An Automated Data-Driven Pipeline for Improving Heterologous Enzyme Expression
0301 basic medicine
Luminescent Agents
Data Science
Green Fluorescent Proteins
Mutation, Missense
Saccharomyces cerevisiae
Protein Engineering
Biosynthetic Pathways
Belladonna Alkaloids
03 medical and health sciences
Solubility
Atropa belladonna
Codon, Nonsense
Mutagenesis
Enzyme Stability
Escherichia coli
Transition Temperature
Acyltransferases
Biotechnology
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 natural enzymes suffer from suboptimal functional expression due to poor intrinsic protein stability. Further, stability enhancing mutations often come at the cost of impaired function. Here we demonstrate an automated protein engineering strategy for stabilizing enzymes while retaining catalytic function using deep mutational scanning coupled to multiple-filter based screening and combinatorial mutagenesis. We validated this strategy by improving the functional expression of a Type III polyketide synthase from the Atropa belladonna biosynthetic pathway for tropane alkaloids. The best variant had a total of 8 mutations with over 25-fold improved activity over wild-type in E. coli cell lysates, an improved melting temperature of 11.5 ± 0.6 °C, and only minimal reduction in catalytic efficiency. We show that the multiple-filter approach maintains acceptable sensitivity with homology modeling structures up to 4 Å RMS. Our results highlight an automated protein engineering tool for improving the stability and solubility of difficult to express enzymes, which has impact for biotechnological applications.
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CITATIONS (27)
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