Spatial confinement of an ethanologenic route into B. subtilis functional membrane microdomains to improve ethanol bioproduction

Abstract

Microorganisms have continuously been used as chassis for the creation of synthetic metabolic pathways. However, the achievement of commercially viable productivity, when generating new metabolic pathways with heterologous enzymes, remains challenging. This is mainly because of cellular toxicity due to the accumulation of intermediates, the loss of flux to undesired byproducts or the leakage and loss of intermediates. However, there is a trend in synthetic biology that proposes the compartmentalization of reactions as a strategy to overcome these difficulties. In fact, bacteria naturally organize different cellular processes into functional membrane microdomains (FMM), which are similar to the eukaryotic lipid rafts. And flotillins, the structural proteins of FMM, play a crucial role in the compartmentalization of key processes, such as cell-wall metabolism or biofilm formation. In this work we have found that by fusing the membrane anchoring region MAR domain (5 – 24 aa) of B. subtilis flotillin (FloT) to the ethanologenic Z. mobilis enzymes PDC and ADH, it is possible to confine the heterologous ethanologenic route into the FMM of B. subtilis. Furthermore, when compartmentalizing the ethanologenic metabolic route, by means of the small MAR tag, higher ethanol titers were obtained in comparison with the control harboring the untagged enzymes. And, by experimenting with different promoters, expression systems, and carbon sources, we managed to modulate and improve the capacity of the gram-positive bacteria B. subtilis of producing ethanol. This project proposes an efficient way of producing biofuels in a non-pathogenic microorganism like B. subtilis that could represent an alternative to the use of fossil fuels