Korean J Urol.  2009 Oct;50(10):1022-1026.

Development of Yvrk Gene-Recombinant E. coli Degrading Oxalate

• Jeong BC
• Park YH
• Kim HH

Abstract

PURPOSE
Recently, the whole DNA sequence of Bacillus subtilis (B. subtilis) was identified, revealing the existence of the YvrK gene encoding a 43 kD oxalate decarboxylase (OXDC), which degrades oxalate by a simple pathway. The objective of this study was to develop recombinant Escherichia coli (E. coli) expressing the Yvrk gene from B. subtilis. MATERIALS AND METHODS: After the extraction of total DNA from B. subtilis, the YvrK gene was cloned by polymerase chain reaction. The cloned DNA encoding OXDC was inserted into the pBAD/gIII-A vector, downstream of the L-arabinose promotor. The plasmid vector was transformed into TOP 10 E. coli, and the transformants were selected with ampicillin. The recombinant E. coli, named pBy, was then analyzed by DNA sequencing and Western blot. To evaluate the oxalate-degrading function of pBy, pBy was cultured in LB broth containing oxalate, and then the amount of oxalate in the medium was assessed. The oxalate-degrading activity of homogenates of pBy was evaluated.
RESULTS
DNA sequencing showed the successful transformation of the YvrK gene into TOP 10 E. coli. Western blot analyses showed that pBy expressed OXDC. pBy removed oxalate during the overnight culture in oxalate-containing LB broth, and the homogenate of pBy degraded 90% of oxalate under acidic conditions.
CONCLUSIONS
A recombinant E. coli expressing the YvrK gene was successfully produced. The bacteria showed potent oxalate-degrading activity. The results of this study will provide a solution to the treatment of calcium oxalate stones and hyperoxaluria, for which there are few medical treatment modalities.

Keyword

Oxalates; Hyperoxaluria; Bacillus subtilis

MeSH Terms

Ampicillin
Arabinose
Bacillus subtilis
Bacteria
Base Sequence
Blotting, Western
Calcium Oxalate
Carboxy-Lyases
Clone Cells
DNA
Escherichia coli
Hyperoxaluria
Oxalates
Plasmids
Polymerase Chain Reaction
Sequence Analysis, DNA
Ampicillin
Arabinose
Calcium Oxalate
Carboxy-Lyases
DNA
Oxalates

Figure

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  Fig. 1. Yvrk gene cloning by polymerase chain reaction (PCR). M: 1 kb plus DNA ladder, PCR using 1μl (1, 2), 3μl (3, 4), 6μl (5, 6), and 0μl (7) genomic DNA showing a 1.2 kb band.

• 

  Fig. 2. Identification of Yvrk transformation by PCR. M: 1 kb plus DNA ladder, Y: Bacillus subtilis, S: pBy. 1.2 kb band of the Yvrk gene is seen in both B. subtilis and pBy.

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  Fig. 3. Western blot for oxalate decarboxylase (OXDC). Using anti-histidine antibody, a 43 kD protein (oxalate decarboxylase) was observed.

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  Fig. 4. Functional assay of pBy. pBy showed oxalate-degrading activity during overnight culture in the LB broth containing oxalate. The activity was dependent on pH.

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  Fig. 5. Functional assay of pBy homogenate. Measurement of the oxalate concentration after mixing 1 mM oxalate and homogenate of pBy for 3 hours showed that most of the oxalate was degraded at pH 5.

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