P102: Effect of puuC overexpression and nitrate addition on glycerol metabolism and anaerobic 3-hydroxypropionic acid production in Klebsiella pneumoniae

3-Hydroxypropionic acid (3-HP), an industrially important platform chemical, is used as a precursor during the production of many commercially important chemicals. Recently, recombinant strains of Klebsiella pneumoniae overexpressing an NAD⁺-dependent g-glutamyl-g-aminobutyraldehyde dehydrogenase (PuuC) enzyme of K. pneumoniae DSM 2026 have been shown to produce 3-HP from glycerol without the addition coenzyme B12, which is expensive. However, 3-HP production in K. pneumoniae is accompanied with NADH generation, and this always results in large accumulation of 1,3-propanediol (1,3-PDO) and lactic acid. In this study, we investigated the potential use of nitrate as an electron acceptor both to regenerate NAD⁺ and to avoid the formation of byproducts during anaerobic production of 3-HP from glycerol. Nitrate could improve NAD⁺ regeneration, but it decreased glycerol flux towards 3-HP production. To divert more glycerol towards 3-HP, a novel recombinant strain, K. pneumoniae DglpKDdhaT (puuC) was developed by disrupting the glpK gene, which encodes glycerol kinase, and the dhaT gene, which encodes 1,3-propanediol oxidoreductase. This strain showed improved cellular NAD⁺ concentrations and a high carbon flux towards 3-HP production. Through anaerobic cultivation in the presence of nitrate, this recombinant strain produced more than 40 ± 3 mM 3-HP with more than 50% yield in glycerol in shake flasks and 250 ± 10 mM 3-HP with approximately 30% yield in glycerol in a fed-batch bioreactor.