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Construction of gene modification system with highly efficient and markerless for Monascus ruber M7
Monascus spp. are traditional medicinal and edible filamentous fungi in China, and can produce various secondary metabolites, such as Monascus pigments (MPs) and citrinin (CIT). Genetic modification methods, such as gene knock-out, complementation, and overexpression, have been used extensively to i...
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| Published in: | Frontiers in microbiology 2022-08, Vol.13, p.952323-952323 |
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| creator | Xu, Na Li, Li Chen, Fusheng |
| description | Monascus
spp. are traditional medicinal and edible filamentous fungi in China, and can produce various secondary metabolites, such as
Monascus
pigments (MPs) and citrinin (CIT). Genetic modification methods, such as gene knock-out, complementation, and overexpression, have been used extensively to investigate the function of related genes in
Monascus
spp.. However, the resistance selection genes that can have been used for genetic modification in
Monascus
spp. are limited, and the gene replacement frequency (GRF) is usually |
| doi_str_mv | 10.3389/fmicb.2022.952323 |
| format | article |
| fullrecord | <record><control><sourceid>proquest_doaj_</sourceid><recordid>TN_cdi_doaj_primary_oai_doaj_org_article_3751c881f53844ec80269b7a3f4858b2</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><doaj_id>oai_doaj_org_article_3751c881f53844ec80269b7a3f4858b2</doaj_id><sourcerecordid>2703983901</sourcerecordid><originalsourceid>FETCH-LOGICAL-c372t-f9336490c08624e0f8fdf1b0501f1fb541743304601ff1113c73834c7d4d776a3</originalsourceid><addsrcrecordid>eNpVkU9vFCEYxonR2KbtB_DG0cuuwMsMcDExG7VNanppE2-EYV52qTNDhRnNfvuyu42x74H335MfkIeQD5ytAbT5FMbou7VgQqxNI0DAG3LO21augImfb_-rz8hVKY-shmSinu_JGTRGGanZOfGbNJU5L36OaaIp0C1OSMfUxxC9Ow7Lvsw40r9x3tFd3O6GPcVQtxGnmbqpp6PLvzAPWAoNKdMfaXLFL4XmpcPaqkvyLrih4NVLviAP377eb65Xt3ffbzZfblcelJhXwQC00jDPdCsksqBDH3jHGsYDD10juZIATLa1D5xz8Ao0SK962SvVOrggNydun9yjfcqxPmxvk4v2OEh5a12eox_Qgmq415qHBrSU6DUTremUgyB1oztRWZ9PrKelG7H39a_ZDa-grzdT3Nlt-mMNqFY2vAI-vgBy-r1gme0Yi8dhcBOmpVihGBgNhh2k_CT1OZWSMfy7hjN78NoevbYHr-3Ja3gG_oqcOQ</addsrcrecordid><sourcetype>Open Website</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>2703983901</pqid></control><display><type>article</type><title>Construction of gene modification system with highly efficient and markerless for Monascus ruber M7</title><source>PubMed Central</source><creator>Xu, Na ; Li, Li ; Chen, Fusheng</creator><creatorcontrib>Xu, Na ; Li, Li ; Chen, Fusheng</creatorcontrib><description>Monascus
spp. are traditional medicinal and edible filamentous fungi in China, and can produce various secondary metabolites, such as
Monascus
pigments (MPs) and citrinin (CIT). Genetic modification methods, such as gene knock-out, complementation, and overexpression, have been used extensively to investigate the function of related genes in
Monascus
spp.. However, the resistance selection genes that can have been used for genetic modification in
Monascus
spp. are limited, and the gene replacement frequency (GRF) is usually <5%. Therefore, we are committed to construct a highly efficient gene editing system without resistance selection marker gene. In this study, using
M. ruber
M7 as the starting strain, we successfully constructed a so-called markerlessly and highly genetic modification system including the mutants Δ
mrpyrG
Δ
mrlig4
and Δ
mrpyrG
Δ
mrlig4
::
mrpyrG
, in which we used the endogenous gene
mrpyrG
from
M. ruber
M7 instead of the resistance marker gene as the screening marker, and simultaneously deleted
mrlig4
related to non-homologous end joining in
M. ruber
M7. Then, the morphology, the growth rate, the production of MPs and CIT of the mutants were analyzed. And the results show that the mutant strains have normal mycelia, cleistothecia and conidia on PDA+Uridine(U) plate, the biomass of each mutant is also no different from
M. ruber
M7. However, the U addition also has a certain effect on the orange and red pigments yield of
M. ruber
M7, which needs our further study. Finally, we applied the system to delete multiple genes from
M. ruber
M7 separately or continuously without any resistance marker gene, and found that the average GRF of Δ
mrpyrG
Δ
mrlig4
was about 18 times of that of
M. ruber
M7. The markerlessly and highly genetic modification system constructed in current study not only will be used for multi-gene simultaneous modification in
Monascus
spp., and also lays a foundation for investigating the effects of multi-genes modification on
Monascus
spp..</description><identifier>ISSN: 1664-302X</identifier><identifier>EISSN: 1664-302X</identifier><identifier>DOI: 10.3389/fmicb.2022.952323</identifier><identifier>PMID: 35979480</identifier><language>eng</language><publisher>Frontiers Media S.A</publisher><subject>genetic modification system ; Microbiology ; Monascus ruber M7 ; mrlig4 ; mrpyrG ; resistance selection marker</subject><ispartof>Frontiers in microbiology, 2022-08, Vol.13, p.952323-952323</ispartof><rights>Copyright © 2022 Xu, Li and Chen. 2022 Xu, Li and Chen</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c372t-f9336490c08624e0f8fdf1b0501f1fb541743304601ff1113c73834c7d4d776a3</citedby><cites>FETCH-LOGICAL-c372t-f9336490c08624e0f8fdf1b0501f1fb541743304601ff1113c73834c7d4d776a3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC9376451/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC9376451/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids></links><search><creatorcontrib>Xu, Na</creatorcontrib><creatorcontrib>Li, Li</creatorcontrib><creatorcontrib>Chen, Fusheng</creatorcontrib><title>Construction of gene modification system with highly efficient and markerless for Monascus ruber M7</title><title>Frontiers in microbiology</title><description>Monascus
spp. are traditional medicinal and edible filamentous fungi in China, and can produce various secondary metabolites, such as
Monascus
pigments (MPs) and citrinin (CIT). Genetic modification methods, such as gene knock-out, complementation, and overexpression, have been used extensively to investigate the function of related genes in
Monascus
spp.. However, the resistance selection genes that can have been used for genetic modification in
Monascus
spp. are limited, and the gene replacement frequency (GRF) is usually <5%. Therefore, we are committed to construct a highly efficient gene editing system without resistance selection marker gene. In this study, using
M. ruber
M7 as the starting strain, we successfully constructed a so-called markerlessly and highly genetic modification system including the mutants Δ
mrpyrG
Δ
mrlig4
and Δ
mrpyrG
Δ
mrlig4
::
mrpyrG
, in which we used the endogenous gene
mrpyrG
from
M. ruber
M7 instead of the resistance marker gene as the screening marker, and simultaneously deleted
mrlig4
related to non-homologous end joining in
M. ruber
M7. Then, the morphology, the growth rate, the production of MPs and CIT of the mutants were analyzed. And the results show that the mutant strains have normal mycelia, cleistothecia and conidia on PDA+Uridine(U) plate, the biomass of each mutant is also no different from
M. ruber
M7. However, the U addition also has a certain effect on the orange and red pigments yield of
M. ruber
M7, which needs our further study. Finally, we applied the system to delete multiple genes from
M. ruber
M7 separately or continuously without any resistance marker gene, and found that the average GRF of Δ
mrpyrG
Δ
mrlig4
was about 18 times of that of
M. ruber
M7. The markerlessly and highly genetic modification system constructed in current study not only will be used for multi-gene simultaneous modification in
Monascus
spp., and also lays a foundation for investigating the effects of multi-genes modification on
Monascus
spp..</description><subject>genetic modification system</subject><subject>Microbiology</subject><subject>Monascus ruber M7</subject><subject>mrlig4</subject><subject>mrpyrG</subject><subject>resistance selection marker</subject><issn>1664-302X</issn><issn>1664-302X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><sourceid>DOA</sourceid><recordid>eNpVkU9vFCEYxonR2KbtB_DG0cuuwMsMcDExG7VNanppE2-EYV52qTNDhRnNfvuyu42x74H335MfkIeQD5ytAbT5FMbou7VgQqxNI0DAG3LO21augImfb_-rz8hVKY-shmSinu_JGTRGGanZOfGbNJU5L36OaaIp0C1OSMfUxxC9Ow7Lvsw40r9x3tFd3O6GPcVQtxGnmbqpp6PLvzAPWAoNKdMfaXLFL4XmpcPaqkvyLrih4NVLviAP377eb65Xt3ffbzZfblcelJhXwQC00jDPdCsksqBDH3jHGsYDD10juZIATLa1D5xz8Ao0SK962SvVOrggNydun9yjfcqxPmxvk4v2OEh5a12eox_Qgmq415qHBrSU6DUTremUgyB1oztRWZ9PrKelG7H39a_ZDa-grzdT3Nlt-mMNqFY2vAI-vgBy-r1gme0Yi8dhcBOmpVihGBgNhh2k_CT1OZWSMfy7hjN78NoevbYHr-3Ja3gG_oqcOQ</recordid><startdate>20220801</startdate><enddate>20220801</enddate><creator>Xu, Na</creator><creator>Li, Li</creator><creator>Chen, Fusheng</creator><general>Frontiers Media S.A</general><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20220801</creationdate><title>Construction of gene modification system with highly efficient and markerless for Monascus ruber M7</title><author>Xu, Na ; Li, Li ; Chen, Fusheng</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c372t-f9336490c08624e0f8fdf1b0501f1fb541743304601ff1113c73834c7d4d776a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2022</creationdate><topic>genetic modification system</topic><topic>Microbiology</topic><topic>Monascus ruber M7</topic><topic>mrlig4</topic><topic>mrpyrG</topic><topic>resistance selection marker</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Xu, Na</creatorcontrib><creatorcontrib>Li, Li</creatorcontrib><creatorcontrib>Chen, Fusheng</creatorcontrib><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>Frontiers in microbiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Xu, Na</au><au>Li, Li</au><au>Chen, Fusheng</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Construction of gene modification system with highly efficient and markerless for Monascus ruber M7</atitle><jtitle>Frontiers in microbiology</jtitle><date>2022-08-01</date><risdate>2022</risdate><volume>13</volume><spage>952323</spage><epage>952323</epage><pages>952323-952323</pages><issn>1664-302X</issn><eissn>1664-302X</eissn><abstract>Monascus
spp. are traditional medicinal and edible filamentous fungi in China, and can produce various secondary metabolites, such as
Monascus
pigments (MPs) and citrinin (CIT). Genetic modification methods, such as gene knock-out, complementation, and overexpression, have been used extensively to investigate the function of related genes in
Monascus
spp.. However, the resistance selection genes that can have been used for genetic modification in
Monascus
spp. are limited, and the gene replacement frequency (GRF) is usually <5%. Therefore, we are committed to construct a highly efficient gene editing system without resistance selection marker gene. In this study, using
M. ruber
M7 as the starting strain, we successfully constructed a so-called markerlessly and highly genetic modification system including the mutants Δ
mrpyrG
Δ
mrlig4
and Δ
mrpyrG
Δ
mrlig4
::
mrpyrG
, in which we used the endogenous gene
mrpyrG
from
M. ruber
M7 instead of the resistance marker gene as the screening marker, and simultaneously deleted
mrlig4
related to non-homologous end joining in
M. ruber
M7. Then, the morphology, the growth rate, the production of MPs and CIT of the mutants were analyzed. And the results show that the mutant strains have normal mycelia, cleistothecia and conidia on PDA+Uridine(U) plate, the biomass of each mutant is also no different from
M. ruber
M7. However, the U addition also has a certain effect on the orange and red pigments yield of
M. ruber
M7, which needs our further study. Finally, we applied the system to delete multiple genes from
M. ruber
M7 separately or continuously without any resistance marker gene, and found that the average GRF of Δ
mrpyrG
Δ
mrlig4
was about 18 times of that of
M. ruber
M7. The markerlessly and highly genetic modification system constructed in current study not only will be used for multi-gene simultaneous modification in
Monascus
spp., and also lays a foundation for investigating the effects of multi-genes modification on
Monascus
spp..</abstract><pub>Frontiers Media S.A</pub><pmid>35979480</pmid><doi>10.3389/fmicb.2022.952323</doi><tpages>1</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | genetic modification system Microbiology Monascus ruber M7 mrlig4 mrpyrG resistance selection marker |
| title | Construction of gene modification system with highly efficient and markerless for Monascus ruber M7 |
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