Molecular cloning of Bacillus sphaericus penicillin V amidase gene and its expression in Escherichia coli and Bacillus subtilis

The Bacillus sphaericus gene coding for penicillin V amidase, which catalyzes the hydrolysis of penicillin V to yield 6-aminopenicillanic acid and phenoxyacetic acid, has been isolated by molecular cloning in Escherichia coli. The gene is contained within a 2.2-kilobase HindIII-PstI fragment and is expressed when transferred into E. coli and Bacillus subtilis. The expression in B. subtilis carrying the recombinant plasmid is approximately two times higher than in the original B. sphaericus strain. A comparison of the purified enzyme from B. sphaericus and the expressed gene product in E. coli minicells suggests that the native enzyme consists of four identical subunits, each with a molecular weight of 35,000.     

Expression of the Arthrobacter viscosus penicillin G acylase gene in Escherichia coli and Bacillus subtilis

The penicillin G acylase gene cloned from Arthrobacter viscosus 8895GU was subcloned into vectors, and the recombinant plasmids were transferred into Escherichia coli or Bacillus subtilis. Both E. coli and B. subtilis transformants expressed the A. viscosus penicillin G acylase. The enzyme activity was found in the intracellular portion of the E. coli transformants or in the cultured medium of the B. subtilis transformants. Penicillin G acylase production in the B. subtilis transformants was 7.2 times higher than that in the parent A. viscosus. The A. viscosus penicillin G acylase was induced by phenylacetic acid in A. viscosus, whereas the enzyme was produced constitutively in both the E. coli and B. subtilis transformants carrying the A. viscosus penicillin G acylase gene.     

Expression of the Arthrobacter viscosus penicillin G acylase gene in Escherichia coli and Bacillus subtilis.

The penicillin G acylase gene cloned from Arthrobacter viscosus 8895GU was subcloned into vectors, and the recombinant plasmids were transferred into Escherichia coli or Bacillus subtilis. Both E. coli and B. subtilis transformants expressed the A. viscosus penicillin G acylase. The enzyme activity was found in the intracellular portion of the E. coli transformants or in the cultured medium of the B. subtilis transformants. Penicillin G acylase production in the B. subtilis transformants was 7.2 times higher than that in the parent A. viscosus. The A. viscosus penicillin G acylase was induced by phenylacetic acid in A. viscosus, whereas the enzyme was produced constitutively in both the E. coli and B. subtilis transformants carrying the A. viscosus penicillin G acylase gene.     

Cloning of the biotin synthetase gene from Bacillus sphaericus and expression in Escherichia coli and Bacilli

Biotin synthetase (BS) catalyses the biotransformation of dethiobiotin (DTB) to biotin. Here we report the cloning, characterization and expression of the gene encoding BS of Bacillus sphaericus. A recombinant plasmid pSB01, containing an 8.2-kb DNA fragment from B. sphaericus, was isolated by phenotypic complementation of an Escherichia coli bioB strain. Nucleotide sequence analysis of this fragment and N-terminal sequence determination of the recombinant protein product revealed that the bioB gene of B. sphaericus consists of a 996-bp open reading frame which is closely associated with at least one other gene. E. coli cells transformed with a bioB expression vector performed efficient bioconversion of DTB to biotin under defined culture conditions. Biotin production from transformed Bacillus subtilis and B. sphaericus recombinant strains was also demonstrated. Comparison of the amino acid sequences of BS from E. coli and B. sphaericus revealed extensive similarity.     

Expression of penicillin G acylase gene from Bacillus megaterium ATCC 14945 in Escherichia coli and Bacillus subtilis.

Penicillin G acylase gene from Bacillus megaterium ATCC 14945 has been isolated. Recombinant Escherichia coli clones were screened for clear halo forming activity on the lawn of Staphylococcus aureus ATCC 6538P using the enzymatic acylating reaction of 7-aminodeacetoxycephalosporanic acid (7-ADCA) and D-(alpha)-phenylglycine methylester. The gene was contained within a 2.8 kb DNA fragment and expressed efficiently when transferred from E. coli to Bacillus subtilis. A twenty times greater amount of enzyme was produced in B. subtilis transformant than that in B. megaterium. The purified enzyme from subcloned B. subtilis showed that the native enzyme consisted of two identical subunits, each with a molecular weight of 57,000. The enzyme was able to react on various cephalosporins, i.e., cephalothin, cefamandole, cephaloridine, cephaloglycin, cephalexin and cephradine.     

Cloning, sequencing, and characterization of the Bacillus subtilis biotin biosynthetic operon.

A 10-kb region of the Bacillus subtilis genome that contains genes involved in biotin-biosynthesis was cloned and sequenced. DNA sequence analysis indicated that B. subtilis contains homologs of the Escherichia coli and Bacillus sphaericus bioA, bioB, bioD, and bioF genes. These four genes and a homolog of the B. sphaericus bioW gene are arranged in a single operon in the order bioWAFDR and are followed by two additional genes, bioI and orf2. bioI and orf2 show no similarity to any other known biotin biosynthetic genes. The bioI gene encodes a protein with similarity to cytochrome P-450s and was able to complement mutations in either bioC or bioH of E. coli. Mutations in bioI caused B. subtilis to grow poorly in the absence of biotin. The bradytroph phenotype of bioI mutants was overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis. The B. subtilis bio operon is preceded by a putative vegetative promoter sequence and contains just downstream a region of dyad symmetry with homology to the bio regulatory region of B. sphaericus. Analysis of a bioW-lacZ translational fusion indicated that expression of the biotin operon is regulated by biotin and the B. subtilis birA gene.     

粪产碱杆菌青霉素G酰化酶在枯草芽孢杆菌中的表达、纯化及其性质

利用PCR技术克隆了粪产碱杆菌 (Alcaligenesfaecalis,CICCAS1.76 7)青霉素G酰化酶 (pencillinGacylase ,PGA)基因 (GenBank登录号AF4 5 5 35 6 )。通过构建工程菌E .coli(pETAPGA) ,该酶在大肠杆菌中获得了表达 ,表达产物分泌到周质空间。进一步构建的工程菌B .subtilis (pMAPGA)和B .subtilis(pBAPGA)实现了该酶的胞外分泌表达。分泌表达的最高表达量为 6 5 3u/L ,比野生型A .faecalis表达量高 10 9倍。表达产物经硫酸铵分级沉淀和DEAE SepharoseCL 6B两步纯化 ,纯度提高 86倍 ,活力回收率达到 81% ,纯化后的PGA活力为 1.4 6 9u/mg。研究表明 ,PGA家族成员中只有粪产碱杆菌PGA和巨大芽孢杆菌PGA可以在枯草芽孢杆菌中分泌表达。与巨大芽孢杆菌PGA相比 ,粪产碱杆菌PGA的最适pH值为 8.0 ,最适温度为 6 0°C ,而且在有机溶剂中具有更强的稳定性。该酶在水相中具有较低的头孢氨苄合成活力。本研究为粪产碱杆菌PGA的获得提供了新的途径。     

Molecular cloning and expression in Escherichia coli of two modification methylase genes of Bacillus subtilis

Two modification methylase genes of Bacillus subtilis R were cloned in Escherichia coli by using a selection procedure which is based on the expression of these genes. Both genes code for DNA-methyltransferases which render the DNA of the cloning host E. coli HB101 insensitive to the BspRI (5'-GGCC) endonuclease of Bacillus sphaericus R. One of the cloned genes is part of the restriction-modification (RM) system BsuRI of B. subtilis R with specificity for 5'-GGCC. The other one is associated with the lysogenizing phage SP beta B and produces the methylase M.BsuP beta BI with specificity for 5'-GGCC. The fragment carrying the SP beta B-derived gene also directs the synthesis in E. coli of a third methylase activity (M.BsuP beta BII), which protects the host DNA against HpaII and MspI cleavage within the sequence 5'-CCGG. Indirect evidence suggests that the two SP beta B modification activities are encoded by the same gene. No cross-hybridization was detected either between the M.BsuRI and M.BsuP beta B genes or between these and the modification methylase gene of B. sphaericus R, which codes for the enzyme M.BspRI with 5'-GGCC specificity.     

Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsH and ptsI genes and evidence for a crr-like gene.

The genes ptsI and ptsH, which encode, respectively, enzyme I and Hpr, cytoplasmic proteins involved in the phosphoenolpyruvate:sugar phosphotransferase system, were cloned from Bacillus subtilis. A plasmid containing a 4.1-kilobase DNA fragment was shown to complement Escherichia coli mutations affecting the ptsH and ptsI genes. In minicells this plasmid expressed two proteins with the molecular weights expected for Hpr and enzyme I. Therefore, ptsH and ptsI are adjacent in B. subtilis, as in E. coli. In E. coli a third gene (crr), involved in glucose translocation and also in catabolite repression, is located downstream from the ptsHI operon. The 4.1-kilobase fragment from B. subtilis was shown to contain a gene that enables an E. coli crr mutant to use glucose. This gene, unlike the E. coli crr gene, was located to the left of ptsH.     

Cloning and expression of the Escherichia coli D-xylose isomerase gene in Bacillus subtilis

A DNA fragment containing the Escherichia coli D-xylose isomerase gene and D-xylulokinase gene had been isolated from an E. coli genomic bank constructed by Clarke and Carbon. The D-xylose isomerase gene coding for the synthesis of an important industrial enzyme, xylose isomerase, was subcloned into a Bacillus-E. coli bifunctional plasmid. It was found that the intact E. coli gene was not expressed in B. subtilis, a host traditionally used to produce industrial enzymes. An attempt was then made to express the E. coli gene in B. subtilis by fusion of the E. coli xylose isomerase structural gene downstream to the promoter of the penicillinase gene isolated from Bacillus licheniformis. Two such fused genes were constructed and they were found able to be expressed in both B. subtilis and E. coli.     

巨大芽孢杆菌青霉素酰化酶在枯草杆菌中的表达及其分离纯化

青霉素酰化酶（ＰＧＡ）在医药工业起着重要的作用,它能够水解青霉素Ｇ产生６－氨基青霉烷酸（６－ＡＰＡ）和苯乙酸,６－ＡＰＡ是半合成青霉素的关键中间体．该酶广泛存在于各种微生物中如真菌和细菌中．国际上对Ｅ．ｃｏｌｉ、Ａｒｔｈｒｏｂａｃｔｅｒｖｉｓｃｏｓｕ...     

Cloning of the Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase gene in Escherichia coli. Nucleotide sequence determination and properties of the plasmid-encoded enzyme

The Bacillus subtilis gene encoding glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) was cloned in pBR322. This gene is designated purF by analogy with the corresponding gene in Escherichia coli. B. subtilis purF was expressed in E. coli from a plasmid promoter. The plasmid-encoded enzyme was functional in vivo and complemented an E. coli purF mutant strain. The nucleotide sequence of a 1651-base pair B. subtilis DNA fragment was determined, thus localizing the 1428-base pair structural gene. A primary translation product of 476 amino acid residues was deduced from the DNA sequence. Comparison with the previously determined NH2-terminal amino acid sequence indicates that 11 residues are proteolytically removed from the NH2 terminus, leaving a protein chain of 465 residues having an NH2-terminal active site cysteine residue. Plasmid-encoded B. subtilis amidophosphoribosyltransferase was purified from E. coli cells and compared to the enzymes from B. subtilis and E. coli. The plasmid-encoded enzyme was similar in properties to amidophosphoribosyltransferase obtained from B. subtilis. Enzyme specific activity, immunological reactivity, in vitro lability to O2, Fe-S content, and NH2-terminal processing were virtually identical with amidophosphoribosyltransferase purified from B. subtilis. Thus E. coli correctly processed the NH2 terminus and assembled [4Fe-4S] centers in B. subtilis amidophosphoribosyltransferase although it does not perform these maturation steps on its own enzyme. Amino acid sequence comparison indicates that the B. subtilis and E. coli enzymes are homologous. Catalytic and regulatory domains were tentatively identified based on comparison with E. coli amidophosphoribosyltransferase and other phosphoribosyltransferase (Argos, P., Hanei, M., Wilson, J., and Kelley, W. (1983) J. Biol. Chem. 258, 6450-6457).     

Dihydrofolate reductase from Bacillus subtilis and its artificial derivatives: expression, purification, and characterization.

The Bacillus subtilis dihydrofolate reductase (DHFR) gene was expressed in Escherichia coli. The gene product was purified to homogeneity by Butyl-Toyopearl, Toyopearl HW55, and DEAE-Toyopearl column chromatographies, and its molecular properties were compared to those of E. coli DHFR. The specific enzyme activity of the B. subtilis DHFR was 240 units/mg under the standard assay conditions, being about four times higher than that of the E. coli DHFR. Km for coenzyme NADPH was 20.7 microM, a value about three times larger than that of E. coli, whereas Km (1.5 microM) for the substrate, dihydrofolate, was similar to that of E. coli DHFR. This seems to reflect the low homology of the amino acid sequence in residues 61-88 of the two DHFRs where one of the NADPH binding sites is located [Bystrof, C. & Kraut, J. (1991) Biochemistry 30, 2227-2239]. Similar to the E. coli DHFR [Iwakura, M. et al. (1992) J. Biochem. 111, 37-45], the extension of amino acid sequences at the C-terminal end of the B. subtilis DHFR could be attained without loss of the enzyme function or decrease of the protein yield. Thus, the DHFR is useful as a carrier protein for expressing small polypeptides, such as leucine enkephalin, bradykinin, and somatostatin.     

Structure of the Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster.

A 10.5-kilobase PstI endonuclease fragment encoding the entire Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster was cloned in Escherichia coli by transformation of a carB strain to uracil-independent growth. The cloned fragment also complemented E. coli pyrB, pyrC, pyrD, pyrE, and pyrF mutants. From the ability of subclones to complement E. coli pyr mutants, the gene order was deduced to be pyrBCADFE. The B. subtilis pyrB gene was shown to be expressed in E. coli, but synthesis of the enzyme was not repressible by the addition of uracil to the growth medium. The approximate molecular weights of the polypeptides encoded by B. subtilis pyrA, pyrB, pyrC, pyrD, pyrE, and pyrF were found to be 110,000, 36,000, 46,000, 34,000, 25,000, and 27,000, respectively.     

Cloning and nucleotide sequencing of a novel 7 beta-(4-carboxybutanamido)cephalosporanic acid acylase gene of Bacillus laterosporus and its expression in Escherichia coli and Bacillus subtilis.

A strain of Bacillus species which produced an enzyme named glutaryl 7-ACA acylase which converts 7 beta-(4-carboxybutanamido)cephalosporanic acid (glutaryl 7-ACA) to 7-amino cephalosporanic acid (7-ACA) was isolated from soil. The gene for the glutaryl 7-ACA acylase was cloned with pHSG298 in Escherichia coli JM109, and the nucleotide sequence was determined by the M13 dideoxy chain termination method. The DNA sequence revealed only one large open reading frame composed of 1,902 bp corresponding to 634 amino acid residues. The deduced amino acid sequence contained a potential signal sequence in its amino-terminal region. Expression of the gene for glutaryl 7-ACA acylase was performed in both E. coli and Bacillus subtilis. The enzyme preparations purified from either recombinant strain of E. coli or B. subtilis were shown to be identical with each other as regards the profile of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were composed of a single peptide with the molecular size of 70 kDa. Determination of the amino-terminal sequence of the two enzyme preparations revealed that both amino-terminal sequences (the first nine amino acids) were identical and completely coincided with residues 28 to 36 of the open reading frame. Extracellular excretion of the enzyme was observed in a recombinant strain of B. subtilis.     

Sequencing and heterologous expression of the gene encoding penicillin V amidase from Bacillus sphaericus

The Bacillus sphaericus gene encoding penicillin V amidase, which catalyzes the hydrolysis of penicillin V, has been characterized. The entire nucleotide sequence of the coding region, as well as 5'- and 3'-flanking regions, was determined using an improved sequencing strategy. The deduced amino acid sequence suggests a protein consisting of 338 residues with an Mr of 37,500. The ATG initiator codon is preceded by a putative ribosome-binding site, typical for genes of Gram-positive origin. High expression of the gene was obtained in Escherichia coli using an inducible promoter, showing that the gene product is stable in this heterologous host.     

Cloning, sequencing, and expression in Escherichia coli of the Bacillus subtilis gene for phosphatidylserine synthase.

The Bacillus subtilis pss gene encoding phosphatidylserine synthase was cloned by its complementation of the temperature sensitivity of an Escherichia coli pssA1 mutant. Nucleotide sequencing of the clone indicated that the pss gene encodes a polypeptide of 177 amino acid residues (deduced molecular weight of 19,613). This value agreed with the molecular weight of approximately 18,000 observed for the maxicell product. The B. subtilis phosphatidylserine synthase showed 35% amino acid sequence homology to the yeast Saccharomyces cerevisiae phosphatidylserine synthase and had a region with a high degree of local homology to the conserved segments in some phospholipid synthases and amino alcohol phosphotransferases of E. coli and S. cerevisiae, whereas no homology was found with that of the E. coli counterpart. A hydropathy analysis revealed that the B. subtilis synthase is very hydrophobic, in contrast to the hydrophilic E. coli counterpart, consisting of several strongly hydrophobic segments that would span the membrane. A manganese-dependent phosphatidylserine synthase activity, a characteristic of the B. subtilis enzyme, was found exclusively in the membrane fraction of E. coli (pssA1) cells harboring a B. subtilis pss plasmid. Overproduction of the B. subtilis synthase in E. coli cells by a lac promoter system resulted in an unusual increase of phosphatidylethanolamine (up to 93% of the total phospholipids), in contrast to gratuitous overproduction of the E. coli counterpart. This finding suggested that the unusual cytoplasmic localization of the E. coli phosphatidylserine synthase plays a role in the regulation of the phospholipid polar headgroup composition in this organism.     

Bacillus subtilis RNase III gene: cloning, function of the gene in Escherichia coli, and construction of Bacillus subtilis strains with altered rnc loci.

The rnc gene of Bacillus subtilis, which has 36% amino acid identity with the gene that encodes Escherichia coli RNase III endonuclease, was cloned in E. coli and shown by functional assays to encode B. subtilis RNase III (Bs-RNase III). The cloned B. subtilis rnc gene could complement an E. coli rnc strain that is deficient in rRNA processing, suggesting that Bs-RNase III is involved in rRNA processing in B. subtilis. Attempts to construct a B. subtilis rnc null mutant were unsuccessful, but a strain was constructed in which only a carboxy-terminal truncated version of Bs-RNase III was expressed. The truncated Bs-RNase III showed virtually no activity in vitro but was active in vivo. Analysis of expression of a copy of the rnc gene integrated at the amy locus and transcribed from a p(spac) promoter suggested that expression of the B. subtilis rnc is under regulatory control.     

Molecular cloning of enantioselective ester hydrolase from Bacillus pumilus DBRL-191

A gene from Bacillus pumilus expressed under its native promoter was cloned in Escherichia coli. Recombinant B. pumilus esterase (BPE) affects the kinetic resolution of racemic mixtures such as unsubstituted and substituted 1-(phenyl)ethanols (E approximately 33-103), ethyl 3-hydroxy-3-phenylpropanoate (E approximately 45-71), trans-4-fluorophenyl-3-hydroxymethyl-N-methylpiperidine (E approximately 10-13) and ethyl 2-hydroxy-4-phenylbutyrate (E approximately 7). The enzyme is composed of a 34-amino acid signal peptide and a 181-amino acid mature protein corresponding to a molecular weight of approximately 19.2kD and pI approximately 9.4. 3-D the structural model of the enzyme built by homology modelling using the atomic coordinates from the crystal structure of B. subtilis lipase (LipA) showed a compact minimal alpha/beta hydrolase fold.