Functional implementation of a linear glycolysis for sugar catabolism in Pseudomonas putida

Pseudomonas putida has many naturally-evolved metabolic and stress-tolerant properties, which makes this organism ideal for many biotechnological applications. Glucose catabolism, a redox reaction that allows to obtain energy from carbohydrates, takes place in P. putida KT2440 through the Entner-Doudoroff (ED) pathway. Through this pathway, glucose is catabolized into pyruvate through a series of enzyme-assisted reactions. At one point, part of the triose pool generated is recycled back to hexoses-P (this is known as the EDEMP cycle, and it is a combination of the ED pathway, with the Embden-Meyerhof-Parnas pathway or EMP, and pentose phosphate pathways), and this allows P. putida to boost catabolic production of NADPH and to interconnect other key metabolic intermediates. This cycle in P. putida KT2440 is a clear example of metabolic adaptation that has evolved to survive in adverse environmental conditions. In fact, this EDEMP cycle results in the generation of only half of the ATP (an energy-carrying molecule) in comparison with the ATP generated through the linear EMP pathway, suggesting that this kind of metabolism favours stress resistance in detriment of biomass formation.

P. putida strain KT2440 does not have the ability to perform glycolysis through the EMP pathway, and this is because it lacks the enzymatic activity known as glycolytic 6-phosphofructo-1-kinase (Pfk). Previous studies have tried to overcome this lack by introducing Escherichia coli’s PfkA enzyme into P. putida, but this was not enough. An alternative is the use of the GlucoBrick platform, a tool for engineering glycolysis in Gram-negative bacteria. This tool was used in the present study to replace the ED pathway (and EDEMP cycle) in P. putida strain KT2440 with a synthetic EMP glycolysis pathway that has been rationally designed.

Read more on this work in the Open Access article available online.

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