Abstract：
2\'-Fucosyllactose (2\'-FL), one of the major human milk oligosaccharides, was produced in several engineered microorganisms. However, the low solubility of α-1,2-fucosyltransferase (α1,2-FucT) often becomes a bottleneck to produce maximum amount of 2\'-FL in the microorganisms. To overcome this solubility issue, the following studies were conducted to improve the soluble expression of α1,2-FucT. Initially, hydrophobic amino acids in the hydrophilic region of the 6 α-helices were mutated, adhering to the α-helix rule. Subsequently, gfp11 was fused to the C-terminal of futC gene encoding α1,2-FucT (FutC), enabling selection of high-fluorescence mutants through split-GFP. Each mutant library was screened via fluorescence activated cell sorting (FACS) to separate soluble mutants for high-throughput screening. As a result, L80C single mutant and A121D/P124A/L125R triple mutant were found, and a combined quadruple mutant was created. Furthermore, we combined mutations of conserved sequences (Q150H/C151R/Q239S) of FutC, which showed positive effects in the previous studies from our lab, with the above quadruple mutants (L80C/A121D/P124A/L125R). The resulting strain produced approximately 3.4-fold higher 2\'-FL titer than that of the wild-type, suggesting that the conserved sequence mutations are an independent subset of the mutations that further improve the solubility of the target protein acquired by random mutagenesis using split-GFP.
摘要：
2'-岩藻糖基乳糖（2'-FL），一种主要的人乳寡糖，是在几种工程微生物中产生的。然而,α-1,2-岩藻糖基转移酶（α1,2-FucT）的低溶解度通常成为微生物中产生最大量2\'-FL的瓶颈。为了克服这个溶解度问题，进行以下研究以改善α1,2-FucT的可溶性表达。最初，6个α-螺旋亲水区的疏水氨基酸发生突变，遵守α-螺旋规则。随后，gfp11与编码α1,2-FucT(FutC)的futC基因的C端融合,能够通过分裂GFP选择高荧光突变体。通过荧光激活细胞分选(FACS)筛选每个突变体文库以分离可溶性突变体用于高通量筛选。因此,发现L80C单突变体和A121D/P124A/L125R三突变体,并创建了一个组合的四重突变体。此外，我们结合了FutC的保守序列（Q150H/C151R/Q239S）的突变，在我们实验室的先前研究中显示出积极的效果，具有上述四重突变体(L80C/A121D/P124A/L125R)。所得菌株产生的2'-FL滴度比野生型高大约3.4倍，这表明保守序列突变是突变的独立子集，可进一步提高通过使用分裂GFP进行随机诱变获得的靶蛋白的溶解度。
