【背景】糖多孢菌属(Saccharopolyspora)是一类重要的稀有放线菌资源,菌株BM8-3分离自马里亚纳海沟沉积物,具有较强的耐盐特性和病原菌拮抗活性。目前尚无对深海来源糖多孢菌的功能基因、基因组学分析以及代谢产物合成的相关研究。【目...【背景】糖多孢菌属(Saccharopolyspora)是一类重要的稀有放线菌资源,菌株BM8-3分离自马里亚纳海沟沉积物,具有较强的耐盐特性和病原菌拮抗活性。目前尚无对深海来源糖多孢菌的功能基因、基因组学分析以及代谢产物合成的相关研究。【目的】分析Saccharopolyspora sp.BM8-3的基因组序列和代谢功能基因,评估次级代谢合成潜力,为深入揭示其深海极端环境适应机制和挖掘新颖次级代谢产物基因资源奠定基础。【方法】利用PacBio平台对菌株BM8-3进行全基因组测序,使用SMRT Link 5.0.1对测序数据进行基因组组装,并通过蛋白质直系同源簇(cluster of orthologous groups of proteins,COG)、京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)等数据库进行基因预测及功能注释。【结果】菌株BM8-3基因组全长6344421 bp,G+C含量为72.5%,共注释到5620个基因、51个tRNA基因和12个rRNA基因。分别有4137、2677个基因在COG、KEGG数据库中提取到注释信息。基因组分析表明,该菌基因组中富含与渗透压、高静水压适应相关的基因,包括海藻糖(otsAB)、甘氨酸甜菜碱(betAB)、四氢嘧啶(ectABCD)、二甲基甘氨酸和肌氨酸(gsmt-sdmt)等相容溶质合成和转运基因,以及与钠钾离子运输系统(如钠离子/质子逆向转运蛋白Mrp)和有机溶质运输通道(如MscL)相关的基因;同时基因组中还注释到了与冷适应相关的基因包括冷休克蛋白编码基因cspA、GroEL-GroES分子伴侣蛋白的编码基因和多种参与不饱和脂肪酸代谢的编码基因(如desC、fabF);此外,KEGG注释结果显示菌株BM8-3具有完整的牛磺酸转运和代谢的TauABCD途径以及硫胺素回收途径(thiamine salvage pathway);基因组中还预测到24个次级代谢产物合成基因簇,50%以上与已知化合物合成基因簇相似度低于60%,说明该菌株可能具备合成多种新颖次级代谢产物的能力。【结论】本研究分析了菌株BM8-3的全基因组信息,揭示了菌株的代谢多样性及其在极端环境中的适应能力,为深海放线菌的环境适应策略及其在硫循环中的生态功能提供了重要参考。展开更多
羽毛山海绵(My ca le p lum ose)来源的糖多孢菌(S accharop oly sp ora sp.nov SP 2-10)具有诱导肿瘤细胞坏死的活性。本文对其发酵产物的活性部位乙酸乙酯层进行活性追踪分离,共得到7个化合物;利用理化性质和波谱学方法鉴定它们的结...羽毛山海绵(My ca le p lum ose)来源的糖多孢菌(S accharop oly sp ora sp.nov SP 2-10)具有诱导肿瘤细胞坏死的活性。本文对其发酵产物的活性部位乙酸乙酯层进行活性追踪分离,共得到7个化合物;利用理化性质和波谱学方法鉴定它们的结构分别为胆甾醇、邻苯二甲酸二异丁酯、4(2,4-二羟基苯甲酰氨基)苯甲酸、苯丙胺酸、脱氧鸟苷、鸟苷和N-乙酰酪胺;并初步评价上述化合物的抗肿瘤活性,结果表明,邻苯二甲酸二异丁酯显示细胞毒活性。展开更多
Comparative transcriptomics uncovered distinct expression patterns of genes associated with cofactor and vitamin metabolism in the high-yielding mutant strain Saccharopolyspora erythraea HL3168 E3,as compared to the w...Comparative transcriptomics uncovered distinct expression patterns of genes associated with cofactor and vitamin metabolism in the high-yielding mutant strain Saccharopolyspora erythraea HL3168 E3,as compared to the wild-type NRRL 2338.An in-depth analysis was conducted on the effects of nine vitamins,and it was determined that thiamine pyrophosphate(TPP),vitamin B2,vitamin B6,vitamin B9,vitamin B12,and hemin are key enhancers in erythromycin production in E3,increasing the erythromycin titer by 7.96-12.66%.Then,the Plackett-Burman design and the path of steepest ascent were applied to further optimize the vitamin combination for maximum production efficiency,enhancing the erythromycin titer in shake flasks by 39.2%.Otherwise,targeted metabolomics and metabolic flux analysis illuminated how vitamin supplementation modulates the central carbon metabolism with notable effects on the TCA cycle and methionine synthesis to augment the provision of energy and precursors essential for erythromycin synthesis.This work highlights the capacity for precise vitamin supplementation to refine metabolic pathways,thereby boosting erythromycin production,and provides valuable directions for application on an industrial scale.展开更多
Microorganisms are often likened to complex production workshops.In Saccharopolyspora spinosa(S.spinosa),the biosynthesis of spinosad is a production line within its intricate workshop.Optimizing the entire production...Microorganisms are often likened to complex production workshops.In Saccharopolyspora spinosa(S.spinosa),the biosynthesis of spinosad is a production line within its intricate workshop.Optimizing the entire production environment and reducing unnecessary metabolic flow is essential to increasing spinosad yield.Pyruvate serves as a crucial precursor for spinosad biosynthesis.Previous studies revealed that the pyc gene is highly expressed in the gluconeogenic pathway,leading to a pyruvate shunt.By downregulating pyc,we enhanced spinosad yield,although the improvement was below expectations.We speculated that most of the accumulated pyruvate following the pyc knockdown entered some synthetic pathways unrelated to spinosad.Through metabolic pathway and qRT-PCR analyses,we found that the expression levels of gltA1 and atoB3 within the pyruvate metabolic tributary,including the TCA cycle and ethylmalonyl-CoA pathway,were significantly increased in the pyc knockdown strain.The combined knockdown of these three genes optimized the spinosad production line,increasing its yield to 633.1±38.6 mg/L,representing a 199.4%increase.This study identifies three key genes for optimizing spinosad biosynthesis and offers insights into gene screening and the efficient construction of Spinosad-producing strains.展开更多
Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inac...Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inaccessibility and lack of effective genome engineering tools.In this work, we characterized the activity of an endogenous type I-B CRISPR-Cas system as well as its recognized protospacer adjacent motifs(PAMs) based on bioinformatics analysis combined with a plasmid interference assay in S. spinosa. By delivering editing plasmids containing a designed mini CRISPR array(repeat+self-targeting spacer+repeat) and repair templates, we achieved 100% editing efficiency for gene deletion. Using this tool, the genetic barrier composed of the restriction-modification(RM) systems was systematically disarmed. We showed that by disarming one type I RM system(encoded by A8926_1903/1904/1905) and two type Ⅱ RM systems(encoded by A8926_1725/1726 and A8926_2652/2653) simultaneously, the transformation efficiency of the replicative and integrative plasmids(pSP01 and pSI01) was increased by approximately 3.9-fold and 4.2-fold, respectively. Using the engineered strain with simultaneous knock-out of these three RM genes as the starting strain, we achieved the deletion of 75-kb spinosyns biosynthetic gene cluster(BGC) as well as gene insertion at high efficiency. Collectively, we developed a reliable and highly efficient genome editing tool based on the endogenous type I CRISPR-Cas system combined with the disarmament of the RM systems in S. spinosa. This is the first time to establish an endogenous CRISPR-Cas-based genome editing tool in the non-model industrial actinomycetes.展开更多
To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the co...To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the control of the promoter for the erythromycin resistance gene by splicing using overlapping extension PCR. This was cloned into the integrating vector pSET152, yielding the Vitreoscilla hemoglobin gene expression plasmid pSET152EVHB. This was then introduced into S. spinosa SP06081 by conjugal transfer, and integrated into the chromosome by site-specific recombination at the integration site ФC31 on pSET152EVHB. The resultant conjugant, S. spinosa S078-1101, was genetically stable. The integration was further confirmed by PCR and Southern blotting analysis. A carbon monoxide differential spectrum assay showed that active Vitreoscilla hemoglobin was successfully expressed in S. spinosa S078-1101. Fermentation results revealed that expression of the Vitreoscilla hemoglobin gene significantly promoted spinosad biosynthesis under normal oxygen and moderately oxygen-limiting conditions （P〈0.01）. These findings demonstrate that integrating expression of the Vitreoscilla hemoglobin gene improves oxygen uptake and is an effective means for the genetic improvement of S. spinosa fermentation. Saccharopolyspora spinosa, spinosad, Vitreoscilla hemoglobin, integrating vector, homologous recombination展开更多
Erythromycin A is a widely used antibiotic produced by Saccharopolyspora erythraea;however,its biosynthetic cluster lacks a regulatory gene,limiting the yield enhancement via regulation engineering of S.erythraea.Here...Erythromycin A is a widely used antibiotic produced by Saccharopolyspora erythraea;however,its biosynthetic cluster lacks a regulatory gene,limiting the yield enhancement via regulation engineering of S.erythraea.Herein,six TetR family transcriptional regulators(TFRs)belonging to three genomic context types were individually inactivated in S.erythraea A226,and one of them,SACE_3446,was proved to play a negative role in regulating erythromycin biosynthesis.EMSA and qRT-PCR analysis revealed that SACE_3446 covering intact N-terminal DNA binding domain specifically bound to the promoter regions of erythromycin biosynthetic gene eryAI,the resistant gene ermE and the adjacent gene SACE_3447(encoding a longchain fatty-acid CoA ligase),and repressed their transcription.Furthermore,we explored the interaction relationships of SACE_3446 and previously identified TFRs(SACE_3986 and SACE_7301)associated with erythromycin production.Given demonstrated relatively independent regulation mode of SACE_3446 and SACE_3986 in erythromycin biosynthesis,we individually and concomitantly inactivated them in an industrial S.erythraea WB.Compared with WB,the WBΔ3446 and WBΔ3446Δ3986 mutants respectively displayed 36%and 65%yield enhancement of erythromycin A,following significantly elevated transcription of eryAI and ermE.When cultured in a 5 L fermentor,erythromycin A ofWBΔ3446 and WBΔ3446Δ3986 successively reached 4095 mg/L and 4670 mg/L with 23%and 41%production improvement relative to WB.The strategy reported here will be useful to improve antibiotics production in other industrial actinomycete.展开更多
Spinosad,a potent broad-spectrum bioinsecticide produced by Saccharopolyspora spinosa,has significant market potential.Despite its effectiveness,the regulatory mechanisms of spinosad biosynthesis remain unclear.Our in...Spinosad,a potent broad-spectrum bioinsecticide produced by Saccharopolyspora spinosa,has significant market potential.Despite its effectiveness,the regulatory mechanisms of spinosad biosynthesis remain unclear.Our investigation identified the crucial role of the LysR family transcriptional regulator ORF-L16,located upstream of spinosad biosynthetic genes,in spinosad biosynthesis.Through reverse transcription PCR(RT-PCR)and 5′-rapid amplification of cDNA ends(5′-Race),we unveiled that the spinosad biosynthetic gene cluster(BGC)contains six transcription units and seven promoters.Electrophoretic mobility shift assays(EMSAs)demonstrated that ORF-L16 bound to seven promoters within the spinosad BGC,indicating its involvement in regulating spinosad biosynthesis.Notably,deletion of ORF-L16 led to a drastic reduction in spinosad production from 1818.73 mg/L to 1.69 mg/L,accompanied by decreased transcription levels of spinosad biosynthetic genes,confirming its positive regulatory function.Additionally,isothermal titration calorimetry(ITC)and EMSA confirmed that spinosyn A,the main product of the spinosad BGC,served as an effector of ORF-L16.Specifically,it decreased the binding affinity between ORF-L16 and spinosad BGC promoters,thus exerting negative feedback regulation on spinosad biosynthesis.This research enhances our comprehension of spinosad biosynthesis regulation and lays the groundwork for future investigations on transcriptional regulators in S.spinosa.展开更多
Triacylglycerol(TAG)is crucial for antibiotic biosynthesis derived from Streptomyces,as it serves as an important carbon source.In this study,the supplementation of exogenous TAG led to a 3.92-fold augmentation in spi...Triacylglycerol(TAG)is crucial for antibiotic biosynthesis derived from Streptomyces,as it serves as an important carbon source.In this study,the supplementation of exogenous TAG led to a 3.92-fold augmentation in spinosad production.The impact of exogenous TAG on the metabolic network of Saccharopolyspora spinosa were deeply analyzed through comparative proteomics.To optimize TAG metabolism and enhance spinosad biosynthesis,the lipase-encoding genes lip886 and lip385 were overexpressed or co-expressed.The results shown that the yield of spinosad was increased by 0.8-fold and 0.4-fold when lip886 and lip385 genes were overexpressed,respectively.Synergistic co-expression of these genes resulted in a 2.29-fold increase in the yield of spinosad.Remarkably,the combined overexpression of lip886 and lip385 in the presence of exogenous TAG elevated spinosad yields by 5.5-fold,led to a drastic increase in spinosad production from 0.036 g/L to 0.234 g/L.This study underscores the modification of intracellular concentrations of free fatty acids(FFAs),short-chain acyl-CoAs,ATP,and NADPH as mechanisms by which exogenous TAG modulates spinosad biosynthesis.Overall,the findings validate the enhancement of TAG catabolism as a beneficial strategy for optimizing spinosad production and provide foundational insights for engineering secondary metabolite biosynthesis pathways in another Streptomyces.展开更多
The microbial genome remains a huge treasure trove for the discovery of diverse natural products.Saccharopolyspora erythraea NRRL23338,the industry producer of erythromycin,has a dozen of biosynthetic gene clusters wh...The microbial genome remains a huge treasure trove for the discovery of diverse natural products.Saccharopolyspora erythraea NRRL23338,the industry producer of erythromycin,has a dozen of biosynthetic gene clusters whose encoding products are unidentified.Heterologous expression of one of the polyketide clusters pks7 in Streptomyces albus B4 chassis resulted in the characterization of its function responsible for synthesizing both 6-methylsalicyclic acid and 6-ethylsalicyclic acid.Meanwhile,two new 6-ethylsalicyclic acid ester derivatives were isolated as shunt metabolites.Their structures were identified by comprehensive analysis of MS and NMR experiments.Putative functions of genes within the pks7 BGC were also discussed.展开更多
采用响应面法对红色糖多孢菌产红霉素发酵培养基进行优化。用Minimum Run Equireolicated Res IV设计对初始发酵培养基添加的6个影响因素的效应进行评价,选择有显著影响的4个因素,即硫酸镁、甜菜碱、硫酸铜和氯化钴。再用最陡爬坡实验...采用响应面法对红色糖多孢菌产红霉素发酵培养基进行优化。用Minimum Run Equireolicated Res IV设计对初始发酵培养基添加的6个影响因素的效应进行评价,选择有显著影响的4个因素,即硫酸镁、甜菜碱、硫酸铜和氯化钴。再用最陡爬坡实验为中心组合实验确定最大响应区间,最后经过响应面分析得到最优化结果,硫酸镁0.106%(w/v),甜菜碱0.0185%(w/v),硫酸铜0.106mmol/L,氯化钴0.0003%(w/v)。优化后红霉素生物效价比优化前提高了30%。展开更多
文摘【背景】糖多孢菌属(Saccharopolyspora)是一类重要的稀有放线菌资源,菌株BM8-3分离自马里亚纳海沟沉积物,具有较强的耐盐特性和病原菌拮抗活性。目前尚无对深海来源糖多孢菌的功能基因、基因组学分析以及代谢产物合成的相关研究。【目的】分析Saccharopolyspora sp.BM8-3的基因组序列和代谢功能基因,评估次级代谢合成潜力,为深入揭示其深海极端环境适应机制和挖掘新颖次级代谢产物基因资源奠定基础。【方法】利用PacBio平台对菌株BM8-3进行全基因组测序,使用SMRT Link 5.0.1对测序数据进行基因组组装,并通过蛋白质直系同源簇(cluster of orthologous groups of proteins,COG)、京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)等数据库进行基因预测及功能注释。【结果】菌株BM8-3基因组全长6344421 bp,G+C含量为72.5%,共注释到5620个基因、51个tRNA基因和12个rRNA基因。分别有4137、2677个基因在COG、KEGG数据库中提取到注释信息。基因组分析表明,该菌基因组中富含与渗透压、高静水压适应相关的基因,包括海藻糖(otsAB)、甘氨酸甜菜碱(betAB)、四氢嘧啶(ectABCD)、二甲基甘氨酸和肌氨酸(gsmt-sdmt)等相容溶质合成和转运基因,以及与钠钾离子运输系统(如钠离子/质子逆向转运蛋白Mrp)和有机溶质运输通道(如MscL)相关的基因;同时基因组中还注释到了与冷适应相关的基因包括冷休克蛋白编码基因cspA、GroEL-GroES分子伴侣蛋白的编码基因和多种参与不饱和脂肪酸代谢的编码基因(如desC、fabF);此外,KEGG注释结果显示菌株BM8-3具有完整的牛磺酸转运和代谢的TauABCD途径以及硫胺素回收途径(thiamine salvage pathway);基因组中还预测到24个次级代谢产物合成基因簇,50%以上与已知化合物合成基因簇相似度低于60%,说明该菌株可能具备合成多种新颖次级代谢产物的能力。【结论】本研究分析了菌株BM8-3的全基因组信息,揭示了菌株的代谢多样性及其在极端环境中的适应能力,为深海放线菌的环境适应策略及其在硫循环中的生态功能提供了重要参考。
文摘羽毛山海绵(My ca le p lum ose)来源的糖多孢菌(S accharop oly sp ora sp.nov SP 2-10)具有诱导肿瘤细胞坏死的活性。本文对其发酵产物的活性部位乙酸乙酯层进行活性追踪分离,共得到7个化合物;利用理化性质和波谱学方法鉴定它们的结构分别为胆甾醇、邻苯二甲酸二异丁酯、4(2,4-二羟基苯甲酰氨基)苯甲酸、苯丙胺酸、脱氧鸟苷、鸟苷和N-乙酰酪胺;并初步评价上述化合物的抗肿瘤活性,结果表明,邻苯二甲酸二异丁酯显示细胞毒活性。
基金financially supported by the National Key Research Development Program of China(2022YFC2105403)the Taishan Scholars Program,the Shanghai Pilot Program for Basic Research(22TQ1400100-14)+1 种基金the Natural Science Foundation of Shanghai(23ZR1416500)the Frontiers Science Center for Materiobiology and Dynamic Chemistry(JKVJ1231036).
文摘Comparative transcriptomics uncovered distinct expression patterns of genes associated with cofactor and vitamin metabolism in the high-yielding mutant strain Saccharopolyspora erythraea HL3168 E3,as compared to the wild-type NRRL 2338.An in-depth analysis was conducted on the effects of nine vitamins,and it was determined that thiamine pyrophosphate(TPP),vitamin B2,vitamin B6,vitamin B9,vitamin B12,and hemin are key enhancers in erythromycin production in E3,increasing the erythromycin titer by 7.96-12.66%.Then,the Plackett-Burman design and the path of steepest ascent were applied to further optimize the vitamin combination for maximum production efficiency,enhancing the erythromycin titer in shake flasks by 39.2%.Otherwise,targeted metabolomics and metabolic flux analysis illuminated how vitamin supplementation modulates the central carbon metabolism with notable effects on the TCA cycle and methionine synthesis to augment the provision of energy and precursors essential for erythromycin synthesis.This work highlights the capacity for precise vitamin supplementation to refine metabolic pathways,thereby boosting erythromycin production,and provides valuable directions for application on an industrial scale.
基金supported by funding from the National Natural Science Foundation of China(31770106,32200062)the Natural Science Foundation of Hunan Province(2024JJ5258).
文摘Microorganisms are often likened to complex production workshops.In Saccharopolyspora spinosa(S.spinosa),the biosynthesis of spinosad is a production line within its intricate workshop.Optimizing the entire production environment and reducing unnecessary metabolic flow is essential to increasing spinosad yield.Pyruvate serves as a crucial precursor for spinosad biosynthesis.Previous studies revealed that the pyc gene is highly expressed in the gluconeogenic pathway,leading to a pyruvate shunt.By downregulating pyc,we enhanced spinosad yield,although the improvement was below expectations.We speculated that most of the accumulated pyruvate following the pyc knockdown entered some synthetic pathways unrelated to spinosad.Through metabolic pathway and qRT-PCR analyses,we found that the expression levels of gltA1 and atoB3 within the pyruvate metabolic tributary,including the TCA cycle and ethylmalonyl-CoA pathway,were significantly increased in the pyc knockdown strain.The combined knockdown of these three genes optimized the spinosad production line,increasing its yield to 633.1±38.6 mg/L,representing a 199.4%increase.This study identifies three key genes for optimizing spinosad biosynthesis and offers insights into gene screening and the efficient construction of Spinosad-producing strains.
基金supported by the National Key Research and Development Program of China(2019YFA0905400)the National Natural Science Foundation of China(32270095)the Open Funding Project(MMLKF23-08)of State Key Laboratory of Microbial Metabolism.
文摘Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inaccessibility and lack of effective genome engineering tools.In this work, we characterized the activity of an endogenous type I-B CRISPR-Cas system as well as its recognized protospacer adjacent motifs(PAMs) based on bioinformatics analysis combined with a plasmid interference assay in S. spinosa. By delivering editing plasmids containing a designed mini CRISPR array(repeat+self-targeting spacer+repeat) and repair templates, we achieved 100% editing efficiency for gene deletion. Using this tool, the genetic barrier composed of the restriction-modification(RM) systems was systematically disarmed. We showed that by disarming one type I RM system(encoded by A8926_1903/1904/1905) and two type Ⅱ RM systems(encoded by A8926_1725/1726 and A8926_2652/2653) simultaneously, the transformation efficiency of the replicative and integrative plasmids(pSP01 and pSI01) was increased by approximately 3.9-fold and 4.2-fold, respectively. Using the engineered strain with simultaneous knock-out of these three RM genes as the starting strain, we achieved the deletion of 75-kb spinosyns biosynthetic gene cluster(BGC) as well as gene insertion at high efficiency. Collectively, we developed a reliable and highly efficient genome editing tool based on the endogenous type I CRISPR-Cas system combined with the disarmament of the RM systems in S. spinosa. This is the first time to establish an endogenous CRISPR-Cas-based genome editing tool in the non-model industrial actinomycetes.
基金supported by the National Basic Research Program of China (Grant Nos. 2012CB722301 and 2011CB111605)the National High Technology Research and Development Project of China (Grant No. 2011AA10A203)the National Natural Science Foundation of China (Grant No. 31070006)
文摘To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the control of the promoter for the erythromycin resistance gene by splicing using overlapping extension PCR. This was cloned into the integrating vector pSET152, yielding the Vitreoscilla hemoglobin gene expression plasmid pSET152EVHB. This was then introduced into S. spinosa SP06081 by conjugal transfer, and integrated into the chromosome by site-specific recombination at the integration site ФC31 on pSET152EVHB. The resultant conjugant, S. spinosa S078-1101, was genetically stable. The integration was further confirmed by PCR and Southern blotting analysis. A carbon monoxide differential spectrum assay showed that active Vitreoscilla hemoglobin was successfully expressed in S. spinosa S078-1101. Fermentation results revealed that expression of the Vitreoscilla hemoglobin gene significantly promoted spinosad biosynthesis under normal oxygen and moderately oxygen-limiting conditions （P〈0.01）. These findings demonstrate that integrating expression of the Vitreoscilla hemoglobin gene improves oxygen uptake and is an effective means for the genetic improvement of S. spinosa fermentation. Saccharopolyspora spinosa, spinosad, Vitreoscilla hemoglobin, integrating vector, homologous recombination
基金the National Program on Key Basic Research Project(973 program,2013CB734000)the National Natural Science Foundation of China(Grant Nos.31300081,31570074)the Initial Foundation of Doctoral Scientific Research in Anhui University(01001904,J01001935).
文摘Erythromycin A is a widely used antibiotic produced by Saccharopolyspora erythraea;however,its biosynthetic cluster lacks a regulatory gene,limiting the yield enhancement via regulation engineering of S.erythraea.Herein,six TetR family transcriptional regulators(TFRs)belonging to three genomic context types were individually inactivated in S.erythraea A226,and one of them,SACE_3446,was proved to play a negative role in regulating erythromycin biosynthesis.EMSA and qRT-PCR analysis revealed that SACE_3446 covering intact N-terminal DNA binding domain specifically bound to the promoter regions of erythromycin biosynthetic gene eryAI,the resistant gene ermE and the adjacent gene SACE_3447(encoding a longchain fatty-acid CoA ligase),and repressed their transcription.Furthermore,we explored the interaction relationships of SACE_3446 and previously identified TFRs(SACE_3986 and SACE_7301)associated with erythromycin production.Given demonstrated relatively independent regulation mode of SACE_3446 and SACE_3986 in erythromycin biosynthesis,we individually and concomitantly inactivated them in an industrial S.erythraea WB.Compared with WB,the WBΔ3446 and WBΔ3446Δ3986 mutants respectively displayed 36%and 65%yield enhancement of erythromycin A,following significantly elevated transcription of eryAI and ermE.When cultured in a 5 L fermentor,erythromycin A ofWBΔ3446 and WBΔ3446Δ3986 successively reached 4095 mg/L and 4670 mg/L with 23%and 41%production improvement relative to WB.The strategy reported here will be useful to improve antibiotics production in other industrial actinomycete.
基金supported by the National Key R&D Program of China(grant number 2018YFA0900400)the National Natural Science Foundation of China(grant number 32100053)the Young Elite Scientists Sponsorship Program by China Association for Science and Technology(grant number YESS20210068).
文摘Spinosad,a potent broad-spectrum bioinsecticide produced by Saccharopolyspora spinosa,has significant market potential.Despite its effectiveness,the regulatory mechanisms of spinosad biosynthesis remain unclear.Our investigation identified the crucial role of the LysR family transcriptional regulator ORF-L16,located upstream of spinosad biosynthetic genes,in spinosad biosynthesis.Through reverse transcription PCR(RT-PCR)and 5′-rapid amplification of cDNA ends(5′-Race),we unveiled that the spinosad biosynthetic gene cluster(BGC)contains six transcription units and seven promoters.Electrophoretic mobility shift assays(EMSAs)demonstrated that ORF-L16 bound to seven promoters within the spinosad BGC,indicating its involvement in regulating spinosad biosynthesis.Notably,deletion of ORF-L16 led to a drastic reduction in spinosad production from 1818.73 mg/L to 1.69 mg/L,accompanied by decreased transcription levels of spinosad biosynthetic genes,confirming its positive regulatory function.Additionally,isothermal titration calorimetry(ITC)and EMSA confirmed that spinosyn A,the main product of the spinosad BGC,served as an effector of ORF-L16.Specifically,it decreased the binding affinity between ORF-L16 and spinosad BGC promoters,thus exerting negative feedback regulation on spinosad biosynthesis.This research enhances our comprehension of spinosad biosynthesis regulation and lays the groundwork for future investigations on transcriptional regulators in S.spinosa.
基金supported by funding from the National Natural Science Foundation of China(31770106,32200062)the Natural Science Foundation of Hunan Province(2024JJ5258).
文摘Triacylglycerol(TAG)is crucial for antibiotic biosynthesis derived from Streptomyces,as it serves as an important carbon source.In this study,the supplementation of exogenous TAG led to a 3.92-fold augmentation in spinosad production.The impact of exogenous TAG on the metabolic network of Saccharopolyspora spinosa were deeply analyzed through comparative proteomics.To optimize TAG metabolism and enhance spinosad biosynthesis,the lipase-encoding genes lip886 and lip385 were overexpressed or co-expressed.The results shown that the yield of spinosad was increased by 0.8-fold and 0.4-fold when lip886 and lip385 genes were overexpressed,respectively.Synergistic co-expression of these genes resulted in a 2.29-fold increase in the yield of spinosad.Remarkably,the combined overexpression of lip886 and lip385 in the presence of exogenous TAG elevated spinosad yields by 5.5-fold,led to a drastic increase in spinosad production from 0.036 g/L to 0.234 g/L.This study underscores the modification of intracellular concentrations of free fatty acids(FFAs),short-chain acyl-CoAs,ATP,and NADPH as mechanisms by which exogenous TAG modulates spinosad biosynthesis.Overall,the findings validate the enhancement of TAG catabolism as a beneficial strategy for optimizing spinosad production and provide foundational insights for engineering secondary metabolite biosynthesis pathways in another Streptomyces.
基金support from the National Key Research and Development Program of China(Nos.2018YFA0900404 and 2022YFC2804105)the National Natural Science Foundation of China(No.82273849).
文摘The microbial genome remains a huge treasure trove for the discovery of diverse natural products.Saccharopolyspora erythraea NRRL23338,the industry producer of erythromycin,has a dozen of biosynthetic gene clusters whose encoding products are unidentified.Heterologous expression of one of the polyketide clusters pks7 in Streptomyces albus B4 chassis resulted in the characterization of its function responsible for synthesizing both 6-methylsalicyclic acid and 6-ethylsalicyclic acid.Meanwhile,two new 6-ethylsalicyclic acid ester derivatives were isolated as shunt metabolites.Their structures were identified by comprehensive analysis of MS and NMR experiments.Putative functions of genes within the pks7 BGC were also discussed.
文摘采用响应面法对红色糖多孢菌产红霉素发酵培养基进行优化。用Minimum Run Equireolicated Res IV设计对初始发酵培养基添加的6个影响因素的效应进行评价,选择有显著影响的4个因素,即硫酸镁、甜菜碱、硫酸铜和氯化钴。再用最陡爬坡实验为中心组合实验确定最大响应区间,最后经过响应面分析得到最优化结果,硫酸镁0.106%(w/v),甜菜碱0.0185%(w/v),硫酸铜0.106mmol/L,氯化钴0.0003%(w/v)。优化后红霉素生物效价比优化前提高了30%。
