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).
Sequence and Feature
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 331
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 331
Illegal NheI site found at 89 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 331
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 331
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 331
- 1000COMPATIBLE 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.
None |