果糖软骨素是酶法合成硫酸软骨素的关键前体物质。目前果糖软骨素的生物合成面临诸多挑战,包括合成效率低、提取工艺复杂以及生产过程中对抗生素的依赖等问题,严重限制了其规模化生产和应用。为了解决上述问题,该研究以可天然合成与转运果糖软骨素的大肠杆菌K4作为底盘细胞,构建了一个无抗性基因质粒表达载体pMUT2 ΔHyp3,该质粒在无抗生素压力下可维持9 d的遗传稳定性。通过重组过表达谷氨酸棒状杆菌来源的尿苷二磷酸(uridine diphosphate,UDP)-葡萄糖脱氢酶编码基因ugdA2与枯草芽孢杆菌来源的UDP-N-乙酰氨基葡萄糖焦磷酸化酶编码基因glmU强化前体合成,果糖软骨素摇瓶产量提升至0.95 g/L。进一步优化发酵培养基组分,产量达到1.35 g/L。最终在3 L发酵罐中无抗生素发酵,48 h产量可达到7.5 g/L,收率达4.4%(质量分数)。该研究不仅实现了果糖软骨素的合成,解决了重组大肠杆菌K4发酵抗生素依赖的问题,也为硫酸软骨素的绿色酶法制造提供了规模化生产的基础。
Fructosylated chondroitin serves as a crucial precursor for the enzymatic synthesis of chondroitin sulfate (CS).However, its current biosynthesis faces significant challenges, such as low synthesis efficiency, complex extraction processes, and reliance on antibiotics during production, which hinder its large-scale production and application.To overcome these limitations, Escherichia coli K4, a strain capable of naturally synthesizing and transporting fructosylated chondroitin, was employed as the chassis cell.A plasmid expression vector, pMUT2 ΔHyp3, was developed, demonstrating genetic stability for up to 9 days without antibiotic pressure.By recombinantly overexpressing the UDP-glucose dehydrogenase gene (ugdA2) from Corynebacterium glutamicum and the UDP-N-acetylglucosamine pyrophosphorylase gene (glmU) from Bacillus subtilis, the precursor synthesis pathway was enhanced, increasing fructosylated chondroitin production in shake-flask cultures to 0.95 g/L.Further optimization of the fermentation medium components elevated the yield to 1.35 g/L.Finally, antibiotic-free fermentation in a 3 L bioreactor achieved a production level of 7.5 g/L within 48 hours, with a yield of 4.4%.This study not only achieved the biosynthesis of fructosylated chondroitin and eliminated antibiotic dependency in recombinant E.coli K4 fermentation systems but also laid the groundwork for scalable, environmentally friendly enzymatic production of chondroitin sulfate.
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