Crocin is a carotenoid compound primarily responsible for the color of saffron. It has been shown to exhibit antioxidant, neuroprotective agent, as well as antiproliferative properties in cancer cells in vitro. The saffron plant requires specific climate conditions to grow and thrive. Additionally, the yield of crocin via extraction from saffron is rather low and the process is costly and labor consuming.
Hence, for many years scientists and research teams have tried to biosynthetically produce lab-based crocin. However, given the complex metabolic pathway required to produce crocin, success has been achieved only to a certain extent. Previous researchers have attempted to produce crocin and its precursors in organisms like Escherichia coli and Saccharomyces cerevisiae and have demonstrated and shown the various involved metabolic pathways [1-3]. Nonetheless, the slow growth of plants, limited yield, and difficulty of constructing and optimising extensive synthetic pathways in microorganisms have limited the potential for industrial scale production. With climate change affecting the range of saffron cultivation and the demand for it increasing in the near future, a novel crocin biosynthesis method is essential.
To overcome these issues, we have decided to build upon the vast knowledge of previous research and of our team members , to focus on Rhodotorula toruloides, an unconventional yeast with a high potential for use in industrial biotechnology. Additionally, the yeast naturally produces β-carotene, which eliminates more than half of the reaction steps needed to be introduced to produce crocin in the organism. Based on literature review [4,5] , only 3 new enzymatic reactions are needed to produce crocin in R. toruloides.
The Biosynthesis Of Crocin and Our Goals
The biosynthesis of crocin in bacteria and yeasts has been an emerging yet narrow field of research in the last decade . It is due to new applications of crocin in medicine, cosmetics, food and other industries. Reliance on crocin from saffron grown in regions already badly affected by climate change is not sustainable. Furthermore, the demand for crocin could increase with time. This raises the need for a cheap and reliable crocin source that we believe could be engineered R. toruloides.
We were motivated to work on crocin biosynthesis because an iGEM team Uppsala 2017 had attempted a similar project before that we knew how to build upon. They attempted to introduce the crocin pathway in E. coli. https://2017.igem.org/Team:Uppsala The collective experience of our student members and advisors on yeast engineering and cultivation would guarantee a successful project.
We plan to achieve higher crocin yields and higher concentrations of it in cell biomass than what the Uppsala team and other researchers observed. Furthermore, since crocin is produced in 5 forms - crocin-1, -2, -3, -4, and -5, of which crocin-2 is the metabolically active compound, we will maximise the synthesis of crocin-2 over the other crocins.
To do that we will use the Golden Gate Assembly method developed for R. toruloides to construct plasmids containing the transcriptional units for crocin synthesis as well as for selectable markers . Crocin genes from bacteria and plants will be optimised and combined in different level 2 constructs to find the best ones for maximal expression and crocin-2 synthesis in our yeast.
We will integrate the constructs in R. toruloides strain NP11. To minimise the use of antibiotics an NP11 ura3 strain will be created to allow the use of uracil auxotrophic marker in the transformation process. The auxotrophic strain will be beneficial for further research of R. toruloides by future iGEM teams or researchers.
Once produced, the crocin would be quantified for different isoforms by HPLC-MS. Growth media optimization, bioreactor experiments and metabolic network modelling are few of the approaches our team will take to increase the yield of crocin-2 in our strain. Our host organism R. glutinis has been shown to naturally increase the accumulation of carotenoids under stress conditions - light, temperature change etc., which will be exploited to increase the overall proportion of crocins over the consumed substrate. Genome scale stoichiometric modelling will be utilised to estimate the achievable crocin yields. Bioreactor experiments will be conducted to test the scale-up of the crocin production process and to characterise growth parameters of our strain.
We are developing a cheap and sustainable crocin production method that would open up new directions in cancer research as well as industrial biotechnology. That would have long-term benefits in the field of medical research (particularly anti-cancer), cosmetics, the food industry, and beyond. It would also boost the relatively dormant Latvian biotechnology innovation ecosystem - the participation in the iGEM competition for the first time ever will give an impulse and inspiration to students, academics and the local science community. Most importantly, our project will open up new research avenues in carotenoid biosynthesis. The open knowledge gained could be used by future iGEM teams that also want to engineer R. toruloides yeast.
 Wang, W., He, P., Zhao, D. et al. Construction of Escherichia coli cell factories for crocin biosynthesis. Microb Cell Fact 18, 120 (2019). https://doi.org/10.1186/s12934-019-1166-1
 Xiangdong Pu, Chunnian He, Yan Yang, Wei Wang, Kaizhi Hu, Zhichao Xu, and Jingyuan Song ACS Synthetic Biology 2020 9 (5), 1160-1168 DOI: 10.1021/acssynbio.0c00039
 Chai, F., Wang, Y., Mei, X. et al. Heterologous biosynthesis and manipulation of crocetin in Saccharomyces cerevisiae . Microb Cell Fact 16, 54 (2017). https://doi.org/10.1186/s12934-017-0665-1
 Taoli Liu, Suize Yu, Zhichao Xu, Jiantao Tan, Bin Wang, Yao-Guang Liu, Qinlong Zhu, Prospects and progress on crocin biosynthetic pathway and metabolic engineering, Computational and Structural Biotechnology Journal, 2020, https://doi.org/10.1016/j.csbj.2020.10.019.
 Hexin Tan, Xianghui Chen, Nan Liang, Ruibing Chen, Junfeng Chen, Chaoyang Hu, Qi Li, Qing Li, Weizhong Pei, Wenhai Xiao, Yingjin Yuan, Wansheng Chen, Lei Zhang, Transcriptome analysis reveals novel enzymes for apo-carotenoid biosynthesis in saffron and allows construction of a pathway for crocetin synthesis in yeast, Journal of Experimental Botany, 2019, https://doi.org/10.1093/jxb/erz211
 Bonturi N, Pinheiro MJ, de Oliveira PM, Rusadze E, Eichinger T, Liudžiūtė G, De Biaggi JS, Brauer A, Remm M, Miranda EA, Ledesma-Amaro R, Lahtvee PJ. Development of a dedicated Golden Gate Assembly Platform (RtGGA) for Rhodotorula toruloides. Metab Eng Commun. 2022 doi: 10.1016/j.mec.2022.e00200.