Part:BBa_K4130004

Choline Oxidase (CodA)

This part encodes the gene for the protein choline oxidase, capable of catalyzing the conversion of choline to glycine betaine.

Biology

Conversion of choline to glycine betaine is an important biological activity, critical to the maintenance of osmotic stability [1]. Without the controlled conversion of choline to glycine betaine, organisms are unable to compensate for high salinity environments [2]. In bacterial microorganisms, glycine betaine is key to survival at human infection sites [3]. In humans, the enzyme associated with the production of glycine betaine has been connected to numerous pathologies including male infertility, metabolic syndrome, cardiovascular disease risk, and breast cancer [4].

Enzymes responsible for conversion of choline to glycine betaine vary across and within the kingdoms of life [4, 5]. In humans, a combination of choline dehydrogenase and betaine aldehyde dehydrogenase catalyze the conversion of choline to glycine betaine [4]. Alternatively, in bacterial microorganisms such as Arthrobacter globiformis, this reaction is catalyzed by a single enzyme: choline oxidase [4,6]. Despite the similarities in reaction substrate and products, the enzymes choline oxidase and choline dehydrogenase vary greatly in in vitro properties. Numerous attempts to purify choline dehydrogenase have resulted in contamination and low product stability [4]. Conversely, choline oxidase has been called a “dream enzyme” for purification and in vitro studies [7]. Accordingly, previous studies have been successful in producing detailed crystal structures and catalytic mechanisms [7].

Usage

Applications of choline oxidase typically involve genetic engineering of crops to increase water and osmotic stress resistance. For example, transformation of Arabidopsis thaliana with choline oxidase resulted in enhanced tolerance to salt and cold stress [8]. Alternatively, engineering expression of choline oxidase in tomato plants resulted in salt and water stress resistance [9]. Finally, similar genetic modifications to rice led to salt and cold tolerance [10].

In our project, choline oxidase was applied for biosensing of choline in maple sap. Towards the end of the maple sap collecting season, increases in choline concentration are accompanied by a cabbage-like taste and flavor, yielding maple syrup unsuitable for human consumption. Currently, this cabbage-like effect can only be detected after sap has been transformed to syrup in a time- and energy-intensive process. Thus we sought to create a method of detecting this sap defect before it is transformed to syrup using choline as a biomarker for the defect.

Design

This BioBrick (BBa_K130005) includes the coding sequence for choline oxidase from Arthrobacter globiformis, an enzyme capable of catalyzing the conversion of choline to glycine betaine.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1477
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

• [1] B. Kempf, E. Bremer. Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments. Arch. Microbiol., 170 (1998), pp. 319-330

• [2] E. Bremer, R. Krämer. Responses of microorganisms to osmotic stress. Annu. Rev. Microbiol., 73 (2019), pp. 313-334

• [3] G. Gadda, N.L. Powell, P. Menon. The trimethylammonium headgroup of choline is a major determinant for substrate binding and specificity in choline oxidase. Arch. Biochem. Biophys., 430 (2004), pp. 264-273

• [4] F. Salvi, G. Gadda. Human choline dehydrogenase: medical promises and biochemical challenges. Arch. Biochem. Biophys., 537 (2013), pp. 243-252

• [5] B. Landfald, A. Strom. Choline-glycine betaine pathway confers a high level of osmotic tolerance in Escherichia coli. Journal of Bacteriology., 165 (1986), pp. 849-855.

• [6] S. Ikuta, S. Imamura, H. Misaki, Y. Horiuti. Purification and characterization of choline oxidase from Arthrobacter globiformis. J. Biochem., 82 (1977), pp. 1741-1749

• [7] G. Gadda. Choline Oxidases. P. Chaiyen, F. Tamanoi (Ed.) The Enzymes (137-166) Academic Press.H. Hayashi, et al.

• [8] H. Hayashi, et. al. Transformation of Arabidopsis thaliana with the codA gene for choline oxidase; accumulation of glycinebetaine and enhanced tolerance to salt and cold stress. Plant J., 12 (1997), pp. 133-142

• [9] D. Goel, et. al. Transformation of tomato with a bacterial codA gene enhances tolerance to salt and water stresses. J. Plant Physiol., 168 (2011), pp. 1286-1294

• [10] A. Sakamoto, N.M. Alia. Metabolic engineering of rice leading to biosynthesis of glycinebetaine and tolerance to salt and cold. Plant Mol. Biol., 38 (1998), pp. 1011-1019

• [11] Godat, B, et al. “MagneHis™ Protein Purification System: Purification of His-Tagged Proteins in Multiple Formats.” Attractive Protein Purification, 2003.

• [12] Taylor, Fred H. Variation in Sugar Content of Maple Sap. University of Vermont and State Agricultural College, Mar. 1956, https://www.uvm.edu/~uvmaple/sapsugarcontentvariation.pdf.