Part:BBa_K2287022

rhXOR

This is the coding sequence of recombinant human xanthine oxidase which is codon optimized for expression in E. coli. The product of this gene, xanthine oxidase, oxidises hypoxanthine to xanthine and xanthine to uric acid in human purine catabolism. With this enzyme, E. coli is able to transfer xanthine and hypoxanthine into uric acid.（fig.1）

Figure 1: the process of generating uric acid in the pathway of de novo purine synthesis

Figure 2: the structure of XOR PDB ID:2ckj

We first cultured E. coli with XOR gene in regular LB medium, but no activity of XOR was detected.

Figure 3: No uric acid was detected when bacteria was cultured in regular LB medium

Since uric acid is reported to cause cell death, we speculated that expression of XOR in large quantity was baneful and thus expression was suppressed. Therefore, we removed IPTG and counted on leakage expression of XOR. XOR activity was tested following the same procedure, but still no uric acid production was detected.

Figure 4: No uric acid was detected when IPTG was removed

When we learnt that XOR contains molybdopterin cofactors as active catalytic site, we added 1mM sodium molybdate into regular LB medium and cultured E. coli with XOR gene under same conditions as before. IPTG was added in experiment group while removed in control group.

Figure 5: E. coli with XOR showed xanthine oxidation activity and uric acid production without IPTG in LB with sodium molybdate (1mM), but not with IPTG. In LB with sodium molybdate (1mM) and without IPTG, XOR could fold correctly and demonstrate catalytic activity.

Then we co-transformed the parts we designed before(prs,purF,purD,purM,purC,purF K326Q) with XOR to test uric acid production ability

Figure 6: Comparison of uric acid production between E. coli with XOR gene over-expressing other enzymes and E. coli with XOR gene with extra xanthine

Figure 7: Comparison of uric acid production between E. coli with XOR gene over-expressing other enzymes and E. coli with XOR gene without extra xanthine

References

1.M. Ferreira Antunes, F.K. Eggimann, M. Kittelmann, et al. Human xanthine oxidase recombinant in E. coli: A whole cell catalyst for preparative drug metabolite synthesis Journal of Biotechnology., 235 (2016), pp. 3-10 Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 612
  Illegal BamHI site found at 707
  Illegal BamHI site found at 848
  Illegal BamHI site found at 1617
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 957
  Illegal NgoMIV site found at 2079
  Illegal AgeI site found at 451
  Illegal AgeI site found at 2458
  Illegal AgeI site found at 2509
  Illegal AgeI site found at 3505
  Illegal AgeI site found at 3859
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1438
  Illegal SapI.rc site found at 1474
  Illegal SapI.rc site found at 2071