Actinoplanes utahensis ZJB-08196 fed-batch fermentation at elevated osmolality for enhancing acarbose production

Acarbose, a potent α-glucosidase inhibitor, is as an oral anti-diabetic drug for treatment of the type two, noninsulin-dependent diabetes. Actinoplanes utahensis ZJB-08196, an osmosis-resistant actinomycete, had a broad osmolality optimum between 309 mOsm kg⁻¹ and 719 mOsm kg⁻¹. Utilizing this unique feature, an fed-batch culture process under preferential osmolality was constructed through intermittently feeding broths with feed medium consisting of 14.0 g l⁻¹ maltose, 6.0 g l⁻¹ glucose and 9.0 g l⁻¹ soybean meal, at 48 h, 72 h, 96 h and 120 h. This intermittent fed-batch culture produced a peak acarbose titer of 4878 mg l⁻¹, increased by 15.9% over the batch culture.     

Production of teicoplanin by Actinoplanes teichomyceticus in continuous fermentation.

Production of the potent antibiotic teicoplanin by Actinoplanes teichomyceticus was studied in batch and in chemostat cultures. It is found that the producing strain deactivates to a non-producing strain named NP-12. This strain is used to find the growth kinetics of the A. teichomyceticus without interference from the product teicoplanin. In batch experiments with NP-12 grown on glucose at different initial concentrations and with different added amounts of teicoplanin, the strong inhibitory effect of teicoplanin was determined. These results obtained on NP-12 were validated in a series of chemostat experiments with the processing strain. All experiments in batch and in chemostat cultures were well represented by Monod kinetics with respect to the carbon and energy source (glucose) and with a substantial inhibitory effect of teicoplanin. Further experiments were made with the producing strain in a continuous reactor coupled to a microfilter that delivers a cell-free permeate. It was found that the derived kinetics almost exactly simulated the behavior of the cell recirculation reactor in addition to when the cell concentration in the reactor was more than four times higher than in the chemostat. For industrial production of teicoplanin, a continuous reactor with cell recirculation and working with a low effluent glucose concentration was by far the best mode of operation. Finally, the deactivation of the producing strain to NP-12 was modeled by a two-step deactivation mechanism. Deactivation was independent of dilution rate but dependent on the inoculum preparation and on the previous history of the inoculum.     

Identification of precursor peptide of aculeacin A acylase as a protein with proteolytic activity

Abstract A protein with the proteolytic activity was isolated from culture filtrate of the aculeacin A acylase producing strain, Actinoplanes utahensis NRRL12052. The purified protein showed a single band of molecular mass of 87 kDa in SDS-PAGE and gel filtration using HPLC, and reacted with anti-aculeacin A acylase antiserum. The 87-kDa protein was degraded to two peptides of molecular mass of 60 kDa and 19 kDa by incubation at 37°C in the presence of 0.1% SDS and the former band also responded to the antiserum. These results indicate that the 87-kDa protein possessing the proteolytic activity is a precursor of aculeacin A acylase.     

Biosynthesis,biotechnological production,and application of teicoplanin: current state and perspectives

The glycopeptide teicoplanin isolated from the fermentation broth of Actinoplanes teichomyceticus is used to treat serious Gram-positive bacterial infections that are resistant to other antibiotics, e.g. β-lactams. The long time frame and progressively broader clinical use of teicoplanin has eventually led to the emergence and spreading of resistance in enterococci and staphylococci towards the antibiotics. Given the structural complexity of the natural product, only fermentative routes are available for bulk production of teicoplanin even though the total synthesis of the antibiotic has been accomplished. Because the low productivity (0.1–3.1 g/L) is a limitation to the commercial production of teicoplanin, substantial effort has been devoted to the strain improvement and process development for enhancing the productivity. This review summarizes the current state of the action mechanism, antibacterial activity, resistance mechanism, biotechnological production, and application of teicoplanin. Hyung-Moo Jung and Marimuthu Jeya equally contributed to this work.     

Application of conjugation using ϕC31 <Emphasis Type="Italic">att/int</Emphasis> system for <Emphasis Type="Italic">Actinoplanes teichomyceticus</Emphasis>, a producer of teicoplanin

Actinoplanes teichomyceticus produces teicoplanin, which is a glycopeptide antibiotic for Gram-positive pathogenic bacteria and methicillin-resistant Staphylococcus aureus (MRSA). For a molecular genetic study of A. teichomyceticus, an effective transformation method using the conjugal transfer of DNA from E. coli to spores of A. teichomyceticus was established for the first time, based on the bacteriophage ϕC31 att/int system, in the genus of Actinoplanes. The high frequency of transconjugation was obtained on MS medium containing 40 mM MgCl₂, using 1.25 × 10⁸ E. coli donor cells and 10⁵ spores without a heat treatment. In addition, by cloning and sequencing the attB site A. teichomyceticus was shown to contain a single attB site within an ORF coding for a pirin homolog. Also, its attB site sequence showed high homology to that of Streptomyces lividans, unlike the case of Kitasatospora setae despite being a non-Streptomyces actinomycete, which seems to be closely related to the high transconjugation frequency of A. teichomyceticus.     

Isolation and characterization of maltokinase (ATP:maltose 1-phosphotransferase) from<Emphasis Type="Italic"> Actinoplanes missouriensis</Emphasis>

Crude extracts of Actinoplanes missouriensis and related strains catalyze the ATP-dependent phosphorylation of maltose to maltose 1-phosphate. The enzyme of A. missouriensis responsible for this reaction was purified and characterized. This protein has an estimated molecular mass of 57 kDa and it is most likely a monomer. The K_m value was 2.6 mM for maltose and 0.54 mM for ATP. Only maltose acted effectively as phosphoryl-group acceptor, and ATP was not replaceable as phosphoryl-group donor. Tryptic peptides of the enzyme were sequenced, and the sequences of these peptides will allow construction of degenerate primers to identify the gene coding for this unique kinase.     

The structures of L-rhamnose isomerase from Pseudomonas stutzeri in complexes with L-rhamnose and D-allose provide insights into broad substrate specificity

Pseudomonas stutzeril-rhamnose isomerase (P. stutzeri L-RhI) can efficiently catalyze the isomerization between various aldoses and ketoses, showing a broad substrate specificity compared to L-RhI from Escherichia coli (E. coli L-RhI). To understand the relationship between structure and substrate specificity, the crystal structures of P. stutzeri L-RhI alone and in complexes with l-rhamnose and d-allose which has different configurations of C4 and C5 from l-rhamnose, were determined at a resolution of 2.0 Å, 1.97 Å, and 1.97 Å, respectively. P. stutzeri L-RhI has a large domain with a (β/α)₈ barrel fold and an additional small domain composed of seven α-helices, forming a homo tetramer, as found in E. coli L-RhI and d-xylose isomerases (D-XIs) from various microorganisms. The β1-α1 loop (Gly60-Arg76) of P. stutzeri L-RhI is involved in the substrate binding of a neighbouring molecule, as found in D-XIs, while in E. coli L-RhI, the corresponding β1-α1 loop is extended (Asp52-Arg78) and covers the substrate-binding site of the same molecule. The complex structures of P. stutzeri L-RhI with l-rhamnose and d-allose show that both substrates are nicely fitted to the substrate -binding site. The part of the substrate-binding site interacting with the substrate at the 1, 2, and 3 positions is equivalent to E. coli L-RhI, and the other part interacting with the 4, 5, and 6 positions is similar to D-XI. In E. coli L-RhI, the β1-α1 loop creates an unique hydrophobic pocket at the the 4, 5, and 6 positions, leading to the strictly recognition of l-rhamnose as the most suitable substrate, while in P. stutzeri L-RhI, there is no corresponding hydrophobic pocket where Phe66 from a neighbouring molecule merely forms hydrophobic interactions with the substrate, leading to the loose substrate recognition at the 4, 5, and 6 positions.     

Regeneration of Mycelial Protoplasts from Lyophyllum shimeji

A new HPLC system for simultaneous analysis of NAD⁺, NADH, NADP⁺, and NADPH was developed and used to measure the transhydrogenase activity of spinach ferredoxin-N ADP ⁺ reductase (EC 1.18.1.2, FNR). The system is based on a reverse-phase HPLC with isocratic elution on an ODS column (4.6 × 50 mm). The four nucleotides were completely separated by developing the column with 0.15 m sodium phosphate/citrate buffer (pH 6.8) containing 1 mm EDTA at 40°C with a flow rate of 1 ml/min. The four nucleotides can be simultaneously assayed within 13 min by monitoring the effluents with a UV detector. It was also indicated that the transhydrogenase activity of spinach FNR can be advantageously assayed by measuring the nucleotides by this method.     

Chemotaxis in Actinoplanes

The chemotactic properties of spores of Actinoplanes brasiliensis were examined. The spores are attracted to chloride and bromide solutions but not to a number of organic compounds that are capable of supporting growth in chemically defined media. Chloride attraction has been tested in several other species of the genus and in a number of Actinoplanes strains isolated from soils. Some are attracted to chloride, but most strains are indifferent to the halide. In dense suspensions, the spores of A. brasiliensis show an apparent microaerophilic behavior. The ecological implications of the chemotactic properties of the A. brasiliensis spores are discussed.Dedicated to Professor R. Y. Stanier on the occasion of his 60th birthday