Skip to main content
SpringerLink
Log in

Broad-specificity amino acid racemase, a novel non-antibiotic selectable marker for transgenic plants

  • Original Article
  • Published:
Plant Biotechnology Reports Aims and scope Submit manuscript

Abstract

The broad-specificity amino acid racemase (Bsar) from Pseudomonas putida catalyzes the racemization of various amino acids, offering a flexible and feasible platform to develop a new non-antibiotic selectable marker system for plant transformation. In the present study, we demonstrated that a Bsar variant, Bsar-R174K, that is useful as a selectable marker gene in Arabidopsis and rice that were susceptible to l-lysine and D-alanine. The introduction of wild-type Bsar, Bsar-R174K or Bsar-R174A into E. coli lysine or asparagine auxotrophs was able to rescue the growth of these microorganisms in minimal media supplemented with selectable amino acid enantiomers. The transformation of Arabidopsis with Bsar or Bsar variants based on d-alanine selection revealed that Bsar-R174K had the greatest efficiency (2.40%), superior to kanamycin selection-based transformation (1.10%). Whereas, l-lysine-based selection exhibited lower efficiency for Bsar-R174K (0.17%). The progenies of selected Bsar-R174K transgenic Arabidopsis revealed normal growth properties. In addition, Bsar-R174K transgenic rice was obtained on l-lysine medium with an efficiency of 0.9%, and the progenies of the transgenic rice revealed morphologically normal phenotypes comparable with their wild-type counterparts. This study presents the first report of broad range amino acid racemase Bsar-R174K as a non-antibiotic selectable marker system applied in transgenic plants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  • Bevan MW, Flavell RB, Chilton MD (1983) A chimaeric antibiotic resistance gene as a selectable marker for plant cell transformation. Nature 304:184–187

    Article  CAS  Google Scholar 

  • Block DM, Brouwer DD, Tenning P (1989) Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the bar and neo genes in the transgenic plants. Plant Physiol 91:694–701

    Article  PubMed  PubMed Central  Google Scholar 

  • Breyer D, Kopertekh L, Reheul D (2014) Alternatives to antibiotic resistance marker genes for in vitro selection of genetically modified plants-scientific developments, current use, operational access and biosafety considerations. Crit Rev Plant Sci 33:286–330

    Article  CAS  Google Scholar 

  • Brookes G, Barfoot P (2014) Economic impact of GM crops: the global income and production effects 1996–2012. GM Crops Food 5:65–75

    Article  PubMed  PubMed Central  Google Scholar 

  • Chang YF, Adams E (1974) d-lysine catabolic pathway in Pseudomonas putida: interrelations with l-lysine catabolism. J Bacteriol 117:753–764

    CAS  PubMed  PubMed Central  Google Scholar 

  • Chen PY, Wang CK, Soong SC, To KY (2003) Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants. Mol Breed 11:287–293. https://doi.org/10.1023/A:1023475710642

    Article  CAS  Google Scholar 

  • Chen IC, Lin WD, Hsu SK, Thiruvengadam V, Hsu WH (2009) Isolation and characterization of a novel lysine racemase from a soil metagenomic library. Appl Environ Microbiol 75:5161–5166

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen IC, Thiruvengadam V, Lin WD, Chang HH, Hsu WH (2010) Lysine racemase: a novel non-antibiotic selectable marker for plant transformation. Plant Mol Biol 72:153–169

    Article  CAS  PubMed  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16 16:735–743

    Article  PubMed  Google Scholar 

  • De Block M, De Brouwer D, Tenning P (1989) Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the bar and neo genes in the transgenic plants. Plant Physiol 91:694–701

    Article  PubMed  PubMed Central  Google Scholar 

  • Dessens JT, Lomonossoff GP (1993) Cauliflower mosaic virus 35S promoter-controlled DNA copies of cowpea mosaic virus RNAs are infectious on plants. J Gen Virol 74:889–892

    Article  CAS  PubMed  Google Scholar 

  • Ebmeier A, Allison L, Cerutti H, Clemente T (2004) Evaluation of the Escherichia coli threonine deaminase gene as a selectable marker for plant transformation. Planta 218:751–758

    Article  CAS  PubMed  Google Scholar 

  • Erikson O, Hertzberg M, Nasholm T (2004) A conditional marker gene allowing both positive and negative selection in plants. Nat Biotechnol 22:455–458

    Article  CAS  PubMed  Google Scholar 

  • Erikson O, Hertzberg M, Nasholm T (2005) The dsdA gene from Escherichia coli provides a novel selectable marker for plant transformation. Plant Mol Biol 57:425–433

    Article  CAS  PubMed  Google Scholar 

  • Fraley RT, Rogers SG, Horsch RB, Sanders PR, Flick JS, Adams SP, Bittner ML, Brand LA, Fink CL, Fry JS, Galluppi GR, Goldberg SB, Hoffmann NL, Woo SC (1983) Expression of bacterial genes in plant cells. Proc Natl Acad Sci USA 80:4803–4807

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gay PB, Gillespie SH (2005) Antibiotic resistance markers in genetically modified plants: a risk to human health? Lancet Infect Dis 5:637–646

    Article  CAS  PubMed  Google Scholar 

  • Gogami Y, Ito K, Kamitani Y, Matsushima Y, Oikawa T (2009) Occurrence of d-serine in rice and characterization of rice serine racemase. Phytochemistry 70:380–387

    Article  CAS  PubMed  Google Scholar 

  • Gordes D, Kolukisaoglu U, Thurow K (2011) Uptake and conversion of d-amino acids in Arabidopsis thaliana. Amino Acids 40:553–563

    Article  PubMed  Google Scholar 

  • Haldrup A, Petersen SG, Okkels FT (1998) The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using d-xylose as the selection agent. Plant Mol Biol 37:287–296

    Article  CAS  PubMed  Google Scholar 

  • Haroun SA, Shukry WM, El-Sawy O (2010) Effect of asparagine or glutamine on growth and metabolic changes in phaseolus vulgaris under in vitro conditions. Biosci Res 7:01–21

    Google Scholar 

  • Herrera-Estrella L, Depicker A, Van Montagu M, Schell J (1983) Expression of chimaeric genes transferred into plant cells using a Ti-plasmid-derived vector. Nature 303:209–213

    Article  CAS  Google Scholar 

  • Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282

    Article  CAS  PubMed  Google Scholar 

  • Huang HT, Davisson JW (1958) Distribution of lysine racemase in bacteria. J Bacteriol 76:495–498

    CAS  PubMed  PubMed Central  Google Scholar 

  • Joersbo M, Kreiberg DI, Petersen J, Brunstedt SG, Okkels J F (1998) Analysis of mannose selection used for transformation of sugar beet. Mol Breed 4:111–117

    Article  CAS  Google Scholar 

  • Joersbo M, Joregensen K, Brunstedt J (2003) A selection system for transgenic plants based on galactose as selective agent and a UDP-glucose: galactose-1-phosphate uridyltransferase gene as selective gene. Mol Breed 11:315–323

    Article  CAS  Google Scholar 

  • Kino K, Sato M, Yoneyama M, Kirimura K (2007) Synthesis of DL-tryptophan by modified broad specificity amino acid racemase from Pseudomonas putida IFO 12996. Appl Microbiol Biotechnol 73:1299–1305

    Article  CAS  PubMed  Google Scholar 

  • Kuan YC, Kao CH, Chen CH, Chen CC, Hu HY, Hsu WH (2011) Biochemical characterization of a novel lysine racemase from Proteus mirabilis BCRC10725. Process Biochem 46:1914–1920

    Article  CAS  Google Scholar 

  • LaFayette PR, Kane PM, Phan BH, Parrott WA (2005) Arabitol dehydrogenase as a selectable marker for rice. Plant Cell Rep 24:596–602. https://doi.org/10.1007/s00299-00005-00015-00293

    Article  CAS  PubMed  Google Scholar 

  • Luo K, Zheng X, Chen Y, Xiao Y, Zhao D, McAvoy R, Pei Y, Li Y (2006) The maize Knotted1 gene is an effective positive selectable marker gene for Agrobacterium-mediated tobacco transformation. Plant Cell Rep 25:403–409

    Article  CAS  PubMed  Google Scholar 

  • Ono K, Yanagida K, Oikawa T, Ogawa T, Soda K (2006) Alanine racemase of alfalfa seedlings (Medicago sativa L.): first evidence for the presence of an amino acid racemase in plants. Phytochemistry 67:856–860

    Article  CAS  PubMed  Google Scholar 

  • Pilone MS (2000) d-Amino acid oxidase: new findings. Cell Mol Life Sci 57:1732–1747

    Article  CAS  PubMed  Google Scholar 

  • Schonbrunn E, Eschenburg S, Shuttleworth WA, Schloss JV, Amrhein N, Evans JN, Kabsch W (2001) Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail. Proc Natl Acad Sci USA 98:1376–1380

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sundar IK, Sakthivel N (2008) Advances in selectable marker genes for plant transformation. J Plant Physiol 165:1698–1716

    Article  CAS  PubMed  Google Scholar 

  • van den Elzen PJM, Townsend J, Kathleen Y, Lee JR, Bedbrook (1985) A chimaeric hygromycin resistance gene as a selectable marker in plant cells. Plant Mol Biol 5:299–302

    Article  PubMed  Google Scholar 

  • Vranova V, Zahradnickova H, Janous D, Skene KR, Matharu AS, Rejsek P, Formanek P (2012) The significance of d-amino acids in soil, fate and utilization by microbes and plants: review and identification of knowledge gaps. Plant Soil 354:21–39

    Article  CAS  Google Scholar 

  • Waldron C, Murphy EB, Roberts JL, Gustafson GD, Armour SL, Malcolm SK (1985) Resistance to hygromycin B. Plant Mol Biol 5:103–108

    Article  CAS  PubMed  Google Scholar 

  • Wu HM, Kuan YC, Chu CH, Hsu WH, Wang WC (2012) Crystal structures of lysine-preferred racemases, the non-antibiotic selectable markers for transgenic plants. PLoS One 7:e48301

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was financially supported through grants from the Ministry of Science and Technology, Taiwan (101-2321-B-007-004, 102-2321-B-007-003, and 103-2321-B-007 -002).

Author information

Authors and Affiliations

  1. Institute of Molecular and Cellular Biology and Department of Life Sciences, National Tsing-Hua University, Hsinchu, 30013, Taiwan

    Yi-Chia Kuan, Jia-Hsin Liu, Tsan-Jan Chen, Hsin-Mao Wu & Wen-Ching Wang

  2. Institute of Molecular Biology, National Chung Hsing University, Taichung, 40227, Taiwan

    Yi-Chia Kuan, Jia-Shin Lin, Wen-Ching Wang & Liang-Jwu Chen

  3. Department of Biotechnology, Hungkuang University, Taichung, 43302, Taiwan

    Yi-Chia Kuan

  4. Department of Plant Genetic Resources, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, 641003, India

    Venkatesan Thiruvengadam

  5. Agricultural Biotechnology Center, National Chung Hsing University, Taichung, 40227, Taiwan

    Liang-Jwu Chen

Authors
  1. Yi-Chia Kuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Venkatesan Thiruvengadam
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jia-Shin Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jia-Hsin Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tsan-Jan Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hsin-Mao Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wen-Ching Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Liang-Jwu Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Wen-Ching Wang or Liang-Jwu Chen.

Ethics declarations

Conflict of interest

The authors declare no competing financial interests.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 13 KB)

Supplementary material 2 (PPTX 9462 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kuan, YC., Thiruvengadam, V., Lin, JS. et al. Broad-specificity amino acid racemase, a novel non-antibiotic selectable marker for transgenic plants. Plant Biotechnol Rep 12, 27–38 (2018). https://doi.org/10.1007/s11816-018-0469-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11816-018-0469-8

Keywords

Search

Navigation