Designing a BioBrick Compatible Pichia Expression System
Laboratory and industrial projects that involve the high volume production of a protein often select E. coli as an expression system due to its rapid growth. However, bacterial expression systems are not always a viable option. In the case where proper folding of the protein of interest requires post-translational modification (such as the addition of disulfide bonds or glycosylation,) a eukaryote must be used. Although several well-defined eukaryotic options exist, yeast is often selected for its ease of use in the laboratory. One yeast species in particular, Pichia pastoris, has gained popularity as an expression system for recombinant human proteins.
P. pastoris has a glycosylation pattern that is more compatible with the human immune system, when compared to the glycosylation pattern of Saccharomyces cerevisiae. P. pastoris is also known for its ability to grow in high densities using methanol as its only food source. Despite the usefulness of yeast species such as P. pastoris there are currently few items in the parts registry that are designed for use within yeast, and none that are specifically designed to be used with P. pastoris. Our team intends on producing pBB3G1 and pBB1Z1, two BioBrick compatible P. pastoris-E. coli shuttle vectors.
The pBB3G1 and pBB1Z1 vectors include features that increase its ease-of-use and versatility, such as: optional inducibility, optional product secretion, trans-kingdom conjugation (TKC), and episomal maintenance of the vector in the host organism. The vectors vary in their expression level. Constitutive expression is achieved in pBB1Z1 by the pGAP promoter. Methanol-induced expression is available in pBB3G1 by means of the pAOX1 promoter.
Product secretion may be had by inserting an alpha secretion signal in the cloning site preceding the have been provided in both vectors.
TKC involves the transfer of DNA from a bacterial cell to a eukaryotic cell, by means of conjugation. Harnessing the ability to shuttle plasmids between E. coli and P. pastoris through TKC would simplify transformation protocols. Currently, transformation methods (using shuttle plasmids cloned in E. coli) are a time consuming process, requiring isolation of the cloned plasmid and transformation into yeast. Transformation using TKC shortens the process by transferring the plasmid directly into the yeast cell. Utilizing TKC as a transformation protocol would translate to faster results in the laboratory and reduced costs in an industrial setting. Currently there are no BioBrick vectors in the parts registry that enable TKC between E. coli and P. pastoris.
TKC functionality is provided by the OriTP sequence which hosts the nick site that will be cleaved during the initiation of conjugation in order to linearize the plasmid, and ligated once transferred to the recipient. Importantly, the OriTP sequence -compared to other variations of OriT- does not require the presence of a helper plasmid within the recipient to complete the final ligation step of conjugal transfer.
One limitation to using P. pastoris as an expression system is that most shuttle plasmids must be integrated into the yeast chromosome. This results in lower expression of desired protein products, as well as lower transformation efficiency. We hope to improve the functionality of the pBB3G1/pBB1Z1 system by allowing the plasmid -once transferred to the yeast cell- to remain as an episomal plasmid. This is made possible by the inclusion of the PARS1 yeast autonomous replication sequence. This sequence ensures that the plasmid is maintained through several (~200) generations.
Transformant selection is simplified by the inclusion of antibiotic resistance genes that are effective in both E. coli and P. pastoris. The backbones that will be submitted to the parts registry will include either Zeocin or Geneticin, however the resistance genes have been cloned into a KpnI site, and may be swapped for any selective marker specific to the user’s needs.
Goal
To design a BioBrick compatible E. coli-P. pastoris shuttle vector.
Background
Methods
Vector Design
Sequences for AOX1, GAP, G418, and Zeocin were obtained from pPICZ, pPICZα, pGAP from Invitrogen. The Wintergreen odor generator (BBa_J45700) was selected as a reporter gene, as well as yeast green fluorescent protein (GFP). In order to improve upon an existing part in the Registry of Standard Biological Parts, we codon-optimized the Wintergreen odor generator for expression in P. pastoris using the Codon Optimization tool provided by IDT. Kozak sequences were added for each open reading frame to be expressed in P. pastoris. The basal vectors pMNBB-ICI and pMNBB-CCI (please refer to Vector Nomenclature, and Figure 1) were assembled from the above sequences in Clone Manager 9.
Parts List
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