Strain K30 Research Articles

Enhancing the heterologous expression of latex clearing protein from Streptomyces sp. strain K30 in Escherichia coli through fermentation condition optimization and molecular modification

Latex clearing protein from Streptomyces sp. strain K30 (LcpK30) is a natural oxidoreductase that can catalyse the cleavage of rubber through dioxygenation. It has significant potential applications in polymer degradation. However, its limited expression in engineered strains restricts its utility. This study aimed to enhance the soluble expression and enzyme activity of LcpK30 in E. coli BL21 (DE3) by optimizing fermentation conditions and making molecular modifications. The enzyme activity reached 5.05 U·mL−1 by optimizing the induction conditions, adding cofactors, and using chemical chaperones, which was 237.1 % of the initial case. Further enhancements in soluble expression were achieved through site mutations guided by the PROSS server, resulting in 8 out of 13 mutants with increased protein expression, a high positive mutation rate of 61.5 %. Subsequently, combined mutants were created by merging single mutants with enhanced protein expression and enzyme activity. The top three double mutants, G91D/S149A, G91D/A210H, and G91D/H296P, displayed expression levels at 173.3 %, 173.3 %, and 153.3 % of the wild-type LcpK30, respectively. These mutants also exhibited enhanced fermentation enzyme activity, reaching 149.5 %, 250.0 %, and 420.2 % compared to the wild-type, along with improved specific activities. This study provides insights for the efficient production of LcpK30 and a practical foundation for its application.

Degradation of UV-pretreated polyolefins by latex clearing protein from Streptomyces sp. Strain K30

Plastic products made of polyethylene (PE), polypropylene (PP), and polystyrene (PS) are widely used in daily life and industrial production. Polyolefins—which have a very stable structure and do not contain any active molecular groups—are difficult to degrade and pose a serious global environment threat. This study selected latex clearing protein (LcpK30) derived from Streptomyces sp. Strain K30. The natural substrate of the enzyme is rubber (cis-1, 4-polyisoprene), and the site of action is the carbon‑carbon double bond. LcpK30 was incubated with UV-irradiated polyolefin PE, PP and PS (UV-PE, UV-PP, and UV-PS containing carbon‑carbon double bonds) for 5 d at 37 °C. The results showed that UV-PE-LcpK30 was more fragmented than UV-PE-blank; the Fourier transform infrared spectroscopy results showed that UV-PE-LcpK30 and UV-PP-LcpK30 produced new active groups (e.g., –OH and –C=O); however, the effect on UV-PS was not significant. Scanning electron microscopy results showed that the treated group had more obvious roughness, cracks, and pits than the control group. The results of high-temperature gel permeation chromatography showed that the average molecular weight (Mw) of UV-PE-LcpK30 and UV-PP-LcpK30 decreased; the Mw of UV-PE5-LcpK30 was reduced by 42.02%. The results of gas chromatography–mass spectrometry showed the production of ketones. Therefore, the LcpK30 latex clearing protein degrade UV-oxidized polyolefin plastics and has great potential for PE and PP degradation but may not be suitable for PS. Furthermore, other Lcps (such as LcpNRRL, LcpNVL3) can also degrade UV-PE.

Determination of virulence and fitness genes associated with the pheU, pheV and selC integration sites of LEE-negative food-borne Shiga toxin-producing Escherichia coli strains

BackgroundIn the current study, nine foodborne “Locus of Enterocyte Effacement” (LEE)-negative Shiga toxin-producing Escherichia coli (STEC) strains were selected for whole genome sequencing and analysis for yet unknown genetic elements within the already known LEE integration sites selC, pheU and pheV. Foreign DNA ranging in size from 3.4 to 57 kbp was detected and further analyzed. Five STEC strains contained an insertion of foreign DNA adjacent to the selC tRNA gene and five and seven strains contained foreign DNA adjacent to the pheU and pheV tRNA genes, respectively. We characterized the foreign DNA insertion associated with selC (STEC O91:H21 strain 17584/1), pheU (STEC O8:H4 strain RF1a and O55:Hnt strain K30) and pheV (STEC O91:H21 strain 17584/1 and O113:H21 strain TS18/08) as examples.ResultsIn total, 293 open reading frames partially encoding putative virulence factors such as TonB-dependent receptors, DNA helicases, a hemolysin activator protein precursor, antigen 43, anti-restriction protein KlcA, ShiA, and phosphoethanolamine transferases were detected. A virulence type IV toxin-antitoxin system was detected in three strains. Additionally, the ato system was found in one strain. In strain 17584/1 we were able to define a new genomic island which we designated GIselC17584/1. The island contained integrases and mobile elements in addition to genes for increased fitness and those playing a putative role in pathogenicity.ConclusionThe data presented highlight the important role of the three tRNAs selC, pheU, and pheV for the genomic flexibility of E. coli.

Cleavage of Rubber by the Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30: Molecular Insights.

Large amounts of rubber waste materials have been permanently released into the environment for more than a century, yet accumulation of rubber particles released, e.g., by abrasion of tires along highways has not been observed. This is indicative of the ubiquitous presence and activity of rubber-degrading microorganisms. Despite increasing research activities on rubber biodegradation during the last 2 decades, the knowledge of the enzymatic cleavage mechanism of rubber by latex clearing protein (Lcp) still is limited. In particular, the catalytic cleavage mechanism and the amino acids of Lcp proteins (Lcps) that are involved have not yet been identified for any Lcp. In this study, we investigated the importance of 10 amino acid residues of Lcp from Streptomyces sp. K30 (LcpK30) by mutagenesis, mutein purification, and biochemical characterization. We identified several essential residues, one of which most likely represents an axial heme ligand in Lcp of Streptomyces sp. K30.

Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30 Is a b-Type Cytochrome and Differs from Rubber Oxygenase A (RoxA) in Its Biophysical Properties.

Specific polyisoprene-cleaving activities of 1.5 U/mg and 4.6 U/mg were determined for purified Strep-tagged latex clearing protein (Lcp) of Streptomyces sp. strain K30 at 23 °C and 37 °C, respectively. Metal analysis revealed the presence of approximately one atom of iron per Lcp molecule. Copper, which had been identified in Lcp1VH2 of Gordonia polyisoprenivorans previously, was below the detection limit in LcpK30. Heme was identified as a cofactor in purified LcpK30 by (i) detection of characteristic α-, β-, and γ (Soret)-bands at 562 nm, 532 nm, and 430 nm in the visible spectrum after chemical reduction, (ii) detection of an acetone-extractable porphyrin molecule, (iii) determination of a heme b-type-specific absorption maximum (556 nm) after chemical conversion of the heme group to a bipyridyl-heme complex, and (iv) detection of a b-heme-specific m/z value of 616.2 via mass spectrometry. Spectroscopic analysis showed that purified Lcp as isolated contains an oxidized heme-Fe(3+) that is free of bound dioxygen. This is in contrast to the rubber oxygenase RoxA, a c-type heme-containing polyisoprene-cleaving enzyme present in Gram-negative rubber degraders, in which the covalently bound heme firmly binds a dioxygen molecule. LcpK30 also differed from RoxA in the lengths of the rubber degradation cleavage products and in having a higher melting point of 61.5 °C (RoxA, 54.3 °C). In summary, RoxA and Lcp both are equipped with a heme cofactor and catalyze an oxidative C-C cleavage reaction but differ in the heme subgroup type and in several biochemical and biophysical properties. These findings suggest differences in the catalytic reaction mechanisms.

Rubber oxygenase and latex clearing protein cleave rubber to different products and use different cleavage mechanisms.

Two types of enzyme for oxidative cleavage of poly(cis-1,4-isoprene) are known. One is rubber oxygenase (RoxA) that is secreted by Xanthomonas sp. strain 35Y and a few other Gram-negative rubber-degrading bacteria during growth on polyisoprene. RoxA was studied in the past, and the recently solved structure showed a structural relationship to bacterial cytochrome c peroxidases (J. Seidel et al., Proc. Natl. Acad. Sci. U. S. A. 110:13833-13838, 2013, http://dx.doi.org/10.1073/pnas.1305560110). The other enzyme is latex-clearing protein (Lcp) that is secreted by rubber-degrading actinomycetes, but Lcp has not yet been purified. Here, we expressed Lcp of Streptomyces sp. strain K30 in a ΔroxA background of Xanthomonas sp. strain 35Y and purified native (untagged) Lcp. The specific activities of Lcp and RoxA were 0.70 and 0.48 U/mg, respectively. Lcp differed from RoxA in the absence of heme groups and other characteristics. Notably, Lcp degraded polyisoprene via endo-type cleavage to tetra-C20 and higher oligo-isoprenoids with aldehyde and keto end groups, whereas RoxA used an exo-type cleavage mechanism to give the main end product 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). RoxA was able to cleave isolated Lcp-derived oligo-isoprenoid molecules to ODTD. Inhibitor studies, spectroscopic investigations and metal analysis gave no indication for the presence of iron, other metals, or cofactors in Lcp. Our results suggest that Lcp could be a member of the growing group of cofactor-independent oxygenases and differs in the cleavage mechanism from heme-dependent RoxA. In conclusion, RoxA and Lcp represent two different answers to the same biochemical problem, the cleavage of polyisoprene, a polymer that has carbon-carbon double bonds as the only functional groups for enzymatic attack.

Historical and Recent Achievements in the Field of Microbial Degradation of Natural and Synthetic Rubber

This review intends to provide an overview of historical and recent achievements in studies of microbial degradation of natural and synthetic rubber. The main scientific focus is on the key enzymes latex-clearing protein (Lcp) from the Gram-positive Streptomyces sp. strain K30 and rubber oxygenase A (RoxA) from the Gram-negative Xanthomonas sp. strain 35Y, which has been hitherto the only known rubber-degrading bacterium that does not belong to the actinomycetes. We also emphasize the importance of knowledge of biodegradation in industrial and environmental biotechnology for waste natural rubber disposal.

Importance of the latex‐clearing protein (Lcp) for poly(cis‐1,4‐isoprene) rubber cleavage in Streptomyces sp. K30

Streptomyces sp. strain K30 induces the formation of an extracellular Lcp (latex-clearing protein) during poly(cis-1,4-isoprene) degradation. To investigate the function of this enzyme in Streptomyces sp. strain K30, the lcp gene was disrupted. This was the first time that the screening for a knock out lcp mutant of Streptomyces sp. strain K30 was successful. The resulting mutant Streptomyces sp. K30_lcpΩKm exhibited reduced growth in liquid mineral salts media containing poly(cis-1,4-isoprene) as the sole carbon and energy source. Additionally, there was no detectable Lcp activity on latex overlay agar plates. When Lcp from Streptomyces sp. strain K30 was heterologously expressed in strains TK23 and TK24 of Streptomyces lividans and a strain of S. erythraea with plasmid pIJ6021::lcp, the recombinant strains acquired the ability to cleave synthetic poly(cis-1,4-isoprene), confirming the involvement of Lcp in initial polymer cleavage. Specific anti-LcpK30 IgGs were employed in Western blot analysis to detect the secretion of Lcp in the supernatant. We have conducted an important experiment to demonstrate Lcp activity using the supernatant of these Lcp-expressing strains in vitro. All three strains obviously secreted a functional Lcp, as indicated by the formation of halo. Functional testing of Lcp with different plasmids in Escherichia coli strains and Pseudomonas strains was, however, not successful.

Isolation and characterization of Streptomyces, Actinoplanes, and Methylibium strains that are involved in degradation of natural rubber and synthetic poly( cis-1,4-isoprene)

Rubber-degrading bacteria were screened for the production of clearing zones around their colonies on latex overlay agar plates. Novel three bacteria, Streptomyces sp. strain LCIC4, Actinoplanes sp. strain OR16, and Methylibium sp. strain NS21, were isolated. To the best of our knowledge, this is the first report on the isolation of a Gram-negative rubber-degrading bacterium other than γ-proteobacteria. Gel permeation chromatography analysis revealed that these strains degraded poly( cis-1,4-isoprene) to low-molecular-weight products. The occurrence of aldehyde groups in the degradation products by NS21 was suggested by staining with Schiff's reagent and 1H-nuclear magnetic resonance spectroscopy. The lcp gene of LCIC4, which showed 99% amino acid sequence identity with that of Streptomyces sp. strain K30, was cloned, and contained a putative twin-arginine motif at its N terminus. It is located next to oxiB, which is estimated to be responsible for oxidation of degradation intermediate of rubber in K30. Southern hybridization analysis using LCIC4 lcp probe revealed the presence of a lcp-homolog in OR16. These results suggest that the lcp-homologs are involved in rubber degradation in LCIC4 and OR16.

Secretion and transcriptional regulation of the latex-clearing protein, Lcp, by the rubber-degrading bacterium Streptomyces sp. strain K30.

About 22,000 1-methyl-3-nitro-1-nitrosoguanidine- and UV-induced mutants of the rubber-degrading bacterium Streptomyces sp. strain K30 were characterized for the ability to produce clear zones on natural rubber latex overlay agar plates. Thirty-five mutants were defective solely in cleavage of rubber and were phenotypically complemented with the wild-type lcp (latex clearing protein) gene. Sixty-nine mutants exhibited a pleiotropic phenotype and were impaired in utilization of rubber and xylan, indicating that the enzymes responsible for the initial cleavage of these polymers are exported by the same secretion pathway (Q. K. Beg, M. Kapoor, L. Mahajan, and G. S. Hoondal, Appl. Microbiol. Biotechnol. 56:326-3381, 2001; U. K. Laemmli, Nature 227:680-685, 1970). Analysis of the amino acid sequence encoded by lcp revealed a twin-arginine motif, indicating that Lcp is a substrate of the twin-arginine translocation (Tat) pathway (K. Dilks, W. Rose, E. Hartmann, and M. Pohlschröder, J. Bacteriol. 185:1478-1483, 2003). A tatC disruption mutant of Streptomyces lividans 10-164 harboring lcp from Streptomyces sp. strain K30 was not capable of forming clear zones on rubber overlay agar plates. Moreover, Lcp and enhanced green fluorescent protein fusion proteins were detected in the supernatant. Using Escherichia coli having the twin-arginine motif in the signal peptide upstream of Lcp, clear evidence that Lcp is secreted was obtained. Transcriptional analysis revealed basal expression of Lcp in glucose-grown cells and that transcription of lcp is obviously induced in the presence of poly(cis-1,4-isoprene). In contrast, oxiB and oxiA, which are located directly downstream of lcp and putatively encode a heteromultimeric aldehyde dehydrogenase oxidizing the primary cleavage products generated by Lcp from poly(cis-1,4-isoprene), were expressed only in the presence of poly(cis-1,4-isoprene). Expression of lcp at a low level is thus required for sensing the polymer in the medium. Rubber degradation products may then induce the transcription of genes coding for enzymes catalyzing the later steps of poly(cis-1,4-isoprene) degradation and the transcription of lcp itself. lcp, oxiB, and oxiA seem to constitute an operon, as a polycistronic mRNA comprising these three genes was detected. The transcriptional start site of lcp was mapped 400 bp upstream of the lcp start codon.

The genomes of the non-clearing-zone-forming and natural-rubber- degrading species Gordonia polyisoprenivorans and Gordonia westfalica harbor genes expressing Lcp activity in Streptomyces strains.

The latex-clearing protein (Lcp(K30)) from the rubber-degrading bacterium Streptomyces sp. strain K30 is involved in the cleavage of poly(cis-1,4-isoprene), yielding isoprenoid aldehydes and ketones. Lcp homologues have so far been detected in all investigated clearing-zone-forming rubber-degrading bacteria. Internal degenerated oligonucleotides derived from lcp genes of Streptomyces sp. strain K30 (lcp(K30)), Streptomyces coelicolor strain A3(2), and Nocardia farcinica strains IFM10152 and E1 were applied in PCR to investigate whether lcp homologues occur also in the non-clearing-zone-forming rubber-utilizing bacteria Gordonia polyisoprenivorans strains VH2 and Y2K, Gordonia alkanivorans strain 44187, and Gordonia westfalica strain Kb1, which grow adhesively on rubber. The 1,230- and 1,224-bp lcp-homologous genes from G. polyisoprenivorans strain VH2 (lcp(VH2)) and G. westfalica strain Kb1 (lcp(Kb1)) were obtained after screening genomic libraries by degenerated PCR amplification, and their translational products exhibited 50 and 52% amino acid identity, respectively, to Lcp(K30). Recombinant lcp(VH2) and lcp(Kb1) harboring cells of the non-rubber-degrading Streptomyces lividans strain TK23 were able to form clearing zones and aldehydes on latex overlay-agar plates, thus indicating that lcp(VH2) and lcp(Kb1) encode functionally active proteins. Analysis by gel permeation chromatography demonstrated lower polymer concentrations and molecular weights of the remaining polyisoprenoid molecules after incubation with these recombinant S. lividans strains. Reverse transcription-PCR analysis demonstrated that lcp(VH2) was transcribed in cells of G. polyisoprenivorans strain VH2 cultivated in the presence of poly(cis-1,4-isoprene) but not in the presence of sodium acetate. Anti-Lcp(K30) immunoglobulin Gs, which were raised in this study, were rather specific for Lcp(K30) and did not cross-react with Lcp(VH2) and Lcp(Kb1). A lcp(VH2) disruption mutant was still able to grow with poly(cis-1,4-isoprene) as sole carbon source; therefore, lcp(VH2) seems not to be essential for rubber degradation in G. polyisoprenivorans.

Identification of Poly( cis -1,4-Isoprene) Degradation Intermediates during Growth of Moderately Thermophilic Actinomycetes on Rubber and Cloning of a Functional lcp Homologue from Nocardia farcinica Strain E1

The enrichment and isolation of thermophilic bacteria capable of rubber [poly(cis-1,4-isoprene)] degradation revealed eight different strains exhibiting both currently known strategies used by rubber-degrading mesophilic bacteria. Taxonomic characterization of these isolates by 16S rRNA gene sequence analysis demonstrated closest relationships to Actinomadura nitritigenes, Nocardia farcinica, and Thermomonospora curvata. While strains related to N. farcinica exhibited adhesive growth as described for mycolic acid-containing actinomycetes belonging to the genus Gordonia, strains related to A. nitritigenes and T. curvata formed translucent halos on natural rubber latex agar as described for several mycelium-forming actinomycetes. For all strains, optimum growth rates were observed at 50 degrees C. The capability of rubber degradation was confirmed by mineralization experiments and by gel permeation chromatography (GPC). Intermediates resulting from early degradation steps were purified by preparative GPC, and their analysis by infrared spectroscopy revealed the occurrence of carbonyl carbon atoms. Staining with Schiff's reagent also revealed the presence of aldehyde groups in the intermediates. Bifunctional isoprenoid species terminated with a keto and aldehyde function were found by matrix-assisted laser desorption ionization-time-of-flight and electrospray ionization mass spectrometry analyses. Evidence was obtained that biodegradation of poly(cis-1,4-isoprene) is initiated by endocleavage, rather than by exocleavage. A gene (lcp) coding for a protein with high homology to Lcp (latex-clearing protein) from Streptomyces sp. strain K30 was identified in Nocardia farcinica E1. Streptomyces lividans TK23 expressing this Lcp homologue was able to cleave synthetic poly(cis-1,4-isoprene), confirming its involvement in initial polymer cleavage.

Identification and Characterization of Genes fromStreptomycessp. Strain K30 Responsible for Clear Zone Formation on Natural Rubber Latex and Poly(cis-1,4-isoprene) Rubber Degradation

Streptomyces sp. strain K30 was isolated from soil next to a city high way in Münster (Germany) according to its ability to degrade natural and synthetic poly(cis-1,4-isoprene) rubber and to form clear zones on natural rubber latex agar plates. The clear zone forming phenotype was used to clone the responsible gene by phenotypic complementation of a clear zone negative mutant. An open reading frame (lcp) of 1,191 bp was identified, which was preceded by a putative signal sequence and restored the capability to form clear zones on natural rubber latex in the mutant. The putative translation product exhibited strong homologies (50% aa identity) to a putative secreted protein from Streptomyces coelicolor strain A3(2), another clear zone forming strain. Heterologous expression of lcp of Streptomyces sp. strain K30 in Streptomyces lividans strain TK23 enabled the latter to form clear zones on latex-overlay agar plates and to accumulate a degradation product of about 12 kDa containing aldehyde groups. Two ORFs putatively encoding a heterodimeric molybdenum hydroxylase (oxiAB) were identified downstream of lcp in Streptomyces sp. strain K30 strain which exerted a positive effect on clear zone formation and enabled the strain to oxidize the resulting aldehydes. Heterologous expression of a fragment harboring lcp plus oxiAB in S. lividans TK23 resulted in accumulation of aldehydes only in the presence of 10 mM tungstate. Determination of protein content during cultivation on poly(cis-1,4-isoprene) revealed an increase of the cellular protein, and gel permeation chromatography analysis indicated a shift of the molecular weight distribution of the rubber to lower values in the transgenic S. lividans strains and in the wild type, thus confirming utilization and degradation of rubber. Therefore, for the first time, genes responsible for clear zone formation on natural rubber latex and synthetic cis-1,4-polyisoprene degradation in Gram-positive bacteria were identified and characterized.