Introduction

“By 2040, 642 million adults will have diabetes” ³

Someone famous in Source Title

By developing tools to genetically engineer Yarrowia lipolytica we aim to create a versatile cell factory, which in the future will be able to produce almost any desired product ranging from complex therapeutic proteins to high value chemical compounds. Besides diversity of products cell factory based on Yarrowia lipolytica offers a wide range of substrate tolerance.

We aim to demonstrate this versatility of our Y. lipolytica strain as a cell factory by producing an extracellular heterologous protein. Our first attempt is the production of hrGFP, followed by proinsulin. Moreover we also attempt to produce the metabolic product beta-carotene from the registered BioBricks. In order to introduce genes necessary to achieve both of mentioned ideas we apply pSB1A8YL plasmid, constructed by our team.

hrGFP

Design

In order to test if we were able to clone a of a construct in E. coli pSB1A8YL, which would be expressed, and ultimately prove that we were able to express genes in Y. lipolytica, we chose to develop our own BioBricks. Namely a constitutive TEF promoter (BBa_K2117000) and a codon optimized hrGFP gene (BBa_K2117003) previously used successfully in Y. lipolytica¹. By combining these two parts in a device (BBa_K2117005), the expression should, in theory, be easily detected due to the fluorescence signal produced. The cloning flow can be seen in Figure 1.

Results

The parts were ordered as gBlocks and assembled in E. coli using A3 assembly. The assembly was confirmed using restriction analysis (See Figure 2), PCR and sequencing (data not shown).

Afterwards the construct was transformed into Y. lipolytica PO1f and grown on plates containing selective media. Single colonies were picked and grown in liquid selective media, and subjected to fluorescence microscopy. Figure 2 shows the Y. lipolytica PO1f cells under a confocal laser microscope with 100x magnification. The high fluorescent output from with the BBa_K2117005 construct, proves that the cells are producing hrGFP, and ultimately that our expression system consisting of pSB1A8YL and the TEF1 promoter (BBa_K2117000) can be used for heterologous protein expression in Y. lipolytica.

Proinsulin

Our experiments told us that our plasmid was a powerful tool, allowing us to conveniently assembling constructs in E. coli, which would be expressed in Y. lipolytica. Now we wanted to use it to take on the ultimate challange: Could we use this tool to produce proinsulin, and potentially increase the sustainability of insulin production?

Role of insulin in our bodies

Insulin is a peptide hormone that plays a crucial role in glucose homeostasis and prevents harmful levels of sugar in the blood. After a meal, beta-cells of pancreas islets release insulin to the blood stream. Then insulin activates the glucose transporters, present on the cells surface, and cells are able to absorb the glucose.

Insulin biosynthesis

At the first stage insulin is synthesized as a chain of 101 amino acids, called preproinsulin, which comprises of signal peptide (pre-peptide) and three short chains: A, B and C. The pre-peptide, responsible for directing a nascent polypeptide, is removed from preproinsulin giving proinsulin. Subsequently, the proinsulin is folded and two disulphide bonds are created between chain A and B and one links chain A. In the last step, the C chain is digested from the proinsulin by an exoprotease - carboxypeptidase E⁵. The mature insulin contains chains A and B linked by 3 disulphide bonds and in total comprises of 51 amino acids.

Insulin is linked with diabetes mellitus, the disease that affects insulin production and results in too high sugar level in the blood stream. The treatment is based on taking insulin from the external sources.

Goal

In order to demonstrate Yarrowia lipolytica as a versatile cell factory we aim to produce proinsulin as an answer to increasing global problem with diabetes.

Numbers of diabetes

Current estimations point out that nowadays 8.5% of the global population stuffers from diabetes (which corresponds to 422 million people). Even more disturbing are the predictions, that show that the diabetes will affect 14% of global population by 2040 will (corresponds to 642 million people, and assuming global population to rise to 9.16 billion) ³.

In 2014 the global insulin market reached $24 billion and it will double by 2020 achieving level of $48 billion.

Denmark has a long tradition of producing insulin to treat diabetes. Novo Nordisk, the company established in Denmark in 1923, is one of the main insulin produces. Nowadays insulin production is based on recombinant technology, which means that insulin gene is introduced to the host organism, either prokaryotic (Escherichia coli) or eukaryotic (Saccharomyces cerevisiae). This approach brings both opportunities as possibility of modifying the coding signals, as limits, since post-translation modifications cannot be carried out in prokaryotic host and production in S. cerevisiae gives lover yield than E.coli.

Design

The human proinsulin sequence was obtain from Sures et al. (1980)). The sequence was codon optimized for Y. lip using the codon optimization tool developed by our team. In order to ensure the high rate of transcription we applied the native promoter of Y. lipolytica, TEF1. By adding the iGEM standard RFC10 prefix and suffix to both proinsulin gene and pTEF we were able to clone them into the standard backbones as well as in our Y. lipolytica shuttle vector using either 3A assembly, Gibson assembly or USER cloning. The literature presents several methods of insulin detection that also seem valid for proinsulin. Screening for the recombinant protein may be performed using SDS-PAGE for the cell lysate. Western blotting is another alternative, widely used for insulin detection.

Results

We succeed to create a construct comprised of the shuttle vector pSB1A8yl with the TEF promoter and the codon optimized proinsulin. The construct was transformed into Y. lip. The slowered growth was observed thus the expression of heterologous protein may be suspected. Component detection was performed by the SDS-PAGE and Western blotting. however none of these methods gave a satisfying results. We suspect that SDS-PAGE failed due to the low detection level. As mentioned before Western blotting is suitable for insulin, however proinsulin is not exported through ER thus it is missing in a secretion signal and as a result may not be folded properly. As a solution another approach could be used, mainly fusion of the proinsulin with either GFP or His followed by a detection of an attached fluorescent protein or peptide instead of the proinsulin itself.

Beta-carotene

Vitamin A has been proved to be important for eyesight in mammals, but we do not have the full pathway for the synthesis of this essential vitamin. Beta-carotene is an important precursor, which must be obtained through the diet.

Numbers of vitamin A

Deficiency of vitamin A affects in the majority of cases children and pregnant women. WHO reports that every year 190 million children suffer from deficiency of this vitamin and 5,2 million among these children struggle with night blindness. This numbers may explain the demand for vitamin A. Estimations for this year demonstrates that the carotenoid market will reach $1.24 billion. It is expected that the carotenoid market will achieve $1.8 billion by 3 years (2019)⁷. Besides increasing demand for vitamin A there is also another motivation, mainly way of the production. Vitamin A has so far mostly been produced by artificial chemical synthesis or by extraction from carrots, however the benefits of using microorganisms for production on an industrial scale is increasing (Barredo 2012).

Biosynthesis of the beta-carotene

Beta-carotene is naturally produced by a range of organisms such as plants and fungi, but neither conventional yeast nor Y. lipolytica has the pathway for biosynthesis. Beta-carotene is produced by four enzymatic steps from farnesyl diphosphate (F-PP), which is naturally produced in Y. lipolytica. In the next step, farnesyl diphosphate is converted to geranylgeranyl diphosphate (GG-PP) in a reaction catalyzed by geranylgeranyl diphosphate synthase (CrtE). GG-PP is transformed to phytoene by CrtYB, which is an enzyme with two domains, one functioning as phytoene synthase and another as lycopene cyclase, in this reaction the first domain plays a crucial role. The next step results in production of lycopene and is catalyzed by carotene desaturase (CrtI). Finally, lycopene is converted by CrtYB with the lycopene cyclase domain into beta-carotene⁶.

Design

The JHU 2011 iGEM team successfully produced beta-carotene in Saccharomyces cerevisiae by constructing three biobricks with the three individual genes encoding the enzymes from the pathway from the fungi Xanthophyllomyces dendrorhous.

In order to remove illegal restriction sites from the beta-carotene biobricks we perform site directed mutagenesis. By designing primers for Gibson assembly of the CrtE, CrtI and CrtYB we combine all three genes with the Y. lip ribosome binding site (RBS), CACA, in front of each ORF. The created construct can be transformed to Y. lip using the shuttle plasmid pSB1A8yl. Since cells overproducing beta-carotene change their color to orange no sophisticated detection method is required.

Results

We improved currently existing biobricks for beta-carotene genes: CrtE, CrtI and CrtYB by inserting ribosome binding site – CACA, in from of each of the gene and removing illegal restriction sites.

Conclusion

TBD

References

1. Blazeck, J., Liu, L., Redden, H., & Alper, H. (2011). Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach. Applied and Environmental Microbiology, 77(22), 7905–7914. doi:10.1128/AEM.05763-11
2. http://www.who.int/
3. Guariguata, L., et al. "Global estimates of diabetes prevalence for 2013 and projections for 2035." Diabetes research and clinical practice 103.2 (2014): 137-149.
4. http://www.idf.org/sites/default/files/IDF_AnnualReport_2015_WEB.pdf
5. http://www.prnewswire.com/news-releases/global-human-insulin-market-size-of-24-billion-in-2014-to-witness-13-cagr-during-2015---2020-518797291.html
6. Baeshen, Nabih A., et al. "Cell factories for insulin production." Microbial cell factories 13.1 (2014): 1.
7. https://2011.igem.org/Team:Johns_Hopkins/Project/VitA
8. http://www.bccresearch.com/market-research/food-and-beverage/carotenoids-global-market-report-fod025e.html