Production of 3‐hydroxypropionic acid from glycerol by recombinant Klebsiella pneumoniae ΔdhaTΔyqhD which can produce vitamin B12 naturally

3‐Hydroxypropionic acid (3‐HP) is an important platform chemical that can be used to synthesize a range of chemical compounds. A previous study demonstrated that recombinant Escherichia coli stains can produce 3‐HP from glycerol in the presence of vitamin B₁₂ (coenzyme B₁₂), when overexpressed with a coenzyme B₁₂‐dependent glycerol dehydratase (DhaB) and an aldehyde dehydrogenase. The present study examined the production of 3‐HP in recombinant Klebsiella pneumoniae strains, which naturally synthesizes vitamin B₁₂ and does not require supplementation of the expensive vitamin. The NAD⁺‐dependent gamma‐glutamyl‐gamma‐aminobutyraldehyde dehydrogenase (PuuC) of K. pneumoniae alone or with its DhaB was overexpressed homologously, and two major oxidoreductases, DhaT and YqhD, were disrupted. Without vitamin B₁₂ addition, the recombinant K. pneumoniae ΔdhaTΔyqhD overexpressing PuuC could produce ～3.8 g/L 3‐HP in 12 h of flask culture. However, this was possible only under the appropriate aeration conditions; 1,3‐propanediol (1,3‐PDO) (instead of 3‐HP) was mainly produced when aeration was insufficient, whereas a very small amount of both 3‐HP and 1,3‐PDO were produced when aeration was too high. The production of a small amount of 3‐HP under improper aeration conditions was attributed to either slow NAD⁺ regeneration (under low aeration) or reduced vitamin B₁₂ synthesis (under high aeration). In a glycerol fed‐batch bioreactor experiment under a constant DO of 5%, the strain, K. pneumoniae ΔdhaTΔyqhD, overexpressing both PuuC and DhaB could produce >28 g/L 3‐HP in 48 h with a yield of >40% on glycerol. Only small amount of 3‐HP was produced when cultivation was carried out at a constant aeration of 1 vvm or constant 10% DO. These results show that K. pneumoniae is potentially useful for the production of 3‐HP in an economical culture medium that does not require vitamin B₁₂. The results also suggest that the aeration conditions should be optimized carefully for the efficient production of 3‐HP while using this strain. Biotechnol. Bioeng. 2013; 110: 511–524. © 2012 Wiley Periodicals, Inc.