Production of 3&#x2010;hydroxypropionic acid from glycerol by recombinant <i>Klebsiella pneumoniae</i> Δ<i>dhaT</i>Δ<i>yqhD</i> which can produce vitamin B₁₂ naturally

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<P><B>Abstract</B></P><P>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 <I>Escherichia coli</I> stains can produce 3‐HP from glycerol in the presence of vitamin B<SUB>12</SUB> (coenzyme B<SUB>12</SUB>), when overexpressed with a coenzyme B<SUB>12</SUB>‐dependent glycerol dehydratase (DhaB) and an aldehyde dehydrogenase. The present study examined the production of 3‐HP in recombinant <I>Klebsiella pneumoniae</I> strains, which naturally synthesizes vitamin B<SUB>12</SUB> and does not require supplementation of the expensive vitamin. The NAD<SUP>+</SUP>‐dependent gamma‐glutamyl‐gamma‐aminobutyraldehyde dehydrogenase (PuuC) of <I>K. pneumoniae</I> alone or with its DhaB was overexpressed homologously, and two major oxidoreductases, DhaT and YqhD, were disrupted. Without vitamin B<SUB>12</SUB> addition, the recombinant <I>K. pneumoniae</I> Δ<I>dhaT</I>Δ<I>yqhD</I> 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<SUP>+</SUP> regeneration (under low aeration) or reduced vitamin B<SUB>12</SUB> synthesis (under high aeration). In a glycerol fed‐batch bioreactor experiment under a constant DO of 5%, the strain, <I>K. pneumoniae</I> Δ<I>dhaT</I>Δ<I>yqhD</I>, 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 <I>K. pneumoniae</I> is potentially useful for the production of 3‐HP in an economical culture medium that does not require vitamin B<SUB>12</SUB>. 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.</P>