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

Abstract

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.

Increased 3-hydroxypropionic acid production from glycerol, by modification of central metabolism in Escherichia coli

“…For example, Rathnasingh et al optimized the expression level of each enzyme in this pathway in E. coli [17]. Ashok et al deleted dhaT and yqhD to reduce the production of byproducts in K. pneumoniae [12]. Kim et al modified glycerol metabolism in 3HP-producing E. coli and developed fed-batch cultivation with simultaneous feeding of glycerol and glucose [19].…”

“…Considering the whole metabolic network, modification of other pathways, as well as the biosynthetic pathway of the target product, is also a key strategy for increasing the metabolic flux, leading to enhanced target production. For example, target production can be enhanced by improving the redox balance and reducing byproduct formation via gene knockout and overexpression [12,20]. …”

Biological Production of 3-Hydroxypropionic Acid: An Update on the Current Status

“…Another consideration when using the dha operon is the inactivation of the glycerol dehydratase during the catalysis of glycerol conversion [17,18], so proper expression of the glycerol dehydratase reactivase is important to maintain the activity of glycerol dehydratase and, in turn, 3HP production. Glycerol dehydratase can also be inactivated by oxygen [19], which on the other hand, is necessary for efficient regeneration of the NAD + [20] that is required for the action of the ald enzymes. …”

Metabolic engineering of Escherichia coli strains for co-production of hydrogen and ethanol from glucose

“…The amplified fragment was cloned into the expression vector pDK7 [15]. This recombinant plasmid was transformed into SH8AE by electroporation, yielding the strain SH8U to overexpress udhA.…”