Team:Linkoping Sweden/Description/light inducible promoter
Inducible System
Knockout of metabolic target genes in C. reinhardtii has been successfully performed many times before. But so far, the outcome of these knockouts has not resulted in algal strains producing enough biomass to be sufficiently profitable for biodiesel production(1). For example, knocking out the sta6 gene in C. reinhardtii, encoding for the starch synthesis enzyme ADP-glucose pyrophosphorylase, results in accumulation of 20 % less biomass during nutrient-replete conditions(2). However, upon nitrogen starvation, the cells turn obese. Their cytoplasm and chloroplast fills up with TAG-bodies due to their increased carbon flux towards lipid synthesis(3). Even though, because of the reduced growth rate of this knockout strain, it is not more productive than other strains from an overall perspective.
We believe the ultimate C. reinhardtii strain for biofuel production would have the growth rate of the wild type and the fatty acid accumulation of the sta6 null strain, and our hypothesis is that this could be accomplished with an inducible knockout system. By inducing the expression of Cas9 in C. reinhardtii before nitrogen starvation, more TAGs would accumulate without affecting the growth phase of the algae.
An inducible system might be especially suitable when working with CRISPR/Cas9 because of strong suggesting evidence for Cas9 toxicity when Cas9 is expressed constitutively in C. reinhardtii (4). Although, the effect of this toxicity in algae with induced Cas9 expression remains to be investigated.
The light inducible promoter
Turning the switch from growth to fatty acid production on an industrial scale requires an effective stimulus. There are several variants of inducible promoters that can be used in C. reinhardii to express a gene such as Cas9 at given stimuli, and many of these promoters include addition and removal of chemicals. Due to the potential complexity of using chemicals for this purpose, we decided to use high-intensity light as promotor stimulus. Despite that the use of a light-inducible knockout promotor in a photosynthetic organism might seem contradictory, it has its advantages due to its manageability. We also believe that a light-inducible promoter could be particularly useful for algae growing on different carbon sources than carbon dioxide, such as sugar fed or green waste algae.
In 2013, Park S. et al. investigated the promoter of the highly inducible promoter gene (LIP) of the marine algae Dunaliella sp.(5). They discovered several light-responsive motifs by experimenting with truncations of the promotor fused with a Renilla luciferase gene introduced in C. reinhardtii. In a more recent study from 2016, they identified sequences over-represented in light-inducible promoters (SORLIP)(6). Duplication of the SORLIP sequence in front of the a 100 bp truncation of the LIP promoter showed promising results in activity when stimulated by high- and medium-intensity light. Therefore, we decided to use the 2xSORLIP promotor sequence to accomplish the inducible expression of Cas9 in C. reinhardtii.
References
1. Goncalves EC, Wilkie AC, Kirst M, Rathinasabapathi B. Metabolic regulation of triacylglycerol accumulation in the green algae: Identification of potential targets for engineering to improve oil yield. Plant Biotechnology Journal. 2016;
2. Krishnan A, Kumaraswamy GK, Vinyard DJ, Gu H, Ananyev G, Posewitz MC, et al. Metabolic and photosynthetic consequences of blocking starch biosynthesis in the green alga Chlamydomonas reinhardtii sta6 mutant. Plant J. 2015;81(6):947–60.
3. Goodenough U, Blaby I, Casero D, Gallaher SD, Goodson C, Johnson S, et al. The path to triacylglyceride obesity in the sta6 strain of Chlamydomonas reinhardtii. Eukaryot Cell [Internet]. 2014 May [cited 2016 Sep 18];13(5):591–613. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24585881
4. Jiang W, Brueggeman AJ, Horken KM, Plucinak TM, Weeks DP. Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii. Eukaryot Cell [Internet]. 2014 Nov [cited 2016 Sep 4];13(11):1465–9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25239977
5. Park S, Lee Y, Lee J-H, Jin E. Expression of the high light-inducible Dunaliella LIP promoter in Chlamydomonas reinhardtii. Planta [Internet]. 2013 Dec [cited 2016 Sep 4];238(6):1147–56. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24043576
6. Baek K, Lee Y, Nam O, Park S, Sim SJ, Jin E. Introducing Dunaliella LIP promoter containing light-inducible motifs improves transgenic expression in Chlamydomonas reinhardtii. Biotechnol J. 2016;11(3):384–92.
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