Shao Jun, Hayashi Takahisa, Wang Peng George
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Appl Environ Microbiol. 2003 Sep;69(9):5238-42. doi: 10.1128/AEM.69.9.5238-5242.2003.
A metabolically engineered Pichia pastoris strain was constructed that harbored three heterologous enzymes: an S11E mutated sucrose synthase from Vigna radiata, a truncated UDP-glucose C4 epimerase from Saccharomyces cerevisiae, and a truncated bovine alpha-1,3-galactosyltransferase. Each gene has its own methanol-inducible alcohol oxidase 1 promoter and transcription terminator on the chromosomal DNA of P. pastoris strain GS115. The proteins were coexpressed intracellularly under the induction of methanol. After permeabilization, the whole P. pastoris cells were used to synthesize alpha-galactosyl (alpha-Gal) trisaccharide (Galalpha1,3Galbeta1,4Glc) with in situ regeneration of UDP-galactose. Up to 28 mM alpha-Gal was accumulated in a 200-ml reaction. The Pichia system described here is simple and flexible. This work demonstrates that recombinant P. pastoris is an excellent alternative to Escherichia coli transformants in large-scale synthesis of oligosaccharides.
构建了一种代谢工程改造的毕赤酵母菌株，该菌株含有三种异源酶：来自绿豆的S11E突变型蔗糖合酶、来自酿酒酵母的截短型UDP-葡萄糖C4差向异构酶和截短型牛α-1,3-半乳糖基转移酶。每个基因在毕赤酵母菌株GS115的染色体DNA上都有其自身的甲醇诱导型醇氧化酶1启动子和转录终止子。这些蛋白质在甲醇诱导下在细胞内共表达。通透化处理后，完整的毕赤酵母细胞用于合成α-半乳糖基（α-Gal）三糖（Galα1,3Galβ1,4Glc）并原位再生UDP-半乳糖。在200毫升反应中积累了高达28 mM的α-Gal。这里描述的毕赤酵母系统简单且灵活。这项工作表明，重组毕赤酵母在大规模合成寡糖方面是大肠杆菌转化体的极佳替代品。
