Antibiotics residues have been accumulating in the environment day by day due to overuse of antibiotics.Recalcitrant antibiotic residues,such as tylosin(TYL),can cause serious environmental problems,which makes it imp...Antibiotics residues have been accumulating in the environment day by day due to overuse of antibiotics.Recalcitrant antibiotic residues,such as tylosin(TYL),can cause serious environmental problems,which makes it important to eliminate TYL from the environment.It is important to eliminate TYL from the environment.In this study,a strain was isolated and purified from fermentation by-product that came from a TYL production factory.The TYL degrading strain was identified by its morphology,physiological and biochemical reactions and sequencing the PCR-amplified fragments of its 16 S r DNAcoding genes.The temperature,shaking speed,initial TYL concentration,p H and inoculum sizes were investigated under simulated conditions by using single factor tests.The results showed that TYL2,a high efficient strain was isolated and was identified as Brevibacillus borstelensis.The degradation rate of TYL by this strain could reach to 75%with an initial concentration of 25 mg L^-1 within 7 days under conditions of 7%B.borstelensis(v/v,2×108 CFU m L^-1)at p H 7.0 and at 35°C.It is interesting that this strain has a very strong ability to degrade the TYL in natural sewage with the degradation rate of 65%within 7 days.This result could be helpful for the degradation of TYL and provide guidance for the degradation of other antibiotics.展开更多
The pharmacokinetics and tissue residue of tylosin in broiler chickens were studied after I.V. and oral administrations in a dose of 50 mg tylosin/kg.b.wt. Tylosin was obeyed a two-compartment open model following I.V...The pharmacokinetics and tissue residue of tylosin in broiler chickens were studied after I.V. and oral administrations in a dose of 50 mg tylosin/kg.b.wt. Tylosin was obeyed a two-compartment open model following I.V. administration at a dose of 50 mg/kg.b.wt. The disposition kinetics of tylosin following I.V. administration revealed that tylosin was highly distributed with V<sub>d(area)</sub> of 6 L/kg and eliminated with half-life (t<sub>1/2β</sub>) equal to 7.29 hours. The disposition kinetics of tylosin following oral administration revealed that the maximum blood concentration (C<sub>max</sub>) was 3.40 μg/ml attained at (t<sub>max</sub>) of 1.08 hour. Tylosin was eliminated with half-life (t<sub>1/2β</sub>) equal to 5.78 hours. The mean systemic bioavailability of tylosin after oral administration was 90.29%. Following repeated oral administration of 50 mg tylosin base/kg.b.wt once daily for 5 consecutive days, the blood (μg/ml) and tissue (μg/g) residues of tylosin showed that liver, kidney and lung contained the highest tylosin residues and completely disappeared from those tissues at 6 days after the last oral dose. Chickens should not be slaughtered for human consumption within the treatment and 6 days after the last oral administrations of tylosin.展开更多
[Objective]To provide a reference for dynamic .regulation of the fermentation process. [Method] Using conditions for tylosin fermentation as feature vectors, final yield during the fermentation process was predicted u...[Objective]To provide a reference for dynamic .regulation of the fermentation process. [Method] Using conditions for tylosin fermentation as feature vectors, final yield during the fermentation process was predicted using the Bayesian method. [ Rgesultl The Bayesian network which learned data of tylosin fermentation could better guide conditions of the fermentation process, and better prediction results were achieved. The yield of fermentation products could be effectively predicted using the obtained Bayesian network and could well guide the fermentation process. E Conclusion] Bayesian network has good predictive performance for microbial fermentation and has good practical yield and application prospects.展开更多
Tylosin is a well-established antibiotic that has been widely employed in human and veterinary medicines. It can act as a potential ligand binding metal ions due to various donor atoms in the structure. Our study on t...Tylosin is a well-established antibiotic that has been widely employed in human and veterinary medicines. It can act as a potential ligand binding metal ions due to various donor atoms in the structure. Our study on the complexation of various metal ions with tylosin ligand revealed that they preferably coordinate with mycaminose fragment to establish Novel trends complexes. Tylosin ligand (TYS) behaves as bidentate for complexation with different metal ions such as Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II) and Zn(II). Various essential metal complexes of tylosin were synthesized and characterized by techniques such as UV, IR, Elemental analysis, magnetic susceptibility and ESR spectra of Cu(II) complex. These techniques are used to know their geometries and mode of bonding, with stoichiometry, 2:2 (M:L). Thermal analysis (TGA and DTA) of ligands and their metal complexes were carried out to distinguish between the coordinate and hydrate solvents and to estimate the stability ranges, peak temperatures. The thermodynamic parameters, such as activation energy (ΔE<sup>*</sup>), the enthalpy of activation (ΔH<sup>*</sup>), entropy of activation (ΔS<sup>*</sup>) and Gibbs free energy (ΔG<sup>*</sup>) are calculated and discussed. Some tylosin complexes show higher activity than tylosin for some bacterial and fungal strains. Low concentration value of minimum inhibitory concentration (MIC) results is 15.625 μg/ml for both complexes [Zn<sub>2</sub>(TYS)<sub>2</sub>Cl<sub>2</sub>(H<sub>2</sub>O)<sub>4</sub>]·25H<sub>2</sub>O and [Cu<sub>2</sub>(TYS)<sub>2</sub>Cl<sub>2</sub>(H<sub>2</sub>O)<sub>4</sub>]·25H<sub>2</sub>O with B. cereus genus maybe a valuable data used to produce novel therapeutic agent. This study constitutes several essential aspects for future research on tylosin metal complexes as antibacterial assessment and as potential medicinal agents.展开更多
Antibiotic production wastewater usually contains high concentrations of antibiotic residues,which can cause instability and deterioration of biological wastewater treatment units and also domestication and proliferat...Antibiotic production wastewater usually contains high concentrations of antibiotic residues,which can cause instability and deterioration of biological wastewater treatment units and also domestication and proliferation of antibiotic-resistance bacteria.An effective pretreatment on antibiotics production wastewater is expected to selectively reduce the concentration of antibiotics and decrease the toxicity,rather than mitigate organic and other contaminants before further treatments.In this work,two polymer-based solid acids,PS-S and CPS-S bearing high concentrations of-SOH_(3)groups (up to 4.57 mmol/g),were prepared and successfully used for hydrolytic mitigation of 100 mg/L tylosin within 20 min.The co-existence of high concentrations of COD and humic substances did not affect the mitigation of tylosin obviously,while more than 500 mg/L of nitrogenous compounds suppressed the hydrolytic efficiency.Recycle and reuse experiments showed that the solid acids performed well in five cycles after regeneration.Three transformation products (P1,P2 and P3)were identified using UPLC-QTOF-MS/MS.Sugar moieties including mycarse,mycaminose,and mycinose detached and released simultaneously or in order from the 16-member lactone ring through desugarization,which led to a dramatic decrease in antibacterial activity as revealed by cytotoxicity evaluations using S.aureus.Ecotoxicity estimation indicated the acute toxicities of the hydrolyzed products to model species (e.g.,fish,daphnid and green algae) were classified as“not harmful”.This work suggested an effective and selective method to pretreat tylosin-contained production wastewater by using polymer-based solid acids.These results will shed light on effective elimination of antibiotics pollution from pharmaceutical industries through strengthening the pretreatments.展开更多
Genome sequencing has revealed that actinomycetes possess the potential to produce many more secondary metabolites than previously thought.The existing challenge is to devise efficient methods to activate these silent...Genome sequencing has revealed that actinomycetes possess the potential to produce many more secondary metabolites than previously thought.The existing challenge is to devise efficient methods to activate these silent biosynthetic gene clusters(BGCs).In Streptomyces ansochromogenes,disruption of wbl A,a pleiotropic regulatory gene,activated the expression of cryptic tylosin analogues and abolished nikkomycin production simultaneously.Overexpressing pathway-specific regulatory genes tylR1 and tylR2 can also trigger the biosynthesis of silent tylosin analogues,in which TylR1 exerted its function via enhancing tylR2 expression.Bacterial one-hybrid system experiments unveiled that Wbl A directly inhibits the transcription of tylR1 and tylR2 to result in the silence of tylosin analogues BGC.Furthermore,Wbl A can activate the nikkomycin production through up-regulating the transcription of pleiotropic regulatory gene adp A.More interestingly,Adp A can activate san G(an activator gene in nikkomycin BGC)but repress wbl A.Our studies provide a valuable insight into the complex functions of pleiotropic regulators.展开更多
In the last few decades,sulfonated carbon materials have garnered significant attention as Brøsted solid acid catalysts.The sulfonation process and catalytic activity of sulfonated biochar can be influenced by th...In the last few decades,sulfonated carbon materials have garnered significant attention as Brøsted solid acid catalysts.The sulfonation process and catalytic activity of sulfonated biochar can be influenced by the aromaticity and degree of condensation exhibited by biochar.However,the relationships between the aromaticity,sulfonating ability,and resultant catalytic activity are not fully understood.In this study,biochar samples pyrolyzed at 300-650℃ exhibiting different aromaticity and degrees of condensation were sulfonated and employed as sulfonate-bearing solid catalysts for hydrolytically removing tylosin.They exhibited excellent hydrolytic performance and their kinetic constants were positively correlated with the total acidity and negatively correlated with their aromaticity.This study has uncovered the relationship between the structure,properties,sulfonating ability,and subsequent hydrolytic performance of biochar samples.It was observed that the aromaticity of biochar decreased as the pyrolysis temperature increased.Lower pyrolysis temperatures resulted in a reduced degree of condensation,smaller ring size,and an increased number of ring edge sites available for sulfonation,ultimately leading to enhanced catalytic performance.These findings provide valuable insights into the fundamental chemistry behind sulfonation upgrading of biochar,with the aim of developing functional catalysts for mitigating antibiotics in contaminated water.展开更多
Carbon-based solid acids have been successfully employed as acidic catalysts for pollutant mitigation in wastewater.To fully tap the potentials of commercially viable carbons for the preparation of solid acids and enh...Carbon-based solid acids have been successfully employed as acidic catalysts for pollutant mitigation in wastewater.To fully tap the potentials of commercially viable carbons for the preparation of solid acids and enhance their catalytic performances is a challenging problem.In this work,three commercialized carbons including biochar,activated carbon and graphite were preprocessed(ball-milling,Hummer exfoliation,HNO3 soaking,and microwave heating in HNO_(3),etc.),sulfonated,and evaluated as solid-acid catalysts for tylosin mitigation.Graphite-originated solid acid performed the best through a balling-milling preprocess,while biochar-originated solid acids behaved well under all preprocessing treatments,in which 40 mg L^(−1) of tylosin was mitigated within 8 min by 1 g L^(−1) of biochar-originated solid acids.The biochar solid acid through the ball-milling preprocess presented high total acidity and large amounts of-SO_(3)H groups,due to dramatically increased surface area and the rise of activation groups(hydroxyl,alkyl and alkoxy groups,etc.)facilitating electrophilic reaction.In addition,decreased particle size and aromaticity and increased structural defects also contributed.Theoretical calculation of average local ionization energy(ALIE)of condensed aromatic model molecules with substituted activation groups confirmed the promoting effects on sulfonation from strong to weak were 8.40-9.06 eV.These findings have deepened the knowledge in tuning carbon surface chemistry for better sulfonation,thus strengthening catalytic degradation of tylosin.The value of this study is in pulling a clear thread for maneuvering solid-acid catalysts using carbons,which holds a novel promise for rationally functionalizing biochar-based catalysts for the remediation of macrolide antibiotics in polluted water.展开更多
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 hyperproduci...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 metK_(cs),the SAM biosynthetic genes adoK_(cs)-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 metK_(cs)and adoK_(cs)-metFcs as two transcripts increased tylosin production by 22%in the resultant strain TLPH11 compared to that in TL01.Furthermore,combinational overexpression of tlrC,metK_(cs),adoK_(cs)-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.展开更多
基金jointly supported by the National Key R&D Program of China(2018YFD0500206)the National Natural Science Foundation of China(31772395)the Fundamental Research Funds for Central Non-profit Scientific Institution,Chinese Academy of Agricultural Sciences(1610132019046)。
文摘Antibiotics residues have been accumulating in the environment day by day due to overuse of antibiotics.Recalcitrant antibiotic residues,such as tylosin(TYL),can cause serious environmental problems,which makes it important to eliminate TYL from the environment.It is important to eliminate TYL from the environment.In this study,a strain was isolated and purified from fermentation by-product that came from a TYL production factory.The TYL degrading strain was identified by its morphology,physiological and biochemical reactions and sequencing the PCR-amplified fragments of its 16 S r DNAcoding genes.The temperature,shaking speed,initial TYL concentration,p H and inoculum sizes were investigated under simulated conditions by using single factor tests.The results showed that TYL2,a high efficient strain was isolated and was identified as Brevibacillus borstelensis.The degradation rate of TYL by this strain could reach to 75%with an initial concentration of 25 mg L^-1 within 7 days under conditions of 7%B.borstelensis(v/v,2×108 CFU m L^-1)at p H 7.0 and at 35°C.It is interesting that this strain has a very strong ability to degrade the TYL in natural sewage with the degradation rate of 65%within 7 days.This result could be helpful for the degradation of TYL and provide guidance for the degradation of other antibiotics.
文摘The pharmacokinetics and tissue residue of tylosin in broiler chickens were studied after I.V. and oral administrations in a dose of 50 mg tylosin/kg.b.wt. Tylosin was obeyed a two-compartment open model following I.V. administration at a dose of 50 mg/kg.b.wt. The disposition kinetics of tylosin following I.V. administration revealed that tylosin was highly distributed with V<sub>d(area)</sub> of 6 L/kg and eliminated with half-life (t<sub>1/2β</sub>) equal to 7.29 hours. The disposition kinetics of tylosin following oral administration revealed that the maximum blood concentration (C<sub>max</sub>) was 3.40 μg/ml attained at (t<sub>max</sub>) of 1.08 hour. Tylosin was eliminated with half-life (t<sub>1/2β</sub>) equal to 5.78 hours. The mean systemic bioavailability of tylosin after oral administration was 90.29%. Following repeated oral administration of 50 mg tylosin base/kg.b.wt once daily for 5 consecutive days, the blood (μg/ml) and tissue (μg/g) residues of tylosin showed that liver, kidney and lung contained the highest tylosin residues and completely disappeared from those tissues at 6 days after the last oral dose. Chickens should not be slaughtered for human consumption within the treatment and 6 days after the last oral administrations of tylosin.
基金funded by the National High Technology Research and Development Program of China (2006AA10A208-2-3)
文摘[Objective]To provide a reference for dynamic .regulation of the fermentation process. [Method] Using conditions for tylosin fermentation as feature vectors, final yield during the fermentation process was predicted using the Bayesian method. [ Rgesultl The Bayesian network which learned data of tylosin fermentation could better guide conditions of the fermentation process, and better prediction results were achieved. The yield of fermentation products could be effectively predicted using the obtained Bayesian network and could well guide the fermentation process. E Conclusion] Bayesian network has good predictive performance for microbial fermentation and has good practical yield and application prospects.
文摘Tylosin is a well-established antibiotic that has been widely employed in human and veterinary medicines. It can act as a potential ligand binding metal ions due to various donor atoms in the structure. Our study on the complexation of various metal ions with tylosin ligand revealed that they preferably coordinate with mycaminose fragment to establish Novel trends complexes. Tylosin ligand (TYS) behaves as bidentate for complexation with different metal ions such as Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II) and Zn(II). Various essential metal complexes of tylosin were synthesized and characterized by techniques such as UV, IR, Elemental analysis, magnetic susceptibility and ESR spectra of Cu(II) complex. These techniques are used to know their geometries and mode of bonding, with stoichiometry, 2:2 (M:L). Thermal analysis (TGA and DTA) of ligands and their metal complexes were carried out to distinguish between the coordinate and hydrate solvents and to estimate the stability ranges, peak temperatures. The thermodynamic parameters, such as activation energy (ΔE<sup>*</sup>), the enthalpy of activation (ΔH<sup>*</sup>), entropy of activation (ΔS<sup>*</sup>) and Gibbs free energy (ΔG<sup>*</sup>) are calculated and discussed. Some tylosin complexes show higher activity than tylosin for some bacterial and fungal strains. Low concentration value of minimum inhibitory concentration (MIC) results is 15.625 μg/ml for both complexes [Zn<sub>2</sub>(TYS)<sub>2</sub>Cl<sub>2</sub>(H<sub>2</sub>O)<sub>4</sub>]·25H<sub>2</sub>O and [Cu<sub>2</sub>(TYS)<sub>2</sub>Cl<sub>2</sub>(H<sub>2</sub>O)<sub>4</sub>]·25H<sub>2</sub>O with B. cereus genus maybe a valuable data used to produce novel therapeutic agent. This study constitutes several essential aspects for future research on tylosin metal complexes as antibacterial assessment and as potential medicinal agents.
基金supported by the National Natural Science Foundation of China (No. 51978052)Beijing Municipal Education Commission through the Innovative Transdisciplinary Program “Ecological Restoration Engineering (No. GJJXK210102)”。
文摘Antibiotic production wastewater usually contains high concentrations of antibiotic residues,which can cause instability and deterioration of biological wastewater treatment units and also domestication and proliferation of antibiotic-resistance bacteria.An effective pretreatment on antibiotics production wastewater is expected to selectively reduce the concentration of antibiotics and decrease the toxicity,rather than mitigate organic and other contaminants before further treatments.In this work,two polymer-based solid acids,PS-S and CPS-S bearing high concentrations of-SOH_(3)groups (up to 4.57 mmol/g),were prepared and successfully used for hydrolytic mitigation of 100 mg/L tylosin within 20 min.The co-existence of high concentrations of COD and humic substances did not affect the mitigation of tylosin obviously,while more than 500 mg/L of nitrogenous compounds suppressed the hydrolytic efficiency.Recycle and reuse experiments showed that the solid acids performed well in five cycles after regeneration.Three transformation products (P1,P2 and P3)were identified using UPLC-QTOF-MS/MS.Sugar moieties including mycarse,mycaminose,and mycinose detached and released simultaneously or in order from the 16-member lactone ring through desugarization,which led to a dramatic decrease in antibacterial activity as revealed by cytotoxicity evaluations using S.aureus.Ecotoxicity estimation indicated the acute toxicities of the hydrolyzed products to model species (e.g.,fish,daphnid and green algae) were classified as“not harmful”.This work suggested an effective and selective method to pretreat tylosin-contained production wastewater by using polymer-based solid acids.These results will shed light on effective elimination of antibiotics pollution from pharmaceutical industries through strengthening the pretreatments.
基金supported by the National Key Research and Development Program of China(2020YFA0907800)the National Natural Science Foundation of China(32170043,82173720)the Beijing Natural Science Foundation(7212153)。
文摘Genome sequencing has revealed that actinomycetes possess the potential to produce many more secondary metabolites than previously thought.The existing challenge is to devise efficient methods to activate these silent biosynthetic gene clusters(BGCs).In Streptomyces ansochromogenes,disruption of wbl A,a pleiotropic regulatory gene,activated the expression of cryptic tylosin analogues and abolished nikkomycin production simultaneously.Overexpressing pathway-specific regulatory genes tylR1 and tylR2 can also trigger the biosynthesis of silent tylosin analogues,in which TylR1 exerted its function via enhancing tylR2 expression.Bacterial one-hybrid system experiments unveiled that Wbl A directly inhibits the transcription of tylR1 and tylR2 to result in the silence of tylosin analogues BGC.Furthermore,Wbl A can activate the nikkomycin production through up-regulating the transcription of pleiotropic regulatory gene adp A.More interestingly,Adp A can activate san G(an activator gene in nikkomycin BGC)but repress wbl A.Our studies provide a valuable insight into the complex functions of pleiotropic regulators.
基金National Natural Science Foundation of China(51978052)State Key Joint Laboratory of Environmental Simulation and Pollution(19K01ESPCR).
文摘In the last few decades,sulfonated carbon materials have garnered significant attention as Brøsted solid acid catalysts.The sulfonation process and catalytic activity of sulfonated biochar can be influenced by the aromaticity and degree of condensation exhibited by biochar.However,the relationships between the aromaticity,sulfonating ability,and resultant catalytic activity are not fully understood.In this study,biochar samples pyrolyzed at 300-650℃ exhibiting different aromaticity and degrees of condensation were sulfonated and employed as sulfonate-bearing solid catalysts for hydrolytically removing tylosin.They exhibited excellent hydrolytic performance and their kinetic constants were positively correlated with the total acidity and negatively correlated with their aromaticity.This study has uncovered the relationship between the structure,properties,sulfonating ability,and subsequent hydrolytic performance of biochar samples.It was observed that the aromaticity of biochar decreased as the pyrolysis temperature increased.Lower pyrolysis temperatures resulted in a reduced degree of condensation,smaller ring size,and an increased number of ring edge sites available for sulfonation,ultimately leading to enhanced catalytic performance.These findings provide valuable insights into the fundamental chemistry behind sulfonation upgrading of biochar,with the aim of developing functional catalysts for mitigating antibiotics in contaminated water.
基金National Natural Science Foundation of China(51978052,42207456)State Key Joint Laboratory of Environmental Simulation and Pollution(19K01ESPCR).
文摘Carbon-based solid acids have been successfully employed as acidic catalysts for pollutant mitigation in wastewater.To fully tap the potentials of commercially viable carbons for the preparation of solid acids and enhance their catalytic performances is a challenging problem.In this work,three commercialized carbons including biochar,activated carbon and graphite were preprocessed(ball-milling,Hummer exfoliation,HNO3 soaking,and microwave heating in HNO_(3),etc.),sulfonated,and evaluated as solid-acid catalysts for tylosin mitigation.Graphite-originated solid acid performed the best through a balling-milling preprocess,while biochar-originated solid acids behaved well under all preprocessing treatments,in which 40 mg L^(−1) of tylosin was mitigated within 8 min by 1 g L^(−1) of biochar-originated solid acids.The biochar solid acid through the ball-milling preprocess presented high total acidity and large amounts of-SO_(3)H groups,due to dramatically increased surface area and the rise of activation groups(hydroxyl,alkyl and alkoxy groups,etc.)facilitating electrophilic reaction.In addition,decreased particle size and aromaticity and increased structural defects also contributed.Theoretical calculation of average local ionization energy(ALIE)of condensed aromatic model molecules with substituted activation groups confirmed the promoting effects on sulfonation from strong to weak were 8.40-9.06 eV.These findings have deepened the knowledge in tuning carbon surface chemistry for better sulfonation,thus strengthening catalytic degradation of tylosin.The value of this study is in pulling a clear thread for maneuvering solid-acid catalysts using carbons,which holds a novel promise for rationally functionalizing biochar-based catalysts for the remediation of macrolide antibiotics in polluted water.
基金the National Key Research and Development Program of China(grant no.2022YFC210540303)the“Major Project”of Haihe Laboratory of Synthetic Biology(22HHSWSS00001).
文摘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 metK_(cs),the SAM biosynthetic genes adoK_(cs)-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 metK_(cs)and adoK_(cs)-metFcs as two transcripts increased tylosin production by 22%in the resultant strain TLPH11 compared to that in TL01.Furthermore,combinational overexpression of tlrC,metK_(cs),adoK_(cs)-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.
