Tylosin Biosynthesis Research Articles

Generation and characterization of two acid-resistant macrocin O-methyltransferase variants with a higher enzyme activity at 30 °C from Streptomyces fradiae

Tylosin is an important macrolide antibiotic produced by Streptomyces fradiae. In the biosynthesis of tylosin, macrocin O-methyltransferase TylF catalyzes the conversion of the side-product tylosin C (macrocin) to the primary component tylosin A (C/A conversion). This conversion is the rate-limiting step in the biosynthesis of tylosin, and affects the quality of the end product. To find a high activity and environment-adapted TylF enzyme, a TylF variant pool has been constructed via protein evolution approach in our previous study (Fan et al., 2023 [41]). In this study, the TylF variants with higher C/A conversion rates were expressed in E. coli and purified. The variants TylFY139F, TylFQ138H, F232Y and TylFT36S, V54A were shown to have a higher C/A conversion rate at 30 °C than that of TylF at 38 °C. Moreover, they had a greater acid resistance and showed more adaptable to the pH change during fermentation. Further protein structural and substrate-binding affinity analyses revealed that the T36S, V54A, Q138H, Y139F, and F232Y mutations enlarged the volume of the substrate-binding pocket, thereby increasing the affinity of enzyme variants for their substrates of SAM and macrocin, and decreasing the inhibition of SAH. Three of the TylF variants were overexpressed in the industrial tylosin-producing S. fradiae strain, and the recombinant strains showed the highest C/A conversion at 30 °C without heating up to 38 °C during the last 24 h of fermentation. This is of great energy-saving significance for tylosin industrial production.

Analysis of Secondary Metabolite Synthesis Potential of Streptomyces fradiae sf106 Based on the Whole Genome and Non-Target Metabolomics and Exploration of the Biosynthesis of Tylosin

Streptomyces fradiae sf106 is a type of actinomycete that can produce abundant secondary metabolites, making it a natural cell factory for drug synthesis. In order to comprehensively understand the genomic profile of Streptomyces fradiae sf106 and its potential for producing secondary metabolites, a combination of several methods was used to perform whole-genome sequencing of sf106. The results showed that sf106 is most closely related to Streptomyces xinghaiensis S187; the average nucleotide identity and average amino acid identity of sf106 and S187 were more than 96%. The genome size of sf106 is approximately 7300 kb, the GC content is greater than 72%, and more than 6700 coding sequences (CDS) were identified. Analysis of mobile genetic elements revealed the presence of a large number of horizontally transferred genes in Streptomyces fradiae sf106, which contribute to microbial diversity. Through antiSMASH prediction, 22 secondary metabolite gene clusters were obtained, which had great potential to generate polyketide metabolites. By examining the data, it was found that the genes contained in cluster 9 were similar to those involved in tylosin synthesis. Non-targeted metabolome sequencing revealed that a total of 1855 identifiable metabolites were produced in the fermentation broth, and the majority of metabolites showed highly significant differences in mean relative abundance between the groups. The identified metabolites were compared against the KEGG compound database to obtain metabolite classifications, mainly including Biological Roles, Phytochemical Compounds, Lipids, and Pesticides. One-way ANOVA indicated that the relative concentration of tylosin differed significantly across all the growth periods, except for the late-logarithmic and stabilization stages. This study provides important basic information on the secondary metabolite research of sf106, which will help us to understand and apply Streptomyces fradiae sf106 more comprehensively.

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.

Cover Picture: Tyl1a, a TDP‐6‐deoxy‐D‐xylo‐4‐hexulose 3,4‐isomerase from Streptomyces fradiae: Structure Prediction, Mutagenesis and Solvent Isotope Incorporation Experiments to Investigate Reaction Mechanism (ChemBioChem 8/2008)

The cover picture shows the modelled structure of Streptomyces fradiae Tyl1a and the isomerisation reaction it catalyses en route to the biosynthesis of the antibiotic tylosin. Specifically, Tyl1a is a TDP-6-deoxy-D-xylo-4-hexulose 3,4-isomerase. Using a combination of homology modelling, site-directed mutagenesis and solvent isotope incorporation experiments, we have identified potential catalytic residues of Tyl1a. This enzyme is predicted to be dimeric, with the core of each subunit comprised of a cupin-like β-barrel where catalysis takes place. A single β-barrel is shown, with the catalytic residues depicted in full atom representation. This study has enabled us to postulate a reaction mechanism for Tyl1a that is similar to that of the structurally related RmlC family of ketosugar nucleotide epimerases. For more details, see the article by R. Field, D. M. Lawson et al. on p. 1295 ff.

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.

Production of hybrid 16-membered macrolides by expressing combinations of polyketide synthase genes in engineered Streptomyces fradiae hosts.

Combinations of the five polyketide synthase (PKS) genes for biosynthesis of tylosin in Streptomyces fradiae (tylG), spiramycin in Streptomyces ambofaciens (srmG), or chalcomycin in Streptomyces bikiniensis (chmG) were expressed in engineered hosts derived from a tylosin-producing strain of S. fradiae. Surprisingly efficient synthesis of compounds predicted from the expressed hybrid PKS was obtained. The post-PKS tailoring enzymes of tylosin biosynthesis acted efficiently on the hybrid intermediates with the exception of TylH-catalyzed hydroxylation of the methyl group at C14, which was efficient if C4 bore a methyl group, but inefficient if a methoxyl was present. Moreover, for some compounds, oxidation of the C6 ethyl side chain to an unprecedented carboxylic acid was observed. By also expressing chmH, a homolog of tylH from the chalcomycin gene cluster, efficient hydroxylation of the 14-methyl group was restored.

Regulation of tylosin production and morphological differentiation in Streptomyces fradiae by TylP, a deduced gamma-butyrolactone receptor.

During promoter-probe analysis carried out in Streptomyces lividans, the TylP protein powerfully inhibited reporter gene expression from the tylP promoter, raising the likelihood that tylP is autoregulated in its native host, Streptomyces fradiae. Also in S. lividans, TylP negatively controlled the tylQ promoter, even though tylQ could still be switched off in S. fradiae strains specifically disrupted in tylP. Under the latter conditions, tylosin production was brought forward and enhanced, whereas overexpression of tylP resulted in reduced levels of the antibiotic, accompanied by barely detectable transcription from multiple genes of the tylosin biosynthetic cluster. Unexpectedly, overexpression of tylP reduced transcription of tylS, a transcriptional activator essential for tylosin production. This was probably a direct effect, as TylP also reduced expression from the tylS promoter in S. lividans. For these several reasons, we conclude that TylP acts as a repressor during tylosin biosynthesis. In addition, TylP influences morphological differentiation in S. fradiae. On solid media, strains in which tylP was disrupted sporulated significantly earlier than wild type and, in liquid culture, displayed hyperfragmentation.

Differential roles of two SARP-encoding regulatory genes during tylosin biosynthesis.

The tylosin biosynthetic gene cluster of Streptomyces fradiae is remarkable in harbouring at least five regulatory genes, two of which (tylS and tylT) encode proteins of the Streptomyces antibiotic regulatory protein (SARP) family. The aim of the present work was to assess the respective contributions of TylS and TylT to tylosin production. A combination of targeted gene disruption, fermentation studies and gene expression analysis via reverse transcriptase-polymerase chain reaction (RT-PCR) suggests that tylS is essential for tylosin production and controls the expression of tylR (previously shown to be a global activator of the biosynthetic pathway) plus at least one other gene involved in polyketide metabolism or regulation thereof. This is the first demonstration of a SARP acting to control another regulatory gene during antibiotic biosynthesis. In contrast, tylT is not essential for tylosin production.

Expression Analysis of the Tylosin-Biosynthetic Gene Cluster: Pivotal Regulatory Role of the tylQ Product

Expression analysis by RT-PCR, applied to the entire tyl cluster, revealed that the pattern of transcription is more complex than expected. For example, the five tylG polyketide synthase genes are not necessarily cotranscribed or even coregulated. Among the regulatory genes, tylQ has emerged as a key factor. Although several genes (including the positive regulator, tylS) were possibly expressed constitutively, only tylQ was silent during secondary metabolism. Analysis of engineered strains, in which tylQ was disrupted or overexpressed, showed that the TylQ protein is a transcriptional repressor that blocks tylosin biosynthesis by controlling expression of the activator, tylR. Before tylosin production can be triggered, tylQ must be switched off, or at least downregulated.

Influence of ancillary genes, encoding aspects of methionine metabolism, on tylosin biosynthesis in Streptomyces fradiae.

The tylosin-biosynthetic (tyl) gene cluster of Streptomyces fradiae contains ancillary genes that encode functions normally associated with primary metabolism. These can be disrupted without loss of viability, since equivalent genes (presumably used for 'housekeeping' purposes) are also present elsewhere in the genome. The tyl cluster also contains two genes that encode products unlike any proteins in the databases. Two ancillary genes, metF (encoding N5,N10-methylenetetrahydrofolate reductase) and metK, encoding S-adenosylmethionine synthase, flank one of the 'unknown' genes (orf9) in the tyl cluster. In a strain of S. fradiae in which all three of these genes were disrupted, tylosin production was reduced, although this effect was obscured in media supplemented with glycine betaine which can donate methyl groups to the tetrahydrofolate pool. Apparently, one consequence of the recruitment of ancillary genes into the tyl cluster is enhanced capacity for transmethylation during secondary metabolism.

The tylosin resistance gene tlrB of Streptomyces fradiae encodes a methyltransferase that targets G748 in 23S rRNA.

tlrB is one of four resistance genes encoded in the operon for biosynthesis of the macrolide tylosin in antibiotic-producing strains of Streptomyces fradiae. Introduction of tlrB into Streptomyces lividans similarly confers tylosin resistance. Biochemical analysis of the rRNA from the two Streptomyces species indicates that in vivo TlrB modifies nucleotide G748 within helix 35 of 23S rRNA. Purified recombinant TlrB retains its activity and specificity in vitro and modifies G748 in 23S rRNA as well as in a 74 nucleotide RNA containing helix 35 and surrounding structures. Modification is dependent on the presence of the methyl group donor, S-adenosyl methionine. Analysis of the 74-mer RNA substrate by biochemical and mass spectrometric methods shows that TlrB adds a single methyl group to the base of G748. Homologues of TlrB in other bacteria have been revealed through database searches, indicating that TlrB is the first member to be described in a new subclass of rRNA methyltransferases that are implicated in macrolide drug resistance.

Multiple regulatory genes in the tylosin biosynthetic cluster of Streptomyces fradiae

The macrolide antibiotic tylosin is composed of a polyketide lactone substituted with three deoxyhexose sugars. In order to produce tylosin efficiently, Streptomyces fradiae presumably requires control mechanisms that balance the yields of the constituent metabolic pathways together with switches that allow for temporal regulation of antibiotic production. In addition to possible metabolic feedback and/or other signalling devices, such control probably involves interplay between specific regulatory proteins. Prior to the present work, however, no candidate regulatory gene(s) had been identified in S. fradiae. DNA sequencing has shown that the tylosin biosynthetic gene cluster, within which four open reading frames utilise the rare TTA codon, contains at least five candidate regulatory genes, one of which (tylP) encodes a gamma-butyrolactone signal receptor for which tylQ is a probable target. Two other genes (tylS and tylT) encode pathway-specific regulatory proteins of the Streptomyces antibiotic regulatory protein (SARP) family and a fifth, tylR, has been shown by mutational analysis to control various aspects of tylosin production. The tyl genes of S. fradiae include the richest collection of regulators yet encountered in a single antibiotic biosynthetic gene cluster. Control of tylosin biosynthesis is now amenable to detailed study, and manipulation of these various regulatory genes is likely to influence yields in tylosin-production fermentations.

The mycinose-biosynthetic genes of Streptomyces fradiae, producer of tylosin.

The tylE-J region of the tylosin-biosynthetic gene cluster of Streptomyces fradiae contains six open reading frames. The products of tylJ and tylD are nucleoside diphospho (NDP)-deoxyhexose 3-epimerase and NDP-deoxyhexose 4-ketoreductase, respectively, involved in the synthesis of NDP-6-deoxyallose from NDP-4-keto, 6-deoxyglucose. After incorporation of deoxyallose at C23-OH of the polyketide lactone, tylosin biosynthesis is completed by the products of tylE and tylF, which convert the deoxyallosyl moiety to mycinose via bis-O-methylation at 2-OH and 3-OH, respectively. Hydroxylation of the polyketide lactone at C23 is catalysed by the cytochrome P450 enzyme, TylHl. The product of tylHll is a ferredoxin of unknown specificity that could conceivably act together with TylHl.

Impact of thioesterase activity on tylosin biosynthesis in Streptomyces fradiae

Background: The polyketide lactone tylactone is produced in Streptomycesfradiae by the TyIG complex of five multifunctional proteins. As with other type I polyketide synthases, the enzyme catalysing the final elongation step (TyIGV) possesses an integral thioesterase domain that is believed to be responsible for chain termination and ring closure to form tylactone, which is then glycosylated to yield tylosin. In common with other macrolide producers, S. fradiae also possesses an additional thioesterase gene ( orf5) located within the cluster of antibiotic biosynthetic genes. The function of the Orf5 protein is addressed here. Results: Disruption of orf5 reduced antibiotic accumulation in S. fradiae by atleast 85%. Under such circumstances, the strain accumulated desmycosin (demycarosyl-tylosin) due to a downstream polar effect on the expression of orf6, which encodes a mycarose biosynthetic enzyme. High levels of desmycosin production were restored in the disrupted strain by complementation with intact orf5, or with the corresponding thioesterase gene, nbmB, from S. narbonensis, but not with DNA encoding the integral thioesterase domain of TyIGV. Conclusions: Polyketide metabolism in S. fradiae is strongly dependent on thethioesterase activity encoded by orf5 ( tylO). It is proposed that the TyIG complex might operate with a significant error frequency and be prone to blockage with aberrant polyketides. A putative editing activity associated with TyIO might be essential to unblock the polyketide synthase complex and thereby promote antibiotic accumulation.

The tylosin biosynthetic cluster from Streptomyces fradiae: genetic organization of the left region.

The genetic organization of the left edge (tyIEDHFJ region) of the tylosin biosynthetic gene cluster from Streptomyces fradiae has been determined. Sequence analysis of a 12.9 kb region has revealed the presence of 11 ORFs, 10 of them belonging to the biosynthetic cluster. The putative functions of the proteins encoded by these genes are as follows: peptidase (ORF1, ddcA), tylosin resistance determinant (ORF2, tlrB), glycosyltransferase (ORF3, tylN), methyltransferase (ORF4, tylE), ketoreductase (ORF5, tylD), ferredoxin (ORF6, tylH2), cytochrome P450 (ORF7, tylH1), methyltransferase (ORF8, tylF), epimerase (ORF9, tylJ), acyl-CoA oxidase (ORF10, tylP) and receptor of regulatory factors (ORF11, tylQ). The functional identification of the genes in the proposed tylosin biosynthetic pathway has been deduced by database searches and previous genetic complementation studies performed with tylosin idiotrophic mutants blocked at various stages in tylosin biosynthesis. The tlrB gene has been shown to be useful as a tylosin resistance marker in Streptomyces lividans, Streptomyces parvulus and Streptomyces coelicolor and the effect of tylF on macrocin depletion has been confirmed. A pathway for the biosynthesis of 6-deoxy-D-allose, the unmethylated mycinose precursor, involving the genes tylD, tylJ and tylN is proposed.

Molecular analysis of tlrB, an antibiotic-resistance gene from tylosin-producing Streptomyces fradiae, and discovery of a novel resistance mechanism.

The tlrB gene, which confers inducible resistance to a range of macrolide antibiotics including biosynthetic precursors of tylosin, was isolated and sequenced. In the genome of Streptomyces fradiae, it lies between pbp, which encodes a putative penicillin-binding protein, and tylN, encoding a glycosyltransferase involved in tylosin biosynthesis. The TlrB protein was produced in E. coli as a fusion to MalE. The fusion protein, but not MalE alone, inactivates macrolides in the presence of S-adenosyl-methionine (SAM) but the modified product(s) has not been characterised.

Characterization and targeted disruption of a glycosyltransferase gene in the tylosin producer, Streptomyces fradiae.

An open reading frame, designated tylN, has been identified by sequence analysis at one end of the tylosin biosynthetic gene cluster of Streptomyces fradiae, alongside a cluster of genes encoding the biosynthesis of dTDP-deoxyallose. This 6-deoxyhexose sugar is converted to mycinose, via bis O-methylation, following attachment to the polyketide lactone during tylosin biosynthesis. The deduced product of tylN is similar to several glycosyltransferases, authentic and putative, and displays a consensus sequence motif that appears to be characteristic of a sub-group of such enzymes. Specific disruption of tylN within the S. fradiae genome resulted in the production of demycinosyl-tylosin, whereas other glycosyltransferase activities involved in tylosin biosynthesis were not affected. Evidently, tylN encodes deoxyallosyl transferase.

Effects of rapessed oil on activity of methylmalonyl-CoA carboxyltransferase in culture of Streptomyces fradiae.

To investigate why more tylosin was produced when Streptomyces fradiae T1558 was cultured in a rapeseed oil medium than in a glucose or starch medium, we measured the activity of methylmalonyl-CoA carboxyltransferase (EC 2.1.3.1) and intracellular propionic acid. The activity of the enzyme, which catalyzes the formation of the precursor of tylosin, protylonolide, was 0.19 U/mg protein in 5 days of culture in rapeseed oil medium, which was 2.5- and 1.3-fold that with the glucose or starch medium, respectively. The intracellular propionic acid concentration was 1.2 g/g of dry weight, which was 4.3- and 2.1-fold that with the glucose or starch medium, respectively. The addition of propionic acid increased tylosin production in batch culture: when 0.2 g/l (final concentration) propionic acid was added to the glucose medium, 3.8 g/l tylosin was produced in 10 days of culture, 4.7-fold the amount without propionic acid. These findings suggest that in glucose medium, intracellular propionic acid is a limiting factor because of the low activity of methylmalonyl-CoA carboxyltransferase of the tylosin biosynthesis pathway.

Stimulation of polyketide metabolism in Streptomyces fradiae by tylosin and its glycosylated precursors

Three glycosyltransferases are involved in tylosin biosynthesis in Streptomyces fradiae. The first sugar to be added to the polyketide aglycone (tylactone) is mycaminose and the gene encoding mycaminosyltransferase is orf2* (tylM2). However, targeted disruption of orf2* did not lead to the accumulation of tylactone under conditions that normally favour tylosin production; instead, the synthesis of tylactone was virtually abolished. This may, in part, have resulted from a polar effect on the expression of genes downstream of orf2*, particularly orf4* (ccr) which encodes crotonyl-CoA reductase, an enzyme that supplies 4-carbon extender units for polyketide metabolism. However, that cannot be the entire explanation, since tylosin production was restored at about 10% of the wild-type level when orf2* was re-introduced into the disrupted strain. When glycosylated precursors of tylosin were fed to the disrupted strain, they were converted to tylosin, confirming that two of the three glycosyltransferase activities associated with tylosin biosynthesis were still intact. Interestingly, however, tylactone also accumulated under such conditions and, to a much lesser extent, when tylosin was added to similar fermentations. It is concluded that glycosylated macrolides exert a pronounced positive effect on polyketide metabolism in S. fradiae.

Evolving enzyme technology for pharmaceutical applications: case studies.

The case studies focus on two types of enzyme applications for pharmaceutical development. Demethylmacrocin O-methyltransferase, macrocin O-methyltransferase (both putatively rate-limiting) and tylosin reductase were purified from Streptomyces fradiae, characterized and the genes manipulated for increasing tylosin biosynthesis in S. fradiae. The rate-limiting enzyme, deacetoxycephalosporin C (DAOC) synthase/hydroxylase (expandase/ hydroxylase), was purified from Cephalosporium acremonium, its gene over-expressed, and cephalosporin C biosynthesis improved in C. acremonium. Also, heterologous expression of penicillin N epimerase and DAOC synthase (expandase) genes of Streptomyces clavuligerus in Penicillium chrysogenum permitted DAOC production in the fungal strain. Second, serine hydroxymethyltransferase of Escherichia coli and phthalyl amidase of Xanthobacter agilis were employed in chemo-enzymatic synthesis of carbacephem. Similarly, echinocandin B deacylase of Actinoplanes utahensis was used in the second-type synthesis of the ECB antifungal agent.