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

Abstract: Popular Abstract in English Since the onset of the 20th century, human society has been using non-renewable resources, mainly oil, for the production of fuels and chemicals that are now an integral part of our everyday life. In the last few decades however, concerns regarding environmental effects, geopolitical issues and the eventual depletion of oil have led to us re-evaluating our dependency on this resource. Current production of a majority of everday chemicals is based on refining petroleum to a small number of other molecules, also known as platform chemicals, which can then be converted to a much larger number of chemicals through various processes. In our efforts to move from a fossil to biobased economy in which renewable resources like sugars or glycerol, obtained through plants, trees, grasses, and/or as residues/wastes of agro-/forestry based industries, will constitute the feedstock for industry, sustainable technologies for processing of the biomass and its components in an environmentally-friendly manner need to be develooped. Biotechnological production of platform chemicals from the biomass feedstocks is mainly based on replacing traditional chemical reactors with microorganisms. Microorganisms can be considered small reactors as they contain mechanisms for the conversion of a large variety of natural as well as synthetic molecules to others. These mechanisms are driven by enzymes which are in turn encoded in their genomes. As microorganisms are present in pretty much every type of environment imaginable on the planet, the number of naturally occurring reactions is very high. Some microorganisms are known to produce biodegradable plastics as a protection mechanism in harsh conditions. Microbial processes for the production of chemicals and materials are based in water and require no organic solvents. They can be performed in lower temperatures and are very specific. As petroleum is cheap however, there is no economic incentive to shift to these greener processes. Thus, it is important to develop microbial methods for the production of these chemicals from cheap resources and in high concentrations and yields. This thesis deals mainly with the production of the platform chemical 3- hydroxypropionic acid (3-HP) and its polymer poly(3-hydroxypropionate). These products are not available commercially. 3-HP is of great promise as it can be further converted into wide array of chemicals, e.g. resins, coatings, lubricants and in the textile industry as anti-static agent. Poly(3-HP) is a biodegradable polymer that can replace certain fossil-based polymers in different applications. 3-HP is produced in smaller amounts by certain microorganisms that grow slowly and/or are expensive to cultivate. Therefore, a copy and paste-strategy has been implemented for moving some of the reactions to well-known organisms that are easier and cheaper to cultivate. Lactobacillus reuteri, a probiotic bacteria, contains a mechanism for the production of 3-hydroxypropionic acid from glycerol, which is currently produced in large amounts as a byproduct from the production of bio-diesel from several plant oils. By copying relevant genes and transferring them to the cheaply cultivated bacterium Escherichia coli, it was proven that 3-HP could be produced with a high yield from glycerol. Such a strategy does require the understanding of the metabolic system in the bacteria in order to avoid any interference and to incorporate strategies for improving the formation of the product in a selective and clean manner. Such strategies were implemented in the present work, thereby increasing the final concentration of 3-HP. Some key enzymes in these processes were studied further in order to gain a better understanding of their function and structure. It the same manner, a relevant gene from Lactobacillus reuteri and a gene from another bacteria, Chromobacterium sp. known to produce a bioplastic, were copied and pasted in Escherichia coli, resulting in a strain with the capacity to produce poly(3-hydroxypropionate) in a cheap and efficient manner.