Functional analysis of arabinofuranosidases and a xylanase of <Emphasis Type="Italic">Corynebacterium alkanolyticum</Emphasis> for arabinoxylan utilization in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Xylooligosaccharides (XOSs) and arabinoxylooligosaccharides (AXOSs) are major oligosaccharides derived from arabinoxylan. In our previous report, Corynebacterium glutamicum was engineered to utilize XOSs by introducing Corynebacterium alkanolyticum xyloside transporter and β-xylosidase. However, this strain was unable to consume AXOSs due to the absence of α-l-arabinofuranosidase activity. In this study, to confer AXOS utilization ability on C. glutamicum, two putative arabinofuranosidase genes (abf51A and abf51B) were isolated from C. alkanolyticum by the combination of degenerate PCR and genome walking methods. Recombinant Abf51A and Abf51B heterologously expressed in Escherichia coli showed arabinofuranosidase activities toward 4-nitrophenyl-α-l-arabinofuranoside with k _cat values of 150 and 63, respectively, with optimum at pH 6.0 to 6.5. However, Abf51A showed only a slight activity toward AXOSs and was more susceptible to product inhibition by arabinose and xylose than Abf51B. Introduction of abf51B gene into the C. glutamicum XOS-utilizing strain enabled it to utilize AXOSs as well as XOSs. The xylI gene encoding a putative xylanase was found upstream of the C. alkanolyticum xyloside transporter genes. A signal peptide was predicted at the N-terminus of the xylI-encoding polypeptide, which indicated XylI was a secreted protein. Recombinant mature XylI protein heterologously expressed in E. coli showed a xylanase activity toward xylans from various plant sources with optimum at pH 6.5, and C. glutamicum recombinant strain expressing native XylI released xylose, xylobiose, xylotriose, and arabino-xylobiose from arabinoxylan. Finally, introduction of the xylI gene into the C. glutamicum AXOS-utilizing strain enabled it to directly utilize arabinoxylan.     

Cloning,purification and characterization of a cellulase-free xylanase from <Emphasis Type="Italic">Geobacillus thermodenitrificans</Emphasis> AK53

Geobacillus thermodenitrificans AK53 xyl gene encoding xylanase was isolated, cloned and expressed in Escherichia coli. After purifying recombinant xylanase from G. thermodenitrificans AK53 (GthAK53Xyl) to homogeneity by ammonium sulfate precipitation and ion exchange chromatography, biochemical properties of the enzyme were determined. The kinetic studies for GthAK53Xyl showed K_M value to be 4.34 mg/mL (for D-xylose) and V_max value to be 2028.9 μmoles mg^–1 min^–1. The optimal temperature and pH for enzyme activity were found out to be 70°C and 5.0, respectively. The expressed protein showed the highest sequence similarity with the xylanases of G. thermodenitrificans JK1 (JN209933) and G. thermodenitrificans T-2 (EU599644). Metal cations Mg²⁺ and Mn²⁺ were found to be required for the enzyme activity, however, Co²⁺, Hg²⁺, Fe²⁺ and Cu²⁺ ions caused inhibitor effect on it. GthAK53Xyl had no cellulolytic activity and degraded xylan in an endo-fashion. The action of the enzyme on xylan from oat spelt produced xylobiose and xylopentose. The reported results are suggestive of a xylanase exhibiting desirable kinetics, stability parameters and metal resistance required for the efficient production of xylobiose at industrial scale.     

<Emphasis Type="Italic">In vitro</Emphasis> Blastogenesis inhibited by <Emphasis Type="Italic">Erwinia carotovora</Emphasis><Emphasis Type="SmallCaps">L</Emphasis>-Asparaginase

Escherichia coliL-asparaginase, an antileukaemic agent in man¹, inhibits in vitro mitogen or antigen-induced blastogenesis in man^2,3 and in animals (M. Bennett, E. G. Mayhew and T. Han, unpublished data) and suppresses bone-marrow derived antibody precursor cells in the mouse⁴. We now report that another L-asparaginase preparation—from Erwinia carotovora—also possesses antileukaemic activity^5,6 and has a more pronounced immunosuppressive effect on in vitro blastogenesis than the E. coli enzyme.     

<Emphasis Type="Italic">C7</Emphasis>-prenylation of tryptophanyl and <Emphasis Type="Italic">O</Emphasis>-prenylation of tyrosyl residues in dipeptides by an <Emphasis Type="Italic">Aspergillus terreus</Emphasis> prenyltransferase

During our search for novel prenyltransferases, a putative gene ATEG_04218 from Aspergillus terreus raised our attention and was therefore amplified from strain DSM 1958 and expressed in Escherichia coli. Biochemical investigations with the purified recombinant protein and different aromatic substrates in the presence of dimethylallyl diphosphate revealed the acceptance of all the tested tryptophan-containing cyclic dipeptides. Structure elucidation of the main enzyme products by NMR and MS analyses confirmed the attachment of the prenyl moiety to C-7 of the indole ring, proving the identification of a cyclic dipeptide C7-prenyltransferase (CdpC7PT). For some substrates, reversely C3- or N1-prenylated derivatives were identified as minor products. In comparison to the known tryptophan-containing cyclic dipeptide C7-prenyltransferase CTrpPT from Aspergillus oryzae, CdpC7PT showed a much higher substrate flexibility. It also accepted cyclo-l-Tyr-l-Tyr as substrate and catalyzed an O-prenylation at the tyrosyl residue, providing the first example from the dimethylallyltryptophan synthase (DMATS) superfamily with an O-prenyltransferase activity towards dipeptides. Furthermore, products with both C7-prenyl at tryptophanyl and O-prenyl at tyrosyl residue were detected in the reaction mixture of cyclo-l-Trp-l-Tyr. Determination of the kinetic parameters proved that (S)-benzodiazepinedione consisting of a tryptophanyl and an anthranilyl moiety was accepted as the best substrate with a K _M value of 204.1 μM and a turnover number of 0.125 s^?1. Cyclo-l-Tyr-l-Tyr was accepted with a K _M value of 1,411.3 μM and a turnover number of 0.012 s^?1.     

Expression and characterization of glutamate decarboxylase from <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> HYE1 isolated from <Emphasis Type="Italic">kimchi</Emphasis>

A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni–NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including K_m and V_max values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme.     

<Emphasis Type="Italic">Paenibacillus bryophyllum</Emphasis> sp. nov., a nitrogen-fixing species isolated from <Emphasis Type="Italic">Bryophyllum pinnatum</Emphasis>

A nitrogen-fixing, endospore-forming bacterium, designated strain L201^T was isolated from the leaves of Bryophyllum pinnatum growing in South China Agricultural University. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain L201^T is affiliated with the genus Paenibacillus, and closely related to Paenibacillus albidus Q4-3^T (97.4%), Paenibacillus odorifer DSM 15391^T (97.3%) and Paenibacillus borealis DSM 13188^T (97.2%). The main fatty acids components was anteiso-C_15:0 (48.1%). The predominant isoprenoid quinone was MK-7. The major polar lipids were found to be diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The G+C content of strain L201^T was 43.9%. DNA–DNA relatedness between L201^T and the reference strain was 29.8%. Biological and biochemical tests, protein patterns, genomic DNA fingerprinting and comparison of cellular fatty acids distinguished strain L201^T from the closely related Paenibacillus species. Based on these data, the novel species Paenibacillus bryophyllum sp. nov. is proposed, with the type strain L201^T(=?KCTC 33951 ^T?=?GDMCC 1.1251 ^T).     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

Synthesis of rosmarinic acid analogues in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To produce rosmarinic acid analogues in the recombinant Escherichia coli BLRA1, harboring a 4-coumarate: CoA ligase from Arabidopsis thaliana (At4CL) and a rosmarinic acid synthase from Coleus blumei (CbRAS).

Results

Incubation of the recombinant E. coli strain BLRA1 with exogenously supplied phenyllactic acid (PL) and analogues as acceptor substrates, and coumaric acid and analogues as donor substrates led to production of 18 compounds, including 13 unnatural RA analogues.

Conclusion

This work demonstrates the viability of synthesizing a broad range of rosmarinic acid analogues in E. coli, and sheds new light on the substrate specificity of CbRAS.

     

Characterization of the biochemical properties of recombinant Xyn10C from a marine bacterium, <Emphasis Type="Italic">Saccharophagus degradans</Emphasis> 2-40

Endo-1,4-β-xylanases are mostly classified into glycoside hydrolase (GH) family 10 or 11. In this study, we examined the catalytic functions of a recombinant endo-1,4-β-xylanase belonging to GH10 (Xyn10C) from a marine bacterium, Saccharophagus degradans 2-40. Optimal activity of this enzyme was evident at 30 °C and pH 7.0, but activity remained even at low temperatures, indicating its adaptation to cold. With respect to other xylanases known to be active in cold temperatures, Xyn10C is unique in that it showed maximal activity in the presence of 2 M of NaCl. The action patterns of recombinant Xyn10C on xylans from hardwood and softwood differed in part, but the enzyme hydrolyzed polysaccharidic substrates primarily to xylobiose and xylotriose through xylo-oligosaccharides, releasing a small amount of xylose. The K _m and V _max values on birchwood xylan were 10.4 mg mL^?1 and 253 µmol mg^?1 min^?1, respectively. The efficient catalytic function of Xyn10C on short-length xylo-oligosaccharide chains was similar to the typical function of other known GH10 xylanases.     

Cloning of <Emphasis Type="Italic">phaCAB</Emphasis> genes from thermophilic <Emphasis Type="Italic">Caldimonas manganoxidans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> for poly(3-hydroxybutyrate) (PHB) production

PHB biosynthesis pathway, consisting of three open reading frames (ORFs) that encode for β-ketothiolase (phaA _Cma , 1179 bp), acetoacetyl-CoA reductase (phaB _Cma , 738 bp), and PHA synthase (phaC _Cma , 1694 bp), of Caldimonas manganoxidans was identified. The functions of PhaA, PhaB, and PhaC were demonstrated by successfully reconstructing PHB biosynthesis pathway of C. manganoxidans in Escherichia coli, where PHB production was confirmed by OD₆₀₀, gas chromatography, Nile blue stain, and transmission electron microscope (TEM). The protein sequence alignment of PHB synthases revealed that phaC _Cma shares at least 60% identity with those of class I PHB synthase. The effects of PhaA, PhaB, and PhaC expression levels on PHB production were investigated. While the overexpression of PhaB is found to be important in recombinant E. coli, performances of PHB production can be quantified as follows: PHB concentration of 16.8 ± 0.6 g/L, yield of 0.28 g/g glucose, content of 74%, productivity of 0.28 g/L/h, and Mw of 1.41 MDa.     

<Emphasis Type="Italic">Paenibacillus seodonensis</Emphasis> sp. nov., isolated from a plant of the genus <Emphasis Type="Italic">Campanula</Emphasis>

Strain DCT-19^T, representing a Gram-stain-positive, rodshaped, aerobic bacterium, was isolated from a native plant belonging to the genus Campanula on Dokdo, the Republic of Korea. Comparative analysis of the 16S rRNA gene sequence showed that this strain was closely related to Paenibacillus amylolyticus NRRL NRS-290^T (98.6%, 16S rRNA gene sequence similarity), Paenibacillus tundrae A10b^T (98.1%), and Paenibacillus xylanexedens NRRL B-51090^T (97.6%). DNADNA hybridization indicated that this strain had relatively low levels of DNA-DNA relatedness with P. amylolyticus NRRL NRS-290^T (30.0%), P. xylanexedens NRRL B-51090^T (29.0%), and P. tundrae A10b^T (24.5%). Additionally, the genomic DNA G + C content of DCT-19^T was 44.8%. The isolated strain grew at pH 6.0–8.0 (optimum, pH 7.0), 0–4% (w/v) NaCl (optimum, 0%), and a temperature of 15–45°C (optimum 25–30°C). The sole respiratory quinone in the strain was menaquinone-7, and the predominant fatty acids were C_15:0 anteiso, C_16:0 iso, and C_16:0. In addition, the major polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. Based on its phenotypic properties, genotypic distinctiveness, and chemotaxonomic features, strain DCT-19^T is proposed as a novel species in the genus Paenibacillus, for which the name Paenibacillus seodonensis sp. nov. is proposed (=KCTC 43009^T =LMG 30888^T). The type strain of Paenibacillus seodonensis is DCT-19^T.     

The <Emphasis Type="Italic">yajC</Emphasis> gene from <Emphasis Type="Italic">Lactobacillus buchneri</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> and its role in ethanol tolerance

The yajC gene (Lbuc_0921) from Lactobacillus buchneri NRRL B-30929 was identified from previous proteomics analyses in response to ethanol treatment. The YajC protein expression was increased by 15-fold in response to 10 % ethanol vs 0 % ethanol. The yajC gene encodes the smaller subunit of the preprotein translocase complex, which interacts with membrane protein SecD and SecF to coordinate protein transport and secretion across cytoplasmic membrane in Escherichia coli. The YajC protein was linked to sensitivity to growth temperatures in E. coli, involved in translocation of virulence factors during Listeria infection, and stimulating a T cell-mediated response of Brucella abortus. In this study, the L. buchneri yajC gene was over-expressed in E. coli. The strain carrying pET28byajC that produces YajC after isopropyl β-d-1-thiogalactopyranoside induction showed tolerance to 4 % ethanol in growth media, compared to the control carrying pET28b. This is the first report linking YajC to ethanol stress and tolerance.     

Engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the synthesis of <Emphasis Type="Italic">N</Emphasis>-hydroxycinnamoyl tryptamine and serotonin

Plants synthesize various phenol amides. Among them, hydroxycinnamoyl (HC) tryptamines and serotonins exhibit antioxidant, anti-inflammatory, and anti-atherogenic activities. We synthesized HC–tryptamines and HC–serotonin from several HCs and either tryptamine or serotonin using Escherichia coli harboring the 4CL (4-coumaroyl CoA ligase) and CaHCTT [hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase] genes. E. coli was engineered to synthesize N-cinnamoyl tryptamine from glucose. TDC (tryptophan decarboxylase) and PAL (phenylalanine ammonia lyase) along with 4CL and CaHCTT were introduced into E. coli and the phenylalanine biosynthetic pathway of E. coli was engineered. Using this strategy, approximately 110.6 mg/L of N-cinnamoyl tryptamine was synthesized. By feeding 100 μM serotonin into the E. coli culture, which could induce the synthesis of cinnamic acid or p-coumaric acid, more than 99 μM of N-cinnamoyl serotonin and N-(p-coumaroyl) serotonin were synthesized.     

Screening and molecular identification of lactic acid bacteria from <Emphasis Type="Italic">gari</Emphasis> and <Emphasis Type="Italic">fufu</Emphasis> and <Emphasis Type="Italic">gari</Emphasis> effluents

Bacterial strains were isolated from cassava-derived food products and, for the first time, from cassava by-products, with a focus on gari, a flour-like product, and the effluents from the production processes for gari and fufu (a dough also made from cassava flour). A total of 47 strains were isolated, all of which were tested to determine their resistance to acidic pH and to bile salt environments. Four of the 47 isolates tested positive in both environments, and these four isolates also showed antibacterial behaviour towards both Gram-positive and Gram-negative microbial pathogens (i.e. Methicillin-resistance Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Salmonella enteritidis, Escherichia coli, Escherichia coli (O157), Yersinia enterocolitica). In most cases, the antibacterial activity was related to bacteriocin production. Molecular identification analysis (16S rDNA and randomly amplified polymorphic DNA-PCR) revealed that the four isolates were different strains of the same species, Lactobacillus fermentum. These results demonstrate that bacteria isolated from cassava-derived food items and cassava by-products have interesting properties and could potentially be used as probiotics.     

Engineering chimeric two-component system into <Emphasis Type="Italic">Escherichia coli</Emphasis> from <Emphasis Type="Italic">Paracoccus denitrificans</Emphasis> to sense methanol

Escherichia coli does not have the methanol sensing apparatus, was engineered to sense methanol by employing chimeric two-component system (TCS) strategy. A chimeric FlhS/EnvZ (FlhSZ) chimeric histidine kinase (HK) was constructed by fusing the sensing domain of Paracoccus denitrificans FlhS with the catalytic domain of E. coli EnvZ. The constructed chimeric TCS FlhSZ/OmpR could sense methanol by the expression of ompC and gfp gene regulated by ompC promoter. Real-time quantitative PCR analysis and GFP-based fluorescence analysis showed the dynamic response of the chimeric TCS to methanol. The expression of ompC and the gfp fluorescence was maximum at 0.01 and 0.5% of methanol, respectively. These results suggested that E. coli was successfully engineered to sense methanol by the introduction of chimeric HK FlhSZ. This strategy can be employed for the construction of several chimeric TCS based bacterial biosensors for the development of biochemical producing recombinant microorganisms.     

1,4-Dioxane degradation potential of members of the genera <Emphasis Type="Italic">Pseudonocardia</Emphasis> and <Emphasis Type="Italic">Rhodococcus</Emphasis>

In recent years, several strains capable of degrading 1,4-dioxane have been isolated from the genera Pseudonocardia and Rhodococcus. This study was conducted to evaluate the 1,4-dioxane degradation potential of phylogenetically diverse strains in these genera. The abilities to degrade 1,4-dioxane as a sole carbon and energy source and co-metabolically with tetrahydrofuran (THF) were evaluated for 13 Pseudonocardia and 12 Rhodococcus species. Pseudonocardia dioxanivorans JCM 13855^T, which is a 1,4-dioxane degrading bacterium also known as P. dioxanivorans CB1190, and Rhodococcus aetherivorans JCM 14343^T could degrade 1,4-dioxane as the sole carbon and energy source. In addition to these two strains, ten Pseudonocardia strains could degrade THF, but no Rhodococcus strains could degrade THF. Of the ten Pseudonocardia strains, Pseudonocardia acacia JCM 16707^T and Pseudonocardia asaccharolytica JCM 10410^T degraded 1,4-dioxane co-metabolically with THF. These results indicated that 1,4-dioxane degradation potential, including degradation for growth and by co-metabolism with THF, is possessed by selected strains of Pseudonocardia and Rhodococcus, although THF degradation potential appeared to be widely distributed in Pseudonocardia. Analysis of soluble di-iron monooxygenase (SDIMO) α-subunit genes in THF and/or 1,4-dioxane degrading strains revealed that not only THF and 1,4-dioxane monooxygenases but also propane monooxygenase-like SDIMOs can be involved in 1,4-dioxane degradation.