庆大霉素及其相关产物在工业底盘细胞中的高效合成

doi:10.12211/2096-8280.2022-004

合成生物学 ›› 2022, Vol. 3 ›› Issue (6): 1277-1291.DOI: 10.12211/2096-8280.2022-004

• 研究论文 • 上一篇

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¹, 常莹莹¹, 邓子新¹^,², 刘天罡¹^,²

^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

收稿日期:2022-01-05 修回日期:2022-02-23 出版日期:2022-12-31 发布日期:2023-01-17
通讯作者: 刘天罡
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
基金资助:
国家重点研发计划“合成生物学”重点专项(2018YFA0900400)

Efficient synthesis of gentamicin and its related products in industrial chassis cells

Liangliang WU¹, Yingying CHANG¹, Zixin DENG¹^,², Tiangang LIU¹^,²

^1.Key Laboratory of Combinatorial Biosynthesis and Drug Discovery，Ministry of Education，Wuhan University School of Pharmaceutical Sciences，Wuhan 430071，Hubei，China
^2.Hubei Engineering Laboratory for Synthetic Microbiology，Wuhan Institute of Biotechnology，Wuhan 430075，Hubei，China

Received:2022-01-05 Revised:2022-02-23 Online:2022-12-31 Published:2023-01-17
Contact: Tiangang LIU

摘要/Abstract

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-cf）、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。

关键词: 庆大霉素, G418, 西索米星, 氨基糖苷类抗生素, 棘孢小单孢菌

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.

Key words: gentamicin, G418, sisomicin, aminoglycoside antibiotics, Micromonospora echinospora

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.

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

图/表 8

参考文献 32

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Strain	Description	Reference
M. echinospora J1-020	Wild-type	This study
DH10B	F^–mcrA ∆(mrr-hsdRMS-mcrBC)	Gibco-BRL
ET12567(pUZ8002)	damdcmhsdS/pUZ8002, for intergeneric conjugation	[21]
ET12567(pUB307)	dam dcm hsdS/pUB307, for intergeneric conjugation	[22]
YC001	genP gene overexpression strain	This study
YC002	genP gene overexpression strain	This study
YC003	genP gene overexpression strain	This study
YC004	genP gene overexpression strain	This study
YC005	genQ gene knockout strain	This study
YC006	genB4 gene knockout strain	This study
YC007	genB4 and genK gene knockout strain	This study
Plasmid
pWHU77	int, att, tsr, ermE*	[18]
pYH7	sti^-, rep, orf85^-, R1^-, tsr, aac(3)IV*	[23]
pXS001	E.coli-actinomycete integrated shuttle plasmid, pWHU77 carries genP under the control of the ermE* promoter	This study
pXS002	E.coli-actinomycete integrated shuttle plasmid, pWHU77 carries genP under the control of the kasOp* promoter	This study
pXS003	E.coli-actinomycete integrated shuttle plasmid, pWHU77 carries genP under the control of the rpsLp-cf promoter	This study
pXS004	E.coli-actinomycete replicating shuttle plasmid, pYH7 carries genP under the control of the rpsLp-cf promoter	This study
pXS005	E.coli-actinomycete replicating shuttle plasmid, genQ gene knockout plasmid	This study
pXS006	E.coli-actinomycete replicating shuttle plasmid, genB4 gene knockout plasmid	This study
pXS007	E.coli-actinomycete replicating shuttle plasmid, genK gene knockout plasmid	This study

Strain	Description	Reference
M. echinospora J1-020	Wild-type	This study
DH10B	F^–mcrA ∆(mrr-hsdRMS-mcrBC)	Gibco-BRL
ET12567(pUZ8002)	damdcmhsdS/pUZ8002, for intergeneric conjugation	[21]
ET12567(pUB307)	dam dcm hsdS/pUB307, for intergeneric conjugation	[22]
YC001	genP gene overexpression strain	This study
YC002	genP gene overexpression strain	This study
YC003	genP gene overexpression strain	This study
YC004	genP gene overexpression strain	This study
YC005	genQ gene knockout strain	This study
YC006	genB4 gene knockout strain	This study
YC007	genB4 and genK gene knockout strain	This study
Plasmid
pWHU77	int, att, tsr, ermE*	[18]
pYH7	sti^-, rep, orf85^-, R1^-, tsr, aac(3)IV*	[23]
pXS001	E.coli-actinomycete integrated shuttle plasmid, pWHU77 carries genP under the control of the ermE* promoter	This study
pXS002	E.coli-actinomycete integrated shuttle plasmid, pWHU77 carries genP under the control of the kasOp* promoter	This study
pXS003	E.coli-actinomycete integrated shuttle plasmid, pWHU77 carries genP under the control of the rpsLp-cf promoter	This study
pXS004	E.coli-actinomycete replicating shuttle plasmid, pYH7 carries genP under the control of the rpsLp-cf promoter	This study
pXS005	E.coli-actinomycete replicating shuttle plasmid, genQ gene knockout plasmid	This study
pXS006	E.coli-actinomycete replicating shuttle plasmid, genB4 gene knockout plasmid	This study
pXS007	E.coli-actinomycete replicating shuttle plasmid, genK gene knockout plasmid	This study

Primers	Sequence(5′ to 3′)	Restriction site
XS001-genP-F	CCACATATGGTGAAGATGGTTGCAGCACC	NdeⅠ
XS001-genP-R	TACGAATTCTCAGAGAAATTCGTCCAGCAG	EcoRⅠ
XS001-YZ-F	GCGAGTGTCCGTTCGAGTGG
XS001-YZ-R	TCAGAGAAATTCGTCCAGCAG
XS002-kasOp*-F	GCAGGTCGACTCTAGTATGCATTCTAGAGGAACGATCGTTGGCTGTGTTC	XbaⅠ
XS002- kasOp*-R	GTGCTGCAACCATCTTCATATGGCGTATCCCCTTTCAGATACC
XS002-genP-F	TCTGAAAGGGGATACGCCATATGAAGATGGTTGCAGCACC
XS002-genP-R	GGAAACAGCTATGACATGATTACGAATTCTCAGAGAAATTCGTCCAGCAG	EcoRⅠ
XS002-YZ-F	GGAACGATCGTTGGCTGTGTTC
XS003-rpsLp-cf-F	AGGTCGACTCTAGTATGCATTCTAGAGGAACGATCGTTGGCTGCCCGCCGCGGGCGCTG	XbaⅠ
XS003-rpsLp-cf-R	GGTGCTGCAACCATCTTCATATGGCGTATCCCCTTTCAGATAC
XS003-genP-F	TCTGAAAGGGGATACGCCATATGAAGATGGTTGCAGCACC
XS003-genP-R	TTTCACACAGGAAACAGCTATGACATGATTACGAATTCTCAGAGAAATTCGTCCAGCAG	EcoRⅠ
XS003-YZ-F	GGAACGATCGTTGGCTGCCCGCCGCGGGCGCTG
XS004-leftarm-F	ACCTGCAGGTCGACTCTAGACACGTCTGAAGCTAGCGCACGTATCCTGGAGAATCCGTC	NheⅠ
XS004-leftarm-R	CCTCCAGCGCCCGCGGCGGGCAGCCAACGATCGTTCCTCACGCCTTGTGGATCGCCACC
XS004-rpsLp-cf-F	CCTGGTGGCGATCCACAAGGCGTGAGGAACGATCGTTGGCTGCCCGCCGCGGGCGCTGG
XS004-genP-R	AGACCACCGCGATCGTCGAGCGCCTCTGGGAGGACTGATCAGAGAAATTCGTCCAGCAG
XS004-rightarm-F	GCTGACCTACATCCAACTGCTGGACGAATTTCTCTGATCAGTCCTCCCAGAGGCGCTCG
XS004-rightarm-R	TAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCAGCGTGGACGGTGTCGAGTGCG	EcoRⅠ
XS004-YZ-F	CCGTTCACCGTGCCCTGGCTGCGCGAGGTG
XS004-YZ-R	CTCGACCCGGCCGTCTGGATCGTGGCGAAG
XS005-leftarm-F	CGGCCATCGTGCCTCCCCACTCCTGCAGATCTGACATCGCGTGCGCGCTGAACCCGTTC	HindⅢ
XS005-leftarm-R	GTGCGGCCTTCCGCGAATTCCGGGACCGGGCCCGACTGGAG
XS005-rightarm-F	CGGGCCCGGTCCCGGAATTCGCGGAAGGCCGCACCGCCGAAG
XS005-rightarm-R	AGCGGAAAAGATCCGTCGACCTGCAGGCATGCAAGCTTCTGGGCGTGCCGTACGTGCTC	BglⅡ
XS005-YZ-F	TCCGCTCGATTCGTTCCGTTCCGAC
XS005-YZ-R	GCACGGGACCACCGGGCAGGTGCTC
XS006-leftarm-F	CTGCAGGTCGACTCTAGACACGTCTGAAGCTAGCATGCAGCGGTTCAAGGGTGCCCACG	NheⅠ
XS006-leftarm-R	AAGCTGACCTACATCCAACTGCTGGACGAATTTCTCTGATCAGCGCTGGTAGGTGCTCG
XS006-rightarm-F	AGCGCTGGCGGCTGCGCGCACCTACCAGCGCTGCTAAGAGAAATTCGTCCAGCAGTTGG
XS006-rightarm-R	GGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCTGCGAGCGGTAAGAGTATGCGGAG	EcoRⅠ
XS006-YZ-F	GGCAGCCGGACTGGGCGACCATCCGGATCG
XS006-YZ-R	CCGAGGACGATCTCGTCGTCTGCCACGGTG
XS007-leftarm-F	CTGCAGGTCGACTCTAGACACGTCTGAAGCTAGCCGGAATGCCCTCACTGGTCAGGATC	NheⅠ
XS007-leftarm-R	CCCCCTACTACGAGAGCGCCTACGAGCTGGCCCGGATGATGGCGCAGCTCGACCCGGAG
XS007-rightarm-F	GTTCCTCCGGGCTCTCCGGGTCGAGCtGCGCCATCATCCGGGCCAGCTCGAGGCGCTC
XS007-rightarm-R	ATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCGTCCGTCGTCACCACGGTTCG	EcoRⅠ
XS007-YZ-F	CGTCACGCGATGGGAGCAGGGCGGAGTATC
XS007-YZ-R	CCGAGCATCACGCTGCCGAAGGAGTTGGAG

Primers	Sequence(5′ to 3′)	Restriction site
XS001-genP-F	CCACATATGGTGAAGATGGTTGCAGCACC	NdeⅠ
XS001-genP-R	TACGAATTCTCAGAGAAATTCGTCCAGCAG	EcoRⅠ
XS001-YZ-F	GCGAGTGTCCGTTCGAGTGG
XS001-YZ-R	TCAGAGAAATTCGTCCAGCAG
XS002-kasOp*-F	GCAGGTCGACTCTAGTATGCATTCTAGAGGAACGATCGTTGGCTGTGTTC	XbaⅠ
XS002- kasOp*-R	GTGCTGCAACCATCTTCATATGGCGTATCCCCTTTCAGATACC
XS002-genP-F	TCTGAAAGGGGATACGCCATATGAAGATGGTTGCAGCACC
XS002-genP-R	GGAAACAGCTATGACATGATTACGAATTCTCAGAGAAATTCGTCCAGCAG	EcoRⅠ
XS002-YZ-F	GGAACGATCGTTGGCTGTGTTC
XS003-rpsLp-cf-F	AGGTCGACTCTAGTATGCATTCTAGAGGAACGATCGTTGGCTGCCCGCCGCGGGCGCTG	XbaⅠ
XS003-rpsLp-cf-R	GGTGCTGCAACCATCTTCATATGGCGTATCCCCTTTCAGATAC
XS003-genP-F	TCTGAAAGGGGATACGCCATATGAAGATGGTTGCAGCACC
XS003-genP-R	TTTCACACAGGAAACAGCTATGACATGATTACGAATTCTCAGAGAAATTCGTCCAGCAG	EcoRⅠ
XS003-YZ-F	GGAACGATCGTTGGCTGCCCGCCGCGGGCGCTG
XS004-leftarm-F	ACCTGCAGGTCGACTCTAGACACGTCTGAAGCTAGCGCACGTATCCTGGAGAATCCGTC	NheⅠ
XS004-leftarm-R	CCTCCAGCGCCCGCGGCGGGCAGCCAACGATCGTTCCTCACGCCTTGTGGATCGCCACC
XS004-rpsLp-cf-F	CCTGGTGGCGATCCACAAGGCGTGAGGAACGATCGTTGGCTGCCCGCCGCGGGCGCTGG
XS004-genP-R	AGACCACCGCGATCGTCGAGCGCCTCTGGGAGGACTGATCAGAGAAATTCGTCCAGCAG
XS004-rightarm-F	GCTGACCTACATCCAACTGCTGGACGAATTTCTCTGATCAGTCCTCCCAGAGGCGCTCG
XS004-rightarm-R	TAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCAGCGTGGACGGTGTCGAGTGCG	EcoRⅠ
XS004-YZ-F	CCGTTCACCGTGCCCTGGCTGCGCGAGGTG
XS004-YZ-R	CTCGACCCGGCCGTCTGGATCGTGGCGAAG
XS005-leftarm-F	CGGCCATCGTGCCTCCCCACTCCTGCAGATCTGACATCGCGTGCGCGCTGAACCCGTTC	HindⅢ
XS005-leftarm-R	GTGCGGCCTTCCGCGAATTCCGGGACCGGGCCCGACTGGAG
XS005-rightarm-F	CGGGCCCGGTCCCGGAATTCGCGGAAGGCCGCACCGCCGAAG
XS005-rightarm-R	AGCGGAAAAGATCCGTCGACCTGCAGGCATGCAAGCTTCTGGGCGTGCCGTACGTGCTC	BglⅡ
XS005-YZ-F	TCCGCTCGATTCGTTCCGTTCCGAC
XS005-YZ-R	GCACGGGACCACCGGGCAGGTGCTC
XS006-leftarm-F	CTGCAGGTCGACTCTAGACACGTCTGAAGCTAGCATGCAGCGGTTCAAGGGTGCCCACG	NheⅠ
XS006-leftarm-R	AAGCTGACCTACATCCAACTGCTGGACGAATTTCTCTGATCAGCGCTGGTAGGTGCTCG
XS006-rightarm-F	AGCGCTGGCGGCTGCGCGCACCTACCAGCGCTGCTAAGAGAAATTCGTCCAGCAGTTGG
XS006-rightarm-R	GGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCTGCGAGCGGTAAGAGTATGCGGAG	EcoRⅠ
XS006-YZ-F	GGCAGCCGGACTGGGCGACCATCCGGATCG
XS006-YZ-R	CCGAGGACGATCTCGTCGTCTGCCACGGTG
XS007-leftarm-F	CTGCAGGTCGACTCTAGACACGTCTGAAGCTAGCCGGAATGCCCTCACTGGTCAGGATC	NheⅠ
XS007-leftarm-R	CCCCCTACTACGAGAGCGCCTACGAGCTGGCCCGGATGATGGCGCAGCTCGACCCGGAG
XS007-rightarm-F	GTTCCTCCGGGCTCTCCGGGTCGAGCtGCGCCATCATCCGGGCCAGCTCGAGGCGCTC
XS007-rightarm-R	ATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCGTCCGTCGTCACCACGGTTCG	EcoRⅠ
XS007-YZ-F	CGTCACGCGATGGGAGCAGGGCGGAGTATC
XS007-YZ-R	CCGAGCATCACGCTGCCGAAGGAGTTGGAG

Type	Morphology of actinomycetes	Plasmid	The mixing ratio (Donor: Receptor)	Incubation time/h	Number of conjugants (Cultivate to 9d)
Diparental conjugation	mycelia	pWHU77	10∶1、4∶1、1∶1	14、16	100～200
Diparental conjugation	mycelia	pYH7	20∶1、10∶1		1
Triparental conjugation	mycelia	pWHU77	4∶4∶1、1∶1∶1		100～200
	mycelia	pYH7	4∶4∶1、1∶1∶1		50～100
	spore	pYH7	10∶10∶1、1∶1∶1	13	>200

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

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