Streamlining Central Catabolism for à La Carte Activation of Glycolysis

The process known as glycolysis encompasses several biochemical sequences, including the Embden-Meyerhof-Parnas (EMP) and the Entner-Doudoroff (ED) pathways. Not all the microorganisms capable of growing on glucose use the canonical EMP glycolytic pathway. In fact, glucose metabolism in bacteria is more diverse than previously thought. Whereas enterobacteria constitute a paradigmatic case of EMP-operating microorganisms, other bacteria favor the use of alternative glycolytic pathways. Modern metabolic engineering endeavors call for adequate microbial chassis to plug-in and -out genetic circuits and new-to-nature functionalities. However, bacteria that are the easiest to manipulate are seldom the most suitable or the most appropriate for specific large-scale endeavors or industrial applications. Among them, environmental Pseudomonas strains, such as P. putida, are interesting microbial platforms since they are pre-endowed with metabolic, physiological, and stress-endurance traits optimal for biotechnological needs. P. putida KT2440 operates an entirely ED-based glycolysis, and the EMP pathway is non-functional due to the absence of a 6-phosphofructokinase activity. Interestingly, EMP outcompetes ED in ATP yield from glucose, making the former preferable over the later from an industrial point of view. Activating an EMP glycolysis in P. putida is therefore of paramount interest. Building on the tenets of synthetic biology, a streamlined glycolytic pathway was designed by taking advantage of the functional glycolytic elements of Escherichia coli. By following a systematic, bottom-up approach, the relevant genes were cured from commonly used restriction enzymes, and all non-coding DNA sequences. The genes were then organized in two independent operons and placed under the transcriptional control of well characterized synthetic parts. As a proof-on-concept, the system was assayed in E. coli and P. putida mutant strains as a first step towards the activation of a flawless linear glycolytic pathway in virtually any bacterium.