Mineralization of low-chlorinated biphenyls by Burkholderia sp. strain LB400 and by a two-membered consortium upon directed interspecies transfer of chlorocatechol pathway genes

The biphenyl-mineralizing bacterium Burkholderia sp. strain LB400 also utilized 3-chloro-, 4-chloro-, 2,3′-dichloro- and 2,4′-dichlorobiphenyl for growth. By the attack of the initial enzyme a chlorine was eliminated dioxygenolytically from position 2 of one of the aromatic rings when hydrogens of both were substituted by chlorine. The strain mineralized 3-chloro- and 2,3′-dichlorobiphenyl via the central intermediate 3-chlorobenzoate through its chlorocatechol pathway enzymes, but excreted stoichiometric amounts of 4-chlorobenzoate from 4-chloro- and 2,4^′-dichlorobiphenyl. These two compounds were mineralized by a co-culture of strain LB400 and a derivative of the (methyl-) benzoate-degrading strain Pseudomonas putida mt-2 (TOL). The complete degradation was achieved upon transfer of a cluster of at least five genes, encoding the regulated chlorocatechol pathway operon, from strain LB400 to strain mt-2. This transfer was demonstrated by the polymerase chain reaction. Received: 15 April 1998 / Received revision: 12 June 1998 / Accepted: 19 June 1998     

Genes controlling hydroxylations of phytosiderophores are located on different chromosomes in barley (Hordeum vulgare L.)

Phytosiderophores, mugineic acids, have been demonstrated to be involved in Fe acquisition in gramineous plants. In this study, chromosomal arm locations of genes encoding for biosynthesis of various phytosiderophores were identified in a cultivar of barley (Hordeum vulgare L. cv. Betzes). Using wheat (Triticum aestivum L. cv. Chinese Spring)-barley (cv. Betzes) ditelosomic addition lines for 4HS and 4HL, a gene for hydroxylation of 2′-deoxymugineic acid to mugineic acid was localized to the long arm of barley chromosome 4H. To locate the gene for hydroxylation of mugineic acid to 3-epihydroxymugineic acid, hybrids between the 4H addition line and other wheat-barley addition lines were studied. Only a hybrid between 4H and 7H addition lines produced 3-epihydroxymugineic acid. The gene was further localized to the long arm of chromosome 7H by feeding mugineic acid to ditelosomic addition lines for 7HS and 7HL. A new phytosiderophore was discovered in both 7H and 7HL addition lines, which was identified to be 3-epihydroxy-2′-deoxymugineic acid by detailed nuclear magnetic resonance studies. These results revealed that in barley there are two pathways from 2′-deoxymugineic acid to 3-epihydroxymugineic acid: 2′-deoxymugineic acid → mugineic acid → 3-epihydroxymugineic acid and 2′-deoxymugineic acid → 3-epihydroxy-2′-deoxymugineic acid → 3-epihydroxymugineic acid. Barley genes encoding for the hydroxylations of phytosiderophores are located in different chromosomes and each gene hydroxylates different C-positions: the long arm of chromosome 4H carries the gene for hydroxylating the C-2′ position and the long arm of chromosome 7H carries the gene for hydroxylating the C-3 position of the azetidine ring. Received: 10 August 1998 / Accepted: 30 September 1998     

Cloning and overexpression in Escherichia coli of the gene encoding dihydroxyacetone kinase isoenzyme I from Schizosaccharomyces pombe, and its application to dihydroxyacetone phosphate production

The gene dak1 encoding a dihydroxyacetone kinase (DHAK) isoenzyme I, one of two isoenzymes in the Schizosaccharomyces pombe IFO 0354 strain, was cloned and sequenced. The dak1 gene comprises 1743 bp and encodes a protein of 62 245 Da. The deduced amino acid sequence showed a similarity to a putative DHAK of Saccharomyces cerevisiae and DHAK of Citrobacter freundii. The dak1 gene was expressed at a high level in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The acetone powder of recombinant E. coli cells was used to produce dihydroxyacetone phosphate. Received: 25 August 1998 / Received revision: 22 September 1998 / Accepted: 11 October 1998     

Transformation of N ′,N ′-dimethyl-N -(hydroxyphenyl)ureas by laccase from the white rot fungus Trametes versicolor

Transformation of N′,N′-dimethyl-N-(hydroxyphenyl)ureas was assayed in the presence of purified laccase produced by the fungus Trametes versicolor. The para- and ortho-hydroxyphenyl derivatives were enzymatically transformed, whereas the meta derivative was not. The performance of laccase-mediated transformation depended on the pH, with an optimum for the para-derivative degradation rate at pH 5. The pH also influenced the nature of the reaction products. The chemical was exclusively oxidised into p-benzoquinone at pH 3 and into mainly N′,N′-dimethyl-N-[(2,5-cyclohexadiene-1-one)-4-ylidene]urea at pH 6. The ortho- derivative was transformed essentially into insoluble purple compounds, probably appearing as polymers resulting from coupling of the parent compound. Received: 14 September 1998 / Received revision: 23 November 1998 / Accepted: 29 November 1998     

High-level expression of the angiotensin-converting-enzyme-inhibiting peptide, YG-1, as tandem multimers in Escherichia coli

To produce a large quantity of the angiotensin-converting-enzyme(ACE)-inhibiting peptide YG-1, which consists of ten amino acids derived from yeast glyceraldehyde-3-phosphate dehydrogenase, a high-level expression was explored with tandem multimers of the YG-1 gene in Escherichia coli. The genes encoding YG-1 were tandemly multimerized to 9-mers, 18-mers and 27-mers, in which each of the repeating units in the tandem multimers was connected to the neighboring genes by a DNA linker encoding Pro-Gly-Arg for the cleavage of multimers by clostripain. The multimers were cloned into the expression vector pET-21b, and expressed in E. coli BL21(DE3) with isopropyl β-d-thiogalactopyranoside induction. The expressed multimeric peptides encoded by the 9-mer, 18-mer and 27-mer accumulated intracellularly as inclusion bodies and comprised about 67%, 25% and 15% of the total proteins in E. coli respectively. The multimeric peptides expressed as inclusion bodies were cleaved with clostripain, and active monomers were purified to homogeneity by reversed-phase high-performance liquid chromatography. In total, 105 mg pure recombinant YG-1 was obtained from 1 l E. coli culture harboring pETYG9, which contained the 9-mer of the YG-1 gene. The recombinant YG-1 was identical to the natural YG-1 in molecular mass, amino acid sequence and ACE-inhibiting activity. Received: 6 January 1998 / Received revision: 23 February 1998 / Accepted: 24 February 1998     

Cloning and characterization of an endo-β-1,3(4)glucanase and an aspartic protease from Phaffia rhodozyma CBS 6938

We describe the identification and expression cloning of two novel enzymes, a β-glucanase and an aspartic protease, secreted from the basidiomycetous yeast Phaffia rhodozyma. A cDNA library from P. rhodozyma CBS 6938 was constructed, and full-length cDNA encoding an endo-1,3(4)-β-glucanase (bg1) and an aspartic protease (pr1) were cloned by expression cloning in Saccharomyces cerevisiae W3124. The bg1 cDNA encodes a 424-residue precursor protein with a putative signal peptide. The pr1 cDNA encodes a 405-residue prepropolypeptide with an 81-residue leader peptide. The aspartic protease was purified and characterized. It has a molecular mass of 36 kDa, an isoelectric point of pH 7.5, a pH activity optimum at 4.0–6.0, and a temperature activity optimum around 40 °C. Both enzymes show only low sequence identity to other known enzymes. Received: 6 August 1998 / Received revision: 29 October 1998 / Accepted: 30 October 1998     

A new arabinofuranohydrolase from Bifidobacterium adolescentis able to remove arabinosyl residues from double-substituted xylose units in arabinoxylan

An arabinofuranohydrolase (AXH-d3) was purified from a cell-free extract of Bifidobacterium adolescentis DSM 20083. The enzyme had a molecular mass of approximately 100 kDa as determined by gel filtration. It displayed maximum activity at pH 6 and 30 °C. Using an arabinoxylan-derived oligosaccharide containing double-substituted xylopyranosyl residues established that the enzyme specifically released terminal arabinofuranosyl residues linked to C-3 of double-substituted xylopyranosyl residues. In addition, this arabinofuranohydrolase released arabinosyl groups from wheat flour arabinoxylan polymer but showed no activity towards p-nitrophenyl α-l-arabinofuranoside or towards sugar-beet arabinan, soy arabinogalactan, arabino-oligosaccharides and arabinogalacto-oligosaccharides. Received: 15 July 1996 / Received revision: 18 October 1996 / Accepted: 18 October 1996     

Conversion of chlorinated propanes by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase

Chlorinated propanes are important pollutants that may show persistent behaviour in the environment. The biotransformation of 1-chloropropane, 1,2-dichloropropane, 1,3-dichloropropane and 1,2,3-trichloropropane was studied using resting cell suspensions of Methylosinus trichosporium OB3b expressing soluble methane monooxygenase. The transformation followed first-order kinetics. The rate constants were in the order 1-chloropropane > 1,3-dichloropropane > 1,2-dichloropropane > 1,2,3-trichloropropane, and varied from 0.07 to 1.03 ml min⁻¹ mg of cells⁻¹ for 1,2,3-trichloropropane and 1-chloropropane respectively. Turnover-dependent inactivation occurred for all of the chloropropanes tested. The inactivation constants were lower for 1-chloropropane and 1,2-dichloropropane than for 1,2,3-trichloropropane and 1,3-dichloropropane. Not all the chloride was released during cometabolic transformation of the chlorinated propanes and production of monochlorinated- and dichlorinated propanols was found by gas chromatography. The reaction pathway of 1,2,3-trichloropropane conversion was studied by mass spectrometric analysis of products formed in ²H₂O, which indicated that 1,2,3-trichloropropane was initially oxidized to 2,3-dichloropropionaldehyde and 1,3-dichloroacetone, depending on whether oxygen insertion occurred on the C-3 or C-2 carbon of 1,2,3,-trichloropropane, followed by reduction to the corresponding propanols. The results show that chloropropanes are susceptible to cometabolic oxidation by methanotrophs, but that the transformation kinetics is worse than with cometabolic conversion of trichloroethylene. Received: 27 November 1997 / Received revision: 27 February 1998 / Accepted: 27 February 1998     

Glycolipids of the smut fungus Ustilago maydis from cultivation on renewable resources

When grown on vegetable oils and their derivatives, the smut fungus Ustilago maydis (DSM 4500 and ATCC 14826) produces several glycolipids under nitrogen-limiting conditions. With 45 g l⁻¹ sunflower oil fatty acids (technical grade) a yield of 30 g l⁻¹ glycolipid was achieved. The resulting mixture contained predominantly mannosylerythritol lipids together with smaller amounts of cellobiose lipids. The production of the more polar cellobiose lipids was enhanced when glucose was used as carbon source. The molecular structure of the main components of the glycolipid mixture were elucidated by a combination of NMR spectroscopic and mass-spectrometric techniques. Received: 22 June 1998 / Received revision: 11 September 1998 / Accepted: 13 September 1998     

New aspects of genes and enzymes for β-lactam antibiotic biosynthesis

Penicillins, cephalosporins and cephamycins are peptide antibiotics synthesized by condensation of l-α-aminoadipic acid, l-cysteine and l-valine to form the tripeptide δ(l-α-aminoadipyl)-l-cysteinyl-d-valine (Aad-Cys-Val) by a non-ribosomal peptide synthetase. The genes pcbAB and pcbC, common to all penicillin and cephalosporin producers, that encode the Aad-Cys-Val synthetase¹ and isopenicillin N (IPN) synthase¹ respectively, have been cloned and the encoded enzymes studied in detail. The IPN synthase has been crystallized and its active center identified, providing evidence for the molecular mechanism of cyclization of the tripeptide Aad-Cys-Val to isopenicillin N. The late genes of the penicillin and cephalosporin pathways have also been characterized although some of the molecular mechanisms catalyzed by the encoded enzymes (e.g. IPN acyltransferase) are still obscure. In cephamycin-producing organisms, biosynthesis of the α-aminoadipic acid precursor proceeds in two steps catalyzed by lysine 6-aminotransferase and piperideine-6-carboxylic acid dehydrogenase. The gene lat for the first of these enzymes is located in the cephamycin gene cluster, providing an interesting example of association of genes encoding enzymes for the formation of a precursor with genes involved in assembly of the antibiotics. Novel enzymes involved in methoxylation at C-7 and carbamoylation at C-3′ of the cephem nucleus were isolated from Nocardia lactamdurans and Streptomyces clavuligerus. The methoxylation system is encoded by two linked genes cmcI-cmcJ and their products (proteins P₇ and P₈) form a complex that is required for hydroxylation at C-7 and for the subsequent methylation of the 7-hydroxycephem derivative to form the methoxyl group. Carbamoylation at the C-3′-hydroxyl group of the cephem nucleus is catalyzed by a specific carbamoyltransferase encoded by the gene cmcH. Finally, genes for a β-lactamase (bla), a penicillin-binding protein (pbp) and a transmembrane protein (cmcT) that appears to be involved in cephamycin exportation, are clustered together with the biosynthetic genes in the cephamycin clusters of S. clavuligerus and N. lactamdurans. Availability of the cloned genes allows metabolic engineering of the β-lactam biosynthetic pathways such as a channelling precursors and directed removal of bottlenecks in the β-lactam biosynthetic pathways. Several new β-lactam antibiotics have been discovered in gram-positive and gram-negative bacteria that will provide new genes for combinatorial synthesis of new molecules. Received: 2 December 1997 / Received revision: 20 February 1998 / Accepted: 24 February 1998     

A Pseudomonas aeruginosa biosensor responds to exposure to ultraviolet radiation

We fused the Pseudomonas aeruginosa recA promoter to a promoterless Vibrio fisherilux operon. This recA–lux fusion (pMOE15) was introduced into wild-type P. aeruginosa strain FRD1 and recA expression was monitored by measuring 490-nm light production. The RM4440 strain responded to increasing doses of ultraviolet radiation by an increase in its bioluminescence. RM4440 has the potential to be useful as a biosensor for the presence of DNA-damaging agents in the environment. Received: 18 February 1998 / Received revision: 18 June 1998 / Accepted: 27 June 1998     

The formation of 1-hydroxymethylnaphthalene and 6-hydroxymethylquinoline by both oxidative and reductive routes in Cunninghamella elegans

Extraction of medium after incubation of the fungus, Cunninghamella elegans, with 0.03% (w/v) 1-methylnaphthalene produced mainly 1-hydroxymethylnaphthalene together with some 1-naphthoic acid and hydroxynaphthoic acid. Higher concentrations of substrate were inhibitory to biotransformation. Similar incubations with 1-naphtoic acid as substrate resulted in reduction of the carboxyl group to give 1-hydroxymethylnaphthalene. When 6-methylquinoline was used, the main product was 6-hydroxymethylquinoline but also some quinoline-6-carboxylic acid and some 6-methylquinoline-N-oxide were identified. In a 2-l fermenter 2.5 g substrate was transformed in 324 h. The 6-hydroxymethylquinoline was also produced by reduction of quinoline-6-carboxylic acid by the organism. Received: 9 March 1998 / Received revision: 15 June 1998 / Accepted: 19 June 1998     

Effect of temperature and pH on growth and product formation of Lactococcus lactis ssp. lactis ATCC 19435 growing on maltose

Lactococcus lactis ssp. lactis ATCC 19435 is known to produce mixed acids when grown on maltose. A change in fermentation conditions only, elevated temperatures (up to 37 °C) and reduced pH values (down to 5.0) resulted in a shift towards homolactic product formation. This was accompanied by decreased growth rate and cell yield. The results are discussed in terms of redox balance and maintenance, and the regulation of lactate dehydrogenase and pyruvate formate-lyase. Received: 14 December 1998 / Received revision: 12 January 1999 / Accepted: 22 January 1999     

Poly-(3-hydroxybutyrate) production from whey by high-density cultivation of recombinant Escherichia coli

Recombinant Escherichia coli strain GCSC 6576, harboring a high-copy-number plasmid containing the Ralstonia eutropha genes for polyhydroxyalkanoate (PHA) synthesis and the E. coli ftsZ gene, was employed to produce poly-(3-hydroxybutyrate) (PHB) from whey. pH-stat fed-batch fermentation, using whey powder as the nutrient feed, produced cellular dry weight and PHB concentrations of 109 g l⁻¹ and 50 g l⁻¹ respectively in 47 h. When concentrated whey solution containing 210 g l⁻¹ lactose was used as the nutrient feed, cellular dry weight and PHB concentrations of 87 g l⁻¹ and 69 g l⁻¹ respectively could be obtained in 49 h by pH-stat fed-batch culture. The PHB content was as high as 80% of the cellular dry weight. These results suggest that cost-effective production of PHB is possible by fed-batch culture of recombinant E. coli using concentrated whey solution as a substrate. Received: 19 December 1997 / Received revision: 17 March 1998 / Accepted: 20 March 1998     

Gratuitous dechlorination of chloroethanes by methanogenic granular sludge

The dechlorinating activity of a methanogenic granular sludge from a methanol-fed upflow anaerobic sludge blanket reactor was investigated with chlorinated ethanes. This unadapted methanogenic consortium degraded all chloroethanes tested. The product formation rates decreased with the number of chlorine substituents. The more highly chlorinated ethanes were also converted, although at a lower rate, in the presence of autoclaved (dead) sludge, indicating the involvement of reduced heat-stable cofactors like vitamin B₁₂ and F₄₃₀. Direct chemical dechlorination of hexa-, penta- and tetrachloroethanes was also observed in medium without sludge, although at a much lower rate. The results show the importance of cometabolic and abiotic (chemical) conversions for the transformation of chlorinated ethanes by the methanogenic consortium. The types of reaction and the products formed were correlated with the Gibbs free-energy change (ΔG ^0′). Reductive hydrogenolysis and dichloroelimination were important dechlorinating mechanisms. Generally, these reactions have a higher ΔG ^0′ value than dehydrochlorination reactions, which occurred less frequently during the transformation of chloroethanes by the methanogenic granular sludge. Received: 8 June 1998 / Received revision: 7 September 1998 / Accepted: 13 September 1998     

Cloning of a new endoglucanase gene from Bacillus sp. BP-23 and characterisation of the enzyme. Performance in paper manufacture from cereal straw

The gene celA, encoding an endoglucanase from the strain Bacillus sp. BP-23, was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1867-bp DNA fragment containing the celA gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44 803 Da. The deduced amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases. The celA gene product synthesized in E. coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel. Activity was enhanced in the presence of 10 mM Mg²⁺ and Ca²⁺ and showed its maximum at 40 °C and pH 4.0. Study of the performance of CelA on paper manufacture from agricultural fibres showed that treatment with the enzyme improved the properties of the pulp and the quality of paper. CelA treatment enhanced the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished. Electron-microscope analysis showed that the surface of straw fibres was modified by CelA. Received: 11 February 1998 / Received revision: 20 March 1998 / Accepted: 20 March 1998     

Engineering Pichia pastoris for stereoselective nitrile hydrolysis by co-producing three heterologous proteins

A Pichia pastoris strain with stereoselective nitrile hydratase activity has been constructed by engineering the co-expression of three genes derived from Pseudomonas putida. Using a technique that could be widely applicable, the genes encoding nitrile hydratase α and β structural subunits and P14K accessory protein were first assembled as individual expression cassettes and then incorporated onto one plasmid, which was integrated into the P. pastoris chromosome. The resulting strain can be used as a catalyst for bioconversions requiring stereospecific nitrile hydrolysis. Received: 3 November 1998 / Received revision: 25 February1999 / Accepted: 14 March 1999     

Manipulation of the DNA coding for the desulphurizing activity in a new isolate of Arthrobacter sp.

A new bacterial strain able to cleave CS bonds from organosulphur heterocyclic compounds through the 4-S pathway and tentatively classified as Arthrobacter sp. was recently isolated. In the present short article we describe the cloning and the characterization of the DNA encoding the enzymes responsible for desulphurization in this microorganism, referred to as Arthrobacter sp. DS7. The desulphurization operon was found to be located in a large plasmid that also bears the genes conferring cadmium and arsenic resistance. By shortening this plasmid, a new cloning vector was prepared and used to obtain a recombinant derivative strain that desulphurizes dibenzothiophene despite of the presence of inorganic sulphur in the growth medium. Received: 25 May 1998 / Received revision: 4 September 1998 / Accepted: 13 September 1998     

Structure-activity analyses reveal the existence of two separate groups of active octadecanoids in elicitation of the tendril-coiling response of Bryonia dioica Jacq.

The Bryonia dioica tendril-coiling assay provides a rapid, sensitive and selective bioassay for jasmonates. Using this assay, a large number of jasmonate and coronatine analogs were analyzed for their biological activities. In a systematic study, C-3 analogs, C-2 analogs, C-1 homologs and -analogs, C-1(1′) analogs of jasmonic acid, as well as analogs of coronatine altered in both the amino acid and the coronafacic acid moiety, were compared. The results demonstrated at least two structurally non-overlapping centers of biological activity, one centered around the structure of jasmonic acid allowing only minor C-1(1′) modifications and a second center around the structure of 12-oxophytodienoic acid and having different structural requirements for activity, thus allowing quite different structural modifications. The C18-group of the jasmonates [12-oxophytodienoic acid and 3-oxo-2(2′ (Z)-pentenyl)-cyclopentane-1-octanoic acid], for which coronatine is a structural mimic, was the much more potent inducer of tendril coiling, when applied externally. The levels of jasmonic acid and 3-oxo-2(2′(Z)-pentenyl)-cyclopentane-1-octanoic acid in mechanically stimulated tendrils remained very low and did not change detectably, while the level of 12-oxophytodienoic acid had earlier been shown to change drastically and transiently during the onset and progression of the coiling reaction. Thus, 12-oxophytodienoic acid, rather than jasmonic acid or 3-oxo-2(2′ (Z)-pentenyl)-cyclopentane-1-octanoic acid, has to be considered as an endogenous signal transducer in B. dioica mechanotransduction. Received: 19 June 1998 / Accepted: 14 September 1998