Engineering Success

Overview

Throughout the project works, we investigated the effect of exogenous addition of AsA on the growth and lipid accumulation of Phaeodactylum tricornutum. qPCR detected significant changes in the expression of APX1, DHAR, UGP and VTC2 genes. We successfully cloned VTC2 gene by Nested PCR and synthesized APX1, DHAR and UGP genes. After that, these four genes were successfully recombined into the plasmid vector to construct the overexpression system. After that, the overexpression system was transferred to Pichia pastoris for functional validation. Finally, the results of the transformation were tested and validated in Phaeodactylum tricornutum. to investigate whether the overexpression system was successfully expressed in Phaeodactylum tricornutum. and its effect on its growth and metabolism, as well as whether its antioxidant stress capacity, lipid content and fucoxanthin content were increased.
There are three engineering iterations in our project. First, in the first cycle, we treat the selected algal species under specific conditions and screen for optimal treatment conditions and target genes. Secondly, in the second cycle, we further define the treatment conditions and perform expression vector construction and expression validation. Finally, in the third cycle, we transferred the vectors into Pichia pastoris and Phaeodactylum tricornutum for further functional validation.

Design Cycle 1: Optimal treatment concentration and target gene screening of AsA in Phaeodactylum tricornutum

The principle of our engineered algal strain is to achieve antioxidant protection of its cellular chloroplasts from oxidative damage by stimulating the production of endogenous ascorbic acid. ROS act as intracellular signaling molecules regulating plant development, programmed cell death, and response to biotic and abiotic stresses. AsA is directly involved in the scavenging of ROS, and the AsA-GSH cycle plays a very important role in the scavenging of ROS. `
At the beginning of the project, we wanted to investigate the effect of high concentrations of ascorbic acid on microalgae and, at the same time, to verify the soundness of the project idea. We treated microalgae with different concentrations of ascorbic acid and measured their physiological and biochemical parameters to reflect the effect of AsA on microalgae cells. (For more details, visit Design)

Fig.1 AsA synthesis pathway and ROS clearance mechanism in plants

Table 1 Measurement indicators and tools used

Fig.2 qPCR analysis of APX1, UGP, VTC2, DHAR gene expression in the VC-250 treatment group

In the first engineering iteration, we found that the addition of certain concentrations of exogenous AsA promoted the photosynthetic efficiency of cell chloroplasts and led to an increase in cell lipids, unsaturated fatty acids, and fucoxanthin content. This led us to great interest. We performed qPCR on the genes of lipid synthesis pathway, rockweed flavin metabolism pathway, ascorbic acid metabolism pathway, and N/P metabolism pathway of the treated microalgae and finally screened four genes that met our expectation for the next engineering operation.

Design Cycle 2: Gene acquisition and expression vector construction

Get gene

Using the cDNA obtained by reverse transcription of Phaeodactylum tricornutum RNA as a template, the VTC2 gene was cloned by Nested PCR. These three genes were synthesized according to the APX1, DHAR and UGP gene sequences obtained from NCBI.

Fig.3 Principle of Nested PCR

Nested PCR has a very high specificity, which can guarantee the accuracy of the genes cloned by our PCR.

Fig.4 Electrophoresis image of the target gene

APX1 and DHAR are the key genes in the AsA-GSH cycle and participate in the regeneration of AsA. VTC2 encodes GGPase, which catalyzes the reaction between GDP-L-galactose and GTP to produce L-galactose-1-phosphate. Studies have proved that VTC2 is the seventh rate-limiting gene in the process of ascorbic acid synthesis. UGPase encoded by UGP catalyzes UTP reaction to produce UDPG, which is an important substance in the synthesis and metabolism of plant organisms.

Ligation

The vector we use is pPink-HC-FHG-cp. pPink-HC-FHG-cp is 8678bp in length, contains Paox1 promoter and CYC1 terminator, and is pre-linked with sfGFP gene.We use T4 ligase to connect APX1, DHAR, UGP and VTC2 to the vector.

Fig.5 pPink-HC-FHG-cp plasmid map

Overexpression system: We inserted APX1, DHAR, UGP, and VTC2 between the promoter and sfGFP, respectively.

Fig.6 Overexpression system

A: APX1 overexpression system. B: DHAR overexpression system. C: UGP overexpression system. D: VTC2 overexpression system.

Transform

The ligated plasmid is transferred to competent cells and cultured. The following is the growth on the medium containing ampicillin.

Fig.7 A: APX1 overexpression system. B: DHAR overexpression system. C: UGP overexpression system. D: VTC2 overexpression system

Positive colony PCR

Potentially positive single colonies were picked for incubation and subjected to PCR, followed by electrophoresis of the correct bands and sequencing.

Fig.8 VTC2. B:UGP. C:DHAR. D:APX1

A: VTC2 overexpression system. B: UGP overexpression system. C: DHAR overexpression system. D: APX1 overexpression system.

Get the plasmid

According to the sequencing results, the bacterial solution was expanded and cultured and the plasmid was extracted, and the plasmid was verified by PCR detection. Before electroporation transformation, we use the recombinant plasmid as a template for PCR detection to verify whether our recombinant plasmid contains the target gene. The results are as follows, the recombinant plasmid contains the target gene.

Fig.9 Plasmid PCR results

Design Cycle 3: Obtain engineered yeast and algae strains

Electric shock conversion and functional verification

1.Use an electroporator to transfer the plasmid into Pichia pastoris and Phaeodactylum tricornutum
2.Use the Yeast Genomic DNA Extraction Kit to extract the genomic DNA of Pichia pastoris , and use the CTAB method to extract the DNA of Phaeodactylum tricornutum.
3.Use the extracted DNA of the chassis organism as a template for PCR detection, and judge whether the recombinant plasmid is introduced into the chassis organism according to the PCR results.

Fig.10 PCR detection of DNA

After electroporation transformation, we expanded Phaeodactylum tricornutum and then extracted its DNA using the CTAB method, and used the DNA as a template to detect whether the recombinant plasmid gene was introduced into the chassis organism by PCR. The results showed that the VTC2 overexpression system and UGP overexpression system we constructed were successfully introduced into Phaeodactylum tricornutum.
4.Use the Total Protein Extraction Kit to extract the proteins of the chassis organisms, and use the Enzyme Activity Detection Kit to detect the activity of DHAR, APX, GGP, UGP, and explore whether the enzyme activity of the chassis organisms that have been successfully introduced into the recombinant plasmid has increased.
5.Detect the expression of genes related to AsA synthesis and regeneration pathways, fucoxanthin synthesis and metabolism pathways by qPCR, and explore the influence of the successful introduction of recombinant plasmids on the molecular mechanism of chassis organism AsA synthesis and regeneration.
6. Use microplate reader, LSM, GC-MS to detect the lipid content and components of the successfully transformed Phaeodactylum tricornutum. Use a microplate reader to detect its fucoxanthin content.

Fig.11 LSM photographs of Phaeodactylum tricornutum containing an overexpression system stained with BODIPY 505/515.Images were acquired randomly from at least 20 cells per sample, and typical images are presented here. Bar was shown in the fifigure.

We added bleomycin to screen Phaeodactylum tricornutum containing UGP overexpression system and VTC2 overexpression system. The screened Phaeodactylum tricornutum was dyed with BODIPY 505/515 fluorescent dye and observed under a laser confocal microscope. The excitation wavelength is 488nm and the emission wavelength is 530nm. The results are as follows. The red area represents chlorophyll and the green area represents lipids. The total oil content of Phaeodactylum tricornutum in the VTC2 overexpression system was significantly higher than that of the wild type. The chlorophyll content of Phaeodactylum tricornutum in the UGP overexpression system was significantly higher than that of the wild type.

Function prediction

Studies have found that the levels of VTC2 and AsA in Chlamydomonas reinhardtii are significantly increased during oxidative stress. VTC2 is a highly regulated enzyme involved in the synthesis of AsA in Chlorella. It works with the ascorbic acid circulation system to help cells resist oxidative stress. ^[1]. VTC2 and VTC5 in Arabidopsis are involved in the synthesis of GGP and are regulated by light ^[2]. Therefore, we speculate that if the composite part BBa_K3812010 is successfully expressed in the chassis organisms, the activity of GGP and the AsA content in the chassis organisms will be significantly increased, and the ability of the chassis organisms to eliminate ROS and resist oxidative stress will also be significantly improved.
It has been studied that after overexpression of DHAR in the cytoplasm of Arabidopsis in tobacco, the AsA content in tobacco increased by nearly 2 times ^[3]. After expressing DHAR in wheat cytoplasm in tobacco and corn, the AsA content of tobacco and corn leaves increased by 4 times, the DHA content decreased, and the AsA/DHA ratio increased ^[4]. Therefore, we speculate that the AsA content in chassis organisms containing composite parts BBa_K3812008 will increase significantly, the ratio of AsA/DHA may also increase, and the regeneration rate of AsA may be significantly accelerated.
Both APX1 and DHAR participate in the recycling process of AsA, and APX1 mainly promotes the degradation of H₂O₂ to H₂O, eliminating the damage of H2O2 to intracellular organelles such as chloroplasts and mitochondria. Therefore, we speculate that the AsA-GSH cycle in the chassis organisms containing the composite part BBa_K3812007 will be accelerated, and the ability to remove ROS will be significantly increased. It is more conducive to cell resistance to oxidative stress, maintaining the stability of the intracellular membrane structure and organelle structure and the improvement of functions.
UGP is mainly involved in sucrose synthesis, cell wall formation, and response to abiotic stress in higher plants ^[5-8]. The purpose of our construction of UGP overexpression system BBa_K3812009 is to promote the synthesis of organic matter and accelerate the growth of chassis biological cells.

References:

[1]Urzi Ca E I, Adler L N, MD Page, et al. Impact of Oxidative Stress on Ascorbate Biosynthesis in Chlamydomonas via Regulation of the VTC2 Gene Encoding a GDP-l-galactose Phosphorylase[J]. Journal of Biological Chemistry, 2012, 287(17):14234-45.
[2]Dowdle J, Ishikawa T, Gatzek S, et al. Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability.[J]. Plant Journal for Cell & Molecular Biology, 2010, 52(4):673-689.
[3]Amin Elsadig, Eltayeb,Naoyoshi, Kawano,Ghaz, et al. Overexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone, salt and polyethylene glycol stresses[J]. Planta, 2007, 225(5):1255-1264.
[4]Chen Z , Young T E , Ling J , et al. Increasing vitamin C content of plants through enhanced ascorbate recycling[J]. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(6).
[5]Winter H, Huber SC. Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes[J]. Crit Rev Plant Sci. 2000,19: 31-67.
[6]Flores-Diaz M. Alape-Giro'n A, Persson B, et al. Cellular UDP-glucose deficiency caused by a single point mutation in the UDP-glucose pyrophosphorylase[J]. J Biol Chem. 1997, 272: 23784-2379.
[7]Bishop JD, Moon BC, Harrow F, et al. A second UDP-glucosepyrophosphorylase is required for differentiation and development in Dictyosteliumdiscoideum[J]. J Biol Chem. 2002, 277: 32430-32437.
[8]Repetto O, Bestel-Corre G, Dumas-Gaudot E, et al. Targeted proteomics to identify cadmiuminduced protein modifications in Glomusmosseae-inoculated pea roots[J]. New Phytol. 2003, 157: 555-567.

Team:CHINA-FAFU/Engineering

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