高分子质量透明质酸具有保湿和抵抗炎症的生理功能,广泛应用于化妆品和医疗领域。其工业生产主要依赖于动物组织提取或兽疫链球菌发酵,而动物材料来源有限,兽疫链球菌具有致病性,因此迫切需要构建安全高效的高分子质量透明质酸生产菌株。该研究首先在食品级表达宿主谷氨酸棒杆菌中表达多杀性巴斯德杆菌来源透明质酸合酶基因(pmhasA),经过密码子和透明质酸提取方法优化,透明质酸分子质量达到1.51 MDa。其次,通过理性改造透明质酸合酶编码基因(pmhasA T104A),透明质酸分子质量提高至1.8 MDa。进一步通过强化代谢途径中UDP-葡萄糖脱氢酶基因(ugdA),透明质酸分子质量提高至3.3 MDa。在此基础上优化最适培养温度为20 ℃,摇瓶中透明质酸分子质量最高为4.8 MDa。最后在3 L发酵罐进行分批补料发酵,高分子质量透明质酸产量达到1.5 g/L。该研究通过透明质酸合酶改造、代谢途径调控和代谢途径强化实现了高分子质量透明质酸的安全、高效合成,为高分子质量透明质酸的生产奠定了基础。
High-molecular-weight hyaluronic acid has biological functions of moisturizing and anti-inflammatory, and widely used in cosmetics and medical fields. In order to produce high-molecular-weight hyaluronic acid, the Pasteurella multocida pmhasA gene encoding hyaluronic acid synthase was expressed in Corynebacterium glutamicum. The molecular weight of hyaluronic acid was 1.51 MDa by optimized the codon and extraction method, 1.8 MDa by site-directed mutation a HA synthase variant (pmhasA T104A), and 3.3 MDa by strengthen the expression of UDP-glucose dehydrogenase gene (ugdA). On this basis, the highest molecular weight of hyaluronic acid in the shake flask reached to 4.8 MDa at 20 ℃. Finally, fed-batch fermentation was carried out in a 3 L fermenter, and the yield of high-molecular-weight hyaluronic acid reached 1.5 g/L. In conclusion, the safe and efficient synthesis of high-molecular-weight hyaluronic acid was achieved through synthase modification, metabolic pathway enhancement and culture condition optimization, which laid a foundation for the industrial production of high-molecular-weight hyaluronic acid.
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