Part:BBa_K4275000

Kluyveromyces marxianus alpha mating-factor secretion signal

K.marxianus mα is a α-mating secretion signal in Kluyveromyces marxianus that encodes for a α-mating factor (αMF) domain[1] fusing with desired protein. The signal peptide in αMF domain expressed direct the fusion protein into endoplasmic reticulum (ER), Golgi body and thus secrete in vitro. K.marxianus αMF is integrated upstream of gene of interest (e.g NpaBGS-t) to express a αMF domain fused with desired protein (enzymes e.g NpaBGS-t), which direct the enzyme to secrete from the host cell. The design eliminates process of lysing host cell and purifying desired proteins therefore reduce the cost of the whole textile degradation process. The integrated part αMF also provides a inspiration of extracellular protein secretion for future iGEM teams, paving the way of improving the efficiency of their protein secretion system.

Usage and Biology

The alpha-mating factor secretion signal consists of two regions: pre- and pro-secretion leader. The pre-secretion leader guides the protein of interest to the Sec61 translocon on the surface of endoplasmic reticulum via its binding with signal-regconition particles (SRPs). The pre-secretion leader is cleaved by the signal peptidase following the translocation of the protein into ER. The pro-secretion leader is further processed by Kex2 endopeptidase[2] in the golgi apparatus at the site KR-EASA. The processed EASA residues are rapidly cleaved off by the Ste13 dipeptidase.

Characterization

Cellulases and cellulase boosters expression

The enzymatic digestion of the polysaccharide chains of cellulose was completed by exoglucanase, endoglucanase and 1-4 betaglucosidase, and this series of reactions are catalysed by LPMO and CDH. We constructed expression vectors for yeast Kluyveromyces marxianus with the unique origin of replication and antibiotic selection marker for the culturing of Kluyveromyces marxianus. Expression vectors were made distinct by the insertion of different sequences coding for the ligated form of the cellulase enzymes, LPMO and CDH. The enzymes were ligated with an alpha-mating factor secretion signal for Kluyveromyces marxianus at the N-terminus and a type I dockerin domain at the C-terminus (Fig.1A).The successful production and secretion of the protein NpaBGS, MtCDH and TrEGIII are examined by SDS-PAGE and western blot analysis (Fig.1D).

Figure 1: Fig.1 Construction of expression vectors for fusion proteins production in yeast Kluyveromyces marxianus and the analysis of the secreted enzymes (A) The design of our expression vector for production of cellulases and cellulase boosters in Kluyveromyces marxianus; the coding sequences for the cellulases and cellulase boosters were ligated with an alpha-mating factor secretion signal for Kluyveromyces marxianus at the N terminus and a type I dockerin domain at the C terminus linked by a flexible linker (B) The growth curve of recombinant yeasts transformed with expression plasmids coding for different enzymes (C) The agarose gel electrophoresis image of coding sequences for different enzymes, respectively NpaBGS, TaLPMO, CBHII, MtCDH and TrEGIII (D) Western blot result for TrEGIII and MtCDH.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. “Mating Factor Alpha-1 [Kluyveromyces Marxianus] - Protein - NCBI.” National Center for Biotechnology Information, U.S. National Library of Medicine, https://www.ncbi.nlm.nih.gov/protein/QGN17207.1.

2. Chahal, Sabreen et al. “Structural characterization of the α-mating factor prepro-peptide for secretion of recombinant proteins in Pichia pastoris.” Gene vol. 598 (2017): 50-62. doi:10.1016/j.gene.2016.10.040