适应性进化与甜菜碱的添加促进安丝菌素的生产
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摘要
【背景】安丝菌素是一类由珍贵束丝放线菌橙色亚种(Actinosynnema pretiosum ssp. auranticu)生产的美登素类衍生抗生素,属于大环内酰胺类物质,根据不同的C-3位基团可以分为一系列衍生物。目前已上市的高效抗癌药物曲妥珠单抗(T-DM1)以AP-3 (Ansamitocin P-3)为生产底物,并表现出很好的乳腺癌治疗效果。然而当前AP-3的产量较低,其过高的成本限制了进一步发展。【目的】应用适应性进化及添加适量甜菜碱策略提高安丝菌素AP-3的产量。【方法】以珍贵束丝放线菌橙色亚种为出发菌株,链霉素和巴龙霉素为胁迫压力进行适应性进化,筛选出安丝菌素积累较高的菌株,随后向进化菌株的发酵培养基中添加0.1%的甜菜碱,AP-3的产量进一步提高,同时分析了进化菌株AP-3相关基因的转录水平,初步探索进化菌株安丝菌素积累提高的原因。【结果】得到2株进化菌株Str16-4-4和Par16-2-1,发酵7d后AP-3产量分别提高了33.4%和31.7%,添加甜菜碱后其AP-3产量相比出发菌株提高了54.6%和47.4%。【结论】通过适应性进化的策略获得了AP-3生产能力提高的珍贵束丝放线菌橙色亚种进化菌株,对促进AP-3的生产提供了新思路,而且为适应性进化策略提高目标产物的产量提供了新的例证。
[Background] Ansamitocins, macrocyclic lactam compounds related to maytansine, are synthesized by Actinosynnema pretiosum ssp. auranticun. Based on the differences of ester side chain moieties in C3 position, ansamitocins contain a series of derivatives. Currently, a novel anticancer drug named Kadcyla(T-DM1), formed from ansamitocin P-3(AP-3), enters the market. T-DM1 had been approved by FDA as an antibody-drug for treating breast cancer. However, the low yield of ansamitocins limits its commercial application. [Objective] To improve AP-3 yield from A. pretiosum ssp. auranticum ATCC 31565, we applied adaptive evolution and addition of betaine. [Methods] The wild-type A. pretiosum ssp. auranticum was used as starting strain to the adverse stress streptomycin and paromomycin. The evolved mutants were screened with better production of AP-3. After 0.1% betaine was added into the fermentation broth of the evolved mutants, the production of AP-3 was further enhanced. Then, gene transcription levels related to AP-3 biosynthesis were analyzed in evolved mutants and starting strain, to investigate the reasons for enhancement of ansamitocin. [Results] Two evolved strains Str16-4-4 and Par16-2-1 have been obtained.After 7 days fermentation, their AP-3 contents were increased by 33.4% and 31.7%, respectively. Addition of0.1% betaine ultimately resulted in an increase of 54.6% and 47.4% AP-3 production compared to the wild strain. [Conclusion] Through the adaptive evolution strategy, AP-3 production was improved in A. pretiosum ssp. auranticum. This study provides a new strategy to improve the production of AP-3, and also gives a new example for adaptive evolutionary strategy to promote the production of target products.
[Background] Ansamitocins, macrocyclic lactam compounds related to maytansine, are synthesized by Actinosynnema pretiosum ssp. auranticun. Based on the differences of ester side chain moieties in C3 position, ansamitocins contain a series of derivatives. Currently, a novel anticancer drug named Kadcyla(T-DM1), formed from ansamitocin P-3(AP-3), enters the market. T-DM1 had been approved by FDA as an antibody-drug for treating breast cancer. However, the low yield of ansamitocins limits its commercial application. [Objective] To improve AP-3 yield from A. pretiosum ssp. auranticum ATCC 31565, we applied adaptive evolution and addition of betaine. [Methods] The wild-type A. pretiosum ssp. auranticum was used as starting strain to the adverse stress streptomycin and paromomycin. The evolved mutants were screened with better production of AP-3. After 0.1% betaine was added into the fermentation broth of the evolved mutants, the production of AP-3 was further enhanced. Then, gene transcription levels related to AP-3 biosynthesis were analyzed in evolved mutants and starting strain, to investigate the reasons for enhancement of ansamitocin. [Results] Two evolved strains Str16-4-4 and Par16-2-1 have been obtained.After 7 days fermentation, their AP-3 contents were increased by 33.4% and 31.7%, respectively. Addition of0.1% betaine ultimately resulted in an increase of 54.6% and 47.4% AP-3 production compared to the wild strain. [Conclusion] Through the adaptive evolution strategy, AP-3 production was improved in A. pretiosum ssp. auranticum. This study provides a new strategy to improve the production of AP-3, and also gives a new example for adaptive evolutionary strategy to promote the production of target products.
引文
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[13]Guo JT,Frost JW.Biosynthesis of 1-deoxy-1-imino-D-erythrose4-phosphate:a defining metabolite in the aminoshikimate pathway[J].Journal of the American Chemical Society,2002,124(4):528-529
[14]Guo JT,Frost JW.Synthesis of aminoshikimic acid[J].Organic Letters,2004,6(10):1585-1588
[15]Spiteller P,Bai LQ,Shang GD,et al.The post-polyketide synthase modification steps in the biosynthesis of the antitumor agent ansamitocin by Actinosynnema pretiosum[J].Journal of the American Chemical Society,2003,125(47):14236-14237
[16]Pan WQ,Kang QJ,Wang L,et al.Asm8,a specific LAL-type activator of 3-amino-5-hydroxybenzoate biosynthesis in ansamitocin production[J].Science China Life Sciences,2013,56(7):601-608
[17]Ng D,Chin HK,Wong VVT.Constitutive overexpression of asm2 and asm39 increases AP-3 production in the actinomycete Actinosynnema pretiosum[J].Journal of Industrial Microbiology and Biotechnology,2009,36(11):1345-1351
[18]Zhang L,Guo LZ,Lu WD.Construction of salt sensitive mutants from Halomonas sp.TTW4 and cloning of the gene involved in cellular osmoregulation[J].Journal of Qingdao Agricultural University(Natural Science),2013,30(3):204-210(in Chinese)张琳,郭立忠,卢伟东.Halomonas sp.TTW4盐敏感突变株的筛选与耐盐基因克隆[J].青岛农业大学学报:自然科学版,2013,30(3):204-210
[19]Qiu LJ,Shen W,Shen ZL,et al.Experimental observation on germicidal efficacy and toxicity of betaine[J].Chinese Journal of Disinfection,1999,16(4):230-231(in Chinese)邱立军,沈芄,沈宗林,等.甜菜碱杀灭微生物效果及毒性的试验观察[J].中国消毒学杂志,1999,16(4):230-231
[2]Martínez MT,Pérez-Fidalgo JA,Martín-Martorell P,et al.Treatment of HER2 positive advanced breast cancer with T-DM1:A review of the literature[J].Critical Reviews in Oncology/Hematology,2016,97:96-106
[3]Brown DR,Barton G,Pan ZS,et al.Nitrogen stress response and stringent response are coupled in Escherichia coli[J].Nature Communications,2014,5:4115
[4]Artsimovitch I,Patlan V,Sekine SI,et al.Structural basis for transcription regulation by alarmone ppGpp[J].Cell,2004,117(3):299-310
[5]Hu HF,Ochi K.Novel approach for improving the productivity of antibiotic-producing strains by inducing combined resistant mutations[J].Applied and Environmental Microbiology,2001,67(4):1885-1892
[6]Wang GJ,Hosaka T,Ochi K.Dramatic activation of antibiotic production in Streptomyces coelicolor by cumulative drug resistance mutations[J].Applied and Environmental Microbiology,2008,74(9):2834-2840
[7]Hagemann M.Molecular biology of cyanobacterial salt acclimation[J].Fems Microbiology Reviews,2011,35(1):87-123
[8]L?est CA,Titgemeyer EC,St-Jean G,et al.Methionine as a methyl group donor in growing cattle[J].Journal of Animal Science,2002,80(8):2197-2206
[9]Chen CF,Huang H,Zhang CQ,et al.Improvement of fermentation technology of rapamycin by Actinoplanes sp.[J].Chinese Journal of Pharmaceuticals,2014,45(10):925-928(in Chinese)陈昌发,黄鹤,张长清,等.游动放线菌产雷帕霉素的发酵工艺改进[J].中国医药工业杂志,2014,45(10):925-928
[10]Li H,Zhang L,Wang X,et al.Lincomycin fermentation process optimization to reduce part B[J].Shandong Chemical Industry,2016,45(8):63-64(in Chinese)李华,张立,王鑫,等.林可霉素发酵工艺优化降低B组分[J].山东化工,2016,45(8):63-64
[11]Fan YX,Gao Y,Zhou J,et al.Process optimization with alternative carbon sources and modulation of secondary metabolism for enhanced ansamitocin P-3 production in Actinosynnema pretiosum[J].Journal of Biotechnology,2014,192:1-10
[12]Jansen G,Barbosa C,Schulenburg H.Experimental evolution as an efficient tool to dissect adaptive paths to antibiotic resistance[J].Drug Resistance Updates,2013,16(6):96-107
[13]Guo JT,Frost JW.Biosynthesis of 1-deoxy-1-imino-D-erythrose4-phosphate:a defining metabolite in the aminoshikimate pathway[J].Journal of the American Chemical Society,2002,124(4):528-529
[14]Guo JT,Frost JW.Synthesis of aminoshikimic acid[J].Organic Letters,2004,6(10):1585-1588
[15]Spiteller P,Bai LQ,Shang GD,et al.The post-polyketide synthase modification steps in the biosynthesis of the antitumor agent ansamitocin by Actinosynnema pretiosum[J].Journal of the American Chemical Society,2003,125(47):14236-14237
[16]Pan WQ,Kang QJ,Wang L,et al.Asm8,a specific LAL-type activator of 3-amino-5-hydroxybenzoate biosynthesis in ansamitocin production[J].Science China Life Sciences,2013,56(7):601-608
[17]Ng D,Chin HK,Wong VVT.Constitutive overexpression of asm2 and asm39 increases AP-3 production in the actinomycete Actinosynnema pretiosum[J].Journal of Industrial Microbiology and Biotechnology,2009,36(11):1345-1351
[18]Zhang L,Guo LZ,Lu WD.Construction of salt sensitive mutants from Halomonas sp.TTW4 and cloning of the gene involved in cellular osmoregulation[J].Journal of Qingdao Agricultural University(Natural Science),2013,30(3):204-210(in Chinese)张琳,郭立忠,卢伟东.Halomonas sp.TTW4盐敏感突变株的筛选与耐盐基因克隆[J].青岛农业大学学报:自然科学版,2013,30(3):204-210
[19]Qiu LJ,Shen W,Shen ZL,et al.Experimental observation on germicidal efficacy and toxicity of betaine[J].Chinese Journal of Disinfection,1999,16(4):230-231(in Chinese)邱立军,沈芄,沈宗林,等.甜菜碱杀灭微生物效果及毒性的试验观察[J].中国消毒学杂志,1999,16(4):230-231