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Computer-assisted multilevel optimization of malonyl-CoA availability in Pseudomonas putida.

Christos Batianis1, Rik P van Rosmalen2, Pedro Moñino Fernández1

  • 1Laboratory of Systems and Synthetic Biology, Wageningen University & Research, Wageningen, 6708 WE, the Netherlands; Bioprocess Engineering, Wageningen University & Research, Wageningen, 6708 PB, the Netherlands.

Metabolic Engineering
|March 19, 2025
PubMed
Summary

This study enhanced malonyl-CoA (a key building block) production in Pseudomonas putida by 5.8-fold. This metabolic engineering advance boosts the industrial synthesis of valuable chemicals like polyketides.

Keywords:
CRISPRiGenome-scale modelingMalonyl-CoAPseudomonas putidaRBS optimization

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Area of Science:

  • Metabolic Engineering
  • Synthetic Biology
  • Industrial Biotechnology

Background:

  • Malonyl-CoA is crucial for producing valuable compounds like fatty acids and polyketides.
  • Limited intracellular malonyl-CoA hinders microbial production of these chemicals.
  • Pseudomonas putida is a versatile host for industrial chemical synthesis.

Purpose of the Study:

  • To systematically increase malonyl-CoA levels in Pseudomonas putida.
  • To develop a multilevel optimization workflow for microbial hosts.
  • To enhance the production of industrially relevant compounds.

Main Methods:

  • Utilized a multilevel optimization workflow involving gene downregulation and chassis selection.
  • Engineered the acetyl-CoA carboxylase complex via ribosome binding site modification.
  • Employed computational tools and high-throughput screening with a malonyl-CoA biosensor.

Main Results:

  • Achieved a 5.8-fold enhancement in the production titer of the polyketide phloroglucinol.
  • Successfully identified and combined beneficial genetic targets for malonyl-CoA overproduction.
  • Demonstrated rapid evaluation of genetic targets using computational and biosensor approaches.

Conclusions:

  • The study effectively integrated computational and genetic engineering for P. putida.
  • The developed workflow significantly increases malonyl-CoA availability in microbial hosts.
  • This research opens new possibilities for industrial strain development and fundamental biological studies.