Studying the function of methyltransferases using nucleotide analogue based random mutagenesis

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient Escherichia coli host strain and report an HTS approach based on colorimetric detection of H2O2-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H2O2 as co-substrate.

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Faculty Opinions recommendation of Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.737201448.793569686 ◽

2020 ◽

Author(s):

Pinghua Liu

Keyword(s):

Random Mutagenesis ◽

Plant Genes ◽

Adenine Base

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Combining random mutagenesis, structure-guided design and next-generation sequencing to mitigate polyreactivity of an anti-IL-21R antibody

mAbs ◽

10.1080/19420862.2021.1883239 ◽

2021 ◽

Vol 13 (1) ◽

pp. 1883239

Author(s):

Sharon M. Campbell ◽

Joseph DeBartolo ◽

James R. Apgar ◽

Lydia Mosyak ◽

Virginie McManus ◽

...

Keyword(s):

Next Generation Sequencing ◽

Random Mutagenesis ◽

Next Generation ◽

Generation Sequencing

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Random mutagenesis to identify novelHelicobacter mustelaevirulence factors

FEMS Immunology & Medical Microbiology ◽

10.1111/j.1574-695x.2006.00207.x ◽

2007 ◽

Vol 50 (2) ◽

pp. 257-263 ◽

Cited By ~ 8

Author(s):

Tadhg Ó Cróinín ◽

Aisling McCormack ◽

Arnoud H.M. van Vliet ◽

Johannes G. Kusters ◽

Billy Bourke

Keyword(s):

Random Mutagenesis

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Evolutionary design of optimal surface topographies for biomaterials

Scientific Reports ◽

10.1038/s41598-020-78777-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Aliaksei Vasilevich ◽

Aurélie Carlier ◽

David A. Winkler ◽

Shantanu Singh ◽

Jan de Boer

Keyword(s):

High Throughput Screening ◽

Random Mutagenesis ◽

Material Design ◽

Fitness Assessment ◽

Surface Topographies ◽

Optimal Surface ◽

Design Production ◽

Brute Force Approach ◽

Survival Of The Fittest ◽

Selection Of

AbstractNatural evolution tackles optimization by producing many genetic variants and exposing these variants to selective pressure, resulting in the survival of the fittest. We use high throughput screening of large libraries of materials with differing surface topographies to probe the interactions of implantable device coatings with cells and tissues. However, the vast size of possible parameter design space precludes a brute force approach to screening all topographical possibilities. Here, we took inspiration from Nature to optimize materials surface topographies using evolutionary algorithms. We show that successive cycles of material design, production, fitness assessment, selection, and mutation results in optimization of biomaterials designs. Starting from a small selection of topographically designed surfaces that upregulate expression of an osteogenic marker, we used genetic crossover and random mutagenesis to generate new generations of topographies.

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Enhanced production of Clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain

Journal of Industrial Microbiology & Biotechnology ◽

10.1093/jimb/kuab004 ◽

2021 ◽

Author(s):

Chang-Hun Shin ◽

Hang Soo Cho ◽

Hyung-Jin Won ◽

Ho Jeong Kwon ◽

Chan-Wha Kim ◽

...

Keyword(s):

Clavulanic Acid ◽

Wild Type Strain ◽

Random Mutagenesis ◽

Streptomyces Clavuligerus ◽

Wild Type ◽

Mutant Selection ◽

Enhanced Production ◽

Industrial Strains ◽

Glycerol Utilization

Abstract Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3 per cent (5.21 ± 0.26 g/L) in a flask culture and 17.4 per cent (6.11 ± 0.36 g/L) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

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