Metabolic engineering of Lactobacillus reuteri DSM 20,016 for improved 1,3-propanediol production from glycerol

The production of 1,3-propanediol (1,3-PDO) from glycerol was studied by GRAS and native 1,3-PDO producer, Lactobacillus reuteri DSM 20016. This strain ferments glucose with production of lactate, acetate, ethanol, and converts glycerol to 1,3-PDO using NADH generated by glucose metabolism. To improve 1,3-PDO production, alcohol dehydrogenases (ADH) were disrupted and 1,3-PDO oxidoreductases (PDOR) were overexpressed. Deletion of ADH (adh2) enhanced 1,3-PDO production yield on glucose by reducing ethanol synthesis, and overexpression of PDOR (pduQ) elevated 1,3-PDO production rate and cell growth rate. The strain with simultaneous adh2 deletion, pduQ overexpression (Delta adh2pduQ++) could produce 687 mM 1,3-PDO with the yield of 1.2 +/- 0.08 mol 1,3-PDO/mol glucose by fed-batch bioreactor cultivation in 48 h. However, the 1,3-PDO production rate was greatly reduced in the late period of bioreactor culture, mainly due to high lactate accumulation. This is the first report on rational metabolic engineering of L. reuteri for improved 1,3-PDO production

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 5.58 Mb. The sequence of a mega plasmid of 291 Kb size is also presented

A novel D(???)-lactic acid-inducible promoter regulated by the GntR-family protein D-LldR of Pseudomonas fluorescens

Lactic acid has two stereoisomers of D(???)- and L(+)-forms, both of which are important monomers of biodegradable plastic, poly-lactic acid. In this study, a novel D-lactate inducible system was identified in Pseudomonas fluorescens A506, partially characterized and tested as biosensor. The D-lactate catabolic operon (lldP-dld-II) was negatively regulated through the inversely transcribed D-lldR (encoding a GntR-type regulator), where the repression is relieved by addition of D-lactate. The derepression was specific to D-lactate and marginally affected by L-lactate. The D-LldR-responsive operator, showing dyad symmetry and separated by one base, was located between +11 and + 27 from the transcription start site of the lldP-dld-II operon. By site-directed mutagenesis, a motif with a dyad symmetry (AATTGGTAtTACCAATT), present in the upstream region of lldP, was identified as essential for the binding of LldR. D-lactate biosensors were developed by connecting the upregulation by D-lactate to a green fluorescent readout. About ~6.0-fold induction by 100 mM D-lactate was observed compared to L-lactate

Improvement of carbon monoxide-dependent hydrogen production activity in Citrobacter amalonaticus Y19 by over-expressing the CO-sensing transcriptional activator, CooA

Citrobacter amalonaticus Y19 (Y19) can produce hydrogen (H-2) from oxidation of carbon monoxide (CO) via the water-gas shift reaction. The reaction is catalyzed by two enzymes, carbon monoxide dehydrogenase (CODH) and carbon monoxide-dependent hydrogenase (CO-Hyd). The contig genome sequencing of Y19 exhibited the presence of unique CO oxidizing gene clusters encoding CODH (cooFS), CO-Hyd (cooMKLXUH) and a putative CO-responsive transcriptional activator (cooA). To improve CO-dependent H-2 production activity, we developed recombinant Y19 by homologously over-expressing cooA. The overexpression of cooA improved the whole-cell CO-dependent H-2 production activity (3.4-fold), and enzyme activities of CODH (5.3-fold) and CO-Hyd (1.2-fold). Furthermore, quantitative PCR analysis revealed a significant increase in the transcription of the genes located in CODH and CO-Hyd operons of recombinant Y19. The high CO-dependent H-2 production activity of the recombinant C. amalonaticus was stably maintained during repeated exposure to CO

Analysis and characterization of coenzyme B12 biosynthetic gene clusters and improvement of B12 biosynthesis in Pseudomonas denitrificans ATCC 13867

Coenzyme B12 is an essential cofactor for many enzymes such as glycerol dehydratase, methionine synthase and methylmalonyl-CoA mutase. Herein, we revisited the B12 biosynthetic gene clusters (I and II) in Pseudomonas denitrificans, a well-known industrial producer of the coenzyme B12, to understand the regulation of gene expression and improve the production of coenzyme B12. There were eight operons, seven in cluster I and one in cluster II, and four operons were regulated by B12-responsive riboswitches with a switch-off concentration at ???5 nM coenzyme B12. DNA sequences of the four riboswitches were partially removed, individually or in combination, to destroy the structures of riboswitches, but no improvement was observed. However, when the whole length of riboswitches in cluster I were completely removed and promoters regulated by the riboswitches were replaced with strong constitutive ones, B12 biosynthesis was improved by up to 2-fold. Interestingly, modification of the promoter region for cluster II, where many (>10) late genes of B12 biosynthesis belong, always resulted in a significant, greater than 6-fold reduction in B12 biosynthesis

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

Production of 3-hydroxypropionic acid from glycerol by recombinant Klebsiella pneumoniae ?dhaT?yqhD 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 ?dhaT?yqhD overexpressing PuuC could produce similar to 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 ?dhaT?yqhD, 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: 511524