Satish Kumar Ainala

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.

Glycerol assimilation and production of 1,3-propanediol by Citrobacter amalonaticus Y19.

Citrobacter amalonaticus Y19 (Y19) was isolated because of its ability for carbon monoxide-dependent hydrogen production (water–gas shift reaction). This paper reports the assimilation of glycerol and the production of 1,3-propanediol (1,3-PDO) by Y19. Genome sequencing revealed that Y19 contained the genes for the utilization of glycerol and 1,2-propanediol (pdu operon) along with those for the synthesis of coenzyme B12 (cob operon). On the other hand, it did not possess the genes for the fermentative metabolism of glycerol of Klebsiella pneumoniae, which consists of both the oxidative (dhaD and dhaK) and reductive (dhaB and dhaT) pathways. In shake-flask cultivation under aerobic conditions, Y19 could grow well with glycerol as the sole carbon source and produced 1,3-PDO. The level of 1,3-PDO production was improved when vitamin B12 was added to the culture medium under aerobic conditions. Under anaerobic conditions, cell growth and 1,3-PDO production on glycerol was also possible, but only when an exogenous electron acceptor, such as nitrate or fumarate, was added. This is the first report of the glycerol metabolism and 1,3-PDO production by C. amalonaticus Y19.

Coenzyme B12 can be produced by engineered Escherichia coli under both anaerobic and aerobic conditions.

Coenzyme B12 (Vitamin B12) is one of the most complex biomolecules and an essential cofactor required for the catalytic activity of many enzymes. Pseudomonas denitrificans synthesizes coenzyme B12 in an oxygen‐dependent manner using a pathway encoded by more than 25 genes that are located in six different operons. Escherichia coli, a robust and suitable host for metabolic engineering was used to produce coenzyme B12. These genes were cloned into three compatible plasmids and expressed heterologously in E. coli BL21 (DE3). Real‐time PCR, SDS–PAGE analysis and bioassay showed that the recombinant E. coli expressed the coenzyme B12 synthetic genes and successfully produced coenzyme B12. However, according to the quantitative determination by inductively coupled plasma‐mass spectrometry, the amount of coenzyme B12 produced by the recombinant E. coli (0.21 ± 0.02 μg/g cdw) was approximately 13‐fold lower than that by P. denitrificans (2.75 ± 0.22 μg/g cdw). Optimization of the culture conditions to improve the production of coenzyme B12 by the recombinant E. coli was successful, and the highest titer (0.65 ± 0.03 μg/g cdw) of coenzyme B12 was obtained. Interestingly, although the synthesis of coenzyme B12 in P. denitrificans is strictly oxygen‐dependent, the recombinant E. coli could produce coenzyme B12 under anaerobic conditions.

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.

Complete Genome Sequence of Pseudomonas denitrificans ATCC 13867

ABSTRACT Pseudomonas denitrificans ATCC 13867, a Gram-negative facultative anaerobic bacterium, is known to produce vitamin B12 under aerobic conditions. This paper reports the annotated whole-genome sequence of the circular chromosome of this organism.

Metabolic flux change in Klebsiella pneumoniae L17 by anaerobic respiration in microbial fuel cell

The metabolic flux in microbial fuel cells (MFCs) is significantly different from conventional fermentation because the electrode in MFCs acts as a terminal electron acceptor. In this study, the difference in the carbon metabolism of Klebsiella pnuemoniae L17 (Kp L17) during growth in MFCs and conventional bioreactors was studied using glucose as the sole carbon and energy source. For metabolic flux analysis (MFA), the in silico metabolic flux model of Kp L17 was also constructed. The MFC bioreactor operated in oxidative mode, where electrons are removed by the anode electrode, generated a smaller quantity of reductive metabolites (e.g., lactate, 2,3-butanediol and ethanol) compared to the conventional fermentative bioreactor (non-MFC). Stoichiometric analysis indicated that the cellular metabolism in MFC had partially (or significantly) shifted to anaerobic respiration from fermentation, the former of which was similar to that often observed under micro-aerobic conditions. Electron balance analysis suggested that 30% of the electrons generated from glucose oxidation were extracted from the microbe and transferred to the electrode. These results highlight the potential use of MFCs in regulating the carbon metabolic flux in a bioprocess.

Characterization of 1,3-propanediol oxidoreductase (DhaT) from Klebsiella pneumoniae J2B

Abstract1,3-propanediol oxidoreductase (DhaT) of Klebsiella pneumoniae converts 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) during microbial production of 1,3-PD from glycerol. In this study, DhaT from newly isolated K. pneumoniae J2B was cloned, expressed, purified, and studied for its kinetic properties. It showed, on its physiological substrate 3-HPA, higher activity than similar aldehydes such as acetaldehyde, propionaldehyde and butyraldehyde. The turnover numbers (kcat, 1/s) were estimated as 59.4 for the forward reaction (3-HPA to 1,3-PD at pH 7.0) and 10.0 for the reverse reaction (1,3-PD to 3-HPA at pH 9.0). The Michaelis constants (Km, mM) were 0.77 (for 3-HPA) and 0.03 (for NADH) for the forward reaction (at pH 7.0), and 7.44 (for 1,3-PD) and 0.23 (for NAD+) for the reverse reaction (at pH 9.0). Between these forward and reverse reactions, the optimum temperature and pH were significantly different (37°C and 7.0 vs. 55°C and 9.0, respectively). These results indicate that, under physiological conditions, DhaT mostly catalyzes the forward reaction. The enzyme was seriously inhibited by heavy metal ions such as Ag+ and Hg2+. DhaT was highly unstable when incubated with its own substrate 3-HPA, indicating the necessity of enhancing its stability for improved 1,3-PD production from glycerol.

Deletion of putative oxidoreductases from Klebsiella pneumoniae J2B could reduce 1,3-propanediol during the production of 3-hydroxypropionic acid from glycerol

Recombinant Klebsiella pneumoniae over-expressing 3-hydroxypropionaldehyde (3-HPA) dehydrogenase can produce 3-hydroxypropionic acid (3-HP), an important platform chemical, from glycerol. However, K. pneumoniae co-produces 1,3-propanediol (1,3-PDO) due to the presence of 1,3-propanediol oxidoreductases, which decreases the titer and yield of 3-HP. Previously, two major oxidoreductases, dhaT and yqhD, were removed from K. pneumoniae; however the mutant still produced a significant amount of 1,3-PDO, indicating the probable existence of other oxidoreductase(s). Genome analysis of K. pneumoniae revealed the presence of five putative oxidoreductases having high amino acid similarities to both DhaT (primary 1,3-propanediol oxidoreductase) and YqhD (aldehyde dehydrogenase). Among them, adhE was highly expressed in the absence of DhaT and YqhD. Additionally, an alkyl hydroperoxide oxidoreductase (ahpF), albeit dissimilar to both DhaT and YqhD, was highly expressed in the absence of DhaT and YqhD. To examine the role of adhE and ahpF in 1,3-PDO production, mutant strains devoid of dhaT, yqhD, ahpF and/or adhE genes were developed. However, these mutants neither reduced the production of 1,3-PDO nor improved the production of 3-HP when engineered to over-express an aldehyde dehydrogenase (KGSADH). These results indicate that, apart from DhaT, YqhD, AhpF and AdhE, K. pneumoniae has other, unknown oxidoreductases that are involved in 1,3-PDO production. It is concluded that complete elimination of 1,3-PDO during 3-HP production from glycerol by K. pneumoniae is highly challenging.

Complete genome sequence of novel carbon monoxide oxidizing bacteria Citrobacter amalonaticus Y19, assembled de novo.

We report here the complete genome sequence of Citrobacter amalonaticus Y19 isolated from an anaerobic digester. PacBio single-molecule real-time (SMRT) sequencing was employed, resulting in a single scaffold of 5.58Mb. The sequence of a mega plasmid of 291Kb size is also presented.

NADH-dependent Lactate Dehydrogenase from Alcaligenes eutrophus H16 Reduces 2-oxoadipate to 2-hydroxyadipate

Adipic acid is an important monomer for the production of nylon-6,6 polyamide. One novel biological route for the synthesis of adipic acid, which combines the lysine synthetic pathway and glutaconic acid production pathway, has been suggested, but this route has suffered from the lack of an efficient 2-oxoadipate reductase connecting the two pathways or converting 2-oxoadipate to 2-hydroxyadipate. In this study, we report that the lactate dehydrogenase of Alcaligenes eutrophus H16 is a promising catalyst for this reaction. The lactate dehydrogenase gene (Ae-ldhO) was cloned, expressed in Escherichia coli, purified, and characterized. The recombinant enzyme, having a molecular weight of 36.7 kDa, exhibited broad substrate specificity for various 2-oxoacids. NADH was the preferred coenzyme over NADPH for all 2-oxoacids tested. The maximum specific activity of Ae-LdhO on 2-oxoadipate was 454.5 ± 20.1 U/mg protein at pH 7.0 and 30℃. The Km values for 2-oxoadipic acid and NADH were 0.32 ± 0.02 and 0.09 ± 0.002 mM, respectively. The activity of Ae-LdhO was enhanced in the presence of some metal ions, such as Mg2+, Co2+ or Ni2+, whereas it was completely inhibited by Hg2+, Ag+, Cu2+ and DTT.