High-level expression in Streptomyces lividans 66 of a gene encoding Streptomyces subtilisin inhibitor from Streptomyces albogriseolus S-3253

A secretory expression system for Streptomyces subtilisin inhibitor (SSI) was established in a heterologous host, Streptomyces lividans 66, by introducing the 1.8-kbp BglII/SalI fragment containing SSI gene into the Streptomyces multicopy vector, pIJ 702. The expression of SSI did not depend on the orientation of the 1.8-kbp BglII/SalI fragment or on the promoter for tyrosinase gene (mel) in pIJ 702, which suggested that this fragment also carries the SSI promoter. The expressed SSI in S.lividans 66 was secreted into the culture medium in a large amount, as observed with the original strain, S. albogriseolus S-3253. Amino acid sequence analysis showed that the SSI secreted from S. lividans 66 contained three additional amino acid residues in the NH2-terminal region. The inhibitory activity toward subtilisin BPN' and the antigenic activity of the SSI secreted from S. lividans 66 were found to be identical with those of authentic SSI.     

The Alcaligenes eutrophus ldh structural gene encodes a novel type of lactate dehydrogenase

Abstract The lactate dehydrogenase gene, ldh , of Alcaligenes eutrophus H16 was identified on a 14-kbp Eco RI restriction fragment of a genomic library in the cosmid pHC79 by hybridization with a 50-mer synthetic oligonucleotide which was derived from the N-terminal amino acid sequence of the purified enzyme. Recombinant strains of Escherichia coli JM83, which harboured a 2.0-kbp Pst I subfragment in pUC9-1, expressed LDH at a high level, if ldh was downstream from and colinear to the E. coli lac promoter. The nucleotide sequence of a region of 4245 bp revealed several open reading frames which might represent coding regions. One represented the ldh gene. The amino acid sequence deduced from ldh exhibited 29% and 36% identity to the L-malate dehydrogenase of Methanothermus fervidus and to the putative translation product of an E. coli sequence of unknown function, respectively. The ldh was separated by short intergenic regions from two other open reading frames: ORF5 was located downstream of and colinear to ldh , and its putative translational product revealed 38 to 56% amino acid identity to penicillin-binding proteins. ORF3 was located upstream of and colinear to ldh , and its putative gene translational product represented a hydrophobic protein. A sequence, which resembled the A. eutrophus alcohol dehydrogenase promoter, was detected upstream of ORF3, which most probably represents the first transcribed gene of an operon consisting of ORF3, ldh and ORF5.     

Sequencing and high expression of aminopeptidase P gene from Escherichia coli HB101

A plasmid pAPP1 with a 4 kbp insert at the PstI site of pBR322, encoding aminopeptidase P gene of Escherichia coli HB101 (Yoshimoto et al. (1988) J. Biochem. 104, 730-734), was subcloned into pUC18 and pUC19. The transformant of E. coli JM83 harboring pAPP4 with a 1.9 kbp fragment showed more than 50-fold higher enzyme activity than that of the host, after cultivation at 37 degrees C for 40 h in LB-medium containing ampicillin. When the gene DNA was inserted reversely in pAPP4, the enzyme productivity decreased markedly. The whole nucleotide sequence of the inserted fragment of plasmid pAPP4 was clarified by the dideoxy chain-terminating method. Within this sequence, the mature enzyme protein-encoding sequence was found to start just after an ATG codon, as judged by comparison with amino-terminal protein sequencing. Eleven bases upstream from the proposed initiation codon was an AGGAGA sequence which seemed to be a ribosome binding site. Thirty-four bases upstream from the proposed start codon was the 6-base sequence TACAAA, the so-called -10 region or Pribnow box. Further, the 6-base sequence TTTACT around 77 bases upstream from the start codon was deduced to be a putative -35 region consensus sequence. The inverted repeat at 1334 was tentatively assumed to be a terminator. The molecular weight of the enzyme was estimated to be 49,650 from the nucleotide sequence. The purified enzyme contained 0.2 gram atom of zinc per subunit. The enzyme activity was inhibited by EDTA and activated 5-fold by Mn2+.     

Analysis of the regulatory region of the protease III (ptr) gene of Escherichia coli K-12

The ptr gene of Escherichia coli encodes protease III (Mr 110,000) and a 50-kDa polypeptide, both of which are found in the periplasmic space. The gene is physically located between the recC and recB loci on the E. coli chromosome. The nucleotide sequence of a 1167-bp EcoRV-ClaI fragment of chromosomal DNA containing the promoter region and 885 bp of the ptr coding sequence has been determined. S1 nuclease mapping analysis showed that the major 5' end of the ptr mRNA was localized 127 bp upstream from the ATG start codon. The open reading frame (ORF), preceded by a Shine-Dalgarno sequence, extends to the end of the sequenced DNA. Downstream from the -35 and -10 regions is a sequence that strongly fits the consensus sequence of known nitrogen-regulated promoters. A signal peptide of 23 amino acids residues is present at the N terminus of the derived amino acid sequence. The cleavage site as well as the ORF were confirmed by sequencing the N terminus of mature protease III.     

地中海拟无枝菌酸菌U32中生物素羧基载体蛋白结构基因的克隆、表达及转录

乙酰辅酶A羧化酶(Acetyl CoA Carboxylase EC 6.4.1.2, ACC)催化依赖于ATP的乙酰辅酶A羧化形成丙二酸单酰辅酶A,该反应是脂肪酸生物合成途径中的第一步,也是受到调控的关键一步。根据结核分枝杆菌(M. tuberculosis)和天蓝色链霉菌(S. coelicolor)中ACC-α亚基的氨基酸保守序列和地中海拟无枝菌酸菌U32对氨基酸密码子的使用偏好,设计简并引物以U32基因组DNA为模板扩增出一条约250bp的片段,并以此片段作探针成功地从U32基因组cosmid文库中克隆到相应的ACC-α亚基的编码基因accA。该基因对应的ORF长1797bp,编码一个598个氨基酸的蛋白,推算出的分子量是63,714Da;基因G+C mol%含量为70.1%,符合U32基因结构特征,距起始密码子GTG上游6个碱基处有链霉菌典型的RBS序列AGGAGG,并有生物素羧化酶特征的ATP结合区。利用pET28(b)系统构建表达载体,在E. coli BL21(DE3)中实现了accA的诱导表达,产物大部分以可溶形式存在,并通过Western Blot证明该蛋白上确有共价结合的生物素。Northern Blot分析了各种氮源对accA基因转录水平的不同影响。