Bio-based C-3 Platform Chemical: Biotechnological Production and -Conversion of 3-Hydroxypropionaldehyde

Abstract: Demands for efficient, greener, economical and sustainable production of chemicals, materials and energy have led to development of industrial biotechnology as a key technology area to provide such products from bio-based raw materials from agricultural-, forestry- and related industrial residues and by-products. For the bio-based industry, it is essential to develop a number of building blocks or platform chemicals for C2-C6 chemicals and even aromatic chemicals. 3-hydroxypropionaldehyde (3HPA) and 3-hydroxypropionic acid (3HP) are potential platform chemicals for C3 chemistry and even for producing polymers. This thesis presents investigations on the biotechnological routes for the production of a C3 platform chemical, 3HPA from glycerol and its conversion to 3HP. Glycerol, was used as the raw material for production of 3HPA using resting cells of the probiotic bacteria, Lactobacillus reuteri, as the biocatalyst. The antimicrobial effect of the bacteria is attributed to the secretion of “reuterin” that is an equilibrium mixture of 3HPA with its dimer and hydrate forms. Glycerol dehydratase, a Vitamin B12-dependent enzyme, presents in L. reuteri, catalyses the dehydration of glycerol to 3HPA. Production of 3HPA at high concentration results in strong inhibition of the enzyme activity and cell viability, which in turn limits the product yield and -productivity. Different means of in situ capture of 3HPA from the reaction were studied. Complexation of 3HPA with bisulfite in a fed-batch biotransformation of glycerol and subsequent removal through binding to an anion exchange resulted in increase in the production of 3HPA to 5.33 g/g biocatalyst from 0.45 g/g in a batch process. In another approach, in situ removal of 3HPA using semicarbazide-functionalized resin in a batch process, productivity was enhanced 2 fold than that without the resin. L. reuteri metabolizes 3HPA further to 1,3-propanediol (1,3PDO) and 3-hydroxypripionic acid (3HP) by reductive and oxidative pathways, respectively. The oxidative pathway, comprises 3 enzymes named propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL) and propionate kinase (PduW). Kinetic characterization and molecular modelling of the first enzyme, PduP, expressed in Escherichia coli was performed. The enzyme had a specific activity of 28.9 U/mg using propionaldehyde as substrate and 18 U/mg with 3HPA as substrate which is the highest specific activity reported up to date. All the Pdu enzymes were then expressed in E. coli in different combinations and used for bioconversion of 3HPA produced by native L. reuteri. Growing cells of the recombinant bacteria with all the three enzymes, E. coli pdu:P:L:W in a fed-batch mode gave 3HP yield of 0.5 mole/mole 3HPA with 1,3PDO as the co-product, while the resting cells gave 3HP yield of 1 mole /mole 3HPA. This showed the possibility of using of Pdu pathway of L. reuteri for production of 3HP.