新霉素主要是由弗氏链霉菌(Streptomyces fradiae)发酵生产的一种氨基糖苷类抗生素,广泛应用于医药、畜牧等领域,能够有效地控制和治疗细菌引起的疾病,提高畜禽的生长速度和生产性能。为了提高新霉素产量,该研究通过比较转录组学分析原始弗氏链霉菌SF-1和诱变高产菌SF-2的转录组数据,挖掘出转录水平差异显著的转录因子,然后过表达构建重组质粒,再通过接合转移构建出工程菌株。经摇瓶发酵验证筛选出对新霉素合成影响显著的工程菌株,进行启动子优化最终得到高产的生产新霉素工程菌株SF-2/pP-NeyR。随后对发酵培养基组分进行正交优化,最终新霉素B效价为11 843 U/mL。综合优化后新霉素B的效价较初始提高了29.6%。该实验结果为新霉素后续代谢机理研究、其和弗氏链霉菌新霉素合成基因簇的相互作用研究、解析调控新霉素合成机理奠定了基础,为进一步提高新霉素效价提供了思路。
Neomycin is an aminoglycoside antibiotic mainly produced by Streptomyces fradiae.It is widely used in medicine, animal husbandry and other fields.It can effectively control and treat diseases caused by bacteria, and improve the growth speed and production performance of livestock and poultry.To improve the production of neomycin, the transcriptome data of the original Streptomyces fradiae SF-1 and the mutagenic high-yield strain SF-2 were analyzed by comparative transcriptomics, and the transcription factors with significant differences in transcription levels were mined, and then the recombinant plasmids were constructed by overexpression, and then the engineered strains were constructed by conjugation transfer.The engineering strain with significant effect on neomycin synthesis was screened by shake flask fermentation, and the high-yield neomycin producing engineering strain SF-2/pP-NeyR was finally obtained by promoter optimization.Then the components of fermentation medium were orthogonal optimized, and the final titer of neomycin B was 11 843 U/mL.After comprehensive optimization, the titer of neomycin B increased by 29.6% compared with the initial one.The experimental results laid a foundation for the follow-up study of metabolic mechanism, the study of its interaction with Streptomyces fradiae neomycin synthesis gene cluster, the analysis and regulation of neomycin synthesis mechanism, and provided ideas for further improving the potency of neomycin.
[1] 胡永彬, 徐礼生.硫酸新霉素的研究[J].广州化工, 2022, 50(12):10-11;19.
HU Y B, XU L S.Study on neomycin sulfate[J].Guangzhou Chemical Industry, 2022, 50(12):10-11;19.
[2] SUBRAMANI P, PIROLO M, HAUGEGAARD S, et al.Neomycin resistance in clinical Escherichia coli from Danish weaner pigs is associated with recent neomycin use and presence of F4 or F18 fimbriaes[J].Preventive Veterinary Medicine, 2023, 212:105852.
[3] XIAO C, LIU J F, YANG A K, et al.Colorimetric determination of neomycin using melamine modified gold nanoparticles[J].Microchimica Acta, 2015, 182(7-8):1501-1507.
[4] PAN D, YU Z T.Intestinal microbiome of poultry and its interaction with host and diet[J].Gut Microbes, 2014, 5(1):108-119.
[5] 刘海燕, 殷瑜.氨基糖苷类抗生素作用机制研究进展[J].中国抗生素杂志, 2019, 44(11):1283-1287.
LIU H Y, YIN Y.Advances in research on the mechanism of aminoglycoside antibiotics[J].Chinese Journal of Antibiotics, 2019, 44(11):1283-1287.
[6] WAKSMAN S A, LECHEVALIER H A.Neomycin, a new antibiotic active against streptomycin-resistant bacteria, including tuberculosis organisms[J].Science, 1949, 109(2830):305-307.
[7] VADASSERY J, REICHELT M, JIMENEZ-ALEMAN G H, et al.Neomycin inhibition of (+)-7-iso-jasmonoyl-L-isoleucine accumulation and signaling[J].Journal of Chemical Ecology, 2014, 40(7):676-686.
[8] YUAN L L, WEI H P, FENG H T, et al.Rapid analysis of native neomycin components on a portable capillary electrophoresis system with potential gradient detection[J].Analytical and Bioanalytical Chemistry, 2006, 385(8):1575-1579.
[9] KUDO F, HOSHI S, KAWASHIMA T, et al.Characterization of a radical S-adenosyl-L-methionine epimerase, NeoN, in the last step of neomycin B biosynthesis[J].Journal of the American Chemical Society, 2014, 136(39):13909-13915.
[10] KUDO F, EGUCHI T.Biosynthetic genes for aminoglycoside antibiotics[J].The Journal of Antibiotics, 2009, 62(9):471-481.
[11] ZHENG J Z, LI Y, GUAN H Y, et al.Enhancement of neomycin production by engineering the entire biosynthetic gene cluster and feeding key precursors in Streptomyces fradiae CGMCC 4.576[J].Applied Microbiology and Biotechnology, 2019, 103(5):2263-2275.
[12] ZHENG J Z, LI Y, GUAN H Y, et al.Component optimization of neomycin biosynthesis via the reconstitution of a combinatorial mini-gene-cluster in Streptomyces fradiae[J].ACS Synthetic Biology, 2020, 9(9):2493-2501.
[13] LI X F, YU F, LIU K, et al.Uncovering the effects of ammonium sulfate on neomycin B biosynthesis in Streptomyces fradiae SF-2[J].Fermentation, 2022,8(12):678.
[14] LI X F, YU F, WANG F, et al.Point mutation of V252 in neomycin C epimerase enlarges substrate-binding pocket and improves neomycin B accumulation in Streptomyces fradiae[J].Bioresources and Bioprocessing, 2022, 9(1):123.
[15] PAN X W, YOU J J, TANG M, et al.Improving prodigiosin production by transcription factor engineering and promoter engineering in Serratia marcescens[J].Frontiers in Microbiology, 2022, 13:977377.
[16] MENG X X, WANG W Z, XIE Z J, et al.Neomycin biosynthesis is regulated positively by AfsA-g and NeoR in Streptomyces fradiae CGMCC 4.7387[J].Science China Life Sciences, 2017, 60(9):980-991.
[17] ZHANG Y Y, ZOU Z Z, NIU G Q, et al.JadR and jadR2 act synergistically to repress jadomycin biosynthesis[J].Science China Life Sciences, 2013, 56(7):584-590.
[18] SCHAFFERT L, SCHNEIKER-BEKEL S, DYMEK S, et al.Essentiality of the maltase AmlE in maltose utilization and its transcriptional regulation by the repressor AmlR in the acarbose-producing bacterium Actinoplanes sp.SE50/110[J].Frontiers in Microbiology, 2019, 10:2448.
[19] ZHU Y P, XU W H, ZHANG J, et al.A hierarchical network of four regulatory genes controlling production of the polyene antibiotic candicidin in Streptomyces sp.strain FR-008[J].Applied and Environmental Microbiology, 2020, 86(9):e00055-20.
[20] LIAO C H, YAO L L, XU Y, et al.Nitrogen regulator GlnR controls uptake and utilization of non-phosphotransferase-system carbon sources in actinomycetes[J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(51):15630-15635.
[21] LU T, WU X H, CAO Q, et al.Sulfane sulfur posttranslationally modifies the global regulator AdpA to influence actinorhodin production and morphological differentiation of Streptomyces coelicolor[J].mBio, 2022, 13(3):e0386221.
[22] XU G B, LI J G, LIU Q, et al.Transcriptional analysis of Myceliophthora thermophila on soluble starch and role of regulator AmyR on polysaccharide degradation[J].Bioresource Technology, 2018, 265:558-562.
[23] LI X F, BAO T, OSIRE T, et al.MarR-type transcription factor RosR regulates glutamate metabolism network and promotes accumulation of L-glutamate in Corynebacterium glutamicum G01[J].Bioresource Technology, 2021, 342:125945.
[24] YU F, ZHANG M, SUN J F, et al.Improved neomycin sulfate potency in Streptomyces fradiae using atmospheric and room temperature plasma (ARTP) mutagenesis and fermentation medium optimization[J].Microorganisms, 2022, 10(1):94.
[25] 余飞, 孙俊峰, 刘鹏飞, 等.弗氏链霉菌产硫酸新霉素高通量选育模型的建立及优化[J].食品与发酵工业, 2019, 45(8):162-167;177.
YU F, SUN J F, LIU P F, et al.A high-throughput screening method for selecting Streptomyces fradiae mutants with improved neomycin yield and its optimization[J].Food and Fermentation Industries, 2019, 45(8):162-167;177.
[26] PLUTA R, ARAGÓN E, PRESCOTT N A, et al.Molecular basis for DNA recognition by the maternal pioneer transcription factor FoxH1[J].Nature Communications, 2022, 13(1):7279-7293.
[27] KANG Z Q, ZHANG M M, GAO K Y, et al.An L-2-hydroxyglutarate biosensor based on specific transcriptional regulator LhgR[J].Nature Communications, 2021, 12(1):3619-3631.
