Fermentation and evaluation of Klebsiella pneumoniae and K. oxytoca on the production of 2,3-butanediol

Klebsiella is one of the genera that has shown unbeatable production performance of 2,3-butanediol (2,3-BD), when compared to other microorganisms. In this study, two Klebsiella strains, K. pneumoniae (DSM 2026) and K. oxytoca (ATCC 43863), were selected and evaluated for 2,3-BD production by batch and fed-batch fermentations using glucose as a carbon source. Those strains' morphologies, particularly their capsular structures, were analyzed by scanning electron microscopy (SEM). The maximum titers of 2,3-BD by K. pneumoniae and K. oxytoca during 10 h batch fermentation were 17.6 and 10.9 g L(-1), respectively; in fed-batch cultivation, the strains showed the maximum titers of 50.9 and 34.1 g L(-1), respectively. Although K. pneumoniae showed higher productivity, SEM showed that it secreted large amounts of capsular polysaccharide, increasing pathogenicity and hindering the separation of cells from the fermentation broth during downstream processing.     

Metabolic engineering of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> and in silico investigation for enhanced 2,3-butanediol production

Objectives

To improve the production of 2,3-butanediol (2,3-BD) in Klebsiella pneumoniae, the genes related to the formation of lactic acid, ethanol, and acetic acid were eliminated.

Results

Although the cell growth and 2,3-BD production rates of the K. pneumoniae ΔldhA ΔadhE Δpta-ackA strain were lower than those of the wild-type strain, the mutant produced a higher titer of 2,3-BD and a higher yield in batch fermentation: 91 g 2,3-BD/l with a yield of 0.45 g per g glucose and a productivity of 1.62 g/l.h in fed-batch fermentation. The metabolic characteristics of the mutants were consistent with the results of in silico simulation.

Conclusions

K. pneumoniae knockout mutants developed with an aid of in silico investigation could produce higher amounts of 2,3-BD with increased titer, yield, and productivity.

     

Development and evaluation of efficient recombinant Escherichia coli strains for the production of 3‐hydroxypropionic acid from glycerol

3‐Hydroxypropionic acid (3‐HP) is a commercially valuable chemical with the potential to be a key building block for deriving many industrially important chemicals. However, its biological production has not been well documented. Our previous study demonstrated the feasibility of producing 3‐HP from glycerol using the recombinant Escherichia coli SH254 expressing glycerol dehydratase (DhaB) and aldehyde dehydrogenase (AldH), and reported that an “imbalance between the two enzymes” and the “instability of the first enzyme DhaB” were the major factors limiting 3‐HP production. In this study, the efficiency of the recombinant strain(s) was improved by expressing DhaB and AldH in two compatible isopropyl‐thio‐β‐galactoside (IPTG) inducible plasmids along with glycerol dehydratase reactivase (GDR). The expression levels of the two proteins were measured. It was found that the changes in protein expression were associated with their enzymatic activity and balance. While cloning an alternate aldehyde dehydrogenase (ALDH), α‐ketoglutaric semialdehyde dehydrogenase (KGSADH), instead of AldH, the recombinant E. coli SH‐BGK1 showed the highest level of 3‐HP production (2.8 g/L) under shake‐flask conditions. When an aerobic fed‐batch process was carried out under bioreactor conditions at pH 7.0, the recombinant SH‐BGK1 produced 38.7 g 3‐HP/L with an average yield of 35%. This article reports the highest level of 3‐HP production from glycerol thus far. Biotechnol. Bioeng. 2009; 104: 729–739 © 2009 Wiley Periodicals, Inc.     

Ribonuclease III cleavage of a bacteriophage T7 processing signal. Divalent cation specificity, and specific anion effects.

Escherichia coli ribonuclease III, purified to homogeneity from an overexpressing bacterial strain, exhibits a high catalytic efficiency and thermostable processing activity in vitro. The RNase III-catalyzed cleavage of a 47 nucleotide substrate (R1.1 RNA), based on the bacteriophage T7 R1.1 processing signal, follows substrate saturation kinetics, with a Km of 0.26 microM, and kcat of 7.7 min.-1 (37 degrees C, in buffer containing 250 mM potassium glutamate and 10 mM MgCl2). Mn2+ and Co2+ can support the enzymatic cleavage of the R1.1 RNA canonical site, and both metal ions exhibit concentration dependences similar to that of Mg2+. Mn2+ and Co2+ in addition promote enzymatic cleavage of a secondary site in R1.1 RNA, which is proposed to result from the altered hydrolytic activity of the metalloenzyme (RNase III 'star' activity), exhibiting a broadened cleavage specificity. Neither Ca2+ nor Zn2+ support RNase III processing, and Zn2+ moreover inhibits the Mg(2+)-dependent enzymatic reaction without blocking substrate binding. RNase III does not require monovalent salt for processing activity; however, the in vitro reactivity pattern is influenced by the monovalent salt concentration, as well as type of anion. First, R1.1 RNA secondary site cleavage increases as the salt concentration is lowered, perhaps reflecting enhanced enzyme binding to substrate. Second, the substitution of glutamate anion for chloride anion extends the salt concentration range within which efficient processing occurs. Third, fluoride anion inhibits RNase III-catalyzed cleavage, by a mechanism which does not involve inhibition of substrate binding.     

Thrombin-reactive polypeptides of platelets may regulate inhibition of thrombin by antithrombin

The central enzyme involved in blood coagulation and activation of platelets is the serine proteinase thrombin. The principal inhibitor of this proteinase in plasma is antithrombin. The mechanism of regulation of the thrombin-antithrombin reaction remains unknown. Two polypeptides of 74 and 55 kDa present on the platelet surface and in plasma are known to specifically enhance the activity of thrombin on different substrates. This study was undertaken to assess the effects of these platelet proteins on thrombin-antithrombin interaction. Direct measurements of residual thrombin activity in mixtures of thrombin and antithrombin, in the presence or absence of the platelet proteins, were made utilizing a specific chromogenic substrate. Under these conditions, when 60% of thrombin activity was inhibited by antithrombin in controls, 100% of enzyme activity was retained in the presence of the platelet proteins. When heparin was used in these assays, the rate of inhibition of thrombin by antithrombin was much more rapid and 62% of thrombin activity remained after 1 min. Under these conditions, the platelet proteins continued to protect thrombin from inactivation with 98% activity remaining at 1 min and 85% activity at 5 min. In contrast, the inhibition of trypsin by antithrombin was not affected by the platelet proteins. Additional studies in platelet aggregation showed that the platelet polypeptides have two effects on thrombin: (i) protection of the enzyme inhibition by antithrombin and (ii) stabilization of thrombin from loss of activity due to aging. The results suggest a novel role for the platelet proteins in hemostasis - regulation of the inhibition of thrombin by antithrombin.     

Isolation of a novel Pseudomonas species SP2 producing vitamin B12 under aerobic condition

Vitamin B₁₂ is a complex biomolecule that acts as a cofactor for a variety of enzymes in microbial metabolism. Pseudomonas denitrificans is exclusively used as an industrial strain for the production of vitamin B₁₂ under aerobic conditions. However, only a few strains of Pseudomonas have been reported to possess the capability of producing this vitamin and they are strongly patent-protected. To improve the applicability of the vitamin B₁₂-producing microorganisms, a new isolate was obtained from municipal waste samples and characterized for its biological properties. The new isolate, designated as SP2, was identified to be a Pseudomonas species based on the sequence homology of its 16S rDNA. Pseudomonas species SP2 had essential genes for vitamin B₁₂ synthesis such as cobB and cobQ and produced a similar amount of vitamin B₁₂ (10.6 ± 0.05 μg/mL) as P. denitrificans ATCC 13867 in 24 h flask culture. SP2 grew well under aerobic condition with the maximum specific growth rate (µ _max ) of 0.91 ± 0.03/h, but showed a poor growth under micro-aerobic conditions. SP2 was resistant to antibiotics like streptomycin, carbenicillin, ampicillin, cefpodoxime, colistin, nalidixic acid and sparfloxacin. The ability of SP2 to grow faster and produce vitamin B₁₂ under aerobic conditions makes it a promising host for the production of some biochemicals requiring a coenzyme B₁₂-dependent enzyme, such as glycerol dehydratase.     

Cloning, expression, and characterization of an aldehyde dehydrogenase from Escherichia coli K-12 that utilizes 3-Hydroxypropionaldehyde as a substrate

For efficient production of isoflavone aglycones from soybean isoflavones, we isolated three novel types of β-glucosidase (BGL1, BGL3, and BGL5) from the filamentous fungi Aspergillus oryzae. Three enzymes were independently displayed on the cell surface of a yeast Saccharomyces cerevisiae as a fusion protein with α-agglutinin. Three β-glucosidase-displaying yeast strains hydrolyzed isoflavone glycosides efficiently but exhibited different substrate specificities. Among these β-glucosidases, BGL1 exhibited the highest activity and also broad substrate specificity to isoflavone glycosides. Although glucose released from isoflavone glycosides are generally known to inhibit β-glucosidase, the residual ratio of isoflavone glycosides in the reaction mixture with BGL1-displaying yeast strain (Sc-BGL1) reached approximately 6.2%, and the glucose concentration in the reaction mixture was maintained at lower level. This result indicated that Sc-BGL1 assimilated the glucose before they inhibited the hydrolysis reaction, and efficient production of isoflavone aglycones was achieved by engineered yeast cells displaying β-glucosidase.     

Release of cytochrome c and activation of pro-caspase-9 following lysosomal photodamage involves Bid cleavage

Photodynamic therapy (PDT) protocols employing lysosomal sensitizers induce apoptosis via a mechanism that causes cytochrome c release prior to loss of mitochondrial membrane potential (DeltaPsi(m)). The current study was designed to determine how lysosomal photodamage initiates mitochondrial-mediated apoptosis in murine hepatoma 1c1c7 cells. Fluorescence microscopy demonstrated that the photosensitizer N-aspartyl chlorin e6 (NPe6) localized to the lysosomes. Irradiation of cultures preloaded with NPe6 induced the rapid destruction of lysosomes, and subsequent cleavage/activation of Bid, pro-caspases-9 and -3. Pro-caspase-8 was not activated. Release of cytochrome c occurred at about the time of Bid cleavage and preceded the loss of DeltaPsi(m). Extracts of purified lysosomes catalyzed the in vitro cleavage of cytosolic Bid, but not pro-caspase-3 activation. Pharmacological inhibition of cathepsin B, L and D activities did not suppress Bid cleavage or pro-caspases-9 and -3 activation. These studies demonstrate that photodamaged lysosomes trigger the mitochondrial apoptotic pathway by releasing proteases that activate Bid.