The microbial production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae involves the formation of various by-products, which are synthesized through the oxidative pathway. To eliminate the by-products synthesis, the oxidative branch of glycerol metabolism was inactivated by constructing two mutant strains. In one of the mutant strains, the structural genes encoding glycerol dehydrogenase and dihydroxyacetone kinase were deleted from the chromosomal DNA, whereas in the second mutant strain dhaR, which is a putative transcription factor that activates, gene expression was deleted from the chromosomal DNA. In the resultant mutant strains lacking the dhaT gene encoding 1,3-PD oxidoreductase, which was simultaneously deleted while replacing the native promoter with the lacZ promoter, the by-product formation except for acetate was eliminated, but it still produced 1,3-PD at a lower level, which might be due to a putative oxidoreductase that catalyzes the production of 1,3-PD. The recombinant strains in which the reductive pathway was recovered produced slightly lower amount of 1,3-PD as compared to the parent strain, which might be due to the reduced activity of DhaB caused by the substitution of the promoter. However, the production yield was higher in the recombinant strain (0.57 mol mol(-1)) than the wild type Cu strain (0.47 mol mol(-1)).
To investigate the effect of cellular fatty acids composition on ethanol tolerance in Escherichia coli, we overexpressed either des, encoding fatty acid desaturase from Bacillus subtilis, or fabA, encoding beta-hydroxydecanoyl thio-ester dehydrase from E. coli, or both genes together, into E. coli. Recombinant E. coli harboring fabA had elevated tolerance against ethanol compared to wild type strain. In contrast, des decreased resistance to ethanol. Co-expression of both genes together complemented ethanol tolerance of E. coli. This result indicates how to engineer bacterial strains to be resistant to higher concentrations of ethanol.
Although the de novo biosynthetic mechanism of 3-hydroxypropionic acid (3-HP) in glycerol-fermenting microorganisms is still unclear, the propanediol utilization protein (PduP) of Lactobacillus species has been suggested to be a key enzyme in this regard. To verify this hypothesis, a pduP gene from Lactobacillus reuteri was cloned and expressed, and the encoded protein was characterized. Recombinant L. reuteri PduP exhibited broad substrate specificity including 3-hydroxypropionaldehyde and utilized both NAD(+) and NADP(+) as a cofactor. Among various aldehyde substrates tested, the specific activity was highest for propionaldehyde, at pH 7.8 and 37 °C. The K(m) and V(max) values for propionaldehyde in the presence of NAD(+) were 1.18 mM and 0.35 U mg⁻¹, respectively. When L. reuteri pduP was overexpressed in Klebsiella pneumoniae, 3-HP production remarkably increased as compared to the wild-type strain (from 0.18 g L⁻¹ to 0.72 g L⁻¹) under shake-flask culture conditions, and the highest titer (1.38 g L⁻¹ 3-HP) was produced by the recombinant strain under batch fermentation conditions in a bioreactor. This is the first report stating the enzymatic properties of PduP protein and the probable role in biosynthesis of 3-HP in glycerol fermentation.
Results to date suggest that microalgal Thraustochytrids family strains can be used to produce high-functional omega-3 rich oil (~ 30-70% of dry cell weight) and carotenoid-based antioxidant pigments simultaneously with value-added bioactive potential. In the present study, we describe the isolation and characterization of a new Thraustochytrid Schizochytrium sp. from the west coastal area of Korea. This newly isolated Thraustochytrid, identified as Schizochytrium sp. through 18S rRNA analysis and named SH104, simultaneously produces high levels of DHA and carotenoid-based antioxidant pigments. An improved Schizochytrium mutant, named SHG104, was obtained from the original host strain by γ-irradiation-induced mutagenesis. Under combined temperature-shift cultivation conditions employing white-light LEDs (light-emitting diodes), Schizochytrium sp. SHG104 yielded 10.8 g L of biomass comprising 45.8% total lipids (32.1% DHA) and 4.6 mg L of astaxanthin. In addition to DHA, the main fatty acids produced by Schizochytrium sp. SHG104 were palmitic acid and a trace of other long-chain fatty acids. The carotenoid profile of SH104 and SHG104 was β-carotene, astaxanthin, canthaxanthin, pheonicoxanthin and echinenone, which analyzed by HPLC and LC/APCI-MS. Furthermore, genomic analysis of Schizochytrium and Aurantiochytrium microalgae confirmed that the presence of carotenogenesis pathway enzymes and genes including geranylgeranyl diphosphate, phytoene synthase, lycopene cyclase, and cytochrome P450 hydroxylase that necessary for the production of antioxidants via a complete biosynthetic KEGG synthesis pathway. This newly isolated Schizochytrium microalga potentially have wide application as a source of antioxidants for astaxanthin-containing pigments, commercial omega-3 lipids and feed additives, such as nutritional supplements for aquaculture.
In the present work, mutant strains of Klebsiella pneumoniae with deletions of the als gene encoding acetolactate synthase involved in synthesis of 2,3-butanediol, the ldhA gene encoding lactate dehydrogenase required for lactate synthesis, or both genes, were prepared. Production of 1,3-propanediol (1,3-PD) from glycerol was enhanced in the ldhA mutant strain (ΔldhA), but lower in Δals or Δals ΔldhA mutant strains compared to the parent strain, concomitant with a reduction in the glycerol consumption rate, indicating that deletion of ldhA alone was useful to improve 1,3-PD production. Fed-batch fermentation analysis revealed that, in the ΔldhA mutant strain, 1,3-PD production was higher at low pH than at neutral pH; the reverse was true for the parent strain. Further optimization of culture conditions, by variation of aeration and glycerol feed rates, dramatically improved the production of 1,3-PD by the mutant strain. The maximum level attained was 102.7 g l(-1) of 1,3-PD from glycerol.
Background1,3-Propanediol (1,3-PDO) is important building blocks for the bio-based chemical industry, Klebsiella pneumoniae can be an attractive candidate for their production. However, 1,3-PDO production is high but productivity is generally low by K. pneumoniae. In this study, repeated fed-batch cultivation by a lactate and 2,3-butanediol (2,3-BDO) deficient mutant of K. pneumoniae were investigated for efficient 1,3-PDO production from industrial by-products such as crude glycerol.ResultsFirst, optimal conditions for repeated fed-batch fermentation of a ΔldhA mutant defective for lactate formation due to deletion of the lactate dehydrogenase gene (ldhA) were determined. Maximal 1,3-PDO production level and productivity obtained by repeated fed-batch fermentation under optimized conditions were 81.1 g/L and 3.38 g/L/h, respectively, and these values were successfully maintained for five cycles of fermentation without any loss of fermentation capacity. This results were much higher than that of the normal fed-batch fermentation. The levels of 2,3-BDO, which is a major by-product, reaching up to ~ 50% of the level of 1,3-PDO, were reduced using a mutant strain [Δ(ldhA als)] containing an additional mutation in the biosynthetic pathway of 2,3-BDO (deletion of the acetolactate synthase gene). The levels of 2,3-BDO were reduced to about 20% of 1,3-PDO levels by repeated fed-batch fermentation of Δ(ldhA als), although maximal 1,3-PDO production and productivity also decreased owing to a defect in the growth of the 2,3-BDO-defective mutant strain.ConclusionThis repeated fed-batch fermentation may be useful for reducing the cost of 1,3-PDO production and may be promising industrialization prospect for the 1,3-PDO production.
In a previous study, we showed that 1,3-propanediol (1,3-PD) was still produced from glycerol by the Klebsiella pneumoniae mutant strain defective in 1,3-PD oxidoreductase (DhaT), although the production level was lower compared to the parent strain. As a potential candidate for another putative 1,3-PD oxidoreductase, we identified and characterized a homolog of Escherichia coli yqhD (88% homology in amino acid sequence), which encodes an alcohol dehydrogenase and is well known to replace the function of DhaT in E. coli. Introduction of multiple copies of the yqhD homolog restored 1,3-PD production in the mutant K. pneumoniae strain defective in DhaT. In addition, by-product formation was still eliminated in the recombinant strain due to the elimination of the glycerol oxidative pathway. An increase in NADP-dependent 1,3-PD oxidoreductase activity was observed in the recombinant strain harboring multiple copies of the yqhD homolog. The level of 1,3-PD production during batch fermentation in the recombinant strain was comparable to that of the parent strain; further engineering can generate an industrial strain producing 1,3-propanediol.
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