Enhancing the soluble expression of α-1,2-fucosyltransferase in E. coli using high-throughput flow cytometry screening coupled with a split-GFP.
J Biotechnol
; 387: 49-57, 2024 May 20.
Article
in En
| MEDLINE
| ID: mdl-38556215
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Green Fluorescent Proteins
/
Escherichia coli
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Flow Cytometry
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Fucosyltransferases
Limits:
Humans
Language:
En
Journal:
J Biotechnol
/
J. biotechnol
/
Journal of biotechnology
Journal subject:
BIOTECNOLOGIA
Year:
2024
Document type:
Article
Country of publication: