An Automated Data-Driven Pipeline for Improving Heterologous Enzyme Expression
Protein Engineering
Synthetic Biology
Heterologous expression
Protein design
DOI:
10.1021/acssynbio.8b00486
Publication Date:
2019-02-05T21:22:26Z
AUTHORS (6)
ABSTRACT
Enzymes are the ultimate entities responsible for chemical transformations in natural and engineered biosynthetic pathways. However, many enzymes suffer from suboptimal functional expression due to poor intrinsic protein stability. Further, stability enhancing mutations often come at cost of impaired function. Here we demonstrate an automated engineering strategy stabilizing while retaining catalytic function using deep mutational scanning coupled multiple-filter based screening combinatorial mutagenesis. We validated this by improving a Type III polyketide synthase Atropa belladonna pathway tropane alkaloids. The best variant had total 8 with over 25-fold improved activity wild-type E. coli cell lysates, melting temperature 11.5 ± 0.6 °C, only minimal reduction efficiency. show that approach maintains acceptable sensitivity homology modeling structures up 4 Å RMS. Our results highlight tool solubility difficult express enzymes, which has impact biotechnological applications.
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