Part:BBa_K3771003

CSAD

Description

L-Cysteine sulfinic acid decarboxylase (CSAD) is an enzyme consisting of 493 amino acids and weighs 55.4 kDa. CSAD functions in the taurine biosynthesis pathway, converting L-Cysteine to taurine [1].


Biology

Fig. 1. Taurine production pathway

CSAD is part of the L-cysteine sulfinic acid pathway, one of two possible taurine synthesis pathways. CSAD catalyzes the decarboxylation of L-Cysteine sulfinic acid into hypotaurine, which is spontaneously oxidized to taurine [1].


Usage

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


Characterization

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

Fig. 2. Confirmation of csad fragment by PCR. M: Marker; Lane 1: csad (1368 bp)

Fig. 3. Confirmation of pSUI-P_trc-csad by digestion. M: Marker; Lane 1~3: Different colonies of pSUI-P_trc-csad (3674 bp)

Fig. 4. Transformation / CSAD in DH5α SDS-PAGE and western blot of CSAD enzyme to confirm protein expression.

Fig. 5. Confirmation of protein expression of CSAD. M: Marker; Lane 1: whole cell of CSAD in DH5α; Lane 2: soluble protein of CSAD in DH5α (~22 kDa)

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

Fig. 6. Taurine production of P_trc-cdo1 + P_lacI-csad + P_trc-cs in different growth mediums.

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

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 AgeI site found at 250
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 35