Synthetic fused sRNA for the simultaneous repression of multiple genes.
Co-repression
Flux control
Metabolic engineering
Microbial cell factory
Synthetic sRNA
Journal
Applied microbiology and biotechnology
ISSN: 1432-0614
Titre abrégé: Appl Microbiol Biotechnol
Pays: Germany
ID NLM: 8406612
Informations de publication
Date de publication:
Apr 2022
Apr 2022
Historique:
received:
22
01
2022
accepted:
05
03
2022
revised:
03
03
2022
pubmed:
16
3
2022
medline:
12
4
2022
entrez:
15
3
2022
Statut:
ppublish
Résumé
Efficient control over multiple gene expression still presents a major challenge. Synthetic sRNA enables targeted gene expression control in trans without directly modifying the chromosome, but its use to simultaneously target multiple genes can often cause cell growth defects because of the need for additional energy for transcription and lowering of their repression efficiency by limiting the amount of Hfq protein. To address these limitations, we present fusion sRNA (fsRNA) that simultaneously regulates the translation of multiple genes efficiently. It is constructed by linking the mRNA-binding modules for multiple targeted genes in one sRNA scaffold via one-pot generation using overlap extension PCR. The repression capacity of fsRNA was demonstrated by the construction of sRNAs to target four endogenous genes: caiF, hybG, ytfR and minD in Escherichia coli. Their cross-reactivity and the effect on cell growth were also investigated. As practical applications, we applied fsRNA to violacein- and protocatechuic acid-producing strains, resulting in increases of 13% violacein and 81% protocatechuic acid, respectively. The developed fsRNA-mediated multiple gene expression regulation system thus enables rapid and efficient development of optimised cell factories for valuable chemicals without cell growth defects and limiting cellular resources.Key points• Synthetic fusion sRNA (fsRNA)-based system was constructed for the repression of multiple target genes.• fsRNA repressed multiple genes by only expressing a single sRNA while minimising the cellular burden.• The application of fsRNA showed the increased production titers of violacein (13%) and protocatechuic acid (81%).
Identifiants
pubmed: 35291022
doi: 10.1007/s00253-022-11867-5
pii: 10.1007/s00253-022-11867-5
doi:
Substances chimiques
Escherichia coli Proteins
0
Host Factor 1 Protein
0
HybG protein, E coli
0
Molecular Chaperones
0
RNA, Bacterial
0
RNA, Small Untranslated
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
2517-2527Subventions
Organisme : National Research Foundation of Korea
ID : NRF-2022R1A2C2004292
Organisme : Chung-Ang University
ID : Research Scholarship Grants in 2021
Informations de copyright
© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.
Références
Ajiboye TO, Habibu RS, Saidu K, Haliru FZ, Ajiboye HO, Aliyu NO, Ibitoye OB, Uwazie JN, Muritala HF, Bello SA, Yusuf II Mohammed AO (2017) Involvement of oxidative stress in protocatechuic acid-mediated bacterial lethality. Microbiologyopen 6 e00472
Apura P, Saramago M, Peregrina A, Viegas SC, Carvalho SM, Saraiva LM, Arraiano CM, Domingues S (2020) Tailor-made sRNAs: a plasmid tool to control the expression of target mRNAs in Pseudomonas putida. Plasmid 109 102503
Bossi L, Figueroa-Bossi N (2016) Competing endogenous RNAs: a target-centric view of small RNA regulation in bacteria. Nat Rev Microbiol 14:775–784
pubmed: 27640758
Chao CY, Yin MC (2009) Antibacterial effects of roselle calyx extracts and protocatechuic acid in ground beef and apple juice. Foodborne Pathog Dis 6:201–206
pubmed: 19099360
Chappell J, Takahashi MK, Lucks JB (2015) Creating small transcription activating RNAs. Nat Chem Biol 11:214–220
pubmed: 25643173
Chappell J, Westbrook A, Verosloff M, Lucks JB (2017) Computational design of small transcription activating RNAs for versatile and dynamic gene regulation. Nat Commun 8:1051
pubmed: 29051490
pmcid: 5648800
Chen S, Zhang A, Blyn LB, Storz G (2004) MicC, a second small-RNA regulator of Omp protein expression in Escherichia coli. J Bacteriol 186:6689–6697
pubmed: 15466019
pmcid: 522180
Cho C, Lee SY (2017) Efficient gene knockdown in Clostridium acetobutylicum by synthetic small regulatory RNAs. Biotechnol Bioeng 114:374–383
pubmed: 27531464
Choi SY, Yoon KH, Lee JI, Mitchell RJ (2015) Violacein: properties and production of a versatile bacterial pigment. Biomed Res Int 2015 465056
Cress BF, Jones JA, Kim DC, Leitz QD, Englaender JA, Collins SM, Linhardt RJ, Koffas MA (2016) Rapid generation of CRISPR/dCas9-regulated orthogonally repressible hybrid T7-lac promoters for modular tuneable control of metabolic pathway fluxes in Escherichia coli. Nucleic Acids Res 44:4472–4485
pubmed: 27079979
pmcid: 4872105
Duran N, Justo GZ, Ferreira CV, Melo PS, Cordi L, Martins D (2007) Violacein: properties and biological activities. Biotechnol Appl Biochem 48:127–133
pubmed: 17927569
Durante-Rodriguez G, de Lorenzo V, Nikel PI (2018) A post-translational metabolic switch enables complete decoupling of bacterial growth from biopolymer production in engineered Escherichia coli. ACS Synth Biol 7:2686–2697
pubmed: 30346720
Eggenhofer F, Tafer H, Stadler PF, Hofacker IL (2011) RNApredator: fast accessibility-based prediction of sRNA targets. Nucleic Acids Res 39:W149-154
pubmed: 21672960
pmcid: 3125805
Fang H, Li D, Kang J, Jiang P, Sun J, Zhang D (2018) Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12. Nat Commun 9:4917
pubmed: 30464241
pmcid: 6249242
Gao W, He Y, Zhang F, Zhao F, Huang C, Zhang Y, Zhao Q, Wang S, Yang C (2019) Metabolic engineering of Bacillus amyloliquefaciens LL3 for enhanced poly-gamma-glutamic acid synthesis. Microb Biotechnol 12:932–945
pubmed: 31219230
pmcid: 6680638
Green AA, Silver PA, Collins JJ, Yin P (2014) Toehold switches: de-novo-designed regulators of gene expression. Cell 159:925–939
pubmed: 25417166
pmcid: 4265554
Gruber AR, Lorenz R, Bernhart SH, Neubock R, Hofacker IL (2008) The Vienna RNA websuite. Nucleic Acids Res 36:W70-74
pubmed: 18424795
pmcid: 2447809
Hao Y, Xu L, Shi H (2011) Theoretical analysis of catalytic-sRNA-mediated gene silencing. J Mol Biol 406:195–204
pubmed: 21145897
Hawkins M, Atkinson J, McGlynn P (2016) Escherichia coli chromosome copy number measurement using flow cytometry analysis. Methods Mol Biol 1431:151–159
pubmed: 27283308
Hussein R, Lim HN (2011) Disruption of small RNA signaling caused by competition for Hfq. Proc Natl Acad Sci U S A 108:1110–1115
pubmed: 21189298
Ishikawa H, Otaka H, Maki K, Morita T, Aiba H (2012) The functional Hfq-binding module of bacterial sRNAs consists of a double or single hairpin preceded by a U-rich sequence and followed by a 3’ poly(U) tail. RNA 18:1062–1074
pubmed: 22454537
pmcid: 3334693
Jones JA, Vernacchio VR, Lachance DM, Lebovich M, Fu L, Shirke AN, Schultz VL, Cress B, Linhardt RJ, Koffas MA (2015) ePathOptimize: a combinatorial approach for transcriptional balancing of metabolic pathways. Sci Rep 5:11301
pubmed: 26062452
pmcid: 4650668
Jost D, Nowojewski A, Levine E (2011) Small RNA biology is systems biology. BMB Rep 44:11–21
pubmed: 21266101
Jung SW, Yeom J, Park JS, Yoo SM (2021) Recent advances in tuning the expression and regulation of genes for constructing microbial cell factories. Biotechnol Adv 50 107767
Konovalova A, Sogaard-Andersen L, Kroos L (2014) Regulated proteolysis in bacterial development. FEMS Microbiol Rev 38:493–522
pubmed: 24354618
Kües U, Stahl U (1989) Replication of plasmids in gram-negative bacteria. Microbiol Rev 53:491–516
pubmed: 2687680
pmcid: 372750
Levine E, Zhang Z, Kuhlman T, Hwa T (2007) Quantitative characteristics of gene regulation by small RNA. PLoS Biol 5 e229
Long M, Xu M, Qiao Z, Ma Z, Osire T, Yang T, Zhang X, Shao M, Rao Z (2020) Directed evolution of ornithine cyclodeaminase using an evolvR-based growth-coupling strategy for efficient biosynthesis of L-proline. ACS Synth Biol 9:1855–1863
pubmed: 32551572
Malecka EM, Strozecka J, Sobanska D, Olejniczak M (2015) Structure of bacterial regulatory RNAs determines their performance in competition for the chaperone protein Hfq. Biochemistry 54:1157–1170
pubmed: 25582129
Mars RA, Nicolas P, Denham EL, van Dijl JM (2016) Regulatory RNAs in Bacillus subtilis: a Gram-positive perspective on bacterial RNA-mediated regulation of gene expression. Microbiol Mol Biol Rev 80:1029–1057
pubmed: 27784798
pmcid: 5116874
Luo ZW, Kim WJ, Lee SY (2018) Metabolic engineering of Escherichia coli for efficient production of 2-pyrone-46-dicarboxylic acid from glucose. ACS Synth Biol 7:2296–2307
pubmed: 30096230
Mancuso F, Bunkenborg J, Wierer M, Molina H (2012) Data extraction from proteomics raw data: an evaluation of nine tandem MS tools using a large Orbitrap data set. J Proteomics 75:5293–5303
pubmed: 22728601
Miscevic D, Mao JY, Kefale T, Abedi D, Moo-Young M, Perry Chou C (2021) Strain engineering for high-level 5-aminolevulinic acid production in Escherichia coli. Biotechnol Bioeng 118:30–42
pubmed: 32860420
Na D, Yoo SM, Chung H, Park H, Park JH, Lee SY (2013) Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nat Biotechnol 31:170–174
pubmed: 23334451
Noh M, Yoo SM, Kim WJ, Lee S (2017) Gene expression knockdown by modulating synthetic small RNA expression in Escherichia coli. Cell Syst 5 418 426 e4
Noh M, Yoo SM, Yang D, Lee SY (2019) Broad-spectrum gene repression using scaffold engineering of synthetic sRNAs. ACS Synth Biol 8:1452–1461
pubmed: 31132322
Overgaard M, Johansen J, Moller-Jensen J, Valentin-Hansen P (2009) Switching off small RNA regulation with trap-mRNA. Mol Microbiol 73:790–800
pubmed: 19682266
Ou C, Shi N, Yang Q, Zhang Y, Wu Z, Wang B, Compans RW, He C (2014) Protocatechuic acid a novel active substance against avian influenza virus H9N2 infection. PLoS One 9 e111004
Panja S, Schu DJ, Woodson SA (2013) Conserved arginines on the rim of Hfq catalyze base pair formation and exchange. Nucleic Acids Res 41:7536–7546
pubmed: 23771143
pmcid: 3753642
Santiago-Frangos A, Woodson SA (2018) Hfq chaperone brings speed dating to bacterial sRNA. Wiley Interdiscip Rev RNA 9:e1475
Sun T, Li S, Song X, Pei G, Diao J, Cui J, Shi M, Chen L, Zhang W (2018) Re-direction of carbon flux to key precursor malonyl-CoA via artificial small RNAs in photosynthetic Synechocystis sp, PCC 6803. Biotechnol Biofuels 11:26
pubmed: 29441124
pmcid: 5798194
Sun D, Chen J, Wang Y, Li M, Rao D, Guo Y, Chen N, Zheng P, Sun J, Ma Y (2019) Metabolic engineering of Corynebacterium glutamicum by synthetic small regulatory RNAs. J Ind Microbiol Biotechnol 46:203–208
pubmed: 30666532
Taniguchi Y, Choi PJ, Li GW, Chen H, Babu M, Hearn J, Emili A, Xie XS (2010) Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science 329:533–538
pubmed: 20671182
pmcid: 2922915
Tian T, Kang JW, Kang A, Lee TS (2019) Redirecting metabolic flux via combinatorial multiplex CRISPRi-mediated repression for isopentenol production in Escherichia coli. ACS Synth Biol 8:391–402
pubmed: 30681833
Tobie WC (1935) The pigment of Bacillus violaceus: I, the production extraction and purification of violacein. J Bacteriol 29:223–227
pubmed: 16559782
pmcid: 543590
Updegrove TB, Zhang A, Storz G (2016) Hfq: the flexible RNA matchmaker. Curr Opin Microbiol 30:133–138
pubmed: 26907610
pmcid: 4821791
Vigouroux A, Bikard D (2020) CRISPR tools to control gene expression in bacteria. Microbiol Mol Biol Rev 84:e00077-e19
pubmed: 32238445
pmcid: 7117552
Wang M, Herrmann CJ, Simonovic M, Szklarczyk D, von Mering C (2015) Version 4.0 of PaxDb: protein abundance data integrated across model organisms tissues and cell-lines. Proteomics 15:3163–3168
pubmed: 25656970
pmcid: 6680238
Yang D, Kim WJ, Yoo SM, Choi JH, Ha SH, Lee MH, Lee SY (2018) Repurposing type III polyketide synthase as a malonyl-CoA biosensor for metabolic engineering in bacteria. Proc Natl Acad Sci U S A 115:9835–9844
pubmed: 30232266
pmcid: 6176597
Yang S, Wang Y, Wei C, Liu Q, Jin X, Du G, Chen J, Kang Z (2018) A new sRNA-mediated posttranscriptional regulation system for Bacillus subtilis. Biotechnol Bioeng 115:2986–2995
pubmed: 30199104
Yang D, Yoo SM, Gu C, Ryu JY, Lee JE, Lee SY (2019) Expanded synthetic small regulatory RNA expression platforms for rapid and multiplex gene expression knockdown. Metab Eng 54:180–190
pubmed: 30999052
Yeom J, Park JS, Jung SW, Lee S, Kwon H, Yoo SM (2021) High-throughput genetic engineering tools for regulating gene expression in a microbial cell factory. Crit Rev Biotechnol:1–18. https://doi.org/10.1080/07388551.2021.2007351
Yoo SM, Na D, Lee SY (2013) Design and use of synthetic regulatory small RNAs to control gene expression in Escherichia coli. Nat Protoc 8:1694–1707
pubmed: 23928502
Zhang J, Zhao Y, Cao Y, Yu Z, Wang G, Li Y, Ye X, Li C, Lin X, Song H (2020) sRNA-based screening chromosomal gene targets and modular designing Escherichia coli for high-titer production of aglycosylated immunoglobulin G. ACS Synth Biol 9:1385–1394
pubmed: 32396719