Strain Improvement and Strain Maintenance Revisited. The Use of Actinoplanes teichomyceticus ATCC 31121 Protoplasts in the Identification of Candidates for Enhanced Teicoplanin Production.

Multicellular cooperation in actinomycetes is a division of labor-based beneficial trait where phenotypically specialized clonal subpopulations, or genetically distinct lineages, perform complementary tasks. The division of labor improves the access to nutrients and optimizes reproductive and vegetative tasks while reducing the costly production of secondary metabolites and/or of secreted enzymes. In this study, we took advantage of the possibility to isolate genetically distinct lineages deriving from the division of labor, for the isolation of heterogeneous teicoplanin producer phenotypes from Actinoplanes teichomyceticus ATCC 31121. In order to efficiently separate phenotypes and associated genomes, we produced and regenerated protoplasts. This approach turned out to be a rapid and effective strain improvement method, as it allowed the identification of those phenotypes in the population that produced higher teicoplanin amounts. Interestingly, a heterogeneous teicoplanin complex productivity pattern was also identified among the clones. This study suggests that strain improvement and strain maintenance should be integrated with the use of protoplasts as a strategy to unravel the hidden industrial potential of vegetative mycelium.

Enzyme Bioprospection of Marine-Derived Actinobacteria from the Chilean Coast and New Insight in the Mechanism of Keratin Degradation in Streptomyces sp. G11C.

Marine actinobacteria are viewed as a promising source of enzymes with potential technological applications. They contribute to the turnover of complex biopolymers, such as pectin, lignocellulose, chitin, and keratin, being able to secrete a wide variety of extracellular enzymes. Among these, keratinases are a valuable alternative for recycling keratin-rich waste, which is generated in large quantities by the poultry industry. In this work, we explored the biocatalytic potential of 75 marine-derived actinobacterial strains, focusing mainly on the search for keratinases. A major part of the strains secreted industrially important enzymes, such as proteases, lipases, cellulases, amylases, and keratinases. Among these, we identified two streptomycete strains that presented great potential for recycling keratin wastes-Streptomyces sp. CHA1 and Streptomyces sp. G11C. Substrate concentration, incubation temperature, and, to a lesser extent, inoculum size were found to be important parameters that influenced the production of keratinolytic enzymes in both strains. In addition, proteomic analysis of culture broths from Streptomyces sp. G11C on turkey feathers showed a high abundance and diversity of peptidases, belonging mainly to the serine and metallo-superfamilies. Two proteases from families S08 and M06 were highly expressed. These results contributed to elucidate the mechanism of keratin degradation mediated by streptomycetes.

The importance of fermentative conditions for the biotechnological production of lignin modifying enzymes from white-rot fungi.

White-rot fungi are the main natural producers of lignin-modifying enzymes, i.e. laccases and peroxidases, whose secretion and activity allows the depolymerization of lignin and the release of polysaccharides contained in lignocellulose. These enzymes are able to oxidize, in addition to lignin, a wide spectrum of natural and synthetic substrates, making their industrial and biotechnological application appealing. However, the complex regulation of the synthesis of lignin-modifying enzymes, as well as the heterogeneous physiology of fungi in response to nutrients, makes the use of white-rot fungi as production platforms challenging. Finally, yet importantly, analytical methods are not fully standardized, making evaluations and comparisons ambiguous. Consequently, robust and cost-effective fermentative processes for the production of lignin-modifying enzymes by fungi have not yet been fully established, limiting their industrial exploitation. In this review, we describe the importance of both the media composition and the fermentative conditions for leveraging the fungal potential in terms of production titer and enzymatic biodiversity of lignin-modifying enzymes.

Production and characterization of a novel antifungal chitinase identified by functional screening of a suppressive-soil metagenome.

BACKGROUND: Through functional screening of a fosmid library, generated from a phytopathogen-suppressive soil metagenome, the novel antifungal chitinase-named Chi18H8 and belonging to family 18 glycosyl hydrolases-was previously discovered. The initial extremely low yield of Chi18H8 recombinant production and purification from Escherichia coli cells (21 µg/g cell) limited its characterization, thus preventing further investigation on its biotechnological potential. RESULTS: We report on how we succeeded in producing hundreds of milligrams of pure and biologically active Chi18H8 by developing and scaling up to a high-yielding, 30 L bioreactor process, based on a novel method of mild solubilization of E. coli inclusion bodies in lactic acid aqueous solution, coupled with a single step purification by hydrophobic interaction chromatography. Chi18H8 was characterized as a Ca2+-dependent mesophilic chitobiosidase, active on chitin substrates at acidic pHs and possessing interesting features, such as solvent tolerance, long-term stability in acidic environment and antifungal activity against the phytopathogens Fusarium graminearum and Rhizoctonia solani. Additionally, Chi18H8 was found to operate according to a non-processive endomode of action on a water-soluble chitin-like substrate. CONCLUSIONS: Expression screening of a metagenomic library may allow access to the functional diversity of uncultivable microbiota and to the discovery of novel enzymes useful for biotechnological applications. A persisting bottleneck, however, is the lack of methods for large scale production of metagenome-sourced enzymes from genes of unknown origin in the commonly used microbial hosts. To our knowledge, this is the first report on a novel metagenome-sourced enzyme produced in hundreds-of-milligram amount by recovering the protein in the biologically active form from recombinant E. coli inclusion bodies.

Exploring the Diversity and Antimicrobial Potential of Marine Actinobacteria from the Comau Fjord in Northern Patagonia, Chile.

Bioprospecting natural products in marine bacteria from fjord environments are attractive due to their unique geographical features. Although, Actinobacteria are well known for producing a myriad of bioactive compounds, investigations regarding fjord-derived marine Actinobacteria are scarce. In this study, the diversity and biotechnological potential of Actinobacteria isolated from marine sediments within the Comau fjord, in Northern Chilean Patagonia, were assessed by culture-based approaches. The 16S rRNA gene sequences revealed that members phylogenetically related to the Micrococcaceae, Dermabacteraceae, Brevibacteriaceae, Corynebacteriaceae, Microbacteriaceae, Dietziaceae, Nocardiaceae, and Streptomycetaceae families were present at the Comau fjord. A high diversity of cultivable Actinobacteria (10 genera) was retrieved by using only five different isolation media. Four isolates belonging to Arthrobacter, Brevibacterium, Corynebacterium and Kocuria genera showed 16S rRNA gene identity <98.7% suggesting that they are novel species. Physiological features such as salt tolerance, artificial sea water requirement, growth temperature, pigmentation and antimicrobial activity were evaluated. Arthrobacter, Brachybacterium, Curtobacterium, Rhodococcus, and Streptomyces isolates showed strong inhibition against both Gram-negative Pseudomonas aeruginosa, Escherichia coli and Salmonella enterica and Gram-positive Staphylococcus aureus, Listeria monocytogenes. Antimicrobial activities in Brachybacterium, Curtobacterium, and Rhodococcus have been scarcely reported, suggesting that non-mycelial strains are a suitable source of bioactive compounds. In addition, all strains bear at least one of the biosynthetic genes coding for NRPS (91%), PKS I (18%), and PKS II (73%). Our results indicate that the Comau fjord is a promising source of novel Actinobacteria with biotechnological potential for producing biologically active compounds.

Proteomics meets blue biotechnology: a wealth of novelties and opportunities.

Blue biotechnology, in which aquatic environments provide the inspiration for various products such as food additives, aquaculture, biosensors, green chemistry, bioenergy, and pharmaceuticals, holds enormous promise. Large-scale efforts to sequence aquatic genomes and metagenomes, as well as campaigns to isolate new organisms and culture-based screenings, are helping to push the boundaries of known organisms. Mass spectrometry-based proteomics can complement 16S gene sequencing in the effort to discover new organisms of potential relevance to blue biotechnology by facilitating the rapid screening of microbial isolates and by providing in depth profiles of the proteomes and metaproteomes of marine organisms, both model cultivable isolates and, more recently, exotic non-cultivable species and communities. Proteomics has already contributed to blue biotechnology by identifying aquatic proteins with potential applications to food fermentation, the textile industry, and biomedical drug development. In this review, we discuss historical developments in blue biotechnology, the current limitations to the known marine biosphere, and the ways in which mass spectrometry can expand that knowledge. We further speculate about directions that research in blue biotechnology will take given current and near-future technological advancements in mass spectrometry.

Methods for the genetic manipulation of Nonomuraea sp. ATCC 39727.

Nonomuraea sp. ATCC 39727 belongs to the Streptosporangiaceae family of filamentous actinomycetes. This microorganism produces the teicoplanin-like glycopeptide A40926, which is the starting material for the synthesis of the second-generation glycopeptide dalbavancin. Notwithstanding the strain's pharmaceutical relevance, the lack or poor efficiency of genetic tools to manipulate Nonomuraea sp. ATCC 39727 has hampered strain and product improvement. Here we report the development of gene transfer systems based on protoplast transformation and intergeneric conjugation from Escherichia coli. Efficiency of transformation and conjugation, followed by site specific or homologous recombination with the Nonomuraea sp. genome, were determined using the integrative plasmid pSET152 (5.7 kb), and the Supercos1 derivative cosmid A40ΔY (30 kb). To our knowledge, this is the first report of the transformation of protoplasts of Nonomuraea sp. ATCC 39727, even though the improved procedure for intergeneric conjugation makes it the method of choice for introducing large segments of DNA into Nonomuraea sp. ATCC 39727.

Novel mechanism of glycopeptide resistance in the A40926 producer Nonomuraea sp. ATCC 39727.

In glycopeptide-resistant enterococci and staphylococci, high-level resistance is achieved by replacing the C-terminal d-alanyl-d-alanine of lipid II with d-alanyl-d-lactate, thus reducing glycopeptide affinity for cell wall targets. Reorganization of the cell wall in these organisms is directed by the vanHAX gene cluster. Similar self-resistance mechanisms have been reported for glycopeptide-producing actinomycetes. We investigated glycopeptide resistance in Nonomuraea sp. ATCC 39727, the producer of the glycopeptide A40926, which is the precursor of the semisynthetic antibiotic dalbavancin, which is currently in phase III clinical trials. The MIC of Nonomuraea sp. ATCC 39727 toward A40926 during vegetative growth was 4 microg/ml, but this increased to ca. 20 microg/ml during A40926 production. vanHAX gene clusters were not detected in Nonomuraea sp. ATCC 39727 by Southern hybridization or by PCR with degenerate primers. However, the dbv gene cluster for A40926 production contains a gene, vanY (ORF7), potentially encoding an enzyme capable of removing the terminal d-Ala residue of pentapeptide peptidoglycan precursors. Analysis of UDP-linked peptidoglycan precursors in Nonomuraea sp. ATCC 39727 revealed the predominant presence of the tetrapeptide UDP-MurNAc-l-Ala-d-Glu-meso-Dap-d-Ala and only traces of the pentapeptide UDP-MurNAc-l-Ala-d-Glu-meso-Dap-d-Ala-d-Ala. This suggested a novel mechanism of glycopeptide resistance in Nonomuraea sp. ATCC 39727 that was based on the d,d-carboxypeptidase activity of vanY. Consistent with this, a vanY-null mutant of Nonomuraea sp. ATCC 39727 demonstrated a reduced level of glycopeptide resistance, without affecting A40926 productivity. Heterologous expression of vanY in a sensitive Streptomyces species, Streptomyces venezuelae, resulted in higher levels of glycopeptide resistance.

Protoplast preparation and reversion to the normal filamentous growth in antibiotic-producing uncommon actinomycetes.

Protoplast preparation, regeneration and fusion represent essential tools for those poorly studied biotechnologically valuable microorganisms inapplicable with the current molecular biology protocols. The protoplast production and regeneration method developed for Planobispora rosea and using the combination of hen egg-white lysozyme (HEWL) and Streptomyces globisporus mutanolysin was applied to a set of antibiotic-producing filamentous actinomycetes belonging to the Streptosporangiaceae, Micromonosporaceae and Streptomycetaceae. 10(7)-10(9) protoplasts were obtained from 100 ml of culture, after incubation times in the digestion solution ranging from a few hours to 1 or 2 days depending on the strain. The efficiency of protoplast reversion to the normal filamentous growth varied from 0.1 to nearly 50%. Analysis of cell wall peptidoglycan in three representative strains (Nonomuraea sp. ATCC 39727, Actinoplanes teichomyceticus ATCC 31121 and Streptomyces coelicolor A3(2)) has evidenced structural variations in the glycan strand and in the peptide chain, which may account for the different response to cell digestion and protoplast regeneration treatments.

Protoplast fusion and gene recombination in the uncommon Actinomycete Planobispora rosea producing GE2270.

An efficient method for protoplast generation for the uncommon actinomycete Planobispora rosea, the producer of the thiazolylpeptide antibiotic GE2270, was developed using a combination of hen egg white lysozyme and Streptomyces globisporus mutanolysin. This method converted more than 70% of vegetative mycelium to protoplasts, which were then regenerated with 50% efficiency in an optimized medium. When P. rosea protoplasts were efficiently fused, recombination between different antibiotic (streptomycin and gentamicin) resistance markers originated sensitive strains (str(s)gen(s)) at frequencies as high as 18% and double resistant fusants (str(r)gen(r)) at frequencies as high as 29%. Double resistant fusants showed GE2270 productivity intermediate between the productivity of the parental strains. Protoplast generation and fusion in P. rosea makes whole genome shuffling feasible as an approach to be used alternately with classical random mutagenesis in industrial strain improvement programs.

Resistance to glycopeptide antibiotics in the teicoplanin producer is mediated by van gene homologue expression directing the synthesis of a modified cell wall peptidoglycan.

Glycopeptide resistance has been studied in detail in enterococci and staphylococci. In these microorganisms, high-level resistance is achieved by replacing the C-terminal D-alanyl-D-alanine of the nascent peptidoglycan with D-alanyl-D-lactate or D-alanyl-D-serine, thus reducing the affinities of glycopeptides for cell wall targets. Reorganization of the cell wall is directed by the expression of the van gene clusters. The identification of van gene homologs in the genomes of several glycopeptide-producing actinomycetes suggests the involvement of a similar self-resistance mechanism to avoid suicide. This report describes a comprehensive study of self-resistance in Actinoplanes teichomyceticus ATCC 31121, the producer of the clinically relevant glycopeptide teicoplanin. A. teichomyceticus ATCC 31121 showed a MIC of teicoplanin of 25 microg/ml and a MIC of vancomycin of 90 microg/ml during vegetative growth. The vanH, vanA, and vanX genes of A. teichomyceticus were found to be organized in an operon whose transcription was constitutive. Analysis of the UDP-linked peptidoglycan precursors revealed the presence of UDP-glycomuramyl pentadepsipeptide terminating in D-alanyl-D-lactate. No trace of precursors ending in d-alanyl-d-alanine was detected. Thus, the van gene complex was transcribed and expressed in the genetic background of A. teichomyceticus and conferred resistance to vancomycin and teicoplanin through the modification of cell wall biosynthesis. During teicoplanin production (maximum productivity, 70 to 80 microg/ml), the MIC of teicoplanin remained in the range of 25 to 35 microg/ml. Teicoplanin-producing cells were found to be tolerant to high concentrations of exogenously added glycopeptides, which were not bactericidal even at 5,000 microg/ml.

Antibiotic production improvement in the rare actinomycete Planobispora rosea by selection of mutants resistant to the aminoglycosides streptomycin and gentamycin and to rifamycin.

During a strain improvement program, spontaneous mutants with single or combined resistance to streptomycin (Str(r)), gentamycin (Gen(r)) or rifamycin (Rif(r)) were selected from the industrial strain of Planobispora rosea, which is the producer of thiazolylpeptide GE2270. Among the mutants resistant to each single antibiotic, higher producers occurred more frequently (60%) among Gen(r) than in Rif(r) (10%) and Str(r) (24%) populations. Two Gen(r) mutants showed up to 1.5-fold improvement in GE2270 production while single resistant mutants Str(r) and Rif(r) produced slightly more than the parental strains. The combination of Str(r) and Rif(r) in the same strain improved GE2270 yield up to 1.7-fold. Finally, a higher GE2270 producing strain (1.8-fold improvement with respect to the parental strain) was selected among those mutants with triple resistance to streptomycin, rifamycin and gentamycin. A hierarchical increase in aerial mycelium and spore formation was observed which paralleled GE2270 production improvement.

Valine influences production and complex composition of glycopeptide antibiotic A40926 in fermentations of Nonomuraea sp. ATCC 39727.

In actinomycetes the catabolism products of branched chain amino acids provide biosynthetic precursors for the formation of several lipid-containing antibiotics. We have determined in Nonomuraea sp. ATCC 39727 the effect of valine on production of glycopeptide antibiotic A40926, which is a complex of factors structurally differing in fatty acid moieties. Addition of valine to minimal medium increased A40926 production and modified complex composition towards a mono-component. Similar results were also obtained in a rich production medium.

Production and characterization of monochlorinated and dechlorinated A40926 derivatives.

Nonomuraea sp. ATCC 39727 is the producer of the A40926 complex of lipoglycopeptide antibiotics which contain chlorine atoms in amino acids 3 and 6 of the peptide backbone. Using a classical mutagenesis and selection approach we have isolated a Nonomuraea sp. ATCC 39727 mutant strain able to direct production towards new A40926 analogues dechloro-A40926 (DDC) lacking two chlorine atoms and the two monochloro-A40926 (MDC1 and MDC2) that are not produced fermenting the wild type strain. Dechlorinated A40926 derivatives were obtained in considerable amount in a standard fermentation process and were purified and chemically characterized. The dechlorinated A40926 derivatives DDC and MDC2 showed improved antimicrobial activity against coagulase negative staphylococci strains in respect to A40926 complex. Dechlorinated derivatives of the related antibiotic teicoplanin are also reported in the literature and are generally less active than the parental products.

The gene cluster for the biosynthesis of the glycopeptide antibiotic A40926 by nonomuraea species.

The glycopeptide A40926 is the precursor of dalbavancin, a second-generation glycopeptide currently under clinical development. The dbv gene cluster, devoted to A40926 biosynthesis, was isolated and characterized from the actinomycete Nonomuraea species ATCC39727. From sequence analysis, 37 open reading frames (ORFs) participate in A40926 biosynthesis, regulation, resistance, and export. Of these, 27 ORFs find a match in at least one of the previously characterized glycopeptide gene clusters, while 10 ORFs are, so far, unique to the dbv cluster. Putative genes could be identified responsible for some of the tailoring steps (attachment of glucosamine, sugar oxidation, and mannosylation) expected during A40926 biosynthesis. After constructing a Nonomuraea mutant by deleting dbv ORFs 8 to 10, the novel compound dechloromannosyl-A40926 aglycone was isolated.

Analysis of transcription of the bph locus of Burkholderia sp. strain LB400 and evidence that the ORF0 gene product acts as a regulator of the bphA1 promoter.

Although gene clusters for the degradation of biphenyls and polychlorobiphenyls have been extensively characterized, comparatively little is known about the regulation of their expression. In the present work, different aspects of transcription of the bph locus of the potent polychlorobiphenyl degrader Burkholderia sp. strain LB400 were investigated. An RNA blot analysis of the entire gene cluster revealed that the transcription of all genes encoding biphenyl catabolic enzymes responded similarly to the presence of biphenyl, succinate or a mixture of the two. One region of the locus, encompassing ORF0, was separately transcribed and differently regulated. A single start position was mapped for this monocistronic transcript. Synthesis of the adjacent RNA, encoding subunits of biphenyl dioxygenase, was strongly biphenyl-inducible. In this case, four major 5'-ends were mapped between 25 and 70 bp upstream of the start codon of gene bphA1. Sequence elements between approximately positions 710 and 1080 upstream were required in cis for full functioning of the respective promoter(s) (P(bphA1)). ORF0(-) mutants of strain LB400 retained the ability to grow on biphenyl, but showed decreased concentrations of bphA1A2 RNA and decreased lacZ expression in strains harbouring a reporter system with a bphA1-lacZ transcriptional fusion. This effect was compensated by the introduction of an intact ORF0 in trans, indicating that the ORF0 gene product mediates activation of P(bphA1).