Part:BBa_K2334013

J23113 4.1.1.97 + J23106 pucM + K2334001

Combination of J23113 + RiboJ + B0034 + 4.1.1.97, OHCU decarboxylase generator, J23106 + RiboJ + B0034 + pucM, HIU hydrolase generator & BBa_K2334001(J23100 + RiboJ + B0034 + pucL, Urate Oxidase Generator)

The stereospecific oxidative degradation of uric acid to (S)-allantoin was recently shown to proceed via three enzymatic steps. The final conversion is a decarboxylation of the unstable intermediate 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) and is catalyzed by OHCU decarboxylase. In our project, the OHCU decarboxylase is used as the important enzyme in urate degradation, whose expression is driven by J23113. RiboJ is used for the quantitive pathway construction.

The ureide pathway, which produces ureides from uric acid, is an essential purine catabolic process for storing and transporting the nitrogen fixed in leguminous plants and some bacteria. PucM from Bacillus subtilis was recently characterized and found to catalyze the second reaction of the pathway, hydrolyzing 5-hydroxyisourate (HIU), a product of uricase in the first step. PucM has 121 amino acid residues and shows high sequence similarity to the functionally unrelated protein transthyretin (TTR), a thyroid hormone-binding protein. Therefore, PucM belongs to the TTRrelated proteins (TRP) family. In our project, PucM is used as the important enzyme in urate degradation, whose expression is driven by J23106 in this part. RiboJ is used for the quantitive pathway construction.

Urate oxidase (uricase, EC 1.7.3.4) is an enzyme with copper bonds that catalyze the oxidative opening of the purine ring of uric acid to form allantoin which is 5–10 times more soluble than uric acid. In Bacilus Subtilis, the gene is coded by pucL gene. This enzyme can be used therapeutically to reduce toxic urate accumulation. In our project, uricase is used as the most important enzyme in urate degradation, whose expression is driven by J23100. RiboJ is used for the quantitive pathway construction.

Figure 1. The metabolic pathway design of the project. The dotted lines represent the enzymes which can accelerate the spontaneous reaction processes. Urate oxidase (uricase, EC 1.7.3.4) is an enzyme with copper bonds that catalyzes the oxidative opening of the purine ring of uric acid to form 5-hydroxyisourate. HIU hydrolase (3.5.2.17) catalyzes the second reaction of the pathway, hydrolyzing 5-hydroxyisourate (HIU), a product of uricase in the first step to form 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU). OHCU is catalyzed by OHCU decarboxylase (4.1.1.97).

We used the crude bacteria extraction to test the partial or the partial pathway(pucL + pucM) function directly. Before we started to react, the total protein quantity of each sample was made the same. 100ul crude extraction was added into 900ul PBS with urate (PH=8.0). HPLC was performed after reaction for 2h and 100 ℃ heat for 10min.The results show that, pucM did work. The tendency can be explained as follows: when the promoter is too strong, it causes excessive consuming of energy in the bacteria, and the expression of the main enzyme pucL is thus limited; when the promoter is too weak, the reaction can’t attain equilibrium as quickly as with a stronger promoter. So when measured at the time, if the equilibrium was not attained, the performance of the pathway with weaker promoter would be worse. Our modeling result is consistent with our experiment result here. However, the result of separated crude extraction mix experiment can't be explained with the same theory, because when the continuous measurement was performed, performances of different reaction systems with PucL protein were always the same when measured at a time.

Figure 2. The result of crude extraction experiment of BBa_K2334012, BBa_K2334013, BBa_K2334014, BBa_K2334015, BBa_K2334016. The numbers in the figure refer to HPLC original peak area urate decrease. The result shows that the expression ability of pucM does influence the performance of urate consuming. J23106 perform best in the group. We compared the effect of pucL together in this group.

To achieve our goal to utilize the urate outside the bacteria, SCU-WestChina cultivated E. coli Nissle 1917 transformed with eGFP, pucL (BBa_K2334001), YgfU+pucL (K2334004 + K2334001), LM4 Full (BBa_K2334017), LM4 Optimum (BBa_K2334017) in M9/LB medium and aerobic & anaerobic environments to explore the paramters may influence the process. The urate concentration measured by HPLC.

The values in different independent repeated experiments are not always the same. But the tendency is the same in every group: For urate utilization ability, M9(Anaerobic)＞M9(Aerobic) ＞LB(Anaerobic)＞LB(Aerobic). It means that bad nutrition environment and anaerobic environment can reduce the urate concentration better. According to our experiment results, our gene constructions (pucL, YgfU + pucL, LM4 Full, LM4 Optimum) all have the ability to reduce the urate concentration outside the cell.

Figure 3. The experiment results of Group 1-3 original HPLC peak area result shown in bar. Green bar is eGFP as control. It's clear that M9 and anaerobic cultivation performed better for urate consuming.

Sequence and Features

Assembly Compatibility:

• 10
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  Illegal NheI site found at 7
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  Illegal NheI site found at 782
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  Illegal NheI site found at 1170
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• 21
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  Illegal NgoMIV site found at 203
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1054