Team:Minnesota/Project/Pichia Expression System

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<h1>Engineering Caffeine Biosynthesis in Baker’s Yeast</h1><br>
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<h1>Designing a BioBrick Compatible Pichia Expression System</h1><br>
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<p>The second project explored during the summer was the cloning of genes from <i>Coffea canephora</i> into <i>Saccharomyces cerevisiae</i> to facilitate caffeine biosynthesis in yeast. Caffeine is the one of the most widely consumed substances in many areas of the U.S. but despite this, many problems exist with the current caffeine production system, such as the plant-based production, which is not only dependent on climate and nutrients but is susceptible to disease and pests; the extraction process which is slow and relatively costly; relatively limited modes of consumption—coffee (bitter and a potential allergen) and energy/ soft drinks (linked to tooth decay, high body weight and Type II Diabetes). Yeast is an ideal organism in which to produce caffeine for a number of reasons. Yeast is used in food products and consumed regularly. It is non-pathogenic and is a model organism preferred in industry use. Working with <i>S. cerevisiae</i> is also a great way to contribute parts to the registry, as parts are lacking for yeast. Providing caffeine biosynthesis genes to the registry will allow future teams to explore and compare this well-characterized caffeine biosynthetic pathway to less characterized pathways (i.e. caffeine biosynthesis in Mate). Additionally, these genes allow for comparison of biosynthesis of other alkaloids in yeast, which is an area that is not yet well studied. In addition to caffeine biosynthesis genes, a yeast-<i>E. coli</i> shuttle vector was also designed and pieced together via Gibson Assembly for use by future iGEM participants. Previously submitted shuttle vectors use auxotrophic markers for selection in yeast, which results in two potential weaknesses: rich media cannot be used, which can hinders yeast biosynthetic capacity, and cell death can cause nutrients from dying cells to diffuse into media, creating the potential for false positives. The use of an antibiotic resistance marker (specifically G418-resistance) as the selectable marker for yeast in our shuttle vector overcomes these two weaknesses. With the help of the parts we’ve developed, a caffeine-producing yeast strain could be used to add caffeine to fermented foods and beverages, such as bread. Don’t have time for that cup of coffee?  Have caffeinated toast instead!</p>
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<p>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, P. 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 with 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.
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pBB3G1 and pBB1Z1 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.
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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.
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Product secretion may be had by inserting an alpha secretion signal in the cloning site preceding the have been provided in both vectors.  
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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 is 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.
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TKC functionality is provided by the OriT¬P sequence which hosts the nick site that will be cleaved at during the initiation of conjugation in order to linearize the plasmid, and ligated once transferred to the recipient. Importantly, the OriT¬P 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.
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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.
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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.
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<p><b>Goal</b><br>  To achieve exogenous production of caffeine in <i>Saccharomyces cerevisiae</i> and to create tools to ease the process of introducing protein expression pathways in yeast for future research.</p>
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<p><b>Goal</b><br>  To design a BioBrick compatible <i>E. coli</i>-<i>P. pastoris</i> shuttle vector.</p>
<p><b>Background</b><br>
<p><b>Background</b><br>
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Caffeine synthesis in most caffeine producing plants requires three enzymes to convert Xanthosine to the final product: Monomethylxanthine methyltransferase (MXMT1), 7-methylxanthosine synthase (XMT1), and 3,7-dimethylxanthine N-methyltransferase (DXMT1). Both the XMT1 and DXMT1 genes produce bifunctional enzymes and this particular pathway was chosen so that one less enzyme would be required in the synthesis (compared to three in <i>Coffea Arabica</i>)</p>
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<p>The induction of this novel pathway in <i>S. cerevisiae</i> requires two additional enzymes from<i> C. canephora</i>: XMT1 and DXMT1. <i>S. cerevisiae</i> will produce the required metabolic precursors to Xanthosine, where after the XMT1 enzyme from <i>C. canephor</i>a will use the substrate to synthesize 7-methylxanthosine, then 7-Methylxanthine. DXMT1 will then convert it to Theobromine and finally caffeine. </p>
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<img style="width:500px;" src="http://i1158.photobucket.com/albums/p607/iGEM_MN/caffeine_synth_resize.png">
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<p><b>Figure 1.  Scheme showing proposed caffeine biosynthetic pathway in S. cerevisiae. </b></p>
<p><b>Figure 1.  Scheme showing proposed caffeine biosynthetic pathway in S. cerevisiae. </b></p>
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<p><b>Methods</b><br>
<p><b>Methods</b><br>
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After settling on the two genes, the sequences were taken from NCBI and finalized for synthetic synthesis (XMT1 accession number DQ422954 and DXMT1 accession number DQ422955). A program was designed to optimize these plant genes for yeast use. Because of the similarity of the two genes (~88% similarity) the program was also designed to identify regions of homology between the two genes and adjust the codon usage such that homologous recombination will not occur at the nucleotide level while the overall protein sequence would be retained. The codons were chosen in descending order of complementary tRNA abundance. Any BioBrick cut sites found within the gene sequences were also removed.</p>
After settling on the two genes, the sequences were taken from NCBI and finalized for synthetic synthesis (XMT1 accession number DQ422954 and DXMT1 accession number DQ422955). A program was designed to optimize these plant genes for yeast use. Because of the similarity of the two genes (~88% similarity) the program was also designed to identify regions of homology between the two genes and adjust the codon usage such that homologous recombination will not occur at the nucleotide level while the overall protein sequence would be retained. The codons were chosen in descending order of complementary tRNA abundance. Any BioBrick cut sites found within the gene sequences were also removed.</p>
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4.&nbsp;&nbsp;pkT127 provided by the Schmidt-Dannert lab containing G418 resistance genes and tTEF1.<br>
4.&nbsp;&nbsp;pkT127 provided by the Schmidt-Dannert lab containing G418 resistance genes and tTEF1.<br>
5.&nbsp;&nbsp;ATCC plasmid provided by the Schmidt-Dannert lab containing the ADH1 promoter to drive the &nbsp;&nbsp;&nbsp;&nbsp;G418 R gene.</p>
5.&nbsp;&nbsp;ATCC plasmid provided by the Schmidt-Dannert lab containing the ADH1 promoter to drive the &nbsp;&nbsp;&nbsp;&nbsp;G418 R gene.</p>
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<p><b>Parts List</b><br>
<p><b>Parts List</b><br>
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BBa_K814000&nbsp;&nbsp;dehydroquinate synthase (DHQS) generator<br>
BBa_K814000&nbsp;&nbsp;dehydroquinate synthase (DHQS) generator<br>
BBa_K814001&nbsp;&nbsp;    ATP-grasp (ATPG) generator<br>
BBa_K814001&nbsp;&nbsp;    ATP-grasp (ATPG) generator<br>
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BBa_K814013&nbsp;&nbsp;    DXMT1 protein generator
BBa_K814013&nbsp;&nbsp;    DXMT1 protein generator
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<p>If the HPLC results cannot confirm the presence of caffeine, the first step would be to sequence our genes and align them with the sequences submitted to IDT for the gBlocks. We could also measure the production of the intermediates compared to the cultures to test whether caffeine synthesis is being arrested in the pathway.</p>
<p>If the HPLC results cannot confirm the presence of caffeine, the first step would be to sequence our genes and align them with the sequences submitted to IDT for the gBlocks. We could also measure the production of the intermediates compared to the cultures to test whether caffeine synthesis is being arrested in the pathway.</p>
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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, P. 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 with 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. pBB3G1 and pBB1Z1 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 is 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 OriT¬P sequence which hosts the nick site that will be cleaved at during the initiation of conjugation in order to linearize the plasmid, and ligated once transferred to the recipient. Importantly, the OriT¬P 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

Parts List

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