酿酒酵母转化半乳糖醇的工程菌株构建及优化

Construction and culture condition optimization of a Saccharomyces cerevisiae strain for production of galactitol

摘要:

半乳糖醇是一种稀少糖醇，广泛应用于食品工业与医药领域。工业上一般通过化学氢化法生产半乳糖醇，但反应条件苛刻、生产成本高、对环境不友好，亟须开发更有效的“绿色”生物合成技术。本研究挖掘了来源于黑曲霉(Aspergillus niger) CBS 513.88的木糖还原酶(Aspergillus niger xylose reductase, AnXR)，该酶属于NADPH依赖的醛酮还原酶家族。对AnXR的酶学性质进行研究，发现其最适温度和pH分别为25 ℃和8.0。以酿酒酵母(Saccharomyces cerevisiae)为底盘细胞，利用基因重组技术敲除半乳糖激酶(galactokinase, GAL1)基因，使得宿主细胞对半乳糖的分解代谢利用大幅降低，并在此底盘菌株中引入AnXR基因构建了催化d-半乳糖生成半乳糖醇的工程菌株。对工程菌株全细胞转化的条件进行优化，半乳糖醇的最高产量达到12.10 g/L。同时，该菌株对其他单糖的还原能力也进行了测试，可生成木糖醇、阿拉伯糖醇等功能性糖醇。利用本研究构建的工程菌株可以实现半乳糖醇等多种功能糖醇的高效生物转化，对稀少糖醇的绿色制造具有借鉴意义。

Abstract:

Galactitol, a rare sugar alcohol, has promising potential in the food industry and pharmaceutical field. The available industrial production methods rely on harsh hydrogenation processes, which incur high costs and environmental concerns. It is urgent to develop environmentally friendly and efficient biosynthesis technologies. In this study, a xylose reductase named AnXR derived from Aspergillus niger CBS 513.88 was identified and characterized for the enzymatic properties. AnXR exhibited the highest activity at 25 ℃ and pH 8.0, and it belonged to the NADPH-dependent aldose reductase family. To engineer a strain for galactitol production, we deleted the galactokinase (GAL1) gene in Saccharomyes cerevisiae by using the recombinant gene technology, which significantly reduced the metabolic utilization of d-galactose by host cells. Subsequently, we introduced the gene encoding AnXR into this modified strain, creating an engineered strain capable of catalyzing the conversion of d-galactose into galactitol. Furthermore, we optimized the whole-cell catalysis conditions for the engineered strain, which achieved a maximum galactitol yield of 12.10 g/L. Finally, we tested the reduction ability of the strain for other monosaccharides and discovered that it could produce functional sugar alcohols such as xylitol and arabinitol. The engineered strain demonstrates efficient biotransformation capabilities for galactitol and other functional sugar alcohols, representing a significant advancement in environmentally sustainable production practices.