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.
Original language | English (US) |
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Pages (from-to) | 474-481 |
Number of pages | 8 |
Journal | ACS Synthetic Biology |
Volume | 8 |
Issue number | 3 |
DOIs | |
State | Published - Mar 15 2019 |
Bibliographical note
Funding Information:We thank Prof. Frank DiMaio for donation of the enzyme decoy sets. This work was supported by Michigan State University funds, by NSF CBET Career Award #1254238 to T.A.W., NIH T32 Biotechnology Training Grant (Award # T32-GM110523) to E.E.W., and NSF award numbers IOS-1546617 and MCB-1714093 to C.S.B. C.S.B. is supported in part by Michigan AgBioResearch and through USDA National Institute of Food and Agriculture, Hatch project number MICL02552.
Publisher Copyright:
© 2019 American Chemical Society.
Keywords
- deep mutational scanning
- enzyme stability
- heterologous pathway expression
- high-throughput screening
- polyketide synthase
- tropane alkaloids