Part:BBa_K3771001

CoaBC

Description

CoaBC is an enzyme that weighs 55.4 kDa. CoaBC functions in the JJU-CoaBC taurine biosynthesis pathway, converting L-cystate to taurine.

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[1,2].

Usage

CoaBC enzyme was used in vitro testing of taurine production. The sequence for CoaBC enzyme and trc promoter were ligated and transformed into E. coli to calculate taurine production using high-performance liquid chromatography (HPLC).

Fig. 1. Taurine pathways in E. coli [1,2].

CoaBC is an enzyme in the JJU-CoaBC pathway, one of three possible taurine synthesis pathways. Its main function is to convert L-cystate to taurine.

Characterization

The coaBC fragment was synthesized by IDT and amplified by PCR. Agarose gel electrophoresis result is shown in Fig. 2.

Fig. 2. Confirmation of coaBC fragment by PCR. M: Marker; Lane 1: coaBC (1221 bp)

SDS-PAGE of the CoaBC enzyme to confirm protein expression.

Fig. 3. Confirmation of protein expression of CoaBC. M: Marker; Lane 1: whole cell of CoaBC in BL21(DE3); Lane 2: soluble protein of CoaBC in BL21(DE3) (~55.4 kDa)

Taurine production yield of CoaBC with other production enzymes calculated by high-performance liquid chromatography (HPLC).

Taurine Production of CoaBC in E. coli BL21(DE3) strain

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

Fig. 4. Confirmation of CoaBC 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) (~55.4 kDa)

Whole cell and supernatant 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 concentrations were lower, two times as much JJU supernatant volume compared to CoaBC supernatant volume was required to produce a significant amount of taurine. As shown in figure 5, 2:1 ratio of JJU to CoaBC supernatant 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 samples and soluble protein

Taurine Production of CoaBC in E. coli BD7G strain

Since CoaBC expression in BL21(DE3) strain was not prominent in the supernatant, 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 [1]. SDS-PAGE results confirm JJU expression in both supernatant and whole cell samples.

Fig. 6. Protein expression of JJU in BD7G and CoaBC in BL21(DE3) in soluble protein samples

The whole cell and supernatant 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 figure 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. Joo Y-C, Ko YJ, You SK, et al. Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine as a Food Additive. Journal of Agricultural and Food Chemistry. 2018;66(51):13454-13463. doi:10.1021/acs.jafc.8b05093 https://pubmed.ncbi.nlm.nih.gov/30516051/

2. CSAD - Cysteine sulfinic acid decarboxylase - Homo sapiens (Human) - CSAD gene & protein. Uniprot.org. Published 2021. Accessed October 22, 2021. https://www.uniprot.org/uniprot/Q9Y600 ‌

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 583
  
• 1000
  COMPATIBLE WITH RFC[1000]