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Assessing and harnessing updated polyketide synthase modules through combinatorial engineering.

Katherine A Ray1, Joshua D Lutgens1, Ramesh Bista1

  • 1Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.

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Summary

We developed a platform for engineering polyketide synthases, enabling the creation of new combinations. This research identifies key challenges in producing functional synthases and offers a path toward designer polyketides.

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Area of Science:

  • Synthetic biology
  • Biochemistry
  • Molecular engineering

Background:

  • Polyketide assembly lines are modular and significant for producing valuable compounds.
  • Combinatorial engineering offers potential for creating novel polyketides.
  • Previous engineering efforts focused on shorter synthases, leaving larger ones unexplored.

Purpose of the Study:

  • To design and implement a BioBricks-like platform for constructing and testing diverse polyketide synthases.
  • To investigate all module combinations of the pikromycin synthase for triketide, tetraketide, and pentaketide synthases.
  • To identify limitations in producing functional synthases and explore hybrid synthase construction.

Main Methods:

  • Development of a modular BioBricks-like platform for rapid assembly of polyketide synthases.
  • Systematic construction of 5 triketide, 25 tetraketide, and 125 pentaketide synthases based on pikromycin synthase.
  • Analysis of product formation and identification of impediments like ketosynthase gatekeeping and module skipping.

Main Results:

  • Successful construction and testing of a large library of pikromycin synthases.
  • Detection of anticipated products from 60% of triketide, 32% of tetraketide, and 6.4% of pentaketide synthases.
  • Identification of ketosynthase gatekeeping and module skipping as primary obstacles to functional synthases.
  • Construction of active hybrid synthases using modules from erythromycin, spinosyn, and rapamycin assembly lines.

Conclusions:

  • The developed platform enables efficient engineering of large polyketide synthases.
  • Ketosynthase gatekeeping and module skipping significantly hinder the production of functional synthases.
  • The platform facilitates the creation of hybrid synthases, paving the way for designer polyketides with tailored properties.