Methylotrophic yeast Pichia pastoris as a new model organism for future iGEM Georgia State University iGEM Team

Biology Department, Georgia State University, Atlanta, GA USA

AcknowledgmentsWe would like to thank GSU Biology Department

Why Pichia pastoris?

Tool Box

Historically, E. coli and S. cerevisiae have been choice organisms for the production of recombinant proteins. However, historical presence does not necessarily vouch for the efficiency of a particular system. While E. coli and S. cerevisiae are acceptable for certain tasks, there are many applications where they are insufficient, and alternative options should be sought. Recently, Pichia pastoris has established itself as a very attractive alternative for protein production. Below, we provide a summary of key points identifying the advantages P. pastoris holds over E. coli and S. cerevisiae :

•Greater Cell Concentrations leading to greater protein production compared to S. cerevisiae and E. coli• It is inexpensive to culture because of the media components (glycerol, methanol, salts, trace elements, and biotin) and it is easy to avert undesirable protease activity by adjusting pH.•High production of foreign protein while low levels of endogenous protein make it easier to isolate the protein of interest.•Post-translational modifications are possible in Pichia compared to E. coli, since it can have better disulfide bonding. • Powerful promoter systems due to their wide range including inducible and constitutive.

Methods

Part: BBa_K431010Category: Coding

Formaldehyde Dehydrogenase(FLD1)FLD1 responsible for the oxidation of formaldehyde to formate, a necessary step in methanol and methylamine utilization. Recently applied in Pichia pastoris for both selectivity and to increase copies of expression cassette. Status: in progress

Part: BBa_K431007Category: Promoter

Alcohol Oxidase 1 promoter (pAOX1)Strong and tightly regulated methanol inducible promoter. This part has been one of the most powerful and successful expression systems in Pichia pastoris. Status: in progress

Part: BBa_K431006Category: Coding

Histidinol Dehydrogenase 4 (HIS4)His4 catalyzes the last step in histidine biosynthesis. This part was designed to be compatible with the His4- auxotrophic mutant strains of Pichia pastoris. This part enables selectivity of transformants and is central to application of our P. pastoris system. Status: in progress

Part: BBa_K431009Category: Promoter

Glyceraldehyde 3-Phosphate Dehydrogenase promoter (pGAP)This is a strong constitutive promoter naturally needed glucose metabolism. This part has been very successful in the literature and reports comparable yields to that of pAOX1. Status: in progress

Part: BBa_K431005Category: Coding

Influenza A hemagglutinin globular head antigen (HA)This is an HA globular head antigen designed from a highly conserved region on the Influenza Flu virus sequence. This part was created to exemplify a practical use for the Pichia pastoris expression system.Status: dry lab

Part: BBa_K431008Category: PromoterTranslational Elongation Factor (pTEF)pTEF is a constitutive promoter which works well in high glucose environments and carbon-limited conditions, leading to greater expression levels of recombinant protein and mass production of heterologuous proteins. This part has been one of the most powerful and successful expression systems in Pichia pastoris.

Status: in progress

Antigen Design:•We compared previously isolated and sequenced Influenza A H1N1 strains. •A conserved region comprised of the Hemagglutinin globular head was identified.•This region of the virus has been shown to produce an immune response.

Future Application

Discussion/Concluisions

Introduction With the parts available from our toolbox, useful systems

can be quickly constructed and utilized in Pichia pastoris. To exemplify this concept, we have designed a BioBrick compatible influenza flu virus antigen (BBa_K431005). We will express the antigen under the control of pGAP in order to obviate difficulties affiliated with methanol utilization while maintaining high theoretical yields. If successful, this construct could be an easily interchangeable system to meet the demands of annual flu vaccines, only requiring the exchange of one part.

In 2009 the World Health Organization raised its pandemic alert for H1N1 influenza A virus to the 6 phase alert. This is the highest alert level and it indicates widespread community transmission of the virus in over two continents. The H1N1 Influenza A virus is a quadruple re-assortment of two swine strains, one human strain, and one avian strain. More than 214 countries and territories have reported laboratory – confirmed cases of pandemic H1N1 Influenza A. The US Centers for Disease Control and Prevention reported between April 2009 and April 2010, approximately 61million cases of pandemic H1N1 occurred, including 274,000 hospitalizations and 12,470 deaths. Efficient and timely vaccine production was a challenge for the scientific community during the pandemic outbreak of H1N1. Another problem with the Influenza viruses is the high variability and mutations that they present with each year. This makes vaccine production time difficult and time consuming. The Pichia system is an ideal construct for producing vaccines. We have designed a standard Pichia system that can be used for the production of various different vaccines. In theory, a high yield circuit can be established that only requires the exchange of the variable antigen, while leaving the remaining factors constant. In the circuit presented below, a vector has been designed to express the HA globular head influenza A antigen under the control of the strong constitutive promoter pGAP.

MethodsPart Design: Using primers that contain Biobrick ends, parts were isolated from Pichia pastoris chromosomal DNA with PCR reactions. To increase the likelihood of gaining a successful product, a systematic approach was applied for primer design. Two sets of primers were constructed for each part using IDT®’s PrimerQuest and Oligoanalyzer programs. The first set added an Xpa I site on the forward and a Spe I site on the reverse. The second set added EcoRI, Not I and Xba I sites on the forward and Spe I, Not I and Pst I sites on the reverse. In this way, hybrid primer combinations were assembled providing a total of four PCR products. With this system, we have successfully acquired a biobrick prospective product for each part.

3. ENX

1 4 L32L

Reverse Primer #1 3’

5’ Spe I

Forward Primer #1 3’

5’ Xba I Primer Set 1

AB

Forward Primer #2 3’

Xba I Not I 5’ EcoRI

Reverse Primer #2 3’

Spe I Not I 5’ Pst 1

Primer Set 2

CD

PCR THERMOCYCLER

1A

+BA

+DC+B C

+D

1. X2. X

S

SNP

SNPS

4. ENX

4 Resulting PCR Products

•The next step was to design this part to fit the registry standard.•Two unwanted restriction sites were identified. •To eliminate these sites, we did a nucleotide swap while maintaining the same amino acid sequence.

•The expected product:

Hemagglutinin Antigen globular head (1045 Base pairs) Virus

Genome

C C

E/N/X HA Globular head Antigen

S/N/PC C

Unwanted restriction sites Virus Genome

C C

E/N/X HA Globular

head Antigen

S/N/P

C CBiobrick part