Part:BBa_K4275051

Kluyveromyces marxianus LAC4 Promoter

The promoter of beta-galactosidase GAL4 in K.marxianus is a galactose-inducible promoter, in which the downtream transcription activity is tightly regulated by the availability of galactose in the medium.The Gene Of Interest will only be expressed at high level in high galactose concentrations (YPG Medium 2% Gal, for example). This promoter is extensively used in our cellulase and booster expression vectors(composite part BBa_K4275036 - BBa_K4275040) for the expression of secretory enzymes in K.marxianus.

Usage and Biology

The sequence of pLAC4 is derived from the Kluyveromyces marxianus strain CBS397, a strain which is known to grow well on lactose as a carbon source.It exhibited a 10-fold induction by lactose relative to glucose alone when used to express YFP in NBRC1777, whilst the GAL1 inducible promoter from S.cerevisiae was inducible by both galactose and lactose, but more strongly for the former [1].

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 Feature

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 331
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 331
  Illegal NheI site found at 89
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 331
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 331
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 331
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Rajkumar, Arun S. et al. "Biological Parts For Kluyveromyces Marxianus Synthetic Biology". Frontiers In Bioengineering And Biotechnology, vol 7, 2019. Frontiers Media SA, https://doi.org/10.3389/fbioe.2019.00097.