Production of L-dihydroxyphenylalanine in Escherichia coli with the tyrosine phenol-lyase gene cloned from Erwinia herbicola.

The gene (tutA) encoding tyrosine phenol-lyase from Erwinia herbicola was cloned into Escherichia coli, and fusions to the lac and tac promoters were constructed. The enzyme was expressed at high levels in E. coli in the presence of isopropyl-beta-D-thiogalactopyranoside or lactose as an inducer. L-Dihydroxyphenylalanine was synthesized in high yield from catechol, pyruvate, and ammonia by induced cells.     

Cloning and expression of the Erwinia chrysanthemi asparaginase gene in Escherichia coli and Erwinia carotovora

A genomic library of Erwinia chrysanthemi DNA was constructed in bacteriophage lambda 1059 and recombinants expressing Er. chrysanthemi asparaginase detected using purified anti-asparaginase IgG. The gene was subcloned on a 4.7 kb EcoRI DNA restriction fragment into pUC9 to generate the recombinant plasmid pASN30. The position and orientation of the asparaginase structural gene was determined by subcloning. The enzyme was produced at high levels in Escherichia coli (5% of soluble protein) and was shown to be exported to the periplasmic space. Purified asparaginase from E. coli cells carrying pASN30 was indistinguishable from the Erwinia enzyme on the basis of specific activity [660-700 units (mg protein)-1], pI value (8.5), and subunit molecular weight (32 X 10(3]. Expression of the cloned gene was subject to glucose repression in E. coli but was not significantly repressed by glycerol. Recombinant plasmids, containing the asparaginase gene, when introduced into Erwinia carotovora, caused increased synthesis of the enzyme (2-4 fold higher than the current production strain).     

Cloning and nucleotide sequence of the bglA gene from Erwinia herbicola and expression of β-glucosidase activity in Escherichia coli

Abstract Genomic DNA fragments encoding β-glucosidase activity from the wild-type strain WD4 of Erwinia herbicola were cloned into Escherichia coli . Two clones containing a common fragment encoded a polypeptide of 58000 Da. Cloned β-glucosidase, expressed in E. coli , showed activity against natural β-glucoside sugars except for cellobiose. An open reading frame of 1442 bp termed bglA was identified by nucleotide sequencing and it coded for a protein of 480 amino acids ( M _r 53896) which showed significant homology with β-glucosidases from glycosyl hydrolase family 1.     

Cloning and expression of pectin lyase gene from Erwinia carotovora in Escherichia coli

A pectin lyase (PNL; EC 4.2.2.10) gene of Erwinia carotovora Er was cloned and expressed in Escherichia coli. The analysis of the nucleotide sequence of the 0.6 kb StuI-EcoRI fragment, which was hybridized with the mixed oligonucleotide probe for PNL gene, revealed the presence of an open reading frame (0RF) and correlated exactly with the known N-terminal 18 amino acid sequence of PNL. When a plasmid pTN2159, which has a BamHI-EcoRI fragment containing this ORF, was introduced into E. coli JM109, PNL was not expressed. When a tac-promoter was inserted in front of the ORF, PNL was efficiently expressed in E. coli. Synthesis of PNL by E. coli was also confirmed by immunoblot analysis.     

Nitrogen Fixation in a Biotype of Erwinia herbicola Resembling Escherichia coli

Two nitrogen-fixing members of the Enterobacteriaceae have been isolated from paper mill process water and compost. Although they closely resembled Escherichia coli , detailed biochemical characterization of these and 7 other isolates established that they should be assigned to a biotype of Erwinia herbicola . They may be distinguished from E. coli by their lack of amino acid decarboxylase activity, their ability to utilize cellobiose and malonate and to ferment cellobiose and amygdalin. In one of them, the capacity to fix nitrogen, ferment cellobiose and utilize malonate was resistant to the effects of ethidium bromide, acridine orange and sodium dodecyl sulphate, and the ability to utilize cellobiose could not be transferred on to E. coli or Salmonella typhi . It is therefore concluded that these characters are not carried on transferable plasmids. Forty-eight strains of E. coli of varying origin were examined for acetylene reducing activity and all were found to be negative. It is concluded that hitherto no naturally occurring strains of E. coli have been shown to fix nitrogen.     

Cloning and regulation of Erwinia herbicola pigment genes.

The genes coding for yellow pigment production in Erwinia herbicola Eho10 (ATCC 39368) were cloned and localized to a 12.4-kilobase (kb) chromosomal fragment. A 2.3-kb AvaI deletion in the cloned fragment resulted in the production of a pink-yellow pigment, a possible precursor of the yellow pigment. Production of yellow pigment in both E. herbicola Eho10 and pigmented Escherichia coli clones was inhibited by glucose. When the pigment genes were transformed into a cya (adenylate cyclase) E. coli mutant, no expression was observed unless exogenous cyclic AMP was provided, which suggests that cyclic AMP is involved in the regulation of pigment gene expression. In E. coli minicells, the 12.4-kb fragment specified the synthesis of at least seven polypeptides. The 2.3-kb AvaI deletion resulted in the loss of a 37K polypeptide and the appearance of a polypeptide of 40 kilodaltons (40K polypeptide). The synthesis of the 37K polypeptide, which appears to be required for yellow pigment production, was not repressed by the presence of glucose in the culture medium, as was the synthesis of other polypeptides specified by the 12.4-kb fragment, suggesting that there are at least two types of gene regulation involved in yellow pigment synthesis. DNA hybridization studies indicated that different yellow pigment genes exist among different E. herbicola strains. None of six pigmented plant pathogenic bacteria examined, Agrobacterium tumefaciens C58, Cornyebacterium flaccumfaciens 1D2, Erwinia rubrifaciens 6D364, Pseudomonas syringae ATCC 19310, Xanthomonas campestris 25D11, and "Xanthomonas oryzae" 17D54, exhibited homology with the cloned pigment genes.     

Identification of carotenoids in Erwinia herbicola and in a transformed Escherichia coli strain

The yellow pigments of Erwinia herbicola Eho 10 and of a transformed Escherichia coli LE392 pPL376 have been identified as carotenoids. HPLC separation, spectra and in some cases mass spectroscopy demonstrated the presence of phytoene (15-cis isomer), beta-carotene (all-trans, 9-cis and 15-cis), beta-cryptoxanthin ( = 3-hydroxy beta-carotene), zeaxanthin (3,3'-dihydroxy beta-carotene) and corresponding carotene glycosides. In addition, lycopene and gamma-carotene accumulated in the presence of the inhibitor 2-(4-chlorophenylthio)-triethylamine.HCl. Carotenoid content in the transformed E. coli was two-fold higher than in E. herbicola. The pattern of the carotenoids was similar in the two organisms. Inactivation of the katF gene in E. coli resulted in an 85% lowering of carotenoid formation, as did the addition of 0.5% glucose to the medium. Suppression of carotenoid formation by inactivation of the katF gene lowered, but did not abolish, the protection offered by carotenoids against inactivation by alpha-terthienyl plus near-ultraviolet light (320-400 nm).     

Cloning and expression in Escherichia coli of pectinase genes of Erwinia carotovora subsp. carotovora

Genes coding for an endo-pectate lyase, an exo-pectate lyase, and an endopolygalacturonase of Erwinia carotovora subsp. carotovora Ecc71 were cloned in Escherichia coli HB101, using the cosmid pHC79. The products of the cloned pectinase genes paralleled their counterparts in strain Ecc71 in isoelectric mobility, mode of substrate degradation, and ability to macerate potato tuber tissue.     

Cloning and expression in Escherichia coli of pectinase genes of Erwinia carotovora subsp. carotovora.

Genes coding for an endo-pectate lyase, an exo-pectate lyase, and an endopolygalacturonase of Erwinia carotovora subsp. carotovora Ecc71 were cloned in Escherichia coli HB101, using the cosmid pHC79. The products of the cloned pectinase genes paralleled their counterparts in strain Ecc71 in isoelectric mobility, mode of substrate degradation, and ability to macerate potato tuber tissue.     

Cloning and expression of the transhydrogenase gene of Escherichia coli.

Based on the rationale that Escherichia coli cells harboring plasmids containing the pnt gene would contain elevated levels of enzyme, we have isolated three clones bearing the transhydrogenase gene from the Clarke and Carbon colony bank. The three plasmids were subjected to restriction endonuclease analysis. A 10.4-kilobase restriction fragment which overlapped all three plasmids was cloned into the PstI site of plasmid pUC13. Examination of several deletion derivatives of the resulting plasmid and subsequent treatment with exonuclease BAL 31 revealed that enhanced transhydrogenase expression was localized within a 3.05-kilobase segment. This segment was located at 35.4 min in the E. coli genome. Plasmid pDC21 conferred on its host 70-fold overproduction of transhydrogenase. The protein products of plasmids carrying the pnt gene were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes from cells containing the plasmids. Two polypeptides of molecular weights 50,000 and 47,000 were coded by the 3.05-kilobase fragment of pDC11. Both polypeptides were required for expression of transhydrogenase activity.     

Functional assignment of Erwinia herbicola Eho10 carotenoid genes expressed in Escherichia coli

Erwinia herbicola is a nonphotosynthetic bacterium that is yellow pigmented due to the presence of carotenoids. When the Erwinia carotenoid biosynthetic genes are expressed in Escherichia coli, this bacterium also displays a yellow phenotype. The DNA sequence of the plasmid pPL376, carrying the entire Erwinia carotenoid gene cluster, has been found to contain 12 open reading frames (ORFs). Six of the ORFs have been identified as carotenoid biosynthesis genes that code for all the enzymes required for conversion of farnesyl pyrophosphate (FPP) to zeaxanthin diglucoside via geranylgeranyl pyrophosphate, phytoene, lycopene, β-carotene, and zeaxanthin. These enzymatic steps were assigned after disruption of each ORF by a specific mutation and analysis of the accumulated intermediates. Carotenoid intermediates were identified by the absorption spectra of the colored components and by high pressure liquid chromatographic analysis. The six carotenoid genes are arranged in at least two operons. The gene coding for β-carotene hydroxylase is transcribed in the opposite direction from that of the other carotenoid genes and overlaps with the gene for phytoene synthase.     

Cloning and expression of the metE gene in Escherichia coli

A lambda-transducing phage was isolated that contains the metE gene. This gene codes for N5-methyl-H4-folate:homocysteine methyltransferase (EC 2.1.1.14), an enzyme that catalyzes the terminal reaction in methionine biosynthesis. A 9.1-kb EcoR1 fragment of this phage, containing the metE gene, was then cloned into pBR325. This plasmid, pJ19, was used to transform Escherichia coli strain 2276, a metE mutant, and restore the MetE+ phenotype. Although the transformed cells produced large amounts of the metE protein in vivo, in vitro studies using pJ19 as template showed low synthesis of the metE protein.     

Cloning and expression of the Escherichia coli K-12 sad gene.

The Escherichia coli K-12 sad gene, which encodes an NAD-dependent succinic semialdehyde dehydrogenase, was cloned into a high-copy-number vector. Minicells carrying a sad+ plasmid produced a 55,000-dalton peptide, the probable sad gene product.     

Cloning and expression of the pepD gene of Escherichia coli

Peptidase D of Escherichia coli, cleaving the unusual dipeptide carnosine, was found to be encoded by the ColE1 hybrid plasmid pLC44-11. From this plasmid the pepD gene was subcloned into small vectors. As shown by successive reduction of the flanking sequences of genomic DNA, the order of genes in the region at 6 min of the E. coli K12 map is phoE, pepD, in the clockwise orientation. Insertional inactivation of the pepD gene and expression of recombinant plasmids in maxicells allowed the identification of the pepD product as a 52 kDa protein. Comparison with the 100 kDa protein molecular mass determined by gel filtration suggests that active peptidase D is probably a dimer.     

Cloning of the pectate lyase genes from Erwinia carotovora and their expression in Escherichia coli

A hybrid cosmid coding for pectate lyase (PL) activity was identified from an Erwinia carotovora genomic library by an immunological screening method. A 7-kb DNA fragment was identified which codes for three proteins identical in size to proteins with PL activity purified from E. carotovora culture supernatants. The three proteins had apparent Mrs of 41, 44 and 44 X 10(3) as estimated by SDS-PAGE. None of the PLs were exported from Escherichia coli strain HB101 but all were found in the periplasmic space. Plant tissue was macerated by the PLs made in E. coli.     

Cloning and expression of the pneumococcal autolysin gene in Escherichia coli

Summary A 7.5 kb BclI-fragment of Streptococcs pneumoniae DNA has been cloned in Escherichia coli HB101 using pBR322 as a vector. The new plasmid (pGL30) of 12.0 kb expresses a protein that has been characterized by biochemical, immunological and genetic methods as the inactive form (E-form) of the pneumococcal N-acetyl-muramyl-l-alanyl amidase (EC 3.5.1.28). Our results demonstrate that the E-form is the primary product of the lyt gene of S. pneumoniae. The inactive E-form can be converted to the active C-form in vitro by incubation of the E-form enzyme with choline-containing pneumococcal cell walls at low temperature in a similar way to enzyme production in the homologous system. The production of this protein in E. coli HB101 was 500-fold higher than in the homologous host. E. coli CSR603 containing pGL30 and labeled with [³⁵S]methionine synthesized a 35 kd protein. pGL30 can transform at high frequency an autolysin-defective mutant of S. pneumoniae to the lyt⁺ phenotype.