Transcriptional control of gene expression in Pichia pastoris by manipulation of terminators
Controlling gene expression is often the foremost goal of most biological endeavors like the production of industrial enzymes and expression of heterologous metabolic pathway genes. The components of the entire “expression cassette” exert control on net protein output. This control is primarily achi...
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| creator | Ramakrishnan, Kamatchi Prattipati, Mahesh Samuel, Premsingh Sankaranarayanan, Meenakshisundaram |
| description | Controlling gene expression is often the foremost goal of most biological endeavors like the production of industrial enzymes and expression of heterologous metabolic pathway genes. The components of the entire “expression cassette” exert control on net protein output. This control is primarily achieved through altering the promoter driving expression and by changing the copy number of the gene. However, there are only a few recent studies on terminators. Terminators are essential components in expression cassettes that influence the 3′ end processing of mRNA, mRNA stability, and translational efficiency, which can modulate protein production. In
Pichia pastoris
(
Komagataella phaffi
), little attention has been paid to the selection of terminator regions in efforts to increase heterologous gene expression. To explore the potential application of the terminator regions on increased secretory production of
Candida antarctica
lipase B (CALB), we assessed the ability of three different classes of terminator regions: (1) terminator regions of methanol oxidation pathway genes of
P. pastoris
; (2) terminator regions of well-expressed and housekeeping genes of
P. pastoris
; and (3) terminators of other yeast genes like
Saccharomyces cerevisiae
. The terminator of dihydroxyacetone synthase (DHAS TT), a high expressing gene in the methanol utilization pathway, shows inducible CALB expression levels similar to the AOX1 terminator (AOX1 TT) under the control of AOX1 promoter and threefold higher in constitutive expression of CALB under the control of GAP promoter. The
Calb
transcript abundance was also found to correlate with protein expression. Furthermore, mRNA half-life determination showed a direct correlation between the stability of transcripts and increased transcription rate. Together, our results emphasize that enhancing transcript stability using the correct choice of transcription terminators (TT) will help in developing robust production strains suitable for scale-up.
Key points
•
Influence of transcription terminators on Calb gene expression
•
Modulation of gene expression by enhancing transcript stability |
| doi_str_mv | 10.1007/s00253-020-10785-8 |
| format | Article |
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Pichia pastoris
(
Komagataella phaffi
), little attention has been paid to the selection of terminator regions in efforts to increase heterologous gene expression. To explore the potential application of the terminator regions on increased secretory production of
Candida antarctica
lipase B (CALB), we assessed the ability of three different classes of terminator regions: (1) terminator regions of methanol oxidation pathway genes of
P. pastoris
; (2) terminator regions of well-expressed and housekeeping genes of
P. pastoris
; and (3) terminators of other yeast genes like
Saccharomyces cerevisiae
. The terminator of dihydroxyacetone synthase (DHAS TT), a high expressing gene in the methanol utilization pathway, shows inducible CALB expression levels similar to the AOX1 terminator (AOX1 TT) under the control of AOX1 promoter and threefold higher in constitutive expression of CALB under the control of GAP promoter. The
Calb
transcript abundance was also found to correlate with protein expression. Furthermore, mRNA half-life determination showed a direct correlation between the stability of transcripts and increased transcription rate. Together, our results emphasize that enhancing transcript stability using the correct choice of transcription terminators (TT) will help in developing robust production strains suitable for scale-up.
Key points
•
Influence of transcription terminators on Calb gene expression
•
Modulation of gene expression by enhancing transcript stability</description><identifier>ISSN: 0175-7598</identifier><identifier>EISSN: 1432-0614</identifier><identifier>DOI: 10.1007/s00253-020-10785-8</identifier><identifier>PMID: 32715362</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer Berlin Heidelberg</publisher><subject>Basidiomycota ; Biomedical and Life Sciences ; Biotechnologically Relevant Enzymes and Proteins ; Biotechnology ; Cassettes ; Copy number ; Dihydroxyacetone synthase ; Fungal Proteins - genetics ; Gene Expression ; Genes ; Genetic aspects ; Genetic engineering ; Genetic research ; Genetic transcription ; Life Sciences ; Lipase ; Messenger RNA ; Metabolic pathways ; Methanol ; Microbial Genetics and Genomics ; Microbiology ; Monosaccharides ; mRNA stability ; Oxidation ; Pichia - genetics ; Pichia pastoris ; Proteins ; Saccharomycetales ; Stability ; Sugars ; Transcription ; Yeast ; Yeasts</subject><ispartof>Applied microbiology and biotechnology, 2020-09, Vol.104 (18), p.7841-7851</ispartof><rights>Springer-Verlag GmbH Germany, part of Springer Nature 2020</rights><rights>COPYRIGHT 2020 Springer</rights><rights>Springer-Verlag GmbH Germany, part of Springer Nature 2020.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c513t-bd7821d5ef94d305d602c3fc32cee3700026f674d1e4f0e854a48990f31d4eac3</citedby><cites>FETCH-LOGICAL-c513t-bd7821d5ef94d305d602c3fc32cee3700026f674d1e4f0e854a48990f31d4eac3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00253-020-10785-8$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00253-020-10785-8$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,776,780,27901,27902,41464,42533,51294</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/32715362$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ramakrishnan, Kamatchi</creatorcontrib><creatorcontrib>Prattipati, Mahesh</creatorcontrib><creatorcontrib>Samuel, Premsingh</creatorcontrib><creatorcontrib>Sankaranarayanan, Meenakshisundaram</creatorcontrib><title>Transcriptional control of gene expression in Pichia pastoris by manipulation of terminators</title><title>Applied microbiology and biotechnology</title><addtitle>Appl Microbiol Biotechnol</addtitle><addtitle>Appl Microbiol Biotechnol</addtitle><description>Controlling gene expression is often the foremost goal of most biological endeavors like the production of industrial enzymes and expression of heterologous metabolic pathway genes. The components of the entire “expression cassette” exert control on net protein output. This control is primarily achieved through altering the promoter driving expression and by changing the copy number of the gene. However, there are only a few recent studies on terminators. Terminators are essential components in expression cassettes that influence the 3′ end processing of mRNA, mRNA stability, and translational efficiency, which can modulate protein production. In
Pichia pastoris
(
Komagataella phaffi
), little attention has been paid to the selection of terminator regions in efforts to increase heterologous gene expression. To explore the potential application of the terminator regions on increased secretory production of
Candida antarctica
lipase B (CALB), we assessed the ability of three different classes of terminator regions: (1) terminator regions of methanol oxidation pathway genes of
P. pastoris
; (2) terminator regions of well-expressed and housekeeping genes of
P. pastoris
; and (3) terminators of other yeast genes like
Saccharomyces cerevisiae
. The terminator of dihydroxyacetone synthase (DHAS TT), a high expressing gene in the methanol utilization pathway, shows inducible CALB expression levels similar to the AOX1 terminator (AOX1 TT) under the control of AOX1 promoter and threefold higher in constitutive expression of CALB under the control of GAP promoter. The
Calb
transcript abundance was also found to correlate with protein expression. Furthermore, mRNA half-life determination showed a direct correlation between the stability of transcripts and increased transcription rate. Together, our results emphasize that enhancing transcript stability using the correct choice of transcription terminators (TT) will help in developing robust production strains suitable for scale-up.
Key points
•
Influence of transcription terminators on Calb gene expression
•
Modulation of gene expression by enhancing transcript stability</description><subject>Basidiomycota</subject><subject>Biomedical and Life Sciences</subject><subject>Biotechnologically Relevant Enzymes and Proteins</subject><subject>Biotechnology</subject><subject>Cassettes</subject><subject>Copy number</subject><subject>Dihydroxyacetone synthase</subject><subject>Fungal Proteins - genetics</subject><subject>Gene Expression</subject><subject>Genes</subject><subject>Genetic aspects</subject><subject>Genetic engineering</subject><subject>Genetic research</subject><subject>Genetic transcription</subject><subject>Life Sciences</subject><subject>Lipase</subject><subject>Messenger RNA</subject><subject>Metabolic pathways</subject><subject>Methanol</subject><subject>Microbial Genetics and Genomics</subject><subject>Microbiology</subject><subject>Monosaccharides</subject><subject>mRNA stability</subject><subject>Oxidation</subject><subject>Pichia - genetics</subject><subject>Pichia pastoris</subject><subject>Proteins</subject><subject>Saccharomycetales</subject><subject>Stability</subject><subject>Sugars</subject><subject>Transcription</subject><subject>Yeast</subject><subject>Yeasts</subject><issn>0175-7598</issn><issn>1432-0614</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNp9kU9rFTEUxQdR7LP6BVzIgBu7mHrzf2ZZitZCQdG6E0Je5uaZMpOMyQy0396Mr1qeiGRxIed3Lpx7quolgVMCoN5mACpYAxQaAqoVTfuo2hDOaAOS8MfVBogSjRJde1Q9y_kGgNBWyqfVEaOKCCbppvp2nUzINvlp9jGYobYxzCkOdXT1DgPWeDslzLmItQ_1J2-_e1NPJs8x-Vxv7-rRBD8tg1n9q2vGNPpgip6fV0-cGTK-uJ_H1df3767PPzRXHy8uz8-uGisIm5ttr1pKeoGu4z0D0UugljnLqEVkCkpM6aTiPUHuAFvBDW-7DhwjPUdj2XH1Zr93SvHHgnnWo88Wh8EEjEvWlFMlKPCWFfT1X-hNXFIJvlJMdkoSph6onRlQ--DinIxdl-ozyZiQnQBeqNN_UOX1OPpyR3S-_B8YTg4M663xdt6ZJWd9-eXzIUv3rE0x54ROT8mPJt1pAnqtX-_r16V-_at-3RbTq_t0y3bE_o_ld98FYHsgFynsMD3E_8_an6vtuI8</recordid><startdate>20200901</startdate><enddate>20200901</enddate><creator>Ramakrishnan, Kamatchi</creator><creator>Prattipati, Mahesh</creator><creator>Samuel, Premsingh</creator><creator>Sankaranarayanan, Meenakshisundaram</creator><general>Springer Berlin Heidelberg</general><general>Springer</general><general>Springer Nature B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>ISR</scope><scope>3V.</scope><scope>7QL</scope><scope>7T7</scope><scope>7WY</scope><scope>7WZ</scope><scope>7X7</scope><scope>7XB</scope><scope>87Z</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8FL</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BEZIV</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FRNLG</scope><scope>FYUFA</scope><scope>F~G</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K60</scope><scope>K6~</scope><scope>K9.</scope><scope>L.-</scope><scope>LK8</scope><scope>M0C</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PQBIZ</scope><scope>PQBZA</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>7X8</scope></search><sort><creationdate>20200901</creationdate><title>Transcriptional control of gene expression in Pichia pastoris by manipulation of terminators</title><author>Ramakrishnan, Kamatchi ; Prattipati, Mahesh ; Samuel, Premsingh ; Sankaranarayanan, Meenakshisundaram</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c513t-bd7821d5ef94d305d602c3fc32cee3700026f674d1e4f0e854a48990f31d4eac3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>Basidiomycota</topic><topic>Biomedical and Life Sciences</topic><topic>Biotechnologically Relevant Enzymes and Proteins</topic><topic>Biotechnology</topic><topic>Cassettes</topic><topic>Copy number</topic><topic>Dihydroxyacetone synthase</topic><topic>Fungal Proteins - genetics</topic><topic>Gene Expression</topic><topic>Genes</topic><topic>Genetic aspects</topic><topic>Genetic engineering</topic><topic>Genetic research</topic><topic>Genetic transcription</topic><topic>Life Sciences</topic><topic>Lipase</topic><topic>Messenger RNA</topic><topic>Metabolic pathways</topic><topic>Methanol</topic><topic>Microbial Genetics and Genomics</topic><topic>Microbiology</topic><topic>Monosaccharides</topic><topic>mRNA stability</topic><topic>Oxidation</topic><topic>Pichia - genetics</topic><topic>Pichia pastoris</topic><topic>Proteins</topic><topic>Saccharomycetales</topic><topic>Stability</topic><topic>Sugars</topic><topic>Transcription</topic><topic>Yeast</topic><topic>Yeasts</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ramakrishnan, Kamatchi</creatorcontrib><creatorcontrib>Prattipati, Mahesh</creatorcontrib><creatorcontrib>Samuel, Premsingh</creatorcontrib><creatorcontrib>Sankaranarayanan, Meenakshisundaram</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>ABI/INFORM Collection</collection><collection>ABI/INFORM Global (PDF only)</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>ABI/INFORM Global (Alumni Edition)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ABI/INFORM Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Business Premium Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Business Premium Collection (Alumni)</collection><collection>Health Research Premium Collection</collection><collection>ABI/INFORM Global (Corporate)</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Business Collection (Alumni Edition)</collection><collection>ProQuest Business Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ABI/INFORM Professional Advanced</collection><collection>ProQuest Biological Science Collection</collection><collection>ABI/INFORM Global</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Business</collection><collection>ProQuest One Business (Alumni)</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>MEDLINE - Academic</collection><jtitle>Applied microbiology and biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ramakrishnan, Kamatchi</au><au>Prattipati, Mahesh</au><au>Samuel, Premsingh</au><au>Sankaranarayanan, Meenakshisundaram</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Transcriptional control of gene expression in Pichia pastoris by manipulation of terminators</atitle><jtitle>Applied microbiology and biotechnology</jtitle><stitle>Appl Microbiol Biotechnol</stitle><addtitle>Appl Microbiol Biotechnol</addtitle><date>2020-09-01</date><risdate>2020</risdate><volume>104</volume><issue>18</issue><spage>7841</spage><epage>7851</epage><pages>7841-7851</pages><issn>0175-7598</issn><eissn>1432-0614</eissn><abstract>Controlling gene expression is often the foremost goal of most biological endeavors like the production of industrial enzymes and expression of heterologous metabolic pathway genes. The components of the entire “expression cassette” exert control on net protein output. This control is primarily achieved through altering the promoter driving expression and by changing the copy number of the gene. However, there are only a few recent studies on terminators. Terminators are essential components in expression cassettes that influence the 3′ end processing of mRNA, mRNA stability, and translational efficiency, which can modulate protein production. In
Pichia pastoris
(
Komagataella phaffi
), little attention has been paid to the selection of terminator regions in efforts to increase heterologous gene expression. To explore the potential application of the terminator regions on increased secretory production of
Candida antarctica
lipase B (CALB), we assessed the ability of three different classes of terminator regions: (1) terminator regions of methanol oxidation pathway genes of
P. pastoris
; (2) terminator regions of well-expressed and housekeeping genes of
P. pastoris
; and (3) terminators of other yeast genes like
Saccharomyces cerevisiae
. The terminator of dihydroxyacetone synthase (DHAS TT), a high expressing gene in the methanol utilization pathway, shows inducible CALB expression levels similar to the AOX1 terminator (AOX1 TT) under the control of AOX1 promoter and threefold higher in constitutive expression of CALB under the control of GAP promoter. The
Calb
transcript abundance was also found to correlate with protein expression. Furthermore, mRNA half-life determination showed a direct correlation between the stability of transcripts and increased transcription rate. Together, our results emphasize that enhancing transcript stability using the correct choice of transcription terminators (TT) will help in developing robust production strains suitable for scale-up.
Key points
•
Influence of transcription terminators on Calb gene expression
•
Modulation of gene expression by enhancing transcript stability</abstract><cop>Berlin/Heidelberg</cop><pub>Springer Berlin Heidelberg</pub><pmid>32715362</pmid><doi>10.1007/s00253-020-10785-8</doi><tpages>11</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0175-7598 |
| ispartof | Applied microbiology and biotechnology, 2020-09, Vol.104 (18), p.7841-7851 |
| issn | 0175-7598 1432-0614 |
| language | eng |
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| source | MEDLINE; SpringerLink Journals |
| subjects | Basidiomycota Biomedical and Life Sciences Biotechnologically Relevant Enzymes and Proteins Biotechnology Cassettes Copy number Dihydroxyacetone synthase Fungal Proteins - genetics Gene Expression Genes Genetic aspects Genetic engineering Genetic research Genetic transcription Life Sciences Lipase Messenger RNA Metabolic pathways Methanol Microbial Genetics and Genomics Microbiology Monosaccharides mRNA stability Oxidation Pichia - genetics Pichia pastoris Proteins Saccharomycetales Stability Sugars Transcription Yeast Yeasts |
| title | Transcriptional control of gene expression in Pichia pastoris by manipulation of terminators |
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