Difference between revisions of "Part:BBa K3771000"

Latest revision as of 02:41, 22 October 2021

JJU

Description

JJU (cysteine lyase) is an enzyme weighing approximately 43 kDa. JJU catalyzes the beta replacement reaction of L-cysteine and sulfite to form L-cysteate and hydrogen sulfide[1].

The three taurine production pathways incorporated into our E. coli include the L-cysteine sulfinic acid pathway, L-cysteine sulfonic acid pathway, and the JJU-CoaBC pathway[1].

Fig. 1. Taurine pathways in E. coli [1]

JJU is an enzyme that is a part of the JJU-CoaBC taurine production pathway, one of three possible taurine synthesis pathways. Its main function is to convert L-cysteine to L-cystate, which then becomes taurine by CoaBC[1].

Fig. 2. Biobrick of P_trc-jju

JJU enzyme was used in in vitro test of taurine production. The sequence for JJU enzyme and trc promoter (BBa_K864400) were ligated and transformed into E. coli. By adding L-cysteine substrate into a reaction solution, JJU can help catalyze the conversion of L-cysteine to taurine. Taurine concentration were determined by high-performance liquid chromatography (HPLC).

The jju sequence synthesized by IDT was amplified by PCR and ligated to trc promoter (BBa_K864400) in the pET28a cloning vector.

Fig. 3. Agarose gel electrophoresis result showing amplified jju gene fragment. M: Marker; Lane 1: jju (1173 bp)

Taurine Production of JJU in E. coli BL21(DE3) strain
In our in vitro test of taurine production by JJU, JJU was transformed into E. coli BL21(DE3) strain. Soluable protein (S) and whole cell (WC) samples were collected to confirm extracellular and intracellular protein expression by SDS-PAGE.

Fig. 4. Confirmation of JJU expression by SDS-PAGE. M: Marker; Lane 1: whole cell of JJU in BL21(DE3); Lane 2: soluble protein of JJU in BL21(DE3) (~43 kDa); Lane 3: whole cell of CoaBC in BL21(DE3); Lane 4: soluble protein of CoaBC in BL21(DE3) (~45 kDa)

Whole cell and soluable protein JJU and CoaBC samples were collected and added in a JJU:CoaBC volume ratio of 1:1, 2:1, and 1:2. High-performance liquid chromatography (HPLC) was conducted to determine taurine concentration. Because JJU concentration was lower, two times as much JJU soluable protein volume compared to CoaBC supernatant volume was required to produce a significant amount of taurine. As shown in Fig. 5, 2:1 ratio of JJU to CoaBC soulable protein volume had the highest taurine concentration of around 95 mg/L.

Fig. 5. Taurine production of both JJU and CoaBC in BL21(DE3) in whole cell and soluble protein samples

Taurine Production of JJU in E. coli BD7G strain
Since JJU expression in BL21(DE3) strain was not prominent in the soluable protein, we performed another in vitro test in which P_T7-jju was transformed into BD7G strain instead of the BL21(DE3) strain. The BD7G strain contains chaperone protein GroELS that aids in protein folding[2]. SDS-PAGE results confirm JJU expression in both soluable protein and whole cell samples.

Fig. 6. Confirmation of JJU expression by SDS-PAGE. M: Marker; Lane 1: whole cell of JJU in BD7G; Lane 2: soluble protein of JJU in BD7G (~43 kDa); Lane 3: whole cell of CoaBC in BL21(DE3); Lane 4: soluble protein of CoaBC in BL21(DE3) (~45 kDa)

The whole cell and soluable protein JJU and CoaBC samples are collected and added in a JJU:CoaBC volume ratio of 1:1, 2:1, and 1:2. When P_T7-jju was transformed into the BD7G strain, 1:1 ratio of JJU to CoaBC supernatant had the highest taurine production, as shown in Fig. 7. This suggests the activity level of JJU does not significantly differ from that of CoaBC, and both are equally crucial and effective in converting L-cysteine to taurine.

Fig. 7. Taurine production of JJU in BD7G and CoaBC in BL21(DE3) in soluble protein samples

References
1. BRENDA - Information on EC 4.4.1.10 - cysteine lyase. Brenda-enzymes.org. Published 2021. Accessed October 21, 2021. https://www.brenda-enzymes.org/enzyme.php?ecno=4.4.1.10 ‌
2. Yu T-H, Yi Y-C, Shih I-Tai, Ng I-Son. Enhanced 5-Aminolevulinic Acid Production by Co-expression of Codon-Optimized hemA Gene with Chaperone in Genetic Engineered Escherichia coli. Applied Biochemistry and Biotechnology. 2019;191(1):299-312. doi:10.1007/s12010-019-03178-9 ‌

Sequence and Features