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PROJECT

Abstract

We extracted genomic DNA from B. subtitlis, S. cerevisiae and S. pombe. We performed PCR amplification of the genes coding these enzymes and cloned into E. coli. Uricase was cloned from B. subtilis and S. pombe. Allantoinase was cloned from E. coli. Allantoicase was cloned from S. cerevisiae. These fragments were inserted into pSB1C3. But we found that only Uricase derived from B. subtilis was in the recombinant E. coli . This was confirmed by SDS-PAGE. In addition, we measured Uricase activity and concluded that there was a possibility that the synthesized Uricase has activity. .

Back Ground

 In Gifu prefecture, Cormorant Fishing on the Nagara river (Ukai) is one of the most summer sights in Japan. Ukai is a traditional night fishing method in which an "usho"(Cormorant Fishing Master) and "u"(cormorant birds) work together to fish by the flames of Kagari-bi (fishing fire lanterns) reflecting on the dark surface of the river, with Mt Kinka and lofty Gifu Castle on its summit providing a dramatic backdrop to the scenery. Ukai has about 1,300 years of history. Now, it is popular as a tourism industry and a special world agricultural culture. On the other hand, damage caused by the cormorant droppings at the riverside is serious in Japan. The damage that we focus on is dieback of trees. In woods on the riverside inhabited by cormorant, some leaves are stained with their droppings and interfered with photosynthesis. Birds’ excrement consists mainly of uric acid which is hardly soluble material. So trees cannot get adequate nutrition and die back. Cormorant is carnivorous animal, and the amount of uric acid in their droppings are larger than that of uric acid in other birds’ droppings. Also, cormorant flocks together and they tend to drop their excrement around their nests. In the city, deterioration of the landscape caused by birds’ droppings is also a serious problem. The droppings fell on cars and window glasses cause corrosion if it is left for a long time. Moreover, acidity of droppings leads to decaying of bridge, highway and signs which may cause serious accidents. So, we plan to solve these problems by expression of birds’ "dropping cleanerase" and degrading uric acid.

Purine metabolism

Purine metabolism is a pathway of synthesizing and degrading purines. To breakdown uric acid into urea, five enzymes, Uricase, HIU hydrolase, OHCU decarboxylase, Allantoinase and Allantoicase are needed. This pathway is shown in Figure.1. The production of allantoin is preceded by two additional distinct unstable intermediate. However, HIU is spontaneously decomposed into OHCU and subsequently into racemic allantoin. So, we decided to reproduce the essential part of this pathway shown in Figure.2 in vitro. Enzymatic activity is required to degrade the intermediate products of purine metabolism. Ammonia, the final product of purine metabolism, is the most assimilated nitrogen source of most fungi and bacteria.

As template genes of Uricase, Allantoinase and Allantoicase, we used four microorganisms. Here, we mention why we used these microorganisms and characteristics.

B. subtilis has eight genes driving uric acid degradation. These genes are located in a cluster with the exception for the gene encoding allantoin permease. B. subtilis can degrade all of five intermediates of uric degradation described above.

According to James and Rudolf(1984), wild type S. pombe is capable of degrading uric acid and allantoin. They confirmed that Uricase, Allantoinase and Allantoicase activities of each mutant are less than those of wild type.

S. cerevisiae has the DAL gene cluster and is good at utilization of allantoin. So, S. ceresisiae uses allantoin as nitrogen source. However, it lacks the gene encoding oxygen-comsuming Uricase and is low-growth with uric acid.

In kegg pathway of purine metabolism in E. coli, it has HIU hydrolase and Allantoinase. It is high-growth with allantoin. Furthermore, E. coli has a special anaerobic pathway to decompose allantoin into urea. This pathway doesn’t need Allantoicase.

We performed growth test in minimal media containing purines of E. coli, B. subtilis, S.cerevisiae to confirm the existence of the target gene. The result is shown in Fig.9, Fig.10 and Fig.13.

Experiments

Growth test in minimal media

E. coli culture was grown in LB liquid at 37℃ overnight with shaking, B. subtilis culture was grown in LB liquid at 30℃, S. pombe culture and S. cerevisiae were grown in YPD liquid at 30℃ in advance. Minimal media containing 2 mM urate or allantoin or ammonium sulfate as a sole nitrogen source and minimal media not containing nitrogen sources were also prepared. Each culture was washed with PBS twice and diluted 100 to 10,000,000,000 times with each minimal media. Cell suspensions were incubated at the appropriate temperatures for one day and spotted onto LB broth or YPD broth. Results were imaged after one day incubation at appropriate temperature.

Plasmid construction

Assay

  Assay for Uricase
 We prepared 0.001% uric acid solution by dissolving in 50.0 mmol/L borate buffer containing 1.0 mmol/L EDTA and detergent(pH8.5). 0.5 mL of distilled water was added and the mixture was preheated at 25℃. E. coli culture was sonicated and diluted 1 to 100 times with the borate buffer. After that, the absorbance at a wavelength of 290.0 nm and 25℃ were continuously measured.
  Assay for Allantoinase
 Enzyme solution was mixed with buffer for allantoinase(50.0 mM Hepes, pH 7.75) containing 45.0 mM allantoin and incubated at 37℃. We withdrew 0.5 mL aliquots at regular timed intervals of 2 min. Each aliquot was quenched with 0.125 ml of 0.20N HCl and heated in a boiling water bath for 4 min to degrade allantoic acid to glyoxylate and urea. After decomposition of allantoate, we degraded urea to ammonia by jack bean urease.(assay for allantoinase)Samples were neutralized with 0.5 ml of 0.4N NaOH and buffered with 0.25 ml of 150.0 mM Hepes buffer, pH 7.75, containing 3.0 mM EDTA. We added 0.125 mg of urease in the reaction mixture. The released ammonia was assayed by Nessler’s reagent.
  Assay for Allantoicase
 We prepared 20.0 mM potassium phosphate buffer containing 15.0 mM allantoic acid. Enzyme solution was mixed with this buffer and incubated at 25℃. After that, we degraded urea and assayed the releasing ammonia by the method mentioned above.

Results

The growth tests in minimal media

The growth tests in minimal media containing purines of B. subtilis , S. cerevisiae were performed. The result is shown below. As expected, Bacillus subtilis grew well with uric acid, allantoin and ammonium sulfate. We confirmed that Bacillus subtilis utilized these intermediate as nitrogen sources. S. cerevisiae was positive growth with allantoin. However, it didn’t grow well with w/o ammonium, uric acid and ammonium sulfate.

  Assay for Uricase from Candida sp. from Wako

SDS-PAGE

Determination of expressed Uricase activity

E. coli culuture were grown in the presence of IPTG. Cell suspensions were sonicated and centrifuged (150rpm, room temperature, 10 min). The top layer was removed, and cells were buffered with 50 mM borate buffer containing 1 mM EDTA and detergent. Uricase from B.subtilis were measured. These crude extractions were diluted 1 to 100 times with the borate buffer. The result is shown in Figure.13 and Table.3. We couldn’t confirm this enzyme catalyzes uric acid into allantoin.

The growth ability of the recombinant E. coli in minimal media containing uric acid

Growth test in minimal media of wild type E. coli and the recombinant E. coli were conducted to confirm Uricase activity which was expressed by the recombinant E. coli. The result of this assay is shown in Figure13. No significant differences were observed in minimal media with 2 mM uric acid and w/o ammonium. However, wild type is more positive growth in minimal media containing ammonia sulfate. We could’t understand why this incident happened.

Determination of recombinant Uricase in the supernatant and the precipitation

In Figure.12, we considered that expressed Uricase is mainly in the precipitation. So, we assayed Uricase in the supernatant and the precipitation of the sonicated cell suspension respectively and tried to verify the activity of the Uricase. This protein extraction and sample preparation was followed the protocol of SDS-PAGE. (Protocols)The result of absorbance change is shown in Fig.15. There is a possibility that device BBa_K2158004 has enzyme activity. Uric acid in the reaction mixture may be catalyzed into allantoin.

Conclusion of assay for Uricase

We combined Figure.13 and Figure.15. The result is shown in Figure.16 Even though the activity is lower than reagent Uricase, the activity of Uricase which expressed by our recombinant E. coli can be detected.

Discussion

Uricase of B. subtilis

In this project, we assayed mainly device BBa_ K2158004. In SDS-PAGE experiment, there is a high possibility that Uricase of B. subtilis was synthesized by the recombinant E. coli. However, in the growth test, the recombinant E. coli didn’t grow well with uric acid. We think IPTG didn’t control lactose operon efficiently and the amount of the expressed enzyme was not enough to grow in the medium. In determination of the recombinant Uricase, the result shows the possibility of expressed functional protein catalyze uric acid to allantoin. So, it is not clear that this protein has the function. This protein should be analyzed particularly.

Allantoinase and Allantoicase

We couldn’t synthesize Allantoinase and Allantoicase. In SDS-PAGE, We confirmed protein that has small molecular weight were expressed instead of Allantoicase. We couldn’t verify the difference between E. coli with device BBa_K2158003 and wild type E. coli. In case of Allantoicase, we ligated BBa_J04500, BBa_K2158003 and BBa_B0015 and tried to make a composite part. According to the sequence data, deletion of one base happened and it changed the reading frame in this composite part. (Basic part, BBa_K2158003 was cloned successfully.) So, we concluded the expressed Allantoicase didn't have appropriate function to degrade allantoic acid. In case of Allantoinase, we couldn’t construct the device accurately.

Uricase of S. pombe

We ligated BBa_J04500, BBa_K2158002 and BBa_B0015 and tried to make a composite part. However, BBa_B0015 was not included in the plasmid. We think this was caused by the low level of the transfer efficiency of the gene encoding Uricase.

Assay of Uricase

We assayed the degradation of uric acid by measuring the absorbance at a wavelength of 290 nm. The absorbance peak of protein is at 280 nm. So, the absorbance of experimental plots is generally higher than that of negative control.

Modeling

  Abstract
 Our goal of this project is to solve problems caused by birds’ droppings with genetically engineered E. coli. To acheive this goal, we manufactured E. coli that synthesizes uricase, which catalyzes uric acid to urea. Therefore, we are interested in "How fast can our E. coli process uric acid theoretically?" We analyzed data obtained from the experiment and calculated theoretically maximum rate of processing uric acid.

  Method
 We made a differential equation which represents time evolution of concentration of uric acid. To making the equation, we used the Michaelis-Menten equation, which is widely used to estimate rate of enzyme reaction. The conversion from uric acid to allantoin is catalyzed by uricase. For this reason, the conversion rate of uric acid is described with the Michaelis-Menten equation. The equation we made is as follows. d [uric acid] / dt = - V_max * [uric acid] / (Km + [Uric acid])…(A) ([X] represents the concentration of a substance X.) V_max is the conversion rate of uric acid under the condition that the ratio of the amount of uric acid to that of uricase is sufficiently high. This parameter is proportional to the concentration of uricase. Then we put the proportionality constant as K_cat. Here, we estimated the two kinetic parameters Vmax and Km in the equation by fitting these parameters to a measurement result of the concentration change of uric acid in the reaction mixture. Besides, we used an algorithm called "differential evolution" in this parameter fitting. A tool which estimates and outputs Vmax and Km by entering the measurement results of the concentration change was developed. We attached the source code of this fitting tool below.

 result
In the following data and description, the unit of concentration of any substances is ng/μL and the unit of time is minute. First, we measured the change of concentration of uric acid in the reaction mixture. Uricase in this mixture was standard reagent. The result of the measurement is summerized in , Table.1 and Figure.11 . Then, we inputted these data into the parameter fitting tool mentioned above. Output of the parameter fitting was as follows. V_max = 0.0083 Km = 0.045 Besides, this graph shows the result of parameter fitting. The blue curve means the fitted curve, and the red plots is the data obtained from the experiment.

The concentration of uricase as a standard reagent was [uricase] = 0.42ng/μL. Using the definition of K_cat, or Vmax = Kcat*[uricase], Kcat was calculated in the following way. Kcat = Vmax/[uricase] = 0.0083/0.42 = 0.020. Now two kinetic parameters which is independent of concentration of enzyme or substrate (Kcat and Km) were estimated. Second, we conducted His-Link purification of protein and measured the absorbance at 280.0 nm to determine the concentration of uricase in the cell lysate. E. coli culture was sonicated to assay the enzyme. Uricase in this solution was synthesized by E. coli which we engineered. The result is below. [uricase] = 156.0ng/μL So Vmax of the producted uricase mixture was calculated in the following way. V_max = K_cat*[uricase] = 0.020*156.0 = 3.1 We can conclude that the uricase mixture made with our engineered E. coli has an ability to process uric acid with theoretically maximum rate of 3.1ng/(μL*min).

 Discussion
Accoding to our modeling, a large amount of Uricase may be needed to solve the problem of birds’ dropping. Highly-improved Uricase should be developed by biosynthesis.

FUTURE WORKS

This year, our purpose is to make it possible for E. coli to degrade urate and solve problems caused by birds’ dropping. So, we cloned genes of uricase, allantoinase and allantoicase into E. coli to assess production and activity of these three enzymes. We could extract genome from three kinds of bacteria, B. subtilis, S.cerevisiae and S. pombe and express three enzymes mentioned above. Meanwhile, this year’s result is not enough to solve the pollution of droppings. Now, we have three plans to improve our project. Firstly, we have to do more accurate assay using the allantoinase-loss-strain of E. coli. E. coli has a gene of allantoinase, and we can consider the possibility of affecting assay. Secondly, we think about the use of total synthesis. This year, we were going to use Candida utilis. However, Candida utilis has a high risk to human health. So, we used B. subtilis and S. pombe in place of that. They have the similar pathway of purine metabolism as Candida utilis . However, Candida utilis has the gene coded uricase which has high enzyme activity. If we use the gene coded uricase of Candida utilis by total synthesis, our project will improve more. Thirdly, each gene is cloned into B. subtilis instead of E. coli . B. subtilis have a function of secreting protein outside a cell. We can utilize expressed enzymes without sonication.

Reference

Schulz AC,Nygaard P,Saxild HH(2001)Functional analysis of 14 genes that constitute the purine catabolic pathway in Bacillus subtilis and evidence for a novel regulon controlled by the PucR transcription activator.J Bacteriol 183:3293-3302.

Wong S,Wolfe KH(2005)Birth of a metabolic gene cluster in yeast by adaptive gene relocation. Nat Genet 37:777-782

Russel Lee, Liting Yang, Gaseene Sebetso, Rebecca Allen, Thi H. N. Doan, Ross Blundell, Edmund Y. L. Lui, Carl A. Morrow, James A. Fraser(2013) Characterization of the Comp,lete Uric Acid Degradation Pathway in the Fungal Pathogen Cryptococcus neoformans.

Gifu Convention and Visitors Bureau, Ukai: Cormorant fishing on the nagara river, http://www.gifucvb.or.jp/en/01_sightseeing/01_01.html

Xin Cheng, Bo Yang, Dong Liu, L Juan He, Gan Chen, Yong Chen, R Fa Huang, Y Sheng Jiang (2012) Genetic Engineering of Bacteria that can Produce Urate Oxidase. ISSN: 2165-8048 IME, an open access journal.

Vladimir Romanov, Ma t thew T. Mer ski, Rober t P. Hausinger (1999) Assays for Allantoinase. Analytical Biochemistry 268:49 –53.

Davide Vigetti, Claudio Monetti, Loredano Pollegioni, Roberto Taramelli, Giovanni Bernardini (2000) Xenopus Allantoicase: Molecular Cloning, Enzymatic Activity and Developmental Expression. Archives of Biochemistry and Biophysics 379: 90-96.

Kyoung Sook Choi, Katharine W. Lee, Susana C. Yu Hico, Allan H. Roush (1968) Assay, Purification and Properties of Allantoicase from Candida utilis. Archives of Biochemistry and Biophysics 126：261-268.

Yasuji Koyama, Toshio Ichikawa, Eiichi Nakano (1996) Cloning, Sequence Analysis, and Expression in Escherichia coli of the Gene Encoding the Candida utilis Urate Oxidase (Uricase).

James R. Kinghorn, Rudolf Fluri (1984) Genetic studies of purine breakdown in the fission yeast Schizosaccharomyces pombe. Current Genetics 8 : 99-105.