Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris
1 University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 18, Vienna, 1190, Austria
2 Austrian Centre of Industrial Biotechnology (ACIB GmbH), Muthgasse 11, Vienna, 1190, Austria
3 University of Applied Sciences FH-Campus Vienna, School of Bioengineering, Muthgasse 18, Vienna, 1190, Austria
4 Lonza AG, Rottenstraße 6, Visp, 3930, Switzerland
Microbial Cell Factories 2013, 12:5 doi:10.1186/1475-2859-12-5Published: 24 January 2013
Inducible high-level expression is favoured for recombinant protein production in Pichia pastoris. Therefore, novel regulated promoters are desired, ideally repressing heterologous gene expression during initial growth and enabling it in the production phase. In a typical large scale fed-batch culture repression is desired during the batch phase where cells grow on a surplus of e.g. glycerol, while heterologous gene expression should be active in the feed phase under carbon (e.g. glucose) limitation.
DNA microarray analysis of P. pastoris wild type cells growing in glycerol-based batch and glucose-based fed batch was used for the identification of genes with both, strong repression on glycerol and high-level expression in the feed phase. Six novel glucose-limit inducible promoters were successfully applied to express the intracellular reporter eGFP. The highest expression levels together with strong repression in pre-culture were achieved with the novel promoters PG1 and PG6.
Human serum albumin (HSA) was used to characterize the promoters with an industrially relevant secreted protein. A PG1 clone with two gene copies reached about 230% of the biomass specific HSA titer in glucose-based fed batch fermentation compared to a PGAP clone with identical gene copy number, while PG6 only achieved 39%. Two clones each carrying eleven gene copies, expressing HSA under control of PG1 and PG6 respectively were generated by post-transformational vector amplification. They produced about 1.0 and 0.7 g L-1 HSA respectively in equal fed batch processes. The suitability in production processes was also verified with HyHEL antibody Fab fragment for PG1 and with porcine carboxypeptidase B for PG6. Moreover, the molecular function of the gene under the control of PG1 was determined to encode a high-affinity glucose transporter and named GTH1.
A set of novel regulated promoters, enabling induction without methanol, was successfully identified by using DNA microarrays and shown to be suitable for high level expression of recombinant proteins in glucose-based protein production processes.