Abstract
Protein rational design has become more and more popular for protein engineering with the advantage of biological big-data. In this study, we described a method of rational design that is able to identify desired mutants by analyzing the coevolution of protein sequence. We employed this approach to evolve an archaeal isopentenyl phosphate kinase that can convert dimethylallyl alcohol (DMA) into precursor of isoprenoids. By designing 9 point mutations, we improved the catalytic activities of IPK about 8-fold in vitro. After introducing the optimal mutant of IPK into engineered E. coli strain for β-carotenoids production, we found that β-carotenoids production exhibited 97% increase over the starting strain. The process of enzyme optimization presented here could be used to improve the catalytic activities of other enzymes.
Highlights
Protein rational design has become more and more popular for protein engineering with the advantage of biological big-data
isopentenyl diphosphate (IPP) would be produced from mevalonate (MVA)[23,24,25] pathway, where phosphomevalonate is decarboxylated to form isopentenyl phosphate (IP) and IP is converted into IPP by isopentenyl phosphate kinase (IPK) (Fig. 1A)
dimethylallyl phosphate (DMAP) can be phosphorylated by IPK to produce DMAPP25
Summary
Protein rational design has become more and more popular for protein engineering with the advantage of biological big-data. We described a method of rational design that is able to identify desired mutants by analyzing the coevolution of protein sequence We employed this approach to evolve an archaeal isopentenyl phosphate kinase that can convert dimethylallyl alcohol (DMA) into precursor of isoprenoids. Substantial effort has been dedicated to investigate the coevolutionary interaction between amino acids through statistical coupling analysis (SCA) or mutual information[14,15,16,17] These co-evolving amino acids were found to be sufficient for recapitulating native folding and function of protein[15,16,17]. We examined the activity of mutants in vitro and in vivo
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