Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Identification and Validation of Four Novel Promoters for Gene Engineering with Broad Suitability across Species

  • Wang, Cai-Yun (School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University) ;
  • Liu, Li-Cheng (School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University) ;
  • Wu, Ying-Cai (School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University) ;
  • Zhang, Yi-Xuan (School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University)
  • Received : 2021.03.29
  • Accepted : 2021.06.27
  • Published : 2021.08.28
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Abstract

The transcriptional capacities of target genes are strongly influenced by promoters, whereas few studies have focused on the development of robust, high-performance and cross-species promoters for wide application in different bacteria. In this work, four novel promoters (Pk.rtufB, Pk.r1, Pk.r2, and Pk.r3) were predicted from Ketogulonicigenium robustum and their inconsistency in the -10 and -35 region nucleotide sequences indicated they were different promoters. Their activities were evaluated by using green fluorescent protein (gfp) as a reporter in different species of bacteria, including K. vulgare SPU B805, Pseudomonas putida KT2440, Paracoccus denitrificans PD1222, Bacillus licheniformis and Raoultella ornithinolytica, due to their importance in metabolic engineering. Our results showed that the four promoters had different activities, with Pk.r1 showing the strongest activity in almost all of the experimental bacteria. By comparison with the commonly used promoters of E. coli (tufB, lac, lacUV5), K. vulgare (Psdh, Psndh) and P. putida KT2440 (JE111411), the four promoters showed significant differences due to only 12.62% nucleotide similarities, and relatively higher ability in regulating target gene expression. Further validation experiments confirmed their ability in initiating the target minCD cassette because of the shape changes under the promoter regulation. The overexpression of sorbose dehydrogenase and cytochrome c551 by Pk.r1 and Pk.r2 resulted in a 22.75% enhancement of 2-KGA yield, indicating their potential for practical application in metabolic engineering. This study demonstrates an example of applying bioinformatics to find new biological components for gene operation and provides four novel promoters with broad suitability, which enriches the usable range of promoters to realize accurate regulation in different genetic backgrounds.

Keywords

Acknowledgement

This work was supported by grants from the National Science and Technology Fundamental Resources Investigation Program of China (2019FY100700), the Scientific Research Fund of Education Department of Liaoning Province (2019LJC10), the Natural Science Foundation of Liaoning Province (XLYC1902072) and the Doctoral Research Start-up Fund Project of Liaoning Provience (2020-BS-122).

References

  1. Rodriguez A, Kildegaard KR, Li M, Borodina I, Nielsen J. 2015. Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis. Metab. Eng. 31: 181-188. https://doi.org/10.1016/j.ymben.2015.08.003
  2. Park SH, Kim HU, Kim TY, Park JS, Kim SS, Lee SY. 2014. Metabolic engineering of Corynebacterium glutamicum for L-arginine production. Nat. Commun. 5: 4618. https://doi.org/10.1038/ncomms5618
  3. Dusseaux S, Croux C, Soucaille P, Meynial-Salles I. 2013. Metabolic engineering of Clostridium acetobutylicum ATCC 824 for the high-yield production of a biofuel composed of an isopropanol/butanol/ethanol mixture. Metab. Eng. 18: 1-8. https://doi.org/10.1016/j.ymben.2013.03.003
  4. Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, et al. 2013. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496: 528-532. https://doi.org/10.1038/nature12051
  5. Chinen A, Kozlov YI, Hara Y, Izui H, Yasueda H. 2007. Innovative metabolic pathway design for efficient L-glutamate production by suppressing CO2 emission. J. Biosci. Bioeng. 103: 262-269. https://doi.org/10.1263/jbb.103.262
  6. Du J, Bai W, Song H, Yuan YJ. 2013. Combinational expression of sorbose/sorbosone dehydrogenases and cofactor pyrroloquinoline quinone increases 2-keto-L-gulonic acid production in Ketogulonigenium vulgare-Bacillus cereus consortium. Metab. Eng. 19: 50-56. https://doi.org/10.1016/j.ymben.2013.05.006
  7. Gao L, Hu Y, Liu J, Du G, Zhou J, Chen J. 2014. Stepwise metabolic engineering of Gluconobacter oxydans WSH-003 for the direct production of 2-keto-L-gulonic acid from D-sorbitol. Metab. Eng. 24: 30-37. https://doi.org/10.1016/j.ymben.2014.04.003
  8. Zhao H, Zhang HM, Chen X, Li T, Wu Q, Ouyang Q, et al. 2017. Novel T7-like expression systems used for Halomonas. Metab. Eng. 39: 128-140. https://doi.org/10.1016/j.ymben.2016.11.007
  9. Shi L, Li K, Zhang H, Liu X, Lin J, Wei D. 2014. Identification of a novel promoter gHp0169 for gene expression in Gluconobacter oxydans. J. Biotechnol. 175: 69-74. https://doi.org/10.1016/j.jbiotec.2014.01.035
  10. Shen R, Yin J, Ye JW, Xiang RJ, Ning ZY, Huang WZ, et al. 2018. Promoter engineering for enhanced P(3HB- co-4HB) production by Halomonas bluephagenesis. ACS Synth. Biol. 7: 1897-1906. https://doi.org/10.1021/acssynbio.8b00102
  11. Theisen MK, Lafontaine Rivera JG, Liao JC. 2016. Stability of ensemble models predicts productivity of enzymatic systems. PLoS Comput. Biol. 12:e1004800. https://doi.org/10.1371/journal.pcbi.1004800
  12. Wang CY, Li Y, Gao ZW, Liu LC, Zhang MY, Zhang TY, et al. 2018. Establishing an innovate carbohydrate metabolic pathway for efficient production of 2-keto-L-gulonic acid in Ketogulonicigenium robustum initiated by intronic promoters. Microb. Cell Fact. 17: 81. https://doi.org/10.1186/s12934-018-0932-9
  13. Wang CY, Li Y, Gao ZW, Liu LC, Wu YC, Zhang MY, et al. 2018. Reconstruction and analysis of carbon metabolic pathway of Ketogulonicigenium vulgare SPU B805 by genome and transcriptome. Sci. Rep. 8: 17838. https://doi.org/10.1038/s41598-018-36038-3
  14. Simon O, Klebensberger J, Mukschel B, Klaiber I, Graf N, Altenbuchner J, et al. 2015. Analysis of the molecular response of Pseudomonas putida KT2440 to the next-generation biofuel n-butanol. J. Proteomics 122: 11-25. https://doi.org/10.1016/j.jprot.2015.03.022
  15. Graf N, Altenbuchner J. 2014. Genetic engineering of Pseudomonas putida KT2440 for rapid and high-yield production of vanillin from ferulic acid. Appl. Microbiol. Biotechnol. 98: 137-149. https://doi.org/10.1007/s00253-013-5303-1
  16. Nikel PI, de Lorenzo V. 2014. Robustness of Pseudomonas putida KT2440 as a host for ethanol biosynthesis. N. Biotechnol. 31: 562-571. https://doi.org/10.1016/j.nbt.2014.02.006
  17. Tiso T, wierckx N, Blank LM. 2014. PanStanford Publishing, pp. 317-366. In Grunwald, P. (eds.)., Industrial Biocatalysis, Non-Pathogenic Pseudomonas as platform for industrial biocatalysis, Singapore.
  18. Kalaiyezhini D, Ramachandran KB. 2015. Biosynthesis of Poly-3-Hydroxybutyrate (PHB) from Glycerol by Paracoccus denitrificansin a batch bioreactor: Effect of process variables. Prep. Biochem. Biotechnol. 45: 69-83. https://doi.org/10.1080/10826068.2014.887582
  19. Yamane T, Chen XF, Ueda S. 1996. Growth-associated production of poly(3-Hydroxyvalerate) from n-pentanol by a methylotrophic bacterium, Paracoccus denitrificans. Appl. Environ. Microbiol. 62: 380-384. https://doi.org/10.1128/aem.62.2.380-384.1996
  20. Ueda S, Matsumoto S, Takagi A, Yamane T. 1992. Synthesis of poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) from methanol and n-amyl alcohol by the methylotrophic bacteria Paracoccus denitrificans and Methylobacterium extorquens. Appl. Environ. Microbiol. 58: 3574-3579. https://doi.org/10.1128/aem.58.11.3574-3579.1992
  21. Song CW, Chelladurai R, Park JM, Song H. 2019. Engineering a newly isolated Bacillus licheniformis strain for the production of (2R,3R)-butanediol. J. Ind. Microbiol. Biotechnol. 47: 97-108. https://doi.org/10.1007/s10295-019-02249-4
  22. Lu C, Ge Y, Cao M, Guo X, Liu P, Gao C, et al. 2020. Metabolic engineering of Bacillus licheniformis for production of acetoin. Front. Bioeng. Biotechnol. 8: 125. https://doi.org/10.3389/fbioe.2020.00125
  23. Falade AO, Mabinya LV, Okoh AI, Nwodo UU. 2019. Biochemical and molecular characterization of a novel dye-decolourizing peroxidase from Raoultella ornithinolytica OKOH-1. Int. J. Biol. Macromol. 121: 454-462. https://doi.org/10.1016/j.ijbiomac.2018.10.045
  24. Hii LS, Rosfarizan M, Ling TC, Ariff AB. 2010. Statistical optimization of pullulanase production by Raoultella planticola DSMZ 4617 using sago starch as carbon and peptone as nitrogen sources. Food Bioprocess Technol. 5: 729-737. https://doi.org/10.1007/s11947-010-0368-7
  25. Hossain GS, Yuan H, Li J, Shin HD, Wang M, Du G, et al. 2017. Metabolic engineering of Raoultella ornithinolytica BF60 for production of 2,5-furandicarboxylic acid from 5-hydroxymethylfurfural. Appl. Environ. Microbiol. 83: e02312-02316.
  26. Kovach ME, Elzer PH, Steven Hill D, Robertson GT, Farris MA, Roop RM, et al. 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166: 175-176. https://doi.org/10.1016/0378-1119(95)00584-1
  27. Solovyev V, Salamov A. 2011. Nova Science Publishers, pp. 61-78. In Li RW (eds.), Metagenomics and its applications in agriculture, biomedicine and environmental studies, Automatic Annotation of Microbial Genomes and Metagenomic Sequences, New York, USA.
  28. de Avila ESS, Echeverrigaray S, Gerhardt GJ. 2011. BacPP: bacterial promoter prediction--a tool for accurate sigma-factor specific assignment in enterobacteria. J. Theor. Biol. 287: 92-99. https://doi.org/10.1016/j.jtbi.2011.07.017
  29. Dominguez W, O'Sullivan DJ. 2013. Developing an efficient and reproducible conjugation-based gene transfer system for bifidobacteria. Microbiology 159: 328-338. https://doi.org/10.1099/mic.0.061408-0
  30. Kotnik T, Frey W, Sack M, Haberl Meglic S, Peterka M, Miklavcic D. 2015. Electroporation-based applications in biotechnology. Trends Biotechnol. 33: 480-488. https://doi.org/10.1016/j.tibtech.2015.06.002
  31. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. 2008. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5: 621-628. https://doi.org/10.1038/nmeth.1226
  32. Elmore JR, Furches A, Wolff GN, Gorday K, Guss AM. 2017. Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440. Metab. Eng. Commun. 5: 1-8. https://doi.org/10.1016/j.meteno.2017.04.001
  33. Fu S, Zhang W, Guo A, Wang J. 2007. Identification of promoters of two dehydrogenase genes in Ketogulonicigenium vulgare DSM 4025 and their strength comparison in K. vulgare and Escherichia coli. Appl. Microbiol. Biotechnol. 75: 1127-1132. https://doi.org/10.1007/s00253-007-0930-z
  34. An G, Friesen JD. 1980. The nucleotide sequence of tufB and four nearby tRNA structurel genes of Escherichia coli. Gene 12: 33-39. https://doi.org/10.1016/0378-1119(80)90013-X
  35. Shin SH, Um Y, Beak JH, Kim S, Lee S, Oh MK,et al. 2013. Complete genome sequence of Raoultella ornithinolytica strain B6, a 2,3-butanediol-producing bacterium isolated from oil-contaminated soil. Genome Announc. 1: e00395-00313.
  36. Rothfield L, Justice S, Garcia-Lara J. 1999. Bacterial cell division. Annu. Rev. Genet. 33: 423-448. https://doi.org/10.1146/annurev.genet.33.1.423
  37. de Boer PA, Crossley RE, Rothfield LI. 1989. A division inhibitor and a topological specificity factor coded for by the minicell locus determine proper placement of the division septum in E. coli. Cell 56: 641-649. https://doi.org/10.1016/0092-8674(89)90586-2
  38. Sass P, Josten M, Famulla K, Schiffer G, Sahl H, Hamoen L, et al. 2011. Antibiotic acyldepsipeptides activate ClpP peptidase to degrade the cell division protein FtsZ. PNAS 108: 17474-17479. https://doi.org/10.1073/pnas.1110385108
  39. Wang P, Zeng W, Xu S, Du G, Zhou J, Chen J. 2018. Current challenges facing one-step production of L-ascorbic acid. Biotechnol. Adv. 36: 1882-1899. https://doi.org/10.1016/j.biotechadv.2018.07.006