N-acetylhexosamine 1-kinase, Nahk)和N,N′-二乙酰壳二糖磷酸化酶(N,N′-diacetylchitobiose phosphorylase, Chbp)的三菌耦合全细胞催化下合成几丁二糖。通过三菌耦合策略得到几丁二糖的产量为1 675 mg/L,后通过单因素优化,得到几丁二糖的最高产量为5 214 mg/L,提高了211%。为了进一步降低成本和简化工艺,构建了几丁二糖生产菌株并通过不同拷贝数的质粒组合平衡途径基因的表达,采用了工程菌Escherichia coli BL21(DE3)(含有质粒pRSFDuet1-Nahk-Chbp、pETDuet1-PPK),达到几丁二糖的产量为1 600 mg/L。该研究为几丁二糖的大规模生物制造提供借鉴与参考。
Owing to the difficulty in isolating single molecules of chitosan oligosaccharides, it becomes challenging to study the functional activities of chitobiose with the same degree of polymerization.Hence, how to obtain oligosaccharides with the same degree of polymerization on a large scale is crucial for the research on the functional activities of chitobiose and its wide-scale popularization and application.The research of this subject aims to utilize relatively inexpensive N-acetylglucosamine as the substrate, with ATP as the cofactor, and synthesize chitobiose under the triculture-coupled whole-cell catalysis of polyphosphorylase (PPK), N-acetylhexosamine kinase (Nahk), and N,N′-diacetylated chitobiose phosphorylase (Chbp).The yield of chitobiose was 1 675 mg/L by three-bacteria coupling.After single-factor optimization, the maximum yield of chitobiose was 5 214 mg/L, an increase of 211%.To further reduce costs and simplify the process, chitosan disaccharide production strains were constructed and the expression of pathway genes was balanced through the combination of plasmids with different copy numbers.The engineered strain Escherichia coli BL21(DE3) (containing plasmids pRSFDuet1-Nahk-Chbp and pETDuet1-PPK) was adopted, achieving a chitosan disaccharide yield of 1 600 mg/L.This study provides references and guidance for the large-scale biological manufacturing of chitobiose.
[1] WALTER A, FRIZ S, MAYER C.Chitin, chitin oligosaccharide, and chitin disaccharide metabolism of Escherichia coli revisited:Reassignment of the roles of ChiA, ChbR, ChbF, and ChbG[J].Microbial Physiology, 2021, 31(2):178-194.
[2] MUKHERJEE S, BEHERA P K, MADHUPRAKASH J.Efficient conversion of crystalline chitin to N-acetylglucosamine and N, N′-diacetylchitobiose by the enzyme cocktail produced by Paenibacillus sp.LS1[J].Carbohydrate Polymers, 2020, 250:116889.
[3] JI X G, ZHU L L, CHANG K L, et al.Chitooligosaccahrides:Digestion characterization and effect of the degree of polymerization on gut microorganisms to manage the metabolome functional diversity in vitro[J].Carbohydrate Polymers, 2022, 275:118716.
[4] QIN X, XIN Y Z, SU X Y, et al.Heterologous expression and characterization of thermostable chitinase and β-N-acetylhexosaminidase from Caldicellulosiruptor acetigenus and their synergistic action on the bioconversion of chitin into N-acetyl-d[J].International Journal of Biological Macromolecules, 2021, 192:250-257.
[5] LIAQAT F, ELTEM R.Chitooligosaccharides and their biological activities:A comprehensive review[J].Carbohydrate Polymers, 2018, 184:243-259.
[6] JIA L G, QI W, WANG K N, et al.Efficient bioconversion of chitinous waste to N-acetylchitobiose and N-acetylglucosamine using a novel salt-tolerant chitinase from Bacillus clausii[J].ACS Sustainable Chemistry & Engineering, 2023, 11(31):11470-11481.
[7] ZHANG J, FENG M, LU X M, et al.Base-free preparation of low molecular weight chitin from crab shell[J].Carbohydrate Polymers, 2018, 190:148-155.
[8] HONG S, YANG Q R, YUAN Y, et al.Sustainable co-solvent induced one step extraction of low molecular weight chitin with high purity from raw lobster shell[J].Carbohydrate Polymers, 2019, 205:236-243.
[9] ARNOLD N D, BRÜCK W M, GARBE D, et al.Enzymatic modification of native chitin and conversion to specialty chemical products[J].Marine Drugs, 2020, 18(2):93.
[10] BEIER S, BERTILSSON S.Bacterial chitin degradation-mechanisms and ecophysiological strategies[J].Frontiers in Microbiology, 2013, 4:149.
[11] JIAN X, LI C, FENG X D.Strategies for modulating transglycosylation activity, substrate specificity, and product polymerization degree of engineered transglycosylases[J].Critical Reviews in Biotechnology, 2023, 43(8):1284-1298.
[12] HULLINGER A C, GREEN V E, KLANCHER C A, et al.Two transmembrane transcriptional regulators coordinate to activate chitin-induced natural transformation in Vibrio cholerae[J].bioRxiv, 2025:2024.09.30.615920.
[13] ZHOU X L, HUANG Y, LIU Y Y, et al.Efficient production of N-acetyl-β-D-glucosamine from shrimp shell powder using chitinolytic enzyme cocktail with β-N-acetylglucosaminidase from domesticated microbiome metagenomes[J].Fermentation, 2024, 10(12):652.
[14] ITOH T, YAGUCHI M, NAKAICHI A, et al.Structural characterization of two solute-binding proteins for N, N′-diacetylchitobiose/N, N′, N″-triacetylchitotoriose of the gram-positive bacterium, Paenibacillus sp.str.FPU-7[J].Journal of Structural Biology:X, 2021, 5:100049.
[15] PARK J K, KEYHANI N O, ROSEMAN S.Chitin catabolism in the marine bacterium Vibrio furnissii[J].Journal of Biological Chemistry, 2000, 275(42):33077-33083.
[16] ITOH T, KIMOTO H.Bacterial chitinase system as a model of chitin biodegradation[M].Targeting Chitin-containing Organisms.Singapore:Springer Singapore, 2019:131-151.
[17] HONDA Y, KITAOKA M, HAYASHI K.Reaction mechanism of chitobiose phosphorylase from Vibrio proteolyticus:Identification of family 36 glycosyltransferase in Vibrio[J].The Biochemical Journal, 2004, 377(Pt 1):225-232.
[18] GUAN J, JAKOB U.The protein scaffolding functions of polyphosphate[J].Journal of Molecular Biology, 2024, 436(14):168504.
[19] CHAROENPOL A, CRESPY D, SCHULTE A, et al.Immobilized chitinase as effective biocatalytic platform for producing bioactive di-N-acetyl chitobiose from recycled chitin food waste[J].Bioresource Technology, 2024, 406:130945.
[20] JUNG W J, PARK R D.Bioproduction of chitooligosaccharides:Present and perspectives[J].Marine Drugs, 2014, 12(11):5328-5356.
[21] SONG J, TAKE L, HYUN L, et al.Highly efficient production of N, N′-diacetylchitobiose according to substrate modification and changes in enzyme kinetics[J].Process Biochemistry, 2023, 133:179-189.
