Exploring the Propanediol Utilization Pathway in Lactobacillus reuteri

Sammanfattning: Concerns regarding environmental issues have led to increasing efforts for investigating sustainable production routes for the future industry that is based on renewable resources. Among the biobased chemicals, the C3-chemicals 3-hydroxypropionaldehyde (3-HPA), 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP), have been identified as top candidates for the chemical industry. Lactobacillus reuteri is a heterofermentative probiotic microorganism that possesses the metabolic pathway called the propanediol utilization (Pdu) pathway that catalyzes dehydration of glycerol to 3-HPA by glycerol dehydratase (PduCDE) and further to 3-HP and 1,3-PDO by a series of reactions catalyzed by propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL), and propionate kinase (PduW), and 1,3-propanediol oxidoreductase (PduQ), respectively. The Pdu pathway is operative inside Pdu microcompartment (MCP) that encapsulates different cofactors and enzymes needed for metabolizing glycerol or 1,2-propanediol, and protects the cells from the toxic effect of the aldehyde intermediate.This thesis focuses mainly on the theoretical investigation of different aspects of the Pdu pathway in L. reuteri, and the MCP shell made up of several proteins. Two of the major shell proteins, PduA and PduJ were investigated, and the effect of small alterations in the pore structures in the respective proteins by generating mutants of the key amino acid residues lining the pores was studied (Paper I). Enzymes of the Pdu pathway, i.e. PduCDE, PduQ and PduP, were characterized with respect to their substrate selectivity (Paper II). In all cases, C3 substrates were the most optimal for the enzyme activities. The substrate range for PduCDE could be increased up to C6 diols by site-directed mutagenesis of two residues of pduC gene, however the passage of the longer substrates through the MCP shell in L. reuteri seemed to be limited. While PduQ has been known to reduce the 3-HPA to 1,3-PDO inside the MCP, the possible role of other alcohol dehydrogenases (ADHs) in transformation of 3-HPA outside the MCP and to maintain redox balance was investigated. It was discovered that while PduQ was the active enzyme in reducing the 3-HPA in the resting cells, it was ADH7, a cytoplasmic enzyme that reduced the hydroxyaldehyde in the growing cells to provide the cofactor NAD+ required for glucose metabolism (Paper III). The heterologously expressed PduQ enzyme was characterized with respect to its activity against its optimal substrates 3-HPA and 1,2-propanediol (Paper IV). Exposure to air resulted in significant loss of PduQ activity and its bound iron content. The identity of the residues on the enzyme binding iron were confirmed by mutational studies.