Difference between revisions of "Part:BBa K731010"

Revision as of 14:01, 12 September 2012

M256I CysE (Serine Acetyltransferase)

CysE is an enzyme involved In cysteine biosynthesis, also known as SAT. It catalyzes the acetylation of serine to give O-acetylserine, the CysE final precursor. Some O-acetylserine is also converted to N-acetylserine, which in turn triggers the assimilation of sulfate through specific genes.

M256I CysE shows enhanced secretion of cysteine and is not inhibited by Cysteine overproduction.

This part has been successfully operated while controlled by araC-pBAD both in pSB1C3 (K731030) and the low copy vector pSB3C5, in which it was characterized. A sfGFP tagged fusion of this part has also been deposited as BBa_K731040 and used to test protein expression levels upon arabinose induction.
This part was cloned by the iGEM Trento 2012 team for the creation of an aerobically engineered pathway for the removal of the black crust disfiguring marble stones. Further information about this part and its characterization can be found in the iGEM Trento 2012 wiki.

Usage and Biology

In cysteine biosynthesis, CysE (a serine acetyltransferase) catalyzes the acetylation of serine to give O-acetylserine, a precursor for the biosynthesis of cysteine. Some O-acetylserine is also converted to N-acetylserine, which in turn triggers the assimilation of sulfate through sulfate assimilation genes.

In Escherichia coli conversion of L-serine to L-cysteine is mediated by the action of two enzymes: serine acetyltransferase [1] catalyses the activation of L-serine by acetyl-CoA. Its product, 0-acetyl-L-serine (OAS), is then subsequently converted to L-cysteine by 0-acetyl-L-serine(thio1)lyase.
The synthesis of OAS-(thio1)-lyase and of the enzymes involved in sulphate uptake and reduction is regulated by induction as well as by repression [2]. The expression of cysE (the SAT structural gene), on the other hand, is constitutive whereas the catalytic activity of the gene product, SAT, is sensitive to feedback inhibition by L-cysteine [3].

Denk and Bock [4], in a work to develop an E. coli strain able to secrete cysteine, isolated a M256I cysE mutant that had a 10-fold decrease in feedback inhibition by cysteine itself, in the end promoting cysteine excretion into the medium.
This particular mutant, thus, would overproduce cysteine, needing and assimiliting more sulfate to satisfy its needs.

The araC-pBAD promoter is active in presence of L-arabinose. L-arabinose binding to the AraC protein inactivates its inhibitory function, permitting RNA polymerase to start transcription on the ara operon.
AraC is also negatively regulated by cAMP via CRP (formerly known as CAP, catabolite activating protein). In presence of glucose, cAMP levels are low, meaning that AraC can still act as a repressor, not allowing transcription. In the original ara operon, this circuit has the function to limit its transcription when arabinose in not needed, which is when it’s not present and/or when glucose (the primary energy source) is available.

Please head over to K731030 for documentation on characterization of this Part.

Sequence and Features