Enhancing the soluble expression of α-1,2-fucosyltransferase in E. coli using high-throughput flow cytometry screening coupled with a split-GFP

• Published in: Journal of biotechnology 2024-05, Vol.387, p.49-57
• Main Authors: Lee, Jun-Min, Kim, Jung Hwa, Kim, Jin Young, Oh, Min-Kyu, Kim, Byung-Gee
• Format: Article
• Language: English
• Subjects: 2’-Fucosyllactose; FACS; Human milk oligosaccharides; split-GFP; α-1,2-fucosyltransferase; α-helix rule
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2024.03.014

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. •This study aimed to increase the production of 2′-FL by enhancing the solubility of α1,2-fucT.•The alpha helix rule and hydropathy contradiction rule were applied to make various mutants.•Increased solubility of the enzyme was screened through split-GFP using FACS.•The α1,2-fucT with improved solubility and higher production capability was selected.