Tylosin Production Research Articles

An efficient method for targeted cloning of large DNA fragments from Streptomyces.

Cloning of large DNA fragments from microorganisms becomes increasingly important but remains seriously challenging due to the complexity and diversity of genetic background. In particular, the methods with high precision and efficiency are in great need for obtaining intact biosynthetic gene clusters (BGCs) of microbial natural products. Here, we report a new strategy for targeted cloning of large DNA fragments (TCLD) from different bacteria. Using this method, precise cloning of desired E. coli chromosomal fragments up to 201kb was achieved with 53% positive rate. Moreover, its application in cloning of large BGCs with high G + C content and multiple repetitive sequences was also demonstrated, including the 98kb tylosin BGC (tyl), 128kb daptomycin BGC (dpt), and 127kb salinomycin BGC (sal). Subsequently, heterologous expression of the cloned tyl BGC in Streptomyces coelicolor M1146 led to the production of tylosins in the resulting recombinant strains. And also, its introduction into Streptomyces fradiae ATCC 19609, a native producer of tylosin, effectively increased tylosin yield to 230%. Hence, TCLD is a powerful tool for cloning large BGCs and would facilitate the discovery of bioactive substances from microbial resources. KEY POINTS: • TCLD is an efficient method for cloning large DNA fragments. • Repeat sequence-mediated intra-molecular cyclization improves the cloning efficiency. • TCLD combined with scarless editing allows unlimited modifications on BGCs.

Enhancing tylosin production by combinatorial overexpression of efflux, SAM biosynthesis, and regulatory genes in hyperproducing Streptomyces xinghaiensis strain

Tylosin is a 16-membered macrolide antibiotic widely used in veterinary medicine to control infections caused by Gram-positive pathogens and mycoplasmas. To improve the fermentation titer of tylosin in the hyperproducing Streptomyces xinghaiensis strain TL01, we sequenced its whole genome and identified the biosynthetic gene cluster therein. Overexpression of the tylosin efflux gene tlrC, the cluster-situated S-adenosyl methionine (SAM) synthetase gene metKcs, the SAM biosynthetic genes adoKcs-metFcs, or the pathway-specific activator gene tylR enhanced tylosin production by 18%, 12%, 11%, and 11% in the respective engineered strains TLPH08-2, TLPH09, TLPH10, and TLPH12. Co-overexpression of metKcs and adoKcs-metFcs as two transcripts increased tylosin production by 22% in the resultant strain TLPH11 compared to that in TL01. Furthermore, combinational overexpression of tlrC, metKcs, adoKcs-metFcs, and tylR as four transcripts increased tylosin production by 23% (10.93g/L) in the resultant strain TLPH17 compared to that in TL01. However, a negligible additive effect was displayed upon combinational overexpression in TLPH17 as suggested by the limited increment of fermentation titer compared to that in TLPH08-2. Transcription analyses indicated that the expression of tlrC and three SAM biosynthetic genes in TLPH17 was considerably lower than that of TLPH08-2 and TLPH11. Based on this observation, the five genes were rearranged into one or two operons to coordinate their overexpression, yielding two engineered strains TLPH23 and TLPH24, and leading to further enhancement of tylosin production over TLPH17. In particular, the production of TLPH23 reached 11.35 g/L. These findings indicated that the combinatorial strategy is a promising approach for enhancing tylosin production in high-yielding industrial strains.

A High-Throughput Method Based on Microculture Technology for Screening of High-Yield Strains of Tylosin-Producing Streptomyces fradiae.

Tylosin is a potent veterinary macrolide antibiotic produced by the fermentation of Streptomyces fradiae; however, it is necessary to modify S. fradiae strains to improve tylosin production. In this study, we established a high-throughput, 24-well plate screening method for identifying S. fradiae strains that produce increased yields of tylosin. Additionally, we constructed mutant libraries of S. fradiae via ultraviolet (UV) irradiation and/or sodium nitrite mutagenesis. A primary screening of the libraries in 24-well plates and UV spectrophotometry identified S. fradiae mutants producing increased yields of tylosin. Mutants with tylosin yield 10% higher than the wild-type strain were inoculated into shake flasks, and the tylosin concentrations produced were determined by high-performance liquid chromatography (HPLC). Joint (UV irradiation and sodium nitrite) mutagenesis resulted in higher yields of mutants with enhanced tylosin production. Finally, 10 mutants showing higher tylosin yield were re-screened in shake flasks. The yield of tylosin A by strains UN-C183 (6767.64 ± 82.43 μg/ml) and UN-C137 (6889.72 ± 70.25 μg/ml) was significantly higher than that of the wild-type strain (6617.99 ± 22.67 μg/ml). These mutant strains will form the basis for further strain breeding in tylosin production.

Discovery of a highly efficient TylF methyltransferase via random mutagenesis for improving tylosin production

Macrolides are currently a class of extensively used antibiotics in human and animal medicine. Tylosin is not only one of the most important veterinary macrolides but also an indispensable material for the bio- and chemo-synthesis of new generations of macrolide antibiotics. Thus, improving its production yield is of great value. As the key rate-limiting enzyme catalyzing the terminal step of tylosin biosynthesis in Streptomyces fradiae (S. fradiae), TylF methyltransferase’s catalytic activity directly affects tylosin yield. In this study, a tylF mutant library of S. fradiae SF-3 was constructed based on error-prone PCR technology. After two steps of screening in 24-well plates and conical flask fermentation and enzyme activity assay, a mutant strain was identified with higher TylF activity and tylosin yield. The mutation of tyrosine to phenylalanine is localized at the 139th amino acid residue on TylF (TylFY139F), and protein structure simulations demonstrated that this mutation changed the protein structure of TylF. Compared with wild-type protein TylF, TylFY139F exhibited higher enzymatic activity and thermostability. More importantly, the Y139 residue in TylF is a previously unidentified position required for TylF activity and tylosin production in S. fradiae, indicating the further potential to engineer the enzyme. These findings provide helpful information for the directed molecular evolution of this important enzyme and the genetic modification of tylosin-producing bacteria.

Assessing the effects of tylosin fermentation dregs as soil amendment on macrolide antibiotic resistance genes and microbial communities: Incubation study

Tylosin fermentation dregs (TFDs) are biosolid waste of antibiotics tylosin production process which contain nutritious components and may be recycled as soil amendments. However, the specific ecological safety of TFDs from the perspective of bacterial resistance in soil microenvironment is not fully explored. In the present study, a series of replicated lab-scale work were performed using the simulated fertilization to gain insight into the potential environmental effects and risks of macrolide antibiotic resistance genes (ARGs) and the soil microbial communities composition via quantitative PCR and 16S rRNA sequencing following the TFDs land application as the soil amendments. The results showed that bio-processes might play an important role in the decomposition of tylosin which degraded above 90% after 20 days in soil. The application of TFDs might induce the development of antibiotic-resistant bacteria, change soil environment and reduce the microbial diversity. Though the abundances of macrolide ARGs exhibited a decreasing trend following the tylosin degradation, other components in TFDs may have a lasting impact on both macrolide ARGs abundance and soil bacterial communities. Thus, this study pointed out the fate of TFDs on soil ecological environment when directly applying into soil, and provide valuable scientific basis for TFDs management.

Biosorption of Pb(II) ions from aqueous solutions by waste biomass of Streptomyces fradiae pretreated with NaOH

Biosorption of Pb(II) ions from a model solution was investigated using Streptomyces fradiae biomass as biosorbent pretreated with sodium hydroxide. The mycelium is a waste product from the biotechnological production of the macrolide antibiotic tylosin in the pharmaceutical industry. The biosorption study was conducted in a batch system with respect to initial pH, initial metal concentration and contact time. For a description of the biosorption equilibrium, Langmuir and Freundlich adsorption models were used. Equilibrium data fitted better to the Langmuir model and the calculated maximum biosorption capacity was 138.88 mg·g−1 at initial pH 5.0, contact time of 120 min, biosorbent dose of 1 g·dm−3 and concentration range for the Pb(II) ions from 10 to 200 mg·dm−3. Pseudo-first and pseudo-second order kinetic models were applied to the experimental data. The results indicated that the Pb(II) uptake process followed the Ho equation. The interference of co-present ions Cu(II) and Zn(II) on the Pb(II) biosorption was also studied. It was determined that at the highest Pb(II) concentration (200 mg·dm−3) Cu(II) and Zn(II) caused 27.22% and 24.88% decreasing in Pb(II) uptake, respectively. The obtained results could be useful in prospective applications of chemically modified waste mycelium of S. fradiae as an alternative biosorbent for Pb(II) removal from aqueous solutions.

A novel Burkholderia vietnamiensis strain for biodegradation of residual tylosin antibiotic in fermentation solid waste

A large amount of fermentation solid waste (FSW) has been generated during the course of tylosin production. This FSW is difficult to be treated and reused because of tylosin residue. In this study, a novel strain TS1 was isolated and identified as Burkholderia vietnamiensis. This isolate exhibited a strong ability to degrade tylosin, and almost 100% of 200 mg kg−1 tylosin was biodegraded after seven days of incubation. Medium pH and temperature had some influences on the biodegradation capacity of strain TS1. The favourable biodegrading conditions were at pH 7.0 and 30-35°C. The behaviour of tylosin degradation followed a first-order kinetic, and the half-lives were 1.54 to 1.70 days when the initial concentrations of tylosin were from 50 mg kg−1 to 400 mg kg−1. These findings suggest that strain TS1 can effectively degrade tylosin residue in FSW and may be used for biotreatment of antibiotic solid waste containing tylosin residue.

Biodegradation of tylosin residue in pharmaceutical solid waste by a novel Citrobacter amalonaticus strain

A large amount of pharmaceutical solid waste (PSW) has been generated in the process of tylosin production using fermentation. This PSW cannot been reused directly because of the existence of residual tylosin. The objective of this study was to investigate the method for biodegradation of tylosin antibiotic in PSW. The results showed that a novel tylosin‐degrading strain was isolated from the soil deposited by PSW and identified as Citrobacter amalonaticus. This strain was capable of degrading almost 100% of tylosin in PSW medium with an initial concentration of 50 mg/L after 72 h of incubation under conditions of the initial pH 6.0 and 30°C. The degradation kinetics for tylosin followed a first‐order model, and half‐lives were 16.16–16.82 h when the initial concentrations of tylosin ranged from 25 to 100 mg/L. These results indicate that the isolated strain has a relatively high ability to degrade tylosin and may be used for bioremediation of the environment contaminated by tylosin waste. © 2014 American Institute of Chemical Engineers Environ Prog, 34: 99–104, 2015

Tolerance of the antibiotic Tylosin on treatment performance of an Up-flowAnaerobic Stage Reactor (UASR)

Tylosin has been considered inhibiting COD removal in anaerobic digestion. In this study it is proven that this is not always the case. Accordingly, elevated concentrations of Tylosin (100-800mgL-1) could be tolerated by the anaerobic system. The influence of Tylosin concentrations on an up-flow anaerobic stage reactor (UASR) was assessed using additions of Tylosin phosphate concentrate. Results showed high efficiency for COD removal (average 93%) when Tylosin was present at concentrations ranging from 0 to 400 mg L-1. However, at Tylosin concentrations of 600 and 800 mg L-1 treatment efficiency declined to 85% and 75% removal respectively. The impact of Tylosin concentrations on archaeal activity were investigated and the analysis revealed that archaeal cells dominated the reactor, confirming that there was no detectable inhibition of the methanogens at Tylosin levels between 100 and 400mg L-1. Nevertheless, the investigation showed a slight reduction in the number of methanogens at Tylosin levels of 600 and 800 mg L-1. These results demonstrated that the methanogens were well adapted to Tylosin. It would not be expected that the process performance of the UASR would be affected, not even at a level well in excess of those appearing in real wastewater from a Tylosin production site.

Genome shuffling: a method to improve biotechnological processes

Genome shuffling is widely used for increasing the production of metabolites by bacterial strains, improving substrate uptake as well as enhancing strain tolerance. This technique combines the advantage of multiparental crossing allowed by DNA shuffling together with the recombination of entire genomes normally associated with conventional breeding, or through protoplast fusion that increases the recombination process. The method of genome shuffling was first presented by Stemmer and co-workers in 2002 when it was used to improve the production of tylosin by Streptomyces fradiae. Nowadays, it is used in many experiments for increasing the production of metabolites by bacterial strains, improving substrate uptake, and enhancing strain tolerance. Genome shuffling is a major milestone in the strain-improvement technology and metabolic engineering.

Production of tylosin in solid-state fermentation byStreptomyces fradiaeNRRL-2702 and its gamma-irradiated mutant (γ-1)

To develop solid-state fermentation system (SSF) for hyper production of tylosin from a mutant gamma-1 of Streptomyces fradiae NRRL-2702 and its parent strain. Various agro-industrial wastes were screened to study their effect on tylosin production in SSF. Wheat bran as solid substrate gave the highest production of 2500 microg of tylosin g(-1) substrate by mutant gamma-1 against parent strain (300 microg tylosin g(-1) substrate). The tylosin yield was further improved to 4500 microg g(-1) substrate [70% moisture, 10% inoculum (v/w), pH 9.2, 30 degrees C, supplemental lactose and sodium glutamate on day 9]. Wild-type strain displayed less production of tylosin (655 microg of tylosin g(-1) substrate) in SSF even after optimization of process parameters. The study has shown that solid-state fermentation system significantly enhanced the tylosin yield by mutant gamma-1. This study proved to be very useful and resulted in 6.87 +/- 0.30-fold increase in tylosin yield by this mutant when compared to that of wild-type strain.

Change in colony morphology and kinetics of tylosin production after UV and gamma irradiation mutagenesis of Streptomyces fradiae NRRL-2702

Tylosin is a macrolide antibiotic used as veterinary drug and growth promoter. Attempts were made for hyper production of tylosin by a strain of Streptomyces fradiae NRRL-2702 through irradiation mutagenesis. Ultraviolet (UV) irradiation of wild-type strain caused development of six morphologically altered colony types on agar plates. After screening using Bacillus subtilis bioassay only morphological mutants indicated the production of tylosin. An increase of 2.7+/-0.22-fold in tylosin production (1500mg/l) in case of mutant UV-2 in complex medium was achieved as compared to wild-type strain (550mg/l). Gamma irradiation of mutant UV-2 using (60)Co gave one morphologically altered colony type gamma-1, which gave 2500mg/l tylosin yield in complex medium. Chemically defined media promoted tylosin production upto 3800mg/l. Maximum value of q(p) (3.34mg/gh) was observed by mutant gamma-1 as compared to wild strain (0.81mg/gh). Moreover, UV irradiation associated changes were unstable with loss of tylosin activity whereas mutant gamma-1 displayed high stability on subsequent culturing.

Fusion PCR-targeted tylCV gene deletion of Streptomyces fradiae for producing desmycosin, the direct precursor of tilmicosin

Tilmicosin is a semisynthetic macrolide antibiotic derived from tylosin. The disruption of tylCV gene from tylosin producer— Streptomyces fradiae would result in the accumulation of desmycosin, the direct precursor of tilmicosin. TylCV gene disruption cassette was constructed by fusion PCR. The tylCV replacement strain of S. fradiae was obtained through intergeneric conjugation between S. fradiae and E. coli. The antibiotic resistance cassette is flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriT RK2 to generate unmarked, nonpolar mutation. After optimization of the culture conditions, the yield of desmycosin reached 7.3 g l −l in 5 l jar fermentor, which could be scale-up for industrial fermentation to produce large amount of desmycosin for semisynthesis of tilmicosin directly.

Regulation of tylosin production: role of a TylP‐interactive ligand

Gamma-butyrolactones regulate secondary metabolism and, sometimes, sporulation in actinomycetes by binding to specific receptor proteins, causing their dissociation from DNA targets and releasing the latter from transcriptional repression. Previously, in engineered strains of Streptomyces lividans, we showed that TylP, a deduced gamma-butyrolactone receptor, downregulated reporter gene expression driven by tylP, tylQ or tylS promoter DNA. These genes all control tylosin production in Streptomyces fradiae. Thus, at early stages of fermentation, TylQ represses tylR whereas TylS is needed for transcriptional activation of tylR. Importantly, TylR is the key activator of tylosin-biosynthetic genes. Here, we show that HIS-tagged TylP binds to specific DNA sequences, similar to the targets for authentic gamma-butyrolactone receptors, in the promoters of tylP, tylQ and tylS. Moreover, such binding is disrupted by material produced in S. fradiae and extractable by organic solvent. That putative gamma-butyrolactone material was not produced when orf18 * was disrupted within the S. fradiae genome and only about 1% of that activity survived inactivation of orf16 *, suggesting roles for the respective gene products in gamma-butyrolactone synthesis. Continued synthesis of tylosin by the disrupted strains contrasts with other reports that loss of gamma-butyrolactones abolishes antibiotic production.

Functional analysis of DesVIII homologues involved in glycosylation of macrolide antibiotics by interspecies complementation

The DesVIII is an auxiliary protein which enhances the transfer of TDP- d-desosamine catalyzed by DesVII glycosyltransferase in the biosynthesis of macrolide antibiotics, neomethymycin, methymycin and pikromycin, in Streptomyces venezuelae ATCC 15439. Homologues of the desVIII gene are present in a number of aminosugar-containing antibiotic biosynthetic gene clusters including eryCII from the erythromycin producer Saccharopolyspora erythraea, oleP1 from the oleandomycin producer Streptomyces antibioticus, dnrQ from the doxorubicin producer Streptomyces peucetius, and tylMIII from the tylosin producer Streptomyces fradiae. In order to gain further insight into the function of these DesVIII homologues, interspecies complementation experiments were carried out by expressing each gene in a desVIII deletion mutant strain of S. venezuelae. Complementation by expressing EryCII, OleP1, and DnrQ in this mutant strain restored the production of glycosylated macrolides to an approximate level of 66%, 26% and 26%, respectively, compared to self-complementation by DesVIII. However, expression of TylMIII did not restore the antibiotic production. These results suggest that the DesVIII homologues (except for TylMIII) can functionally replace the native DesVIII for glycosylation to proceed in vivo and their functions are similar in acting as glycosyltransferase auxiliary proteins. The requirement of glycosyltransferase auxiliary protein seems to be more widespread in polyketide biosynthetic pathways than previously known.

Regulation of tylosin biosynthesis involving ‘SARP‐helper’ activity

Tylosin production in Streptomyces fradiae is regulated via interplay between a repressor, TylQ, and an activator of the SARP family, TylS, during regulation of tylR. The latter encodes the pathway-specific activator of the tylosin-biosynthetic (tyl) genes. Also controlled by TylS is a hitherto unassigned gene, tylU, whose product is shown here to be important for tylosin production. Thus, targeted disruption of tylU reduced tylosin yields by about 80% and bioconversion analysis with the resultant strain revealed defects in both polyketide metabolism and deoxyhexose biosynthesis. Such defects were completely eliminated by engineered overexpression of tylR (but not tylS) and Western analysis revealed significantly reduced levels of TylR in the tylU-disrupted strain. These results are consistent with a model in which TylS and TylU act in concert to facilitate expression of tylR, for which TylU (but not TylS) is nonessential. Activator proteins of the SARP family, such as TylS, are widespread among Streptomyces spp. and are important regulators of antibiotic production. Their action has been widely studied with no prior indication of associated 'helper' activity, the prevalence of which now remains to be established.

Increase in tylosin production by a commercial strain of Streptomyces fradiae

Conventional mutagenesis (UV irradiation and exposure to nitrosoguanidine) were used to produce and regenerate protoplasts, aiming at increasing the antibiotic activity of a Streptomycesfradiae strain producing tylosin. Variants exceeding the activity of the initial producer strain by 0.5-28.3% were obtained. The most active variants were produced by a combined exposure to UV and nitrosoguanidine, as well as upon regeneration of protoplasts formed from the cells of clones produced by UV irradiation. Unstable inheritance of the trait of increased tylosin production was demonstrated.

Degradation and Metabolite Production of Tylosin in Anaerobic and Aerobic Swine‐Manure Lagoons

Watershed contamination from antibiotics is becoming a critical issue because of increased numbers of confined animal-feeding operations and the use of antibiotics in animal production. To understand the fate of tylosin in manure before it is land-applied, degradation in manure lagoon slurries at 22 degrees C was studied. Tylosin disappearance followed a biphasic pattern, where rapid initial loss was followed by a slow removal phase. The 90% disappearance times for tylosin, relomycin (tylosin D), and desmycosin (tylosin B) in anaerobically incubated slurries were 30 to 130 hours. Aerating the slurries reduced the 90% disappearance times to between 12 and 26 hours. Biodegradation and abiotic degradation occur, but strong sorption to slurry solids was probably the primary mechanism of tylosin disappearance. Dihydrodesmycosin and an unknown degradate with molecular mass of m/z 934.5 were detected. Residual tylosin remained in slurry after eight months of incubation, indicating that degradation in lagoons is incomplete and that residues will enter agricultural fields.

Positive control of tylosin biosynthesis: pivotal role of TylR

Control of tylosin production in Streptomyces fradiae features interplay between a repressor, TylQ, and an activator, TylS, during regulation of tylR. The latter encodes a pathway-specific activator that controls most of the tylosin-biosynthetic (tyl) genes that are subject to regulation. This was established by targeted gene disruption applied separately to tylR and tylS together with transcript analysis involving reverse transcription polymerase chain reaction (RT-PCR). TylR controls multiple genes that encode the synthesis or addition of all three tylosin sugars, plus polyketide ring oxidation, and at least one of the polyketide synthase (PKS) megagenes, tylGI. (Expression of a few tyl genes, plus the resistance determinants tlrB and tlrD, together with some ancillary or unassigned genes, is not apparently regulated during fermentation, consistent with constitutive expression.) In contrast, the only gene known for sure to be directly controlled by TylS is tylR, and there are very few additional candidates. These include the mycinose-biosynthetic gene, tylJ, and two previously unassigned genes, ORF12* (tylU) plus ORF11* (tylV). TylS also controls the PKS genes [tylGIII-tylGIV-tylGV] although not in obligatory fashion. These genes can be transcribed (i.e. tylosin can be produced) in a tylS-KO strain by forcing overexpression of tylR using a foreign promoter. We therefore suspect that TylS might control the PKS genes indirectly, although this remains to be established unequivocally. Conceivably, the direct effects of TylS are exerted exclusively on other regulators. Tylosin production levels were elevated when tylS or (especially) tylR was overexpressed in S. fradiae wild-type and yield increments of industrial significance were generated by similar manipulation of an enhanced production strain.

Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains.

Development of natural products for therapeutic use is often hindered by limited availability of material from producing organisms. The speed at which current technologies enable the cloning, sequencing, and manipulation of secondary metabolite genes for production of novel compounds has made it impractical to optimize each new organism by conventional strain improvement procedures. We have exploited the overproduction properties of two industrial organisms- Saccharopolyspora erythraea and Streptomyces fradiae, previously improved for erythromycin and tylosin production, respectively-to enhance titers of polyketides produced by genetically modified polyketide synthases (PKSs). An efficient method for delivering large PKS expression vectors into S. erythraea was achieved by insertion of a chromosomal attachment site ( attB) for phiC31-based integrating vectors. For both strains, it was discovered that only the native PKS-associated promoter was capable of sustaining high polyketide titers in that strain. Expression of PKS genes cloned from wild-type organisms in the overproduction strains resulted in high polyketide titers whereas expression of the PKS gene from the S. erythraea overproducer in heterologous hosts resulted in only normal titers. This demonstrated that the overproduction characteristics are primarily due to mutations in non-PKS genes and should therefore operate on other PKSs. Expression of genetically engineered erythromycin PKS genes resulted in production of erythromycin analogs in greatly superior quantity than obtained from previously used hosts. Further development of these hosts could bypass tedious mutagenesis and screening approaches to strain improvement and expedite development of compounds from this valuable class of natural products.