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Published on: February 8, 2017
Creation of a shikimate pathway variant.
Ningqing Ran1, K M Draths, J W Frost
1Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Journal of the American Chemical Society
|June 4, 2004
Summary
Engineered enzymes overcome competition for a key metabolite, increasing yields of microbially produced natural products. This advance in the shikimate pathway enhances the biosynthesis of essential compounds.
Area of Science:
- Metabolic Engineering
- Synthetic Biology
- Microbial Biotechnology
Background:
- The shikimate pathway is crucial for producing aromatic compounds in microbes.
- Competition for phosphoenolpyruvate (PEP) by the carbohydrate phosphotransferase system limits microbial production yields.
- 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase is a key enzyme in the shikimate pathway that consumes PEP.
Purpose of the Study:
- To engineer a novel shikimate pathway variant to circumvent PEP competition.
- To enhance the production of DAHP and downstream natural products.
- To assess the functionality of the engineered pathway in microbial growth and metabolism.
Main Methods:
- Directed evolution of 2-Keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases from E. coli and K. pneumoniae.
- Characterization of evolved KDPGal aldolase isozymes for improved DAHP synthesis activity.
- Construction and analysis of E. coli strains lacking native DAHP synthase but relying on evolved KDPGal aldolase.
Main Results:
- Evolved KDPGal aldolase isozymes showed 4-8 fold higher specific activity in catalyzing DAHP formation.
- The engineered pathway successfully produced DAHP from pyruvate and d-erythrose 4-phosphate.
- E. coli constructs utilizing the evolved KDPGal aldolase supported microbial growth and 3-dehydroshikimate synthesis.
Conclusions:
- The engineered shikimate pathway variant effectively bypasses PEP competition.
- Directed evolution of KDPGal aldolases provides a viable strategy for enhancing microbial production of shikimate pathway products.
- This approach holds potential for improving the industrial biosynthesis of various natural products.
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