该研究通过同源重组方法构建了exoK基因缺失菌株At-ΔexoK,分析At-ΔexoK菌株可得然胶产率和凝胶性质。以农杆菌(Agrobacterium sp.) ATCC31749的exoK基因缺失菌株At-ΔexoK为出发菌株,通过高碳源筛选,获得一株耐高糖发酵菌株LQC22G。采用单因素、Plackett-Burman、最陡爬坡和响应面试验对发酵培养基组成进行了优化。结果显示,At-ΔexoK菌株可得然胶产率为(32.6±1.8) g/L,比野生菌株降低了10.4%;可得然胶凝胶强度为938.7 g/L,相对于野生型菌株增加了17.6%,分子质量为4.04×106 Da,分子质量增加了53%。通过响应面实验优化得发酵培养基为:蔗糖122.0 g/L、磷酸氢二铵5.8 g/L、碳酸钙1.0 g/L、磷酸二氢钾4.7 g/L、玉米浆粉1.0 g/L、硫酸镁2.0 g/L,在此条件下可得然胶产率达(83.17±0.25) g/L,比培养基优化前产率提高了27.2%,比采用野生菌株基础培养基发酵产率更是提高了155%,显著提高了可得然胶的发酵生产效率,为后续在工业化生产中应用提供了良好的基础。
As the application market for curdlan continues to expand, the demand for its yield and properties is gradually increasing.In order to address the insufficient gel strength and yield of curdlan at current stage, this study employed homologous recombination to construct an Agrobacterium sp.ATCC31749 exoK gene knockout strain (At-ΔexoK), and analyzed the yield and gel properties of curdlan produced by the engineered strain.Starting with the engineered strain, a high-sugar-tolerant strain, LQC22G, was further obtained through high-concentration carbon source screening.The composition of the fermentation medium was optimized using various methods including single-factor, Plackett-Burman, steepest ascent, and response surface.The results revealed that the curdlan yield of At-ΔexoK was (32.6±1.8) g/L, which was 10.4% lower than that of wild-type strain.The gel strength of curdlan produced by At-ΔexoK was 938.7 g/L, representing a 17.6% increase over the wild-type strain.The molecular weight of curdlan produced by At-ΔexoK was (4.04 × 106) Da, indicating a 53% increase in molecular weight.The fermentation medium optimized through response surface experiments was determined as follows:sucrose 122.0 g/L, (NH4)2HPO4 5.8 g/L, CaCO31.0 g/L, KH2PO4 4.7 g/L, corn syrup 1.0 g/L, MgSO4 2.0 g/L.With this medium, the curdlan yield reached (83.17 ± 0.25) g/L, which indicated a 27.2% increase over the unoptimized medium and a 155% increase over the yield obtained with the wild-type strain in the unoptimized medium.This work enhances the production efficiency of curdlan significantly and provides a solid foundation for its industrial application in the future.
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