Microbial production of 3-hydroxypropionic acid and poly(3-hydroxypropionate): Investigation of Lactobacillus reuteri propanediol utilization pathway enzymes

Abstract: Concerns regarding environmental issues such as greenhouse gas emissions and climate change have led to a shift within the research community and chemical and energy industry sectors for finding sustainable routes for producing fuels and chemicals from renewable resources, thereby minimizing our dependence on petroleum. The C3-chemical 3-hydroxypropionic acid has been identifed as a top candidate for the biobased chemical industry. This platform chemical is a ?-hydroxy acid containing two functional groups (hydroxyl and carboxyl) enabling its conversion into value-added chemicals such as 1,3-propanediol, acrolein, malonic acid, acrylamide and acrylic acid, which can be used in resins, coatings, paints, adhesives, lubricants, and in the textile industry as anti-static agent. Polymerized 3- HP, poly(3-hydroxypropionate) (poly(3-HP)), is a biodegradable and stable polymer which, besides its potential role as a biomaterial, can be degraded to 3-HP monomer. In recent years, a dramatic increase in the interest for microbial production of 3-HP and poly(3-HP) has been observed. Metabolic engineering and recombinant expression of various enzymatic pathways in a number of bacterial strains have been suggested and implemented, with mainly renewable glucose and glycerol as substrates. This thesis presents a novel pathway called the propanediol utilization pathway present in Lactobacillus reuteri that catalyzes dehydration of glycerol to 3- hydroxypropionaldehyde (3-HPA) and further to 3-HP by a series of reactions catalyzed by propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL) and propionate kinase (PduW). Through structural modeling and kinetic characterization of PduP, its 3-HPA consuming ability was confirmed and catalytic mechanism proposed. PduP, PduL and PduW-mediated conversion of 3-HPA to 3- HP was confirmed through their recombinant expression in Escherichia coli. 3-HPA produced from glycerol by L. reuteri was used as a substrate for conversion to 3-HP by the recombinant E. coli. A yield of 1 mol/mol was reached with a titer of 12 mM 3-HP. Depletion of the cofactor NAD+ required for the catalysis of 3-HP to 3-HPCoA, was deemed responsible for the low titer. Regeneration of NAD+, used up in PduP catalyzed reaction, was achieved by recombinant expression of NADH oxidase (Nox) from L. reuteri in E. coli expressing PduP, PduL and PduW. The final 3-HP titer by this recombinant strain was at least twice that of E. coli carrying solely PduP, PduL and PduW. For the production of poly(3-HP), PduL and PduW in the recombinant strain were replaced by polyhydroxyalkanoate synthase of Chromobacterium sp. that converts 3- HP-CoA to poly(3-HP). A poly(3-HP) content of up to 40% (w/w) cell dry weight was reached in an efficient and cheap process requring no additivies or expensive cofactors.