Part:BBa_K4605011

Expressing PPTase to activate bpsA

C. glutamicum has the native pcpS gene, which expresses PPTase(4'-phosphopantetheinyl transferase). The PPTase is of great significance because it converts the apo-form of the BpsA into its active holo-form by attaching coenzyme A to the peptide carrier domain (PCP).However,in K.xylinus,there is no pcpS exist.Thus,we need to build a bpsA-pcpS fragment so that bpsA can be activated to catalyze the reaction.

Here is an example of PPTase for its function[1].It is responsible for the transfer of 4-phosphopantetheine from coenzyme A to PCP and ACP domains in non-ribosomal peptide and polyketide synth(et)ases, respectively.

Because K. xylinus does not have the native PPTase that is necessary for activating apo-form of indigoidine synthase into its active holo-form by adding coenzyme A to the peptide carrier domain (PCP), we need to transfect the target gene both bpsA and pcpS (encoding PPTase)into K. xylinus using pSB1A2 as a plasmid vector, and synthesize indigoidine fibers using K. xylinus which is capable of producing cellulose in high yield.With previous basic explorations, we will use PSB1A2 plasmid backbone, ligated with promoters such as strong promoters (J23104,J23100,J23119 etc.), and CDS sequences to express bpsA and pcpS in K. xylinus while binding to bacterial cellulose membranes.

References

[1]J. Kipchirchir Bitok, Christophe Lemetre, Melinda A. Ternei, Sean F. Brady, Identification of biosynthetic gene clusters from metagenomic libraries using PPTase complementation in a Streptomyces host, FEMS Microbiology Letters, Volume 364, Issue 16, August 2017.

[2]Sword TT, Barker JW, Spradley M, Chen Y, Petzold CJ, Bailey CB. Expression of blue pigment synthetase a from Streptomyces lavenduale reveals insights on the effects of refactoring biosynthetic megasynthases for heterologous expression in Escherichia coli. Protein Expr Purif. 2023 Oct;210:106317.

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 142
  Illegal NgoMIV site found at 156
  Illegal NgoMIV site found at 261
  Illegal NgoMIV site found at 294
  Illegal AgeI site found at 285
  
• 1000
  COMPATIBLE WITH RFC[1000]