Shengfang Zhou

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

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 K. pneumoniae overexpressing an NAD(+)-dependent γ-glutamyl-γ-aminobutyraldehyde dehydrogenase (PuuC) enzyme of K. pneumoniae DSM 2026 were shown to produce 3-HP from glycerol without the addition coenzyme B(12), 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 prevent the formation of byproducts during anaerobic production of 3-HP from glycerol. Nitrate addition could improve NAD(+) regeneration, but decreased glycerol flux towards 3-HP production. To divert more glycerol towards 3-HP, a novel recombinant strain K. pneumoniae ΔglpKΔdhaT (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±3mM 3-HP with more than 50% yield on glycerol in shake flasks and 250±10mM 3-HP with approximately 30% yield on glycerol in a fed-batch bioreactor.

Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol using resting cells of the lactate dehydrogenase-deficient recombinant Klebsiella pneumoniae overexpressing an aldehyde dehydrogenase

In the present study, the lactate dehydrogenase-deficient (ldhA(-)) recombinant Klebsiella pneumoniae overexpressing an ALDH (KGSADH) was developed and the co-production of 3-HP and PDO from glycerol by this recombinant under resting cell conditions was examined. The new recombinant did not produce any appreciable lactate, which seriously inhibits the production of 3-HP and PDO. The final titers of 3-HP and PDO by the ldhA(-) recombinant strain at 60 h were 252.2 mM and 308.7 mM, respectively, which were improved by approximately 30% and 50%, respectively, compared to those by the counterpart recombinant strain, which was the wild type for ldhA. In addition, after deleting ldhA, the cumulative yield on glycerol and specific production rate of these two metabolites (3-HP and PDO) were enhanced by 41.4% and 52%, respectively.

Production of 3‐hydroxypropionic acid from glycerol by recombinant Pseudomonas denitrificans

3‐Hydroxypropionic acid (3‐HP) can be produced from glycerol through two sequential enzymatic reactions that are catalyzed by a coenzyme B12‐dependent glycerol dehydratase and an NAD(P)+‐dependent aldehyde dehydrogenase (ALDH), respectively. Pseudomonas denitrificans synthesizes coenzyme B12 under aerobic conditions, where NAD(P)+ is regenerated efficiently. Hence, it is considered an ideal host for the production of 3‐HP from glycerol under aerobic conditions. In this study, recombinant strains of P. denitrificans were developed and their potential for the production of 3‐HP from glycerol was evaluated. When the enzymes, glycerol dehydratase (DhaB) and glycerol dehydratase reactivase (GdrAB), of Klebsiella pneumoniae were expressed heterologously, P. denitrificans could produce 3‐HP at 37.7 mmol/L with 62% (mol/mol) yield on glycerol. Glucose was required as the carbon and energy sources for cell growth. The overexpression of heterologous ALDH was not essential; however, the titer and yield of 3‐HP were improved to 54.7 mmol/L and 67% (mol/mol), respectively, when an ALDH gene (puuC) from K. pneumoniae was overexpressed. One serious drawback hindering the use of P. denitrificans as a recombinant host for 3‐HP production is that it oxidizes 3‐HP to malonate and utilizes 3‐HP as a carbon source for growth. This is the first report on the development and use of recombinant P. denitrificans for 3‐HP production from glycerol. Biotechnol. Bioeng. 2013;110: 3177–3187.

Inducible gene expression system by 3-hydroxypropionic acid.

Background3-Hydroxypropionic acid (3-HP) is an important platform chemical that boasts a variety of industrial applications. Gene expression systems inducible by 3-HP, if available, are of great utility for optimization of the pathways of 3-HP production and excretion.ResultsHere we report the presence of unique inducible gene expression systems in Pseudomonas denitrificans and other microorganisms. In P. denitrificans, transcription of three genes (hpdH, mmsA and hbdH-4) involved in 3-HP degradation was upregulated by 3-HP by the action of a transcriptional regulator protein, LysR, and a cis-acting regulatory site for LysR binding. Similar inducible systems having an LysR transcriptional regulator were identified in other microorganisms that also could degrade 3-HP. A docking study showed that the 3-HP binding pocket is located between the N-terminal helix-turn-helix motif and the C-terminal cofactor-binding domain.ConclusionsThis LysR-regulated 3-HP-inducible system should prove useful for control of the level of gene expression in response to 3-HP.

3-Hydroxyisobutyrate dehydrogenase-I from Pseudomonas denitrificans ATCC 13867 degrades 3-hydroxypropionic acid

This study examined the role and physiological relevance of 3-hydroxyisobutyrate dehydrogenase-I (3HIBDHI) of Pseudomonas denitrificans ATCC 13867 in the degradation of 3-hydroxypropionic acid (3-HP) during 3-HP production. The gene encoding 3HIBDH-I of P. denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant 3HIBDH-I was then purified on a Ni-NTA-HP column and characterized for its choice of substrates, cofactors, metals, reductants, and the optimal temperature and pH. The recombinant 3HIBDH-I showed a high catalytic constant (kcat/Km) of 604.1 ± 71.1 mM/S on (S)-3-hydroxyisobutyrate, but no detectable activity on (R)-3-hydroxyisobutyrate. 3HIBDH-I preferred NAD+ over NADP+ as a cofactor for its catalytic activity. The kcat/Km determined for 3-HP was 15.40 ± 1.43 mM/S in the presence of NAD+ at 37°C and pH 9.0. In addition to (S)-3-hydroxyisobutyrate and 3-HP, 3HIBDH-I utilized l-serine, methyl-d,l-serine, and methyl-(S)-(+)-3-hydroxy-2-methylpropionate; on the other hand, the kcat/Km values determined for these substrates were less than 5.0mM/S. Ethylenediaminetetraacetic acid, 2-mercaptoethanol, dithiothreitol and Mn2+ increased the activity of 3HIBDHI significantly, whereas the presence of Fe2+, Hg2+ and Ag+ in the reaction mixture at 1.0 mM completely inhibited its activity. This study revealed the characteristics of 3HIBDH-I and its significance in 3-HP degradation.

Cloning, Expression and Characterization of 3-Hydroxyisobutyrate Dehydrogenase from Pseudomonas denitrificans ATCC 13867

The gene encoding an NAD+-dependent, 3-hydroxyisobutyrate dehydrogenase (3HIBDH-IV) from Pseudomonas denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL 21 (DE3) and characterized to understand its physiological relevance in the degradation of 3-hydroxypropionic acid (3-HP). The deduced amino acid sequence showed high similarity to other 3-hydroxyisobutyrate dehydrogenase isozymes (3HIBDHs) of P. denitrificans ATCC 13867. A comparison of 3HIBDH-IV with its relevant enzymes along with molecular docking studies suggested that Lys171, Asn175 and Gly123 are important for its catalytic function on 3-hydroxyacids. The recombinant 3HIBDH-IV was purified to homogeneity utilizing a Ni-NTA-HP resin column in high yield. 3HIBDH-IV was very specific to (S)-3-hydroxyisobutyrate, but also catalyzed the oxidation of 3-HP to malonate semialdehyde. The specific activity and half-saturation constant (K m) for 3-HP at 30°C and pH 9.0 were determined to be 17 U/mg protein and 1.0 mM, respectively. Heavy metals, such as Ag+ and Hg2+, completely inhibited the 3HIBDH-IV activity, whereas dithiothreitol, 2-mercaptoethanol and ethylenediaminetetraacetic acid increased its activity 1.5–1.8-fold. This paper reports the characteristics of 3HIBDH-IV as well as its probable role in 3-HP degradation.