Overview

    We conducted experiments in the lab strictly according to the "design-build-test-learn" cycle. To achieve the goal of building a high-yield GABA longan cell factory, we first conducted the design of experiments, mainly by reviewing a large amount of literature to determine the appropriate GABA synthesis pathway and using HMM search. In the process, we created five composite parts, one of which was created based on the existing basic parts, and two parts were not available due to the confidentiality principle. We then transferred the vector to Agrobacterium tumefaciens-infested longan healing tissues and treated them with both blue light and high temperature to test whether the system we constructed could achieve a real increase in GABA content in longan healing tissues and form a long-term stable succession culture of high-yielding GABA cell system to expand the culture using fermenters. After passing the test, we will reflect and summarize our experience from the above three parts to better carry out the next ground engineering cycle and expect to extract the GABA from the high GABA-yielding longan cell line to better serve the pharmaceutical industry and achieve our ultimate goal. Fig.1 iGEM engineering cycle Fig.2 Engineering Cycle Diagram

Identification of genes

Determination of the pathway
    In higher plants, the metabolism of GABA is mainly accomplished by three enzymes, firstly, the irreversible decarboxylation of L-glutamic acid (Glu) at the α-position under the action of GAD to form GABA, and then the reaction of GABA with pyruvate and α-ketoglutarate catalyzed by GABA transaminase (GABA-T) to form succinate. Then, GABA reacts with pyruvate and α-ketoglutarate to form succinic semialdehyde, and finally succinic semialdehyde dehydrogenase (SSADH) catalyzes the oxidative dehydrogenation of succinic semialdehyde to form succinic acid, which finally enters the tricarboxylic acid cycle (krebs circle). This metabolic pathway constitutes a branch of the TCA cycle, called the GABA branch. Fig3. Plant GABA synthesis pathway     After comparing the three synthesis pathways, we chose the pathway of GABA generation from glutamate catalyzed by GAD in the GABA branch.
GAD&CaM gene screening
    By means of sequence alignment, phylogenetic affinity analysis, structural domain validation and transcriptome analysis, we successfully screened DlGAD2,DlGAD5,DlCaM1 and DlCaM2 as candidate genes for constructing the vector system. Fig.4 Evolutionary tree of CaM genes in longan, Oryza sativa L. and Arabidopsis Fig.5 Conservative structural domain of CaM genes in longan, Oryza sativa L. and Arabidopsis. Fig.6 Expression of DlCaM in the early stage of somatic embryogenesis in Longan GAD gene screening Fig.7 Conservative structural domain of DlGAD genes in longan Fig.8 Expression of DlGADs in the early stage of somatic embryogenesis in Longan     During the previous screening of genes, we took many detours, such as initially misclassifying DlCMLs within DlCaMs and not screening DlGADs precisely enough. However, after correcting the mistakes one by one, we finally identified the target genes.

Construction of vector

    We selected the pCAMBIA1301SN vector after modification of pCAMBIA1301 to construct the Longan DlGAD overexpression vector, and we selected the pGW3 (Genovo-w-3, Source: The New Zealand Institute for Plant and Food Research Limited) vector to construct the sgRNA of Longan DlCaM gene editing vector.
Construction of pCAMBIA1301SN-DlGAD overexpression vector
    The constructed recombinant plasmid pCAMBIA1301SN was used as a template for PCR using specific primers for the DlGAD2 gene and DlGAD5 gene of Longan, and the results were shown in Fig10 and Fig11. The sizes of PCR amplification products were about 1428bp and 1473bp, which were consistent with the sizes of DlGAD2 gene and DlGAD5 gene of Longan.
Fig.9 Flow chart of constructing Longan gene overexpression vector(1) Fig.10 Flow chart of constructing Longan gene overexpression vector(2)     In order to further verify the accuracy of the insertion site of the DlGAD2 gene in Longan, the recombinant plasmid pCAMBIA1301-DlGAD2 was verified by double digestion using the restriction endonucleases FastDigest KpnⅠ and FastDigest SalⅠ, and the recombinant plasmid pCAMBIA1301SN-DlGAD5 was verified by double digestion using the restriction endonucleases FastDigest KpnⅠ and FastDigest PstⅠ, and the digested products were imaged by electrophoresis gel, and four bands were present as shown in Fig12. The serial number ① represents the band of the product after double digestion of DlGAD5 with a length of 1473bp, serial number ② represents the band of the product after double digestion of DlGAD2 with a length of 1428bp, serial number ③represents the band of the product after double digestion of the empty plasmid pCAMBIA1301SN with a length of 12596bp by the restriction endonucleases FastDigest KpnⅠ and The band of the product of double digestion of FastDigest PstⅠ and FastDigest SalⅠ by restriction endonucleases FastDigest KpnⅠ and FastDigest PstⅠ, and the band of the product of double digestion of pCAMBIA1301SN by restriction endonucleases FastDigest KpnⅠ and FastDigest SalⅠ, indicating the correct insertion site of DlGAD2 and DlGAD5 on the vector. The insertion site was correct. The results of the combined PCR assay and double digestion validation showed that the DlGAD5 and DlGAD2 genes were successfully inserted into the polyclonal site of the intermediate expression vector pCAMBIA1301SN and could be used for subsequent studies. Fig.11:PCR amplification product of DlGAD2 Fig.12 PCR amplification product of DlGAD5 Fig.13 Enzyme-digested product Construction of pGW3-ΔDlCaM
    The basic flow of this section is as follows: Fig.14 Flow chart of constructing pGW3-ΔDlCaM ①Plasmids
The vector we used in our experiments was Genovo-W-3, commonly referred to as pGW3. pGW3 has a fragment length of 16,654 bp. We cleaved it with BsaⅠ and gum recovered a fragment of size 15,429 bp, the pGW3 backbone. We used T4 ligase to ligate the sgRNA to the pGW3 backbone to obtain the recombinant plasmid. Fig.15: Genovo-W-3 plasmid mapping ②Enzyme digestion
Plasmid pGW3 was digested with Bsa I: Fig.16 Enzyme-digested product ③Identification of positive clones
After 10 -12h, single clones were selected for colony PCR identification and sequencing to confirm whether the target was attached to the vector. The bacteriophage with correct PCR bands will be selected and a tube of large shake extracted plasmids will be sequenced. Fig.17 Bacteriophage PCR result ④Transfer of recombinant plasmid into Agrobacterium
Fig.18: pGW3-ΔDlCaM1 recombinant vector and pGW3-ΔDlCaM2 recombinant vector Agrobacterium bacteriophage PCR

Agrobacterium infestion

    Due to the epidemic, our access to the laboratory was somewhat hampered during the summer and the culture of the material took longer, with the first successful infestation only in mid-August and one in September. The infestation process requires a lot of attention, and one mistake may cause material contamination leading to experimental failure, we experienced many failures, and finally achieved success in the case of transient 6 days.

Exogenous treatments

1.Blue light treatment
Material: Longan callus (+ Ag) subcultured for 20 days was directly treated with blue light in culture flask
Number of treatments: 27 bottles of materials with relatively consistent growth
Blue light parameters: 32umol·m-2·S-2
Treatment time: 30min, 60min, 90min, 120min, 150min, 180min, 210min. Take out 3 bottles at each time point mix the samples on the filter paper, put them into the freezing tube, make corresponding marks, put them into liquid nitrogen, and store them in the refrigerator at -80 ℃.
The specific method was as follows: the materials treated for 20 days were transferred with black bags and placed in a blue light incubator, and the samples were mixed every 3 bottles when they were collected. The materials treated by blue light at different times were collected 0.1g per tube, and 6 tubes were collected respectively, and then put into liquid nitrogen immediately
2.High temperature stress treatment:
Material: Longan callus (+ Ag) subcultured for 20 days was directly treated with high temperature stress in culture flask
Number of treatments: 30 bottles of materials with relatively consistent growth
Treatment temperature: 40 ℃
Treatment time: 30min, 60min, 90min, 120min,150min, 180min. Take out 3 bottles at each time point, mix the samples on the filter paper, put them into the freezing tube, make corresponding marks, put them into liquid nitrogen, and store them in the refrigerator at -80 ℃.
The specific method was as follows: the materials treated for 20 days were transferred with black bags and placed in the temperature treatment incubator, and the samples were mixed every 3 bottles when the samples were collected. The materials treated at high temperature for different times were collected 0.1g per tube, and 6 tubes were collected respectively, and then put into liquid nitrogen immediately

GABA content

High temperature and blue light treatment
By observing the trend of GABA content change with time under different culture environments, we found that the GABA content changed with time approximately normally distributed, and the GABA content reached a higher level when the time reached about 30 min. Fig.21: Blue light processing Fig.22 High temperature treatment GABA content in longan after knockdown of DlCaM1
Fig.23 GABA content in longan after knockdown of DlCaM1     As shown in the Fig., the endogenous GABA content of longan healing tissues was increased after knockdown of DlCaM1.

Transfer to fermentor

    Under the influence of the COVID-19, we were not able to enter the laboratory on time for the experiments. In the last crunch, we decided to simulate the fermenter culture with suspension culture of longan healing tissue, and the GABA content of the suspension culture material was measured for 6 d of continuous sample collection, and the results are shown below: Fig.24 GABA content of suspension culture material Fig.25 Predicted content of GABA per yield Fig.26 Predicted annual GABA production content     The GABA content of longan healing tissues cultured in suspension showed an overall trend of increasing and then slowly decreasing from day 6 to day 11 of the initial culture, and reached a peak of 6440.567926 μg/g at day 7 of the suspension culture. The annual yield of longan healing tissue in 5L and 30L fermenters was estimated. The single yield of 5L was 30447784.87 μg and the annual yield was 1587634497 μg, and the single yield of 30L was 182686709.23 μg and the annual yield was 9525806981.3 μg. Depending on this conclusion, the production of high yield GABA cell factories can be guided as a way to increase the GABA content.