Team:FAFU-CHINA/Design
Industrial Production
Through interviews with experts and algae factories9, we learned about the approximate process of the industrialization of microalgae biodiesel.
By reading the literature[1], we learn that the price of microalgae biodiesel is now too high, which limits its development.
Among them, the lipid content of algae (which is determined by biomass and single-cell oil content) is low, and the recovery technology is backward, intracellular products are difficult to isolate and the conversion technology is immature to limit its cost. This year we are trying to improve the oil content, harvesting techniques and culture methods of algae.
Experiment Design
This year we tried to improve the lipid content, harvesting technology and culture methods of algae.
We chose the cell wall defect mucous strain CC-849 as the chassis organism.
Why?
1. Relatively high biomass and grease content
2. Gene editing is easy to operate
3. It is cell wall defect type, intracellular products are easy to obtain
4. Normal growth in sewage and high nitrogen and phosphorus removal rate
In the pathway of C. reinhardtii, we selected several genes of key enzymes that are related to lipid accumulation or biomass accumulation. We built their carriers and transferred their electricity to algae. Then we measured their expression level and its effect on physiological indexes of algae.
1.Isocitrate dehydrogenase(IDH) is a key enzyme in the TCA cycle, which can catalyze isocitrate to 2-ketoglutaric acid, provide reducing power for cell growth, and participate in algal energy metabolism and anabolism[4].
2. Malate dehydrogenase(MDH)is one of the key enzymes in bioglucose metabolism and plays an important role in the metabolism of reactive oxygen species[5].
3. Carbamol phosphate synthase(CMPS) plays a role in the urea cycle of C.reinhardtii, affecting the nitrogen and carbon content in algae cells, and plays an important role in the impact of oils and fats[6].
4. Argininosuccinate lyase (ASL), Arginine metabolism can be changed to improve the acid stress resistance, so that the growth of chlamydomonas rhinolepis in acidic wastewater is less affected[7]
In addition, we transferred the exogenous gene Flo from Saccharomyces cerevisiae into algae, which enabled Chlamydomonas reinhardtii to express flocculating protein and increase the self-flocculating function of algae[8].
We modified the gene flo1 from Tianjin-2018. Additionally, the codon optimized flo5 gene was selected, and the three genes were electrically transferred to algae.
Through literature reading[9] and consultation with teachers in our lab, and consulted with other teams, we used the promoter in front of ChR gene as the photosensitive promoter. It can be activated in blue light to promote expression.
We also plan to use sewage as an additional nutrient source to culture microalgae. The extra nitrogen and phosphorus in sewage can increase the biomass of microalgae without controlling the cost.
Expectation
Wild-type Chlamydomonas reinharadtil CC-849 has no function of self-flocculation, and the lipid production is not ideal.
So we hope that through our genetic modification of the algae strain, the last genetically modified algae strain can reach our requirements: it can increase the lipid production and has the function of self-flocculation.
In sewage culture, we hope that engineered algae plants have similar or higher anti-fouling properties than wild-type plants, and can grow normally and accumulate grease in the after-treatment of domestic sewage.
Expectation
Wild-type Chlamydomonas reinharadtil CC-849 has no function of self-flocculation, and the lipid production is not ideal.
So we hope that through our genetic modification of the algae strain, the last genetically modified algae strain can reach our requirements: it can increase the lipid production and has the function of self-flocculation.
In sewage culture, we hope that engineered algae plants have similar or higher anti-fouling properties than wild-type plants, and can grow normally and accumulate grease in the after-treatment of domestic sewage.
Reference
[1] Ma F, Hanna M A. Biodiesel production: a review[J]. Bioresource Technology, 1999, 70(1): 1-15.
[2] Park W K , Yoo G , Moon M , et al. Phytohormone Supplementation Significantly Increases Growth of Chlamydomonas reinhardtii Cultivated for Biodiesel Production[J]. Applied Biochemistry and Biotechnology, 2013, 171(5):1128-1142.
[3]付艳. 利用城市生活污水高效培养微藻的优化研究[D]. 2015.
[4] Lu C, Ward P S, Kapoor G S, et al. IDH mutation impairs histone demethylation and results in a block to cell differentiation.[J]. Nature, 2012, 483(7390):474-8.
[5] Boyd E F, Nelson K, Wang F S, et al. Molecular genetic basis of allelic polymorphism in malate dehydrogenase (mdh) in natural populations of Escherichia coli and Salmonella enterica.[J]. Proceedings of the National Academy of Sciences of the United States of America, 1994, 91(4):1280-1284.
[6] Nakagawa T, Lomb D, Haigis M, et al. SIRT5 Deacetylates Carbamoyl Phosphate Synthetase 1 and Regulates the Urea Cycle[J]. Cell, 2009, 137(3):560-570.
[7] ZHANG Ming-Yang, ZHANG Juan, LIU Long, DU Guo-Cheng, CHEN Jian. Influence of key acid-resistant genes in arginine metabolism on stress tolerance in Lactococcus lactis NZ9000[J]. Microbiology China, 2017, 44(2): 314-324.
[8] Diazsantos E, Vila M, Vigara J, et al. A new approach to express transgenes in microalgae and its use to increase the flocculation ability of Chlamydomonas reinhardtii[J]. Journal of Applied Phycology, 2016, 28(3): 1611-1621.
[9] NagelG,OligD,Fuhrmann M,etal.Channelrhodopsin-1:a light-gated proton channel in gre en algae [J ].Science , 2002 ,296(5577):2395
