Part:BBa_K3196036

AOX1

Characterization

It is a strong promoter induced by methanol and is often used to produce recombinant protein in Pichia pastoris expression system.

Usage and Biology

AOX1 gene is the main source of alcohol oxidase (Aox). AOX1 promoter is strictly induced by methanol and inhibited in the presence of glucose, glycerol, fructose and other carbon sources.

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]

Team: SMS_Shenzhen 2022

Advantages of AOX1 promoter

AOX1 promoter is highly repressed in cells grown on glucose, glycerol, and most other carbon sources, but it is strongly induced by methanol.[1-4] Expression of AOX1 is tightly regulated at the transcriptional level [5] and appears to be controlled by both repression/derepression and induction mechanisms (Fig. 1).[6]

Fig.1 | Specific AOX activity during the transition phase in four P. pastoris high cell density fed-batch cultivations.[6]

Besides, for P. pastoris, being an obligate aerobe when growing on methanol does not switch its metabolism under oxygen-limiting conditions as S. cereVisiae and other facultatively aerobic organisms do. This makes it possible to run processes to high cell density also under oxygen limitation.[7-9]

References

[1] Cereghino, G. P. L.; Cereghino, J. L.; Ilgen, C.; Cregg, J. M. Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr. Opin. Biotechnol. 2002, 13, 329-332.
[2] Ellis, S. B.; Brust, P. F.; Koutz, P. J.; Waters, A. F.; Harpold, M. M.; Gingeras, T. R. Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris. Mol. Cell. Biol. 1985, 5, 1111-1121.
[3] Tschopp, J. F.; Brust, P. F.; Cregg, J. M.; Stillman, C. A.; Gingeras, T. R. Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res. 1987, 15, 3859-3876.
[4] Cregg, J. M.; Madden, K. R. Development of yeast trnsformation systems and construction of methanol-utilization-defective mutants of Pichia pastoris by gene disruption. Biol. Res. Ind. Yeasts 1988, 2, 1-18.
[5] Couderec, R.; Baretti, J. Oxidation of methanol by the yeast Pichia pastoris. Purification and properties of alcohol oxidase. Agric. Biol. Chem. 1980, 44, 2279-2289.
[6] Jahic, M. Process techniques for production of recombinant proteins with Pichia pastoris. Ph.D. thesis, Stockholm, 2003. ISBN 91-7283.-5222-2., pp 1-123.
[7] Jahic, M.; Rotticci-Mulder, J. C.; Martinelle, M.; Hult, K.; Enfors, S. O. Modeling of growth and energy metabolism of Pichia pastoris producing a fusion protein. Bioprocess Biosyst. Eng. 2002, 24, 385- 393.
[8] Charoenrat, T.; Ketudat-Cairns, M.; Stendahl-Andersen, H.; Jahic, M.; Enfors, S. O. Oxygen-limited fed-batch process: An alternative control for Pichia pastoris recombinant protein processes. Bioprocess Biosyst. Eng. 2005, 27, 399-406.
[9] Trentmann, O.; Khatri, N. K.; Hoffmann, F. Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris. Biotechnol. Prog. 2004, 20, 1766-1775.