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C(1) compounds as auxiliary substrate for engineered Pseudomonas putida S12.

Frank W Koopman¹, Johannes H de Winde, Harald J Ruijssenaars

¹B-Basic, Delft, The Netherlands. F.W.Koopman@tudelft.nl

Applied Microbiology and Biotechnology

|March 13, 2009

Summary

Engineered bacteria Pseudomonas putida S12 efficiently use methanol and formaldehyde. Introducing the ribulose monophosphate (RuMP) pathway significantly boosted biomass yield, enhancing its bioconversion capabilities.

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Area of Science:

Metabolic Engineering
Synthetic Biology
Microbial Biotechnology

Background:

Pseudomonas putida S12 is a solvent-tolerant bacterium with potential as a bioconversion platform.
Efficient utilization of C(1) compounds like methanol and formaldehyde is crucial for sustainable bioprocesses.
Formaldehyde, a methanol oxidation intermediate, is toxic and requires effective metabolic pathways for detoxification and assimilation.

Purpose of the Study:

To engineer Pseudomonas putida S12 for efficient co-utilization of glucose with formaldehyde and methanol.
To enhance biomass yield by introducing the ribulose monophosphate (RuMP) pathway for formaldehyde assimilation.
To evaluate the performance of the engineered strain using different concentrations of auxiliary C(1) substrates.

Main Methods:

Genetic engineering of P. putida S12 by introducing the Bacillus brevis hps and phi genes, key enzymes of the RuMP pathway.
Cultivation of engineered and control strains in C-limited chemostats with mixtures of glucose and formaldehyde or methanol.
Measurement of biomass yield (C-mol biomass/C-mol substrate) under varying substrate feed concentrations.

Main Results:

The engineered P. putida S12 strain showed significantly increased biomass yield when formaldehyde was used as an auxiliary substrate, reaching 91% compared to 35% without formaldehyde.
The RuMP pathway-expressing strain demonstrated improved growth at higher relative formaldehyde concentrations.
Replacing formaldehyde with methanol, leveraging endogenous methanol oxidation activity, resulted in an 84% biomass yield.

Conclusions:

Introduction of the RuMP pathway enzymes enables efficient formaldehyde assimilation in P. putida S12.
Co-utilization of renewable C(1) compounds like methanol enhances biomass yield and strengthens P. putida S12's role as a bioconversion host.
This metabolic engineering strategy contributes to the cost-effective use of P. putida S12 for biotechnological applications.