β-葡聚糖是存在于酵母细胞壁中的一种多糖,具有抗炎抗氧化等重要的作用。为提高酿酒酵母中β-葡聚糖的产量,该研究采用代谢工程中常用的“推-拉-抑制”策略。首先,通过在酿酒酵母中过表达β-葡聚糖合成途径关键酶及其调节亚基“拉”动关键前体二磷酸尿苷(uridine diphosphate,UDP)-葡萄糖流向β-葡聚糖合成途径;之后通过强化前体UDP-葡萄糖途径酶,将代谢通量“推”向前体UDP-葡萄糖的有效合成;最后抑制蛋白质糖基化等UDP-葡萄糖的其他消耗途径,积累更多的UDP-葡萄糖用于目的产物β-葡聚糖的合成。除此之外,通过引入木糖还原酶基因xyl1,木糖醇脱氢酶基因xyl2和木酮糖激酶基因xyl3,构建了木糖氧化还原途径;进一步通过过表达转运蛋白Hxt7(F79S),敲除snf1基因,提高了对木糖的利用能力,并最终将β-葡聚糖的产量提高至86.09 mg/g。最后,利用半乳糖诱导型启动子Pgal1动态调控内切葡聚糖酶基因(eng1)表达,使酿酒酵母中β-葡聚糖的分子质量从1×106~2×106 g/mol降低至1.26×105 g/mol,实现了低分子质量β-葡聚糖的高效合成,为β-葡聚糖的生产提供了重要参考。
β-glucan is a polysaccharide found in the cell wall of yeast. It has important anti-inflammatory and antioxidant properties. To increase the production of β-glucan in Saccharomyces cerevisiae, this study used a ‘push-pull-inhibit’ strategy, which is commonly used in metabolic engineering. Firstly, we have overexpressed the key enzyme FKS1 and its regulatory subunits Rho1 in in β-glucan synthesis pathway in S. cerevisiae. After increasing their copy numbers, the key precursor UDP-glucose were ‘pulled’ towards the β-glucan synthesis pathway. Secondly, via enhancing the precursor UDP-glucose pathway enzymes, the metabolic flux was ‘pushed’ towards the pathway. Finally, other UDP-glucose consumption pathways such as protein glycosylation were inhibited. More UDP-glucose were accumulated for the synthesis of the target product β-glucan, and the β-glucan titer has improved to 49.07 mg/g. In addition, the xylose metabolic pathway was constructed by introducing xylose reductase gene xyl1, xylitol dehydrogenase gene xyl2 and xylulose kinase gene xyl3. Furthermore, by overexpressing the transporter protein Hxt7 (F79S) and knocking down the gene snf1, the utilization of xylose was improved. The yield of β-glucan on glucose and xylose showed a significant improvement and had increased to 86.09 mg/g. Finally, the galactose-inducible promoter Pgal1 was used to regulate the expression of the endoglucanase gene eng1 dynamically, which reduced the molecular weight of β-glucan in yeast from 1×106-2×106 g/mol to 1.26×105 g/mol. This work achieving the efficient synthesis of low molecular weight β-glucan and provided an important reference for the production of β-glucan.
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