Secretion by Streptomyces lividans of a cloned Gram-negative beta-lactamase

Abstract The OXA-2 β-lactamase gene was first found on a conjugative plasmid R46 from a clinical isolate of Salmonella typhimurium . To transfer the gene to Streptomyces lividans a shuttle vector was created by fusing an Escherichia coli plasmid carrying the OXA-2 β-lactamase gene with the S. lividans vector pIJ61. The OXA-2 β-lactamase gene was expressed in S. lividans , although with a much reduced efficiency; virtually all of the β-lactamase activity was found in the culture supernatant. The identity of the enzyme was established by substrate specificity and isoelectric focusing.     

Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase from Streptomyces cacaoi

A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine beta-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces lividans TK24 using the high-copy-number plasmid pIJ702 as vector. A 30-fold higher yield of beta-lactamase was obtained from S. lividans strain ML1, carrying the recombinant plasmid pDML51, than from S. cacaoi grown under optimal production conditions. In all respects (molecular mass, isoelectric point, kinetics of inhibition by beta-iodopenicillanate) the overproduced S. lividans ML1 beta-lactamase was identical to the original S. cacaoi enzyme. A considerable reduction of beta-lactamase production was caused by elimination of a 12.8 kb portion of the 19 kb DNA fragment by cleavage at an internal SphI site located more than 3 kb upstream of the beta-lactamase structural gene. The beta-lactamase gene was located within a 1.8 NcoI-BclI fragment but when this fragment was cloned in S. lividans pIJ702, the resulting strain produced hardly any more beta-lactamase than the original S. cacaoi.     

Cloning and amplified expression in Streptomyces lividans of a gene encoding extracellular beta-lactamase from Streptomyces albus G

A 4.9-kb DNA fragment containing the bla gene for the extracellular beta-lactamase (BLA) of Streptomyces albus G was cloned in Streptomyces lividans using the conjugative, low-copy-number plasmid pIJ61 as vector. No expression of bla was observed when this DNA fragment was introduced into Escherichia coli HB101 on a plasmid vector. A 1.5-kb PstI-SstI fragment containing the bla gene was cloned in S. lividans on the nonconjugative, high-copy-number plasmid pIJ702. A tenfold higher yield of BLA was obtained from S. lividans carrying this plasmid than from S. albus G grown under optimal production conditions. The BLA from the clone reacts with beta-iodopenicillanate according to a branched pathway which is characteristic of the original S. albus G BLA enzyme.     

Improved Production of Mannanase by Streptomyces lividans

Replacement of the natural promoter of the (beta)-mannanase gene of Streptomyces lividans by lacp resulted in a 15-fold increase in enzyme production over that of the previously reported clone S. lividans IAF36, a clone carrying multiple copies of manA, and a 350-fold increase over that of the wild-type strain S. lividans 1326. In addition, the use of lacp in the shuttle vector pIAF199 allowed synthesis of the enzymes on carbon sources that did not contain mannan, such as xylan and whey, which offers interesting possibilities for industrial production of the enzyme.     

Structure of an amplifiable DNA sequence in Streptomyces lividans 66

Summary Spontaneous chloramphenicol-sensitive mutants of Streptomyces lividans 66 had previously been shown to be very unstable and to yield arginine auxotrophic mutants at a frequency of 25% of spores; the Arg^- mutants had amplified a particular 5.7 kb DNA sequence to over one hundred tandem copies per genome. In this paper we report the cloning of the amplifiable region from amplified and wild-type strains. This showed that the amplifiable fragment is already present as a duplication in wild type cells. Hybridisation experiments also demonstrated that in the amplified strains there was a deletion of neighbouring DNA sequences to one side of the amplifiable element; sequences to the other side remain intact.     

Synthesis and secretion of hirudin by Streptomyces lividans

Summary To examine the secretory production of heterologous proteins by Streptomyces lividans, we fused the DNA encoding the signal peptide of the -amylase inhibitor tendamistat, derived from S. tendae with a synthetic gene encoding the thrombin inhibitor hirudin. The analysis of secretion by immunoblots revealed an efficient translocation of hirudin through the membrane, with no detectable immunoreaction among the cellular proteins. The secreted hirudin was stable in the shaking culture for about 6 days. A comparison of the hirudin secreted by S. lividans and recombinant reference hirudin from yeast by immunoblots and thrombin inhibition assays shows that hirudin from Streptomyces has a lower specific activity, which may be due to a different aminoterminal sequence or to inexact processing of the precursor.Offprint requests to: J. Engels     

Synthesis of bovine growth hormone by Streptomyces lividans

Streptomyces lividans 66 was transformed with a plasmid containing the regulatory region of the Streptomyces fradiae aph gene and a structural gene that specifies bovine growth hormone (bGH). When grown in liquid culture the transformant contained a protein identical to authentic bGH, as judged by radioimmunoassay and immuno-blotting (Western analysis). The bGH was present in cells that had been in culture for up to four weeks but was not found in the medium. The strategy employed should be generally applicable to the expression of foreign genes in actinomycetes.     

Stability of plasmid vector plJ303 in Streptomyces lividans TK24 during laboratory-scale fermentations

Plasmid plJ303 stability in Streptomyces lividans cultures has been studied by measuring plasmid copy number under various growth conditions. An increase in mean plasmid copy number was normally seen during early rapid growth in both shaken culture and stirred vessel fermentations at 28 degrees C. Maximum copy numbers were consistently attained in early stationary phase followed by a decline (of variable amount) upon further incubation. The imposition of environmental stress (high growth temperature, i.e., 37 degrees C, and low dissolved oxygen tension, i.e., <5% air saturation) led to a plasmid copy number of zero and a 50% reduction, respectively. Interestingly, the relative proportions of plasmid topoisomers changed with time since progressively more supercoiled forms were observed throughout the stationary phase. Plasmid dimers were also observed in some cultures, and no evidence of structural plasmid instability was found. In general, this host-vector system seemed remarkably stable under normal growth conditions. However, copious organic acid production by the host was observed and was thought to be undesirable for good heterologous gene expression of a secreted protein. (c) 1993 John Wiley & Sons, Inc.     

Segregational stability of pSG5-derived vector plasmids in continuous cultures of Streptomyces lividans 66

Summary The pSG5-derived vector plasmids pGM4, pGM9 and pGM12 as well as the pIJ101 derivatives pIJ303 and pIJ702 are stably inherited at non-selective conditions in N-limited chemostats of S. Iividans 66. In glucose-limited chemostats only pGM12 and the shuttle vector pGM120 which carry the minus origin of replication are stably maintained. Small vectors lacking the minus origin as pGM4, pGM9, pGM11 and the shuttle vectors pGM103 and pGM160 were lost from the host strain.     

Expression in Streptomyces lividans of Nonomuraea genes cloned in an artificial chromosome

A bacterial artificial chromosomal library of Nonomuraea sp. ATCC39727 was constructed using Escherichia coli–Streptomyces artificial chromosome (ESAC) and screened for the presence of dbv genes known to be involved in the biosynthesis of the glycopeptide A40926. dbv genes were cloned as two large, partially overlapping, fragments and transferred into the host Streptomyces lividans, thus generating strains S. lividans∷NmESAC50 and S. lividans∷NmESAC57. The heterologous expression of Nonomuraea genes in S. lividans was successfully demonstrated by using combined RT–PCR and proteomic approaches. MALDI-TOF analysis revealed that a Nonomuraea ABC transporter is expressed as two isoforms in S. lividans. Moreover, its expression may not require a Nonomuraea positive regulator at all, as it is present at similar levels in both clones even though S. lividans∷NmESAC57 lacks regulatory genes. Considered together, these results show that S. lividans expresses Nonomuraea genes from their own promoters and support the idea that S. lividans can be a good host for genetic analysis of Nonomuraea.     

Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase of Actinomadura R39.

By using the promoter-probe plasmid pIJ424, genomic DNA fragments of Actinomadura R39 were shown to have promoter activity in Streptomyces lividans. The same 100-200-copy-number plasmid was used to clone in S. lividans TK24, the gene that encodes the Actinomadura R39 beta-lactamase. Gene cloning resulted in an amplified expression of the beta-lactamase when compared with the amounts of enzyme produced by the original strain (1 mg versus 0.008 mg.litre of culture-1).