Optimization of the yield of the final products by genetic engineering
In the beginning, we intended to insert the four genes as a cassette to reduce the number of cloning steps and to reduce the number of markers used for transformation. We have chosen five different promoters of different strength to use them in various combinations with the genes to maximize the yield of the final products by controlling the flux of intermediates through the pathway.
Initially, it was planned to assemble the genetic parts using Vegas , Gibson  and CPEC  methods in parallel. However, we have faced some problems making large assemblies. Finally, we have switched to conventional genetic engineering techniques. It was decided to express genes under various promoters from centromeric plasmids carrying different selection markers. Non-identical selection would allow us to insert several genes simultaneously using a double, triple and quadruple selection plates. We would like to create yeast strains with different number and combinations of promoter-gene cassettes. Based on the yield of shinorine and porphyra-334, the optimal combination will be chosen. This work is currently underway.
The increase of sedoheptulose-7-phosphate (S7P) concentration
One of the precursors for shinorine and porphyra-334 biosynthesis is S7P. S7P is one of the intermediates of the pentose-phosphate pathway (PPP). It was reported that the concentration of this substance in yeast cells is very low . Therefore, the increase in the level of S7P in the cells potentially may lead to a rise in the shinorine and porphyra-334 yield.
The change of carbon source from glucose to xylulose increases the inner cell concentration of S7P . However, due to the fact we plan to scale-up shinorine and porphyra-334 production in the bioreactor, it is inconvenient to switch from glucose to more expensive sugar.
Eventually, we expect the rise of S7P concentration will enhance the flux through the enzymes of shinorine and porphyra-334 biosynthesis and will lead to the optimal target products’ yield.
Optimization of the yield of the final products by cultivation conditions
Except for genetic engineering, the yield of target MAAs can be increased by optimization of the growth conditions. In order to get an insight into how variations of different parameters may affect the product yield, we have contacted several researchers working in the field.For example, Prof. S. Churio proposed us to use not only UV but also visible light to favor MAAs biosynthesis.
Dr. K. P. Lawrence suggested us in the interview to apply some other types of environmental stress to induce shinorine and porphyra-334, such as osmotic stress, thermal stress or enhanced salinity. Also, Prof. R. Sommaruga and Prof. S. Churio proposed to use nitrogen-enriched media.
MAAs-enriched yeast extract
In nature, shinorine and porphyra-334 are MAAs, which are synthesized by auto- and heterotrophic organisms and protects them from UV radiation     and some other types of the stress  . However, a limited number of organisms have industrial importance.
Our approach of obtaining shinorine- and porphyra-334-enriched yeast extract would allow us to combine valuable properties of both yeast extracts and MAAs. In this case, there is no need in a purification of the final products, since it can be time- and cost-consuming or even impossible. Also, short life cycle, heterotrophic growth, and well-developed manipulation techniques make yeast extremely attractive as potential producers of MAAs.
As a future goal, in order to increase the effectiveness of the sunscreen, the possibility of incorporation of more diverse MAAs with distinct spectral characteristics can be considered. It was also recommended by the researchers in the field (Dr. K. P. Lawrence, Prof. R. Sommaruga, Dr. R. Garcesa). Although, this will require a thorough investigation of the information available since the genes of the enzymes for the biosynthesis of many MAAs are not yet identified.
- Mitchell, L. A., Chuang, J., Agmon, N., Khunsriraksakul, C., Phillips, N. A., Cai, Y., ... & Blomquist, P. (2015). Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae. Nucleic acids research, 43(13), 6620-6630.
- Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison III, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature methods, 6(5), 343.
- Quan, J., & Tian, J. (2011). Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nature protocols, 6(2), 242.
- Senac, T., & Hahn-Hägerdal, B. (1990). Intermediary metabolite concentrations in xylulose-and glucose-fermenting Saccharomyces cerevisiae cells. Applied and environmental microbiology, 56(1), 120-126.
- Schaaff, I., Hohmann, S., & Zimmermann, F. K. (1990). Molecular analysis of the structural gene for yeast transaldolase. European journal of biochemistry, 188(3), 597-603.
- Clasquin, M. F., Melamud, E., Singer, A., Gooding, J. R., Xu, X., Dong, A., ... & Rabinowitz, J. D. (2011). Riboneogenesis in yeast. Cell, 145(6), 969-980.
- Miyamoto, K. T., Komatsu, M., & Ikeda, H. (2014). Discovery of gene cluster for mycosporine-like amino acid biosynthesis from Actinomycetales microorganisms and production of a novel mycosporine-like amino acid by heterologous expression. Applied and environmental microbiology, AEM-00727.
- Sinha, R. P., Singh, S. P., & Häder, D. P. (2007). Database on mycosporines and mycosporine-like amino acids (MAAs) in fungi, cyanobacteria, macroalgae, phytoplankton and animals. Journal of Photochemistry and Photobiology B: Biology, 89(1), 29-35.
- Conde, F. R., Churio, M. S., & Previtali, C. M. (2000). The photoprotector mechanism of mycosporine-like amino acids. Excited-state properties and photostability of porphyra-334 in aqueous solution. Journal of Photochemistry and Photobiology B: Biology, 56(2-3), 139-144.
- Singh, S. P., Kumari, S., Rastogi, R. P., Singh, K. L., & Sinha, R. P. (2008). Mycosporine-like amino acids (MAAs): chemical structure, biosynthesis and significance as UV-absorbing/screening compounds.
- Lawrence, K. P., Long, P. F., & Young, A. R. (2017). Mycosporine-like amino acids for skin photoprotection. Curr Med Chem, 24, 1-16.
- Oren, A., & Gunde-Cimerman, N. (2007). Mycosporines and mycosporine-like amino acids: UV protectants or multipurpose secondary metabolites?. FEMS microbiology letters, 269(1), 1-10.