Team:CHINA-FAFU/Design

Design

Pioneer of Carbon Sequestration- Phaeodactylum tricornutum

Phaeodactylum tricornutum is a single-celled diatom that lives in the ocean. Diatoms have very efficient photosynthesis and carbon sequestration capabilities, accounting for about 40% of global carbon sequestration and 10-50 times that of forests. In addition, Phaeodactylum tricornutum is often used as a raw material for the production of biodiesel. The intracellular lipid content of wild-type Phaeodactylum tricornutum can reach 20% to 30% of the dry cell weight. Phaeodactylum tricornutum can continuously accumulate oil while absorbing carbon dioxide. Therefore, we hope to further explore the molecular mechanism of Phaeodactylum tricornutumphotosynthetic carbon fixation and genetically improve the wild-type Phaeodactylum tricornutum to construct a more efficient The photosynthetic carbon fixation engineering algae strain.

Overview

We first chose to add ascorbic acid to culture the Phaeodactylum tricornutum cells to explore the effect of exogenous AsA on the growth and metabolism of Phaeodactylum tricornutum. It is a relatively common strategy to add plant growth regulators to promote the production of microalgae or accumulate oil, but plant growth regulators have very strict requirements. Most plant growth regulators are synthetic compounds and are included in the category of pesticides internationally. Unreasonable use may cause serious environmental pollution problems, and may also endanger the life and health of organisms ^[1].

Ascorbic acid is a natural organic compound with antioxidant function, which is a kind of vitamin. It is widely found in plants, such as citrus, tomatoes, etc. . It mainly plays the role of removing ROS in plant cells and ensuring cell life activities^[2]. Orderly. Synthetic ascorbic acid is a very safe compound, which is often used as medicine or health food, food additives^[3]^[4], and will not disrupt the environment like plant growth regulation^[5].

Therefore, we chose to use ascorbic acid-containing medium to cultivate Phaeodactylum tricornutum, hoping to increase the growth rate of microalgae and the ability of photosynthetic carbon fixation. At the same time, we overexpress the key genes related to AaA anabolism that we have screened out in the chassis organisms, trying to construct an engineered algae strain that can more efficiently produce lipids and other high-value by-products.

Feature selection

APX (Ascorbate Peroxidase) is one of the key enzymes in AsA-GSH in plant cells. It catalyzes the reaction of AsA (Ascorbic acid) and H2O2 to generate MDA (Monodehydroascorbic acid) and H2O. APX1 is a kind of APX isoenzyme. It exists in the cytoplasm and plays a very important role in the regulation of H2O2 levels in the cytoplasm, nucleus and organelles. It is one of the core enzymes of the cellular ROS scavenging network ^[6] .

DHAR (didehydroascorbate reductase) exists in the chloroplast, mitochondria and cytoplasm of plant cells, and is a very important enzyme in AsA-GSH. DHAR catalyzes the reaction of DHA (didehydroascorbic acid) and GSH (reduced glutathione) to produce AsA and GSSG (oxidized glutathione). APX and DHAR, as the key genes in the AsA-GSH cycle of plants, are extremely important for plants to eliminate intracellular ROS and maintain the normal operation of cell physiological metabolism [7]. Therefore, we plan to link the APX1 and DHAR genes to the pPink-HC-FHG-cp vector through the construction of exogenous gene overexpression technology, and finally introduce them into Pichia pastoris. This may significantly improve the scavenging ability of intracellular ROS, and the cell growth rate may be increased to a certain extent. Our plan is that if Pichia pastoris. cells overexpressing APX1 and DHAR genes grow faster, we will overexpress these two genes in Phaeodactylum tricornutum to increase the growth rate of Phaeodactylum tricornutum. Explore the effect on its lipid and fucoxanthin content.

GGPase (GDP-L galactose phosphorylase) encoded by VTC2 is a key enzyme in the L-galactose (Smirnoff-Wheeler) pathway in the AsA synthesis pathway of plants. In Arabidopsis, GGPase catalyzes the GDP-L-galactose reaction to produce L-galactose-1-P, which is the first step in the biosynthesis of AsA. VTC2 is a highly regulated enzyme for the biosynthesis of ascorbic acid inChlamydomonas reinhardtii. It also participates in the ascorbic acid circulation system and is the main way for algae cells to provide ascorbic acid to eliminate ROS. In order to alleviate ROS stress, higher plants will further increase the expression of VTC2 by increasing the transcription levels of all enzymes in the AsA-GSH cycle such as APX, MDAR, and GSHR. In algae cells, for example, when Chlamydomonas reinhardtii is under oxidative stress, the transcription level of VTC2 and the transcription abundance of all enzymes in the AsA-GSH cycle increase, and the total content of intracellular ascorbic acid also increases [8].Therefore, we plan to overexpress this gene in Phaeodactylum tricornutum cells to improve its ability to resist oxidative stress.

UGP-encoded UGPase (UDP-glucose pyrophosphorylase, UGPase) catalyzes UTP reaction to generate UDPG (uridine diphosphate glucose). UDPG is the precursor molecule of sucrose, glycogen, glycoprotein, cellulose and other substances in plants, and it is plant organic matter An important substance in the anabolic pathway [9]. We use exogenous gene overexpression technology to link UGP with pPink-HC-FHG-cp vector, and introduce it into Pichia pastoris. cells, and then into Phaeodactylum tricornutum. We predict that the carbohydrate content of these two chassis organisms may increase significantly. Among them, for Phaeodactylum tricornutum, it may improve its photosynthetic carbon fixation ability, which is more conducive to the accumulation of organic matter by Phaeodactylum tricornutum.

Overexpression vector construction strategy

We use STUI and KPNI restriction enzymes to digest the pPink-HC-FHG-cp vector, which will be used to introduce into Pichia pastoris. for expression.
In this vector, we added Paox1 promoter, CYC1 terminator, sfGFP coding sequence and ampicillin resistance gene. The AOX1 promoter is a common and efficient promoter in Pichia pastoris.. It is a methanol-inducible promoter. It is activated in the methanol carbon source and induces the transcription and translation of the CDS region. When a repressive carbon source such as glucose and glycerol exists, it is activated. The promoter is turned off, inhibiting the transcription and translation of the CDS region. sfGFP encodes green fluorescent protein, which can make our transformed Pichia pastoris. cells have green fluorescence.
In the vector used for Phaeodactylum tricornutum transformation, we added ampicillin resistance gene and bleomycin resistance gene. The ampicillin resistance gene can make our transformed Escherichia coli have ampicillin resistance. The bleomycin resistance gene will be that our transformed Phaeodactylum tricornutum has bleomycin resistance, which is convenient for us to screen the transformed Phaeodactylum tricornutum.

References:

[1] Le, V.N., Nguyen, Q.T., Nguyen, T.D. et al. The potential health risks and environmental pollution associated with the application of plant growth regulators in vegetable production in several suburban areas of Hanoi, Vietnam. BIOLOGIA FUTURA 71, 323–331. (2020).
[2] Bilska K, Wojciechowska N, Alipour S, Kalemba EM. Ascorbic Acid—The Little-Known Antioxidant in Woody Plants. Antioxidants (2019).
[3] Gęgotek, A., Ambrożewicz, E., Jastrząb, A. et al. Rutin and ascorbic acid cooperation in antioxidant and antiapoptotic effect on human skin keratinocytes and fibroblasts exposed to UVA and UVB radiation. Arch Dermatol Res 311, 203–219 (2019).
[4] Ravetti, S.; Clemente, C.; Brignone, S.; Hergert, L.; Allemandi, D.; Palma, S. Ascorbic Acid in Skin Health. Cosmetics (2019).
[5] Xiaojing Hou, et al."Ascorbic acid enhanced activation of oxygen by ferrous iron: A case of aerobic degradation of rhodamine B." Journal of Hazardous Materials 308. (2016).
[6] Davletova S, Rizhsky L, Liang H, et al. Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis [J]. Plant Cell, 2005, 17(1): 268–281.
[7]Shan, C., Zhang, S. & Ou, X. The roles of H2S and H2O2 in regulating AsA-GSH cycle in the leaves of wheat seedlings under drought stress. Protoplasma 255, 1257–1262 (2018).
[8]Urzi Ca , E. I. , et al. "Impact of Oxidative Stress on Ascorbate Biosynthesis in Chlamydomonas via Regulation of the VTC2 Gene Encoding a GDP-l-galactose Phosphorylase." Journal of Biological Chemistry 287.17(2012):14234-45.
[9]Bao-Hua Zhu, et al."Silencing UDP-glucose pyrophosphorylase gene in Phaeodactylum tricornutum affects carbon allocation." New BIOTECHNOLOGY 33.1(2016): doi:10.1016/j.nbt.2015.06.003.