代谢改造熊蜂生假丝酵母提高酸型槐糖脂产量

Metabolic engineering of Starmerella bombicola for enhanced acid-type sophorolipid production

摘要:

[目的] 槐糖脂是一类生物表面活性剂，不仅具有常规表面活性剂所具有的增溶、乳化、润湿、发泡、分散、降低表面张力等通用性能，且对环境的耐受性极强。熊蜂生假丝酵母（Starmerella bombicola）能够发酵生产槐糖脂，但槐糖脂具有酸型、内酯型和乙酰化型等不同类型，结构多样，难以分离。本文拟通过代谢工程改造，构建高产酸型槐糖脂的熊蜂生假丝酵母工程菌株。[方法] 利用潮霉素抗性基因构建了标记基因重复利用系统Rec-six基因编辑系统，在此基础上将合成内酯型槐糖脂的关键基因——内酯酶基因SBLE敲除获得一株只产酸型槐糖脂的工程菌株Δsble，进一步同源过量表达葡萄糖基转移酶基因UGTB并敲除过氧化物酶体膜转运蛋白编码基因PXA1，构建了高产酸型槐糖脂的酵母工程菌。[结果] 与出发菌株相比，重组熊蜂生假丝酵母发酵油酸能够合成单一的酸型槐糖脂，而不再合成内酯型槐糖脂，同时酸型槐糖脂的产量由20 g/L提高到44 g/L，提高了2.1倍。[结论] 通过敲除PXA1、SBLE和过表达UGTB来改造熊蜂生假丝酵母，能够有效提高重组菌的酸型槐糖脂产量，为发酵法生产酸型槐糖脂奠定了基础。

Abstract:

[Objective] Sophorolipids are biosurfactants with properties such as solubilization, emulsifying, wetting, foaming, dispersing, and reducing surface tension of conventional surfactants, and tolerance to the environment. Starmerella bombicola can produce sophorolipids, but the components of sophorolipids are complex and difficult to separate. In order to obtain the single high-yield acid type sophorolipid, the strain S. bombicola producing only acid sophorolipid was constructed by metabolic engineering.[Methods] The Rec-six gene editing system was developed using the hygromycin resistance gene as a selection marker. Based on this system, the critical lactonase gene SBLE for lactone-type sophorolipid was knocked out to obtain an engineered strain producing only acid-type sophorolipid. The expression cassette of the UGTB gene (glucosyltransferase) was further integrated into the genome of the recombinant strain and the PXA1 gene (peroxisome membrane transporter) was knocked out to construct a metabolic process yeast strain with high acid-producing sophorolipids.[Results] Compared with the original strain, the yield of oleic acid synthase was increased from 20 g/L to 44 g/L, and the lactone type of sophorolipid was undetectable.[Conclusion] The recombinant S. bombicola can effectively increase the yield of acid-type sophorolipids by over-expressing UGTB and knocking out PXA1 and SBLE, for enhanced the production of acid-type sophorolipid by fermentation.