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Identification and application of a novel strong constitutive promoter in Corynebacterium glutamicum

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Abstract

The replacement of promoters with various strengths is an effective strategy to fine-tune gene expression. More available promoters with a broad range of transcription efficiency are needed in metabolic engineering. In this study, a putative protein coding gene CP_2454 was identified with a stable and high transcriptional level from an l-leucine-producing strain Corynebacterium glutamicum CP, which was absent in wild-type C. glutamicum ATCC 13032. The transcriptional level of CP_2454 was about 80.0% those of tuf and sod, and was 1.6 and 3.2 times those of ilvB and gapA, respectively. These promoters were cloned into C. glutamicum ATCC 13032 to control the expression of green fluorescent protein (GFP). While, the expressional level of GFP under the control of PCP_2454 was close to that of Ptuf and Psod, which was significantly higher than that of other tested promoters. The native promoter of ilvB controlling the expression of a feedback-resistant acetolactate synthase was replaced by PCP_2454, resulting in an increase of l-valine titer by 58.5%. A further 24.9% increase of l-valine titer was achieved after replacement of the promoter of ilvD encoding dihydroxyacid dehydratase with PCP_2454. Identification of the constitutive promoter PCP_2454 expands the promoter library of C. glutamicum and provides essential reference for tunable expression of target genes in C. glutamicum.

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References

  • Akyol I, Comlekcioglu U, Karakas A, Serdaroglu K, Ekinci MS, Ozkose E (2008) Regulation of the acid inducible rcfB promoter in Lactococcus lactis subsp. lactis. Ann Microbiol 58:269–273

    Article  CAS  Google Scholar 

  • Baumgart M, Frunzke J (2015) The manganese-responsive regulator MntR represses transcription of a predicted ZIP family metal ion transporter in Corynebacterium glutamicum. FEMS Microbiol Lett 362:1–10

    Article  PubMed  CAS  Google Scholar 

  • Chung SC, Park JS, Yun J (2017) Improvement of succinate production by release of end-product inhibition in Corynebacterium glutamicum. Metab Eng 40:157–164

    Article  PubMed  CAS  Google Scholar 

  • Davey JW, Hohenlohe PA, Etter PD, Boone JQ, Catchen JM, Blaxter ML (2011) Genome-wide genetic marker discovery and genotyping using next-generation sequencing. Nat Rev Genet 12:499–510

    Article  PubMed  CAS  Google Scholar 

  • Gerstmeir R, Wendisch VF, Schnicke S, Ruan H, Farwick M, Reinscheid D, Eikmanns BJ (2003) Acetate metabolism and its regulation in Corynebacterium glutamicum. J Biotechnol 104:99–122

    Article  PubMed  CAS  Google Scholar 

  • Gui Y, Ma Y, Xu Q, Zhang C, Xie X, Chen N (2016) Complete genome sequence of Corynebacterium glutamicum CP, a Chinese l-leucine producing strain. J Biotechnol 220:64–65

    Article  PubMed  CAS  Google Scholar 

  • Harnpicharnchai P, Promdonkoy P, Sae-Tang K, Roongsawang N, Tanapongpipat S (2014) Use of the glyceraldehyde-3-phosphate dehydrogenase promoter from a thermotolerant yeast, Pichia thermomethanolica, for heterologous gene expression, especially at elevated temperature. Ann Microbiol 64:1457–1462

    Article  CAS  Google Scholar 

  • Ikeda M, Mizuno Y, Awane S, Hayashi M, Mitsuhashi S, Takeno S (2011) Identification and application of a different glucose uptake system that functions as an alternative to the phosphotransferase system in Corynebacterium glutamicum. Appl Microbiol Biotechnol 90:1443–1451

    Article  PubMed  CAS  Google Scholar 

  • Jakoby M, Ngouoto-Nkili CE, Burkovski A (1999) Construction and application of new Corynebacterium glutamicum vectors. Biotechnol Tech 13:437–441

    Article  CAS  Google Scholar 

  • Kind S, Neubauer S, Becker J, Yamamoto M, Völkert M, Abendroth GV, Zelder O, Wittmann C (2014) From zero to hero—production of bio-based nylon from renewable resources using engineered Corynebacterium glutamicum. Metab Eng 25:113–123

    Article  PubMed  CAS  Google Scholar 

  • Liu Y, Zhuang Y, Ding D, Xu Y, Sun J, Zhang D (2017) Biosensor-based evolution and elucidation of a biosynthetic pathway in Escherichia coli. ACS Synth Biol 6:837–848

    Article  PubMed  CAS  Google Scholar 

  • Malakar P, Venkatesh KV (2012) Effect of substrate and IPTG concentrations on the burden to growth of Escherichia coli on glycerol due to the expression of Lac proteins. Appl Microbiol Biotechnol 93:2543–2549

    Article  PubMed  CAS  Google Scholar 

  • Nešvera J, Pátek M (2011) Tools for genetic manipulations in Corynebacterium glutamicum and their applications. Appl Microbiol Biotechnol 90:1641–1654

    Article  PubMed  CAS  Google Scholar 

  • Neuner A, Heinzle E (2015) Mixed glucose and lactate uptake by Corynebacterium glutamicum through metabolic engineering. Biotechnol J 6:318–329

    Article  CAS  Google Scholar 

  • Okibe N, Suzuki N, Inui M, Yukawa H (2010) Isolation, evaluation and use of two strong, carbon source-inducible promoters from Corynebacterium glutamicum. Lett Appl Microbiol 50:173–180

    Article  PubMed  CAS  Google Scholar 

  • Pátek M, Nešvera J (2011) Sigma factors and promoters in Corynebacterium glutamicum. J Biotechnol 154:101–113

    Article  PubMed  CAS  Google Scholar 

  • Plassmeier JK, Busche T, Molck S, Persicke M, Pühler A, Rückert C, Kalinowski J (2013) A propionate-inducible expression system based on the Corynebacterium glutamicum prpD2, promoter and PrpR activator and its application for the redirection of amino acid biosynthesis pathways. J Biotechnol 163:225–232

    Article  PubMed  CAS  Google Scholar 

  • Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73

    Article  PubMed  Google Scholar 

  • Scheele S, Oertel D, Bongaerts J, Evers S, Hellmuth H, Maurer KH, Bott M, Freudl R (2013) Secretory production of an FAD cofactor-containing cytosolic enzyme (sorbitol–xylitol oxidase from Streptomyces coelicolor) using the twin-arginine translocation (Tat) pathway of Corynebacterium glutamicum. Microb Biotechnol 6:202–206

    Article  PubMed  CAS  Google Scholar 

  • Siebert D, Wendisch VF (2015) Metabolic pathway engineering for production of 1,2-propanediol and 1-propanol by Corynebacterium glutamicum. Biotechnol Biofuels 8:91

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Solovyev V, Salamov A (2011) Automatic annotation of microbial genomes and metagenomic sequences. Metagenomics Appl Agric Biomed Environ 2011:61–78

    Google Scholar 

  • Sun Y, Guo W, Wang F, Peng F, Yang Y, Dai X, Liu X, Bai Z (2016) Transcriptome and multivariable data analysis of Corynebacterium glutamicum under different dissolved oxygen conditions in bioreactors. PLoS One 11:e0167156

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Tateno T, Fukuda H, Kondo A (2007) Direct production of l-lysine from raw corn starch by Corynebacterium glutamicum secreting Streptococcus bovis alpha-amylase using cspB promoter and signal sequence. Appl Microbiol Biotechnol 77:533–541

    Article  PubMed  CAS  Google Scholar 

  • Teramoto H, Inui M, Yukawa H (2011) Transcriptional regulators of multiple genes involved in carbon metabolism in Corynebacterium glutamicum. J Biotechnol 154:114–125

    Article  PubMed  CAS  Google Scholar 

  • Toyoda K, Inui M (2016) Regulons of global transcription factors in Corynebacterium glutamicum. Appl Microbiol Biotechnol 100:45–60

    Article  PubMed  CAS  Google Scholar 

  • Vogt M, Haas S, Klaffl S, Polen T, Eggeling L, van Ooyen J, Bott M (2013) Pushing product formation to its limit: metabolic engineering of Corynebacterium glutamicum for l-leucine overproduction. Metab Eng 22:40–52

    Article  PubMed  CAS  Google Scholar 

  • Yim SS, An SJ, Kang M, Lee J, Jeong KJ (2013) Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum. Biotechnol Bioeng 110:2959–2969

    Article  PubMed  CAS  Google Scholar 

Download references

Funding

This study was supported by the National Natural Science Foundation of China (31470211 and 31770053), Natural Science Foundation of Tianjin (17JCQNJC09500), and Tianjin Municipal Science and Technology Commission (17YFZCSY01050).

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Author notes

  1. Hongbo Wei and Yuechao Mao contributed equally to this work and should be considered as co-first authors.

Authors and Affiliations

  1. National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science & Technology, Tianjin, 300457, People’s Republic of China

    Hongbo Wei, Yuechao Ma, Qixin Chen, Yi Cui, Lihong Du, Qian Ma, Yanjun Li, Xixian Xie & Ning Chen

  2. Key Laboratory of Microbial Engineering of China Light Industry, Tianjin University of Science & Technology, Tianjin, 300457, People’s Republic of China

    Yuechao Ma, Qian Ma, Yanjun Li, Xixian Xie & Ning Chen

  3. Tianjin Engineering Lab of Efficient and Green Amino Acid Manufacture, Tianjin University of Science & Technology, Tianjin, 300457, People’s Republic of China

    Qian Ma, Yanjun Li, Xixian Xie & Ning Chen

  4. College of Biotechnology, Tianjin University of Science & Technology, No. 29, 13 Main Street, Tianjin Economic and Technological Development Area, Tianjin, 300457, People’s Republic of China

    Xixian Xie & Ning Chen

Authors
  1. Hongbo Wei
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  2. Yuechao Ma
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  4. Yi Cui
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  7. Yanjun Li
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  8. Xixian Xie
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  9. Ning Chen
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Correspondence to Xixian Xie or Ning Chen.

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Wei, H., Ma, Y., Chen, Q. et al. Identification and application of a novel strong constitutive promoter in Corynebacterium glutamicum. Ann Microbiol 68, 375–382 (2018). https://doi.org/10.1007/s13213-018-1344-0

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  • DOI: https://doi.org/10.1007/s13213-018-1344-0

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