Ruei-En Hu , Wen-Chi Leu , Yu-Chieh Lin , I-Son Ng
{"title":"A novel heat-inducible plasmid-driven T7 system for enhancing carbonic anhydrase in engineered Escherichia coli strains","authors":"Ruei-En Hu , Wen-Chi Leu , Yu-Chieh Lin , I-Son Ng","doi":"10.1016/j.procbio.2024.07.010","DOIUrl":null,"url":null,"abstract":"<div><p>Over the past decades, the heat-inducible (HI) promoter, derived from cI857 phage system has been widely utilized. However, the developments of alternative temperature-controlled promoters are limited. In this study, we explored a novel HI promoter through PCR amplification using the degenerate primer design. BLAST analysis identified this sequence as a partial <em>pls</em>B on <em>Escherichia coli</em> chromosome. Antisense RNAs targeting distinct regions of HI promoter were applied to examine the mechanism. The fluorescence of super-folder green fluorescent protein (sfGFP) driven by the HI promoter showed 5.5-fold increase from 30 to 42<!--> <sup>o</sup>C, whereas the truncated HI yielded a trace amount of sfGFP. Among various <em>E. coli</em> strains, BL21 exhibited the highest fluorescence, reaching 3976 a.u. at 42°C. Subsequently, the novel HI promoter was employed to drive T7RNA polymerase within a plasmid-driven T7 (PDT7) plasmid, serving its capability to express sfGFP and carbonic anhydrases (CA), respectively. The maximum intensity of sfGFP reached 38702 a.u., and CA activity surged to 14286 WAU/mL in W3110 among other strains. Finally, the highest CA activity was 27521 WAU/mL at alkali pH 9. The promising results from the novel heat-inducible promoter-driven T7RNAP, incorporating the T7 terminator as HI-PDT7-TT, demonstrate potential for expressing more heterogeneous proteins across various chassis in the future.</p></div>","PeriodicalId":20811,"journal":{"name":"Process Biochemistry","volume":null,"pages":null},"PeriodicalIF":3.7000,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Process Biochemistry","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1359511324002307","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Over the past decades, the heat-inducible (HI) promoter, derived from cI857 phage system has been widely utilized. However, the developments of alternative temperature-controlled promoters are limited. In this study, we explored a novel HI promoter through PCR amplification using the degenerate primer design. BLAST analysis identified this sequence as a partial plsB on Escherichia coli chromosome. Antisense RNAs targeting distinct regions of HI promoter were applied to examine the mechanism. The fluorescence of super-folder green fluorescent protein (sfGFP) driven by the HI promoter showed 5.5-fold increase from 30 to 42 oC, whereas the truncated HI yielded a trace amount of sfGFP. Among various E. coli strains, BL21 exhibited the highest fluorescence, reaching 3976 a.u. at 42°C. Subsequently, the novel HI promoter was employed to drive T7RNA polymerase within a plasmid-driven T7 (PDT7) plasmid, serving its capability to express sfGFP and carbonic anhydrases (CA), respectively. The maximum intensity of sfGFP reached 38702 a.u., and CA activity surged to 14286 WAU/mL in W3110 among other strains. Finally, the highest CA activity was 27521 WAU/mL at alkali pH 9. The promising results from the novel heat-inducible promoter-driven T7RNAP, incorporating the T7 terminator as HI-PDT7-TT, demonstrate potential for expressing more heterogeneous proteins across various chassis in the future.
期刊介绍:
Process Biochemistry is an application-orientated research journal devoted to reporting advances with originality and novelty, in the science and technology of the processes involving bioactive molecules and living organisms. These processes concern the production of useful metabolites or materials, or the removal of toxic compounds using tools and methods of current biology and engineering. Its main areas of interest include novel bioprocesses and enabling technologies (such as nanobiotechnology, tissue engineering, directed evolution, metabolic engineering, systems biology, and synthetic biology) applicable in food (nutraceutical), healthcare (medical, pharmaceutical, cosmetic), energy (biofuels), environmental, and biorefinery industries and their underlying biological and engineering principles.