庆大霉素及其相关产物在工业底盘细胞中的高效合成

吴亮亮; 常莹莹; 邓子新; 刘天罡

doi:10.12211/2096-8280.2022-004

您当前的位置：

首页 >

文章列表页 >

庆大霉素及其相关产物在工业底盘细胞中的高效合成

研究论文 | 更新时间：2026-01-19

- 庆大霉素及其相关产物在工业底盘细胞中的高效合成
- Efficient synthesis of gentamicin and its related products in industrial chassis cells
- 合成生物学 2022年3卷第6期页码：1277-1291
- 作者机构：
  
  1.武汉大学药学院，组合生物合成与新药发现教育部重点实验室，湖北武汉 430071
  2.武汉生物技术研究院，合成微生物技术湖北省工程实验室，湖北武汉 430075
- 作者简介：
  
  [ "吴亮亮（1996—），男，硕士研究生。研究方向为定向合成代谢指导工业菌株中庆大霉素的产量提升。E-mail：wuliangliang@whu.edu.cn" ]
  [ "常莹莹（1994—），女，工程师。研究方向为定向合成代谢指导工业菌株中庆大霉素的产量提升。E-mail：yingyingchang@whu.edu.cn" ]
  [ "刘天罡（1979—），男，教授，博士生导师。研究方向为天然产物高效合成与创新发现。E-mail：liutg@whu.edu.cn" ]
- 基金信息：
  
  国家重点研发计划“合成生物学”重点专项(2018YFA0900400)
- DOI：10.12211/2096-8280.2022-004
  中图分类号：
- 收稿：2022-01-05，
  
  修回：2022-02-23，
  
  纸质出版：2022-12-31
- 稿件说明：
移动端阅览
吴亮亮，常莹莹，邓子新，刘天罡. 庆大霉素及其相关产物在工业底盘细胞中的高效合成［J］. 合成生物学， 2022， 3（6）： 1277-1291

WU Liangliang， CHANG Yingying， DENG Zixin， LIU Tiangang. Efficient synthesis of gentamicin and its related products in industrial chassis cells［J］. Synthetic Biology Journal， 2022， 3（6）： 1277-1291
吴亮亮，常莹莹，邓子新，刘天罡. 庆大霉素及其相关产物在工业底盘细胞中的高效合成［J］. 合成生物学， 2022， 3（6）： 1277-1291 DOI： 10.12211/2096-8280.2022-004.

WU Liangliang， CHANG Yingying， DENG Zixin， LIU Tiangang. Efficient synthesis of gentamicin and its related products in industrial chassis cells［J］. Synthetic Biology Journal， 2022， 3（6）： 1277-1291 DOI： 10.12211/2096-8280.2022-004.

摘要

庆大霉素是一种氨基糖苷类抗生素，在临床上广泛应用于治疗由革兰氏阴性菌引起的严重感染。它可由棘孢小单孢菌

Micromonospora echinospora

产生，生物合成途径清晰。为了提高庆大霉素的产量，本文以工业菌株

M. echinospora

J1-020为基础，确定庆大霉素合成基因簇信息，建立了稳定的遗传操作方法。在此基础上，使用强（

kasOp*

）、中（

rpsLp

）、弱（

ermE*

）三种强度的启动子评估磷酸转移酶GenP的最适过表达水平，构建对应

attB/attP

位点整合突变株YC002、YC003、YC001。摇瓶发酵结果显示，YC001、YC002、YC003菌株的庆大霉素C组分的产量较原始菌株［（1008±57） mg/L］分别提高了16.9%［（1178±39） mg/L］、30.8 %［（1319±29） mg/L］和18.8 %［（1198±46） mg/L］；同时，结合杂质含量，在以上三株菌株中确定了中强度启动子控制

genP

过表达的效果最佳，以此构建对应的稳定整合在基

因组上的

genP

过表达菌株YC004。使得庆大霉素C组分摇瓶发酵产量提高了34.5 %［（1427±37） mg/L］。此外，以工业菌株

M. echinospora

J1-020为底盘，构建

genQ

敲除菌株，获得了只产生G418单一组分的菌株YC005，其摇瓶发酵产量为460 mg/L。以YC004为出发菌株，依次敲除

genB4、genK

，获得了只产生西索米星单一组分的菌株YC007，其摇瓶发酵产量达1046 mg/L。综上，以该工业菌株

M. echinospora

J1-020为底盘，借助合理的代谢工程策略有望快速获得多种氨基糖苷类抗生素的高产菌株。

Abstract

Gentamicin is a kind of aminoglycoside antibiotic

widely used to treat severe Gram-negative bacterial infections. As an important secondary metabolite produced by

Micromonospora echinospora

its biosynthetic pathway has been studied for years and scientists have a clear understanding for the biosynthetic gene cluster. In order to increase the titer of gentamicin

this study used the industrial strain

M. echinospora

J1-020 to determine the gentamicin synthetic gene cluster and established a stable genetic manipulation method. On this basis

three promoters with strong (

kasOp*

)

medium (

rpsLp-cf

)

and weak (

ermE*

) strengths were used to evaluate the optimal overexpression level of phosphotransferase GenP

and the corresponding

attB/attP

site integration mutant strains YC002

YC003

YC001 was then constructed. After shaking flask fermentation

the results showed that the titer of gentamicin C component of YC001

YC002 and YC003 strains were increased by 16.9 % [(1178±39)mg/L

]

30.8 % [(1319±29)mg/L

]

and 18.8%[(1198±46)mg/L

]

respectively

compared with the original strain [(1008±57)mg/L

]

. At the same time

combined with the impurity content

it was determined that the medium-strength promoter has the best effect on controlling the overexpression of gene

genP

in the above three strains

so that the corresponding overexpression strain YC004 of the

genP

stably integrated in the genome was constructed through homologous recombination. After shaking flask

fermentation

the results showed that the titer of gentamicin C component increased by 34.5 %[(1427±37)mg/L

]

. Then

using

M. echinospora

J1-020 as the chassis

the

genQ

knockout strain YC005 was constructed to produce the G418 as the single component. The results showed that the titer of G418 was 460 mg/L. Finally

the gene

genP

overexpression strain YC004 as the starting strain

in which

genB4

and

genK

were knocked out

was used to construct a double knockout mutant YC007 in order to produce sisomicin as a single component. After shaking flask fermentation

the titer of sisomicin was 1046 mg/L. It is expected that overproduction strains of various aminoglycoside antibiotics can be readily constructed by rational metabolic engineering strategies in the industrial chassis.

关键词

Keywords

references

LLEWELLYN N M , SPENCER J B . Biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics [J ] . Natural Product Reports , 2006 , 23 ( 6 ): 864 - 874 .

Waksman SA . Antibiotics and chemotherapy [J ] . California medicine , 1953 , 78 ( 5 ): 417 - 423 .

SWART E A , HUTCHISON D , WAKSMAN S A . Neomycin, recovery and purification [J ] . Archives of Biochemistry , 1949 , 24 ( 1 ): 92 - 103 .

UMEZAWA H , UEDA M , MAEDA K , et al . Production and isolation of a new antibiotic: kanamycin [J ] . The Journal of Antibiotics , 1957 , 10 ( 5 ): 181 - 188 .

WEINSTEIN M J , LUEDEMANN G M , ODEN E M , et al . Gentamicin, a new antibiotic complex from micromonospora [J ] . Journal of Medicinal Chemistry , 1963 , 6 : 463 - 464 .

FOURMY D , RECHT M I , BLANCHARD S C , et al . Structure of the A site of Escherichia coli 16S ribosomal RNA complexed with an aminoglycoside antibiotic [J ] . Science , 1996 , 274 ( 5291 ): 1367 - 1371 .

TESTA R T , TILLEY B C . Biotransformation, a new approach to aminoglycoside biosynthesis: II. Gentamicin [J ] . The Journal of Antibiotics , 1976 , 29 ( 2 ): 140 - 146 .

KHAREL M K , BASNET D B , LEE H C , et al . Molecular cloning and characterization of a 2-deoxystreptamine biosynthetic gene cluster in gentamicin-producing Micromonospora echinospora ATCC 15835 [J ] . Molecules and Cells , 2004 , 18 ( 1 ): 71 - 78 .

PARK J W , HONG J S J , PARAJULI N , et al . Genetic dissection of the biosynthetic route to gentamicin A2 by heterologous expression of its minimal gene set [J ] . Proceedings of the National Academy of Sciences of the United States of America , 2008 , 105 ( 24 ): 8399 - 8404 .

KIM J Y , SUH J W , KANG S H , et al . Gene inactivation study of gntE reveals its role in the first step of pseudotrisaccharide modifications in gentamicin biosynthesis [J ] . Biochemical and Biophysical Research Communications , 2008 , 372 ( 4 ): 730 - 734 .

HONG W R , YAN L B . Identification of gntK , a gene required for the methylation of purpurosamine C-6' in gentamicin biosynthesis [J ] . The Journal of General and Applied Microbiology , 2012 , 58 ( 5 ): 349 - 356 .

KIM H J , MCCARTY R M , OGASAWARA Y , et al . GenK-catalyzed C-6′ methylation in the biosynthesis of gentamicin: isolation and characterization of a cobalamin-dependent radical SAM enzyme [J ] . Journal of the American Chemical Society , 2013 , 135 ( 22 ): 8093 - 8096 .

HUANG C , HUANG F L , MOISON E , et al . Delineating the biosynthesis of gentamicin X2, the common precursor of the gentamicin C antibiotic complex [J ] . Chemistry & Biology , 2015 , 22 ( 2 ): 251 - 261 .

GUO J H , HUANG F L , HUANG C , et al . Specificity and promiscuity at the branch point in gentamicin biosynthesis [J ] . Chemistry & Biology , 2014 , 21 ( 5 ): 608 - 618 .

SHAO L , CHEN J S , WANG C X , et al . Characterization of a key aminoglycoside phosphotransferase in gentamicin biosynthesis [J ] . Bioorganic & Medicinal Chemistry Letters , 2013 , 23 ( 5 ): 1438 - 1441 .

ZHOU S T , CHEN X T , NI X P , et al . Pyridoxal-5′-phosphate-dependent enzyme GenB3 catalyzes C-3′, 4′-dideoxygenation in gentamicin biosynthesis [J ] . Microbial Cell Factories , 2021 , 20 ( 1 ): 65 .

CHEN X T , ZHANG H , ZHOU S T , et al . The bifunctional enzyme, GenB4, catalyzes the last step of gentamicin 3′, 4′-di-deoxygenation via reduction and transamination activities [J ] . Microbial Cell Factories , 2020 , 19 ( 1 ): 62 .

LI S , GUO J , REVA A , et al . Methyltransferases of gentamicin biosynthesis [J ] . Proceedings of the National Academy of Sciences of the United States of America , 2018 , 115 ( 6 ): 1340 - 1345 .

BAN Y H , SONG M C , HWANG J Y , et al . Complete reconstitution of the diverse pathways of gentamicin B biosynthesis [J ] . Nature Chemical Biology , 2019 , 15 ( 3 ): 295 - 303 .

CHANG Y Y , CHAI B Z , DING Y K , et al . Overproduction of gentamicin B in industrial strain Micromonospora echinospora CCTCC M 2018898 by cloning of the missing genes genR and genS [J ] . Metabolic Engineering Communications , 2019 , 9 : e00096 .

MACNEIL D J , OCCI J L , GEWAIN K M , et al . Complex organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase [J ] . Gene , 1992 , 115 ( 1/2 ): 119 - 125 .

FLETT F , MERSINIAS V , SMITH C P . High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes [J ] . FEMS Microbiology Letters , 1997 , 155 ( 2 ): 223 - 229 .

SUN Y H , HONG H , SAMBORSKYY M , et al . Organization of the biosynthetic gene cluster in Streptomyces sp. DSM 4137 for the novel neuroprotectant polyketide meridamycin [J ] . Microbiology , 2006 , 152 ( Pt 12 ): 3507 - 3515 .

WEBER T , BLIN K , DUDDELA S , et al . antiSMASH 3.0—a comprehensive resource for the genome mining of biosynthetic gene clusters [J ] . Nucleic Acids Research , 2015 , 43 ( W1 ): W237 - W243 .

BILYK O , LUZHETSKYY A . Metabolic engineering of natural product biosynthesis in actinobacteria [J ] . Current Opinion in Biotechnology , 2016 , 42 : 98 - 107 .

SASAKI K , MIZUSAWA H , ISHIDATE M , et al . Regulation of G418 selection efficiency by cell-cell interaction in transfection [J ] . Somatic Cell and Molecular Genetics , 1992 , 18 ( 6 ): 517 - 527 .

BAIAZITOV R Y , FRIESEN W , JOHNSON B , et al . Chemical modifications of G418 (geneticin): synthesis of novel readthrough aminoglycosides results in an improved in vitro safety window but no improvements in vivo [J ] . Carbohydrate Research , 2020 , 495 : 108058 .

NI X P , SUN Z P , ZHANG H Y , et al . Genetic engineering combined with random mutagenesis to enhance G418 production in Micromonospora echinospora [J ] . Journal of Industrial Microbiology & Biotechnology , 2014 , 41 ( 9 ): 1383 - 1390 .

SHAEER K M , ZMARLICKA M T , CHAHINE E B , et al . Plazomicin: a next-generation aminoglycoside [J ] . Pharmacotherapy , 2019 , 39 ( 1 ): 77 - 93 .

NI X P , SUN Z P , GU Y W , et al . Assembly of a novel biosynthetic pathway for gentamicin B production in Micromonospora echinospora [J ] . Microbial Cell Factories , 2016 , 15 : 1 .

WU Z , GAO W L , ZHOU S T , et al . Improving gentamicin B and gentamicin C1a production by engineering the glycosyltransferases that transfer primary metabolites into secondary metabolites biosynthesis [J ] . Microbiological Research , 2017 , 203 : 40 - 46 .

LI D , LI H , NI X P , et al . Construction of a gentamicin C1a-overproducing strain of Micromonospora purpurea by inactivation of the gacD gene [J ] . Microbiological research , 2013 , 168 ( 5 ): 263 - 267 .

浏览量

下载量

CSCD

文章被引用时，请邮件提醒。

提交

工具集

关联资源

暂无数据