14 results on '"Yue Zhong"'
Search Results
2. Engineering the acyltransferase domain of epothilone polyketide synthase to alter the substrate specificity
- Author
-
Qianwen Yue, Yue-zhong Li, Huimin Wang, Yan Shi, Junheng Liang, Guosong Chen, Xiaoming Ding, Guoping Zhao, Long Li, Xiaoying Bian, and Youming Zhang
- Subjects
Stereochemistry ,Substrate specificity ,Bioengineering ,Epothilone ,medicine.disease_cause ,Protein Engineering ,01 natural sciences ,Applied Microbiology and Biotechnology ,Microbiology ,03 medical and health sciences ,Polyketide ,Bacterial Proteins ,Polyketide synthase ,Catalytic Domain ,medicine ,030304 developmental biology ,0303 health sciences ,Mutation ,biology ,Bacteria ,010405 organic chemistry ,Chemistry ,Research ,QR1-502 ,0104 chemical sciences ,Protein Structure, Tertiary ,Acyl carrier protein ,Acyltransferase ,Dehydratase ,biology.protein ,AT ,Domain swap ,Linker ,Polyketide Synthases ,Biotechnology ,medicine.drug - Abstract
Background Polyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal. Results The substitution of module 4 drastically diminished the activity of epothilone PKS. However, with careful design of the KS-AT linker and the post-AT linker, replacing EPOAT4 with EPOAT2, EPOAT6, EPOAT7 or EPOAT8 (specifically incorporating methylmalonyl-CoA (MMCoA)) significantly increased the ratio of epothilone D (4) to epothilone C (3) (the highest ratio of 4:3 = 4.6:1), whereas the ratio of 4:3 in the parental strain Schlegelella brevitalea 104-1 was 1.4:1. We also obtained three strains by swapping EPOAT4 with EPOAT3, EPOAT5, or EPOAT9, which specifically incorporate malonyl-CoA (MCoA). These strains produced only epothilone C, and the yield was increased by a factor of 1.8 compared to that of parental strain 104-1. Furthermore, mutations of five residues in the AT domain identified Ser310 as the critical factor for MMCoA recognition in EPOAT4. Then, the mutation of His308 to valine or tyrosine combined with the mutation of Phe310 to serine further altered the product ratios. At the same time, we successfully deleted the inactive module 9 DH and ER domains and fused the ΨKR domain with the KR domain through an ~ 25-residue linker to generate a productive and simplified epothilone PKS. Conclusions These results suggested that the substitution and deletion of catalytic domains effectively produces desirable compounds and that selection of the linkers between domains is crucial for maintaining intact PKS catalytic activity.
- Published
- 2021
3. CRISPR/dCas9-mediated transcriptional improvement of the biosynthetic gene cluster for the epothilone production in Myxococcus xanthus
- Author
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Zhi-feng Li, Duo-hong Sheng, Xiao-zhuang Hu, Yue-zhong Li, Jiang-he Chen, Wan-wan Feng, Ye Wang, Xin-Jing Yue, Ran Peng, and Youming Zhang
- Subjects
0301 basic medicine ,Transcriptional Activation ,Myxococcus xanthus ,Transcription, Genetic ,030106 microbiology ,lcsh:QR1-502 ,Secondary Metabolism ,Bioengineering ,Biosynthetic gene cluster ,Applied Microbiology and Biotechnology ,sgRNAs ,lcsh:Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,Transcription (biology) ,Sigma factor ,RNA polymerase ,CRISPR-Associated Protein 9 ,Gene cluster ,CRISPR ,Promoter Regions, Genetic ,Gene ,biology ,Chemistry ,Cas9 ,Transcriptional improvement ,Research ,Promoter ,CRISPR/dCas9 activation ,biology.organism_classification ,Recombinant Proteins ,Cell biology ,030104 developmental biology ,Epothilones ,Multigene Family ,Activator proteins ,CRISPR-Cas Systems ,Biotechnology ,RNA, Guide, Kinetoplastida - Abstract
Background The CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites. Results In this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation. We firstly optimized the cas9 codon to fit the M. xanthus cells, mutated the gene to inactivate the nuclease activity, and constructed the dCas9-activator system in an epothilone producer. We compared the improvement efficiency of different sgRNAs on the production of epothilones and the expression of the biosynthetic genes. We also compared the improvement effects of different activator proteins, the ω and α subunits of RNA polymerase, and the sigma factors σ54 and CarQ. By using a copper-inducible promoter, we determined that higher expressions of dCas9-activator improved the activation effects. Conclusions Our results showed that the CRISPR/dCas-mediated transcription activation is a simple and broadly applicable technique to improve the transcriptional efficiency for the production of secondary metabolites in microorganisms. This is the first time to construct the CRISPR/dCas9 activation system in myxobacteria and the first time to assay the CRISPR/dCas9 activations for the biosynthesis of microbial secondary metabolites. Electronic supplementary material The online version of this article (10.1186/s12934-018-0867-1) contains supplementary material, which is available to authorized users.
- Published
- 2018
4. Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus
- Author
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Wen-chao Hu, Yingjie Yang, Ye Wang, Yue-zhong Li, Duo-hong Sheng, Peng Zhang, Ya Gong, and Zhi-feng Li
- Subjects
0301 basic medicine ,Myxococcus xanthus ,030106 microbiology ,Mutant ,lcsh:QR1-502 ,Bioengineering ,Computational biology ,Spacer sequence ,Applied Microbiology and Biotechnology ,Genome ,lcsh:Microbiology ,03 medical and health sciences ,RNA, Transfer ,Genome editing ,CRISPR ,Deletion of large genome fragments ,Free energy ,CRISPR/Cas9 ,Gene ,Sequence Deletion ,Genetics ,Base Sequence ,biology ,Cas9 ,Research ,biology.organism_classification ,030104 developmental biology ,Genes, Bacterial ,Epothilones ,Multigene Family ,On-target cleavage efficiency ,CRISPR-Cas Systems ,GC-content ,Plasmids ,RNA, Guide, Kinetoplastida ,Biosynthetic gene clusters for secondary metabolites ,Biotechnology - Abstract
Background The CRISPR/Cas9 system is a powerful tool for genome editing, in which the sgRNA binds and guides the Cas9 protein for the sequence-specific cleavage. The protocol is employable in different organisms, but is often limited by cell damage due to the endonuclease activity of the introduced Cas9 and the potential off-target DNA cleavage from incorrect guide by the 20 nt spacer. Results In this study, after resolving some critical limits, we have established an efficient CRISPR/Cas9 system for the deletion of large genome fragments related to the biosynthesis of secondary metabolites in Myxococcus xanthus cells. We revealed that the high expression of a codon-optimized cas9 gene in M. xanthus was cytotoxic, and developed a temporally high expression strategy to reduce the cell damage from high expressions of Cas9. We optimized the deletion protocol by using the tRNA–sgRNA–tRNA chimeric structure to ensure correct sgRNA sequence. We found that, in addition to the position-dependent nucleotide preference, the free energy of a 20 nt spacer was a key factor for the deletion efficiency. Conclusions By using the developed protocol, we achieved the CRISPR/Cas9-induced deletion of large biosynthetic gene clusters for secondary metabolites in M. xanthus DK1622 and its epothilone-producing mutant. The findings and the proposals described in this paper were suggested to be workable in other organisms, for example, other Gram negative bacteria with high GC content. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0758-x) contains supplementary material, which is available to authorized users.
- Published
- 2017
5. A bacterial negative transcription regulator binding on an inverted repeat in the promoter for epothilone biosynthesis
- Author
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Xin-Jing Yue, Zheng Zhang, Yue-zhong Li, Peng Zhang, Xiao-wen Cui, Ran Peng, and Zhi-feng Li
- Subjects
0301 basic medicine ,inorganic chemicals ,Transcription, Genetic ,Inverted repeat ,030106 microbiology ,lcsh:QR1-502 ,Secondary Metabolism ,Bioengineering ,Epothilone ,Applied Microbiology and Biotechnology ,Genome ,lcsh:Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,Suppression, Genetic ,Myxobacteria ,Biosynthesis ,Gene cluster ,Proteobacteria ,medicine ,Myxococcales ,Secondary metabolism ,Promoter Regions, Genetic ,Gene ,Genetics ,biology ,organic chemicals ,Research ,Inverted Repeat Sequences ,technology, industry, and agriculture ,Gene Expression Regulation, Bacterial ,biology.organism_classification ,Transcription inhibition ,Inverted repeat sequence in promoter ,030104 developmental biology ,Biochemistry ,chemistry ,Epothilones ,Multigene Family ,lipids (amino acids, peptides, and proteins) ,Negative transcription regulator ,Epothilone synthesis ,Biotechnology ,medicine.drug - Abstract
Background Microbial secondary metabolism is regulated by a complex and mostly-unknown network of global and pathway-specific regulators. A dozen biosynthetic gene clusters for secondary metabolites have been reported in myxobacteria, but a few regulation factors have been identified. Results We identified a transcription regulator Esi for the biosynthesis of epothilones. Inactivation of esi promoted the epothilone production, while overexpression of the gene suppressed the production. The regulation was determined to be resulted from the transcriptional changes of epothilone genes. Esi was able to bind, probably via the N-terminus of the protein, to an inverted repeat sequence in the promoter of the epothilone biosynthetic gene cluster. The Esi-homologous sequences retrieved from the RefSeq database are all of the Proteobacteria. However, the Esi regulation is not universal in myxobacteria, because the esi gene exists only in a few myxobacterial genomes. Conclusions Esi binds to the epothilone promoter and down-regulates the transcriptional level of the whole gene cluster to affect the biosynthesis of epothilone. This is the first transcription regulator identified for epothilone biosynthesis. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0706-9) contains supplementary material, which is available to authorized users.
- Published
- 2017
6. CRISPR/dCas9-mediated transcriptional improvement of the biosynthetic gene cluster for the epothilone production in Myxococcus xanthus
- Author
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Peng, Ran, primary, Wang, Ye, additional, Feng, Wan-wan, additional, Yue, Xin-jing, additional, Chen, Jiang-he, additional, Hu, Xiao-zhuang, additional, Li, Zhi-feng, additional, Sheng, Duo-hong, additional, Zhang, You-ming, additional, and Li, Yue-zhong, additional
- Published
- 2018
- Full Text
- View/download PDF
7. Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus
- Author
-
Yang, Ying-jie, primary, Wang, Ye, additional, Li, Zhi-feng, additional, Gong, Ya, additional, Zhang, Peng, additional, Hu, Wen-chao, additional, Sheng, Duo-hong, additional, and Li, Yue-zhong, additional
- Published
- 2017
- Full Text
- View/download PDF
8. A bacterial negative transcription regulator binding on an inverted repeat in the promoter for epothilone biosynthesis
- Author
-
Yue, Xin-jing, primary, Cui, Xiao-wen, additional, Zhang, Zheng, additional, Peng, Ran, additional, Zhang, Peng, additional, Li, Zhi-feng, additional, and Li, Yue-zhong, additional
- Published
- 2017
- Full Text
- View/download PDF
9. Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus
- Author
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Hailong Wang, Lian-Shuai Zheng, Xin-Jing Yue, Xiu-Nan Yi, Li-Ping Zhu, Yue-zhong Li, Zhi-feng Li, Kui Han, and Youming Zhang
- Subjects
Myxococcus xanthus ,Gene Expression ,Mutagenesis (molecular biology technique) ,Bioengineering ,Applied Microbiology and Biotechnology ,Transcriptome ,Site-directed integration ,Bacterial Proteins ,Gene expression ,Transposition ,Chloramphenicol acetyl transferase ,Epothilone biosynthetic gene cluster ,Gene ,Genetics ,biology ,Host (biology) ,Research ,Chromosome ,biology.organism_classification ,Mutagenesis, Insertional ,Epothilones ,Heterologous expression ,Expression efficiency ,Biotechnology - Abstract
Background Exotic genes, especially clustered multiple-genes for a complex pathway, are normally integrated into chromosome for heterologous expression. The influences of insertion sites on heterologous expression and allotropic expressions of exotic genes on host remain mostly unclear. Results We compared the integration and expression efficiencies of single and multiple exotic genes that were inserted into Myxococcus xanthus genome by transposition and attB-site-directed recombination. While the site-directed integration had a rather stable chloramphenicol acetyl transferase (CAT) activity, the transposition produced varied CAT enzyme activities. We attempted to integrate the 56-kb gene cluster for the biosynthesis of antitumor polyketides epothilones into M. xanthus genome by site-direction but failed, which was determined to be due to the insertion size limitation at the attB site. The transposition technique produced many recombinants with varied production capabilities of epothilones, which, however, were not paralleled to the transcriptional characteristics of the local sites where the genes were integrated. Comparative transcriptomics analysis demonstrated that the allopatric integrations caused selective changes of host transcriptomes, leading to varied expressions of epothilone genes in different mutants. Conclusions With the increase of insertion fragment size, transposition is a more practicable integration method for the expression of exotic genes. Allopatric integrations selectively change host transcriptomes, which lead to varied expression efficiencies of exotic genes. Electronic supplementary material The online version of this article (doi:10.1186/s12934-015-0294-5) contains supplementary material, which is available to authorized users.
- Published
- 2015
10. Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus
- Author
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Zhu, Li-Ping, primary, Yue, Xin-Jing, additional, Han, Kui, additional, Li, Zhi-Feng, additional, Zheng, Lian-Shuai, additional, Yi, Xiu-Nan, additional, Wang, Hai-Long, additional, Zhang, You-Ming, additional, and Li, Yue-Zhong, additional
- Published
- 2015
- Full Text
- View/download PDF
11. Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus.
- Author
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Li-Ping Zhu, Xin-Jing Yue, Kui Han, Zhi-Feng Li, Lian-Shuai Zheng, Xiu-Nan Yi, Hai-Long Wang, You-Ming Zhang, and Yue-Zhong Li
- Subjects
CHROMOSOMAL translocation ,CHLORAMPHENICOL ,MYXOCOCCUS xanthus ,TRANSFERASES ,BIOSYNTHESIS - Abstract
Background: Exotic genes, especially clustered multiple-genes for a complex pathway, are normally integrated into chromosome for heterologous expression. The influences of insertion sites on heterologous expression and allotropic expressions of exotic genes on host remain mostly unclear. Results: We compared the integration and expression efficiencies of single and multiple exotic genes that were inserted into Myxococcus xanthus genome by transposition and attB-site-directed recombination. While the site-directed integration had a rather stable chloramphenicol acetyl transferase (CAT) activity, the transposition produced varied CAT enzyme activities. We attempted to integrate the 56-kb gene cluster for the biosynthesis of antitumor polyketides epothilones into M. xanthus genome by site-direction but failed, which was determined to be due to the insertion size limitation at the attB site. The transposition technique produced many recombinants with varied production capabilities of epothilones, which, however, were not paralleled to the transcriptional characteristics of the local sites where the genes were integrated. Comparative transcriptomics analysis demonstrated that the allopatric integrations caused selective changes of host transcriptomes, leading to varied expressions of epothilone genes in different mutants. Conclusions: With the increase of insertion fragment size, transposition is a more practicable integration method for the expression of exotic genes. Allopatric integrations selectively change host transcriptomes, which lead to varied expression efficiencies of exotic genes. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
12. Engineering the acyltransferase domain of epothilone polyketide synthase to alter the substrate specificity
- Author
-
Huimin Wang, Junheng Liang, Qianwen Yue, Long Li, Yan Shi, Guosong Chen, Yue-zhong Li, Xiaoying Bian, Youming Zhang, Guoping Zhao, and Xiaoming Ding
- Subjects
Polyketide synthase ,AT ,Epothilone ,Domain swap ,Substrate specificity ,Microbiology ,QR1-502 - Abstract
Abstract Background Polyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal. Results The substitution of module 4 drastically diminished the activity of epothilone PKS. However, with careful design of the KS-AT linker and the post-AT linker, replacing EPOAT4 with EPOAT2, EPOAT6, EPOAT7 or EPOAT8 (specifically incorporating methylmalonyl-CoA (MMCoA)) significantly increased the ratio of epothilone D (4) to epothilone C (3) (the highest ratio of 4:3 = 4.6:1), whereas the ratio of 4:3 in the parental strain Schlegelella brevitalea 104-1 was 1.4:1. We also obtained three strains by swapping EPOAT4 with EPOAT3, EPOAT5, or EPOAT9, which specifically incorporate malonyl-CoA (MCoA). These strains produced only epothilone C, and the yield was increased by a factor of 1.8 compared to that of parental strain 104-1. Furthermore, mutations of five residues in the AT domain identified Ser310 as the critical factor for MMCoA recognition in EPOAT4. Then, the mutation of His308 to valine or tyrosine combined with the mutation of Phe310 to serine further altered the product ratios. At the same time, we successfully deleted the inactive module 9 DH and ER domains and fused the ΨKR domain with the KR domain through an ~ 25-residue linker to generate a productive and simplified epothilone PKS. Conclusions These results suggested that the substitution and deletion of catalytic domains effectively produces desirable compounds and that selection of the linkers between domains is crucial for maintaining intact PKS catalytic activity.
- Published
- 2021
- Full Text
- View/download PDF
13. CRISPR/dCas9-mediated transcriptional improvement of the biosynthetic gene cluster for the epothilone production in Myxococcus xanthus
- Author
-
Ran Peng, Ye Wang, Wan-wan Feng, Xin-jing Yue, Jiang-he Chen, Xiao-zhuang Hu, Zhi-feng Li, Duo-hong Sheng, You-ming Zhang, and Yue-zhong Li
- Subjects
CRISPR/dCas9 activation ,sgRNAs ,Activator proteins ,Promoter ,Transcriptional improvement ,Biosynthetic gene cluster ,Microbiology ,QR1-502 - Abstract
Abstract Background The CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites. Results In this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation. We firstly optimized the cas9 codon to fit the M. xanthus cells, mutated the gene to inactivate the nuclease activity, and constructed the dCas9-activator system in an epothilone producer. We compared the improvement efficiency of different sgRNAs on the production of epothilones and the expression of the biosynthetic genes. We also compared the improvement effects of different activator proteins, the ω and α subunits of RNA polymerase, and the sigma factors σ54 and CarQ. By using a copper-inducible promoter, we determined that higher expressions of dCas9-activator improved the activation effects. Conclusions Our results showed that the CRISPR/dCas-mediated transcription activation is a simple and broadly applicable technique to improve the transcriptional efficiency for the production of secondary metabolites in microorganisms. This is the first time to construct the CRISPR/dCas9 activation system in myxobacteria and the first time to assay the CRISPR/dCas9 activations for the biosynthesis of microbial secondary metabolites.
- Published
- 2018
- Full Text
- View/download PDF
14. Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus
- Author
-
Ying-jie Yang, Ye Wang, Zhi-feng Li, Ya Gong, Peng Zhang, Wen-chao Hu, Duo-hong Sheng, and Yue-zhong Li
- Subjects
CRISPR/Cas9 ,On-target cleavage efficiency ,Spacer sequence ,Free energy ,Deletion of large genome fragments ,Biosynthetic gene clusters for secondary metabolites ,Microbiology ,QR1-502 - Abstract
Abstract Background The CRISPR/Cas9 system is a powerful tool for genome editing, in which the sgRNA binds and guides the Cas9 protein for the sequence-specific cleavage. The protocol is employable in different organisms, but is often limited by cell damage due to the endonuclease activity of the introduced Cas9 and the potential off-target DNA cleavage from incorrect guide by the 20 nt spacer. Results In this study, after resolving some critical limits, we have established an efficient CRISPR/Cas9 system for the deletion of large genome fragments related to the biosynthesis of secondary metabolites in Myxococcus xanthus cells. We revealed that the high expression of a codon-optimized cas9 gene in M. xanthus was cytotoxic, and developed a temporally high expression strategy to reduce the cell damage from high expressions of Cas9. We optimized the deletion protocol by using the tRNA–sgRNA–tRNA chimeric structure to ensure correct sgRNA sequence. We found that, in addition to the position-dependent nucleotide preference, the free energy of a 20 nt spacer was a key factor for the deletion efficiency. Conclusions By using the developed protocol, we achieved the CRISPR/Cas9-induced deletion of large biosynthetic gene clusters for secondary metabolites in M. xanthus DK1622 and its epothilone-producing mutant. The findings and the proposals described in this paper were suggested to be workable in other organisms, for example, other Gram negative bacteria with high GC content.
- Published
- 2017
- Full Text
- View/download PDF
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