Urea cooler

1. Abstract

We made urea cycle in E.coli by introducing of arginase encoded by rocF gene and get urea to make urea cooler. To make urea cooler, we need large amount of urea. But just by introducing rocF, only a little amount of urea can be produced because arginine biosynthesis is repressed. Therefore, we tried to derepress the effect of repression.
Furthermore, we researched flux to provide more urea. As a result, we found that the artificial urea production system, as well as natural one, is robust in a stoichometrically point of view. The analysis also found that supplementation of Arg, Glu and Asp would increase urea production rate.

Fig. The reactions related with the urea cycle Fig.4 Urea concentration in growth media 1 hour after IPTG induction

2.1 Introduction

Coolers can be made by adding urea to water, since dissolving urea in water is an endothermic reaction (-57.8 cal/g). However, E. coli does not synthetize urea naturally, so we attempted to complete the urea cycle inside E. coli and get urea.
Originally, E.coli has all enzymes of the urea cycle except for the arginase. In this work, introduction of the Bacillus subtilis rocF gene on a standardized plasmid completed urea cycle and enabled E.coli to produce urea as reported by TUCHMAN et al., (1997)
(Fig.1).

Fig.1 Addition of a gene which codes arginase completes urea cycle in E.coli

However, just by introducing arginase , E.coli, produces only a little amount of urea. TUCHMAN et al proposed that catabolite repression in arginine biosynthesis pathway is the main reason for the low efficiency of production(TUCHMAN et al., 1997) The bacterial arginine biosynthetic genes are all regulated via a common repressor protein encoded by the argR gene and activated in the presence of arginine . (Fig.3)They circumvented the arginine repression by introduction of arginine operator sequences (Arg boxes), which bind the arginine repressor. Upon arginine repressor binding to Arg boxes, the amount of the arginine repressor which can repress arginine biosynthesis is reduced. In this work, we tried two ways of solving this problem. One way is introducing the Arg boxes as previous work. The other way is using an E. coli that has an argR deletion genotype so that the repressor is not synthetized.

Fig.2 Arginine biosynthesis is repressed by arginine repressor and its co-repressor

2.2 Results

Bacterial strains and plasmids The bacterial strains and plasmids used in this study are listed in Table 1 and Table 2, and the constructions are shown in Fig.3.

TABLE 1. E.coli strains used in this study

Strain	argR
MG1655	+
JD24293	-

JD24293 was obtained from National Institute of Genetics.

TABLE2. Expression plasmids used in this study

Designation	vector	rocF	Arg box
pTrc-rocF	pSB3K3	+	-
pTrc-rocF-Arg Box	pSB3K3	+	+

Fig.3 Plasmids used in this study
The details of the constructions are here.

MG1655 and JD24293 were transformed separately with pSB3K3, pTrc-rocF or pTRC-rocF-Arg box. A detailed method is described here.

Urea concentrations detected in growth media of bacterial samples 1 hour after IPTG induction are shown in Fig.5. Detialed procedure is described here

Fig.4 Urea concentration in growth media 1 hour after IPTG induction

In MG1655(ArgR+), addition of Trc promoter-rocF led to more production of urea compared to the bare backbone pSB3K3 as expected. These results show that insertion of rocF resulted in arginase production as expected, therefore completing the urea cycle in E.coli. In the same strain, however, addition of Arg box sequence led to little change in urea production. The reason why the effect of Arg boxes was not apparent is probably that pSB3K3 is a low-copy-number plasmid, in contrast to high-copy number used in the previous report. A low-copy-number plasmid is not capable of introducing enough number of Arg boxes to effectively deactivate the arginine repressor. Both of the plasmids containing rocF gene in the stain JD24293(Arg-) produce urea more efficiently than those in MG1655.

These results are in line with the fact that JD24293 carries argR (a gene which codes arginine repressor) loss-of-function mutant, which means deactivation of arginine repressor by Arg boxes is not needed and addition of the Arg box does not result in a significant increase of urea production.