The seasonal expansion of Achatina Fulica and Angiostrongyliasis Cantonensis causes severe diseases to local people and damage to local environments. However, the current solution for the issue is time-consuming and detrimental due to the use of toxic chemicals, which would pose negative externalities on surrounding plants and animals. GreatBay-SCIE aims to propose an environmentally friendly and efficient method using synthetic biology to engineer Saccharomyces Cerevisiae to attract the snails and eliminate the nematodes, with a kill switch to ensure biosafety. Firstly, as Achatina Fulica favors banana-like odor, we are determined to modify S.cerevisiae to increase the production of isoamyl acetate to produce the smell by overexpressing six endogenous genes in the corresponding pathway. In pursuance to produce a papaya oil odor, we decided to synthesize α-pinene in S.cerevisiae as well by overexpressing three endogenous genes and one heterologous gene in the original given pathway. Subsequently, for the elimination pathway, we transformed sequence encoding for Cry1518-35 into S.cerevisiae for the production of the toxic protein to eliminate nematodes. In order to achieve secretory expression, we have tested several signal peptides that can function in S. cerevisiae. Lastly, considering biosafety and potential leakage of genetically modified yeasts, Bax and BI-1 genes which are responsible for a toxin-antitoxin kill switch system were transformed into our engineered S.cerevisiae. Meanwhile, for the genetically engineered S. cerevisiae to be implemented in reality, we have designed the hardware to fulfil our ALERTS solution.
Fig.1 Schematic design of engineered S. cerevisiae in GAStroPurifier, aiming to produce isoamyl acetate and alpha-pinene to express banana-like and papaya oil-like odor for attraction, to produce Cry1518-35 crystal protein for elimination, and to insert Bax and BI-1 for kill switch.
We decided to test the attracting effects of odors that we planned to synthesize. Therefore, we placed fermented dough mixed with different fruit in a private yard, and recorded the number of giant African snails at different durations. The results demonstrate that giant African snails can be attracted by dough with banana, papaya, and dough only.
Table.1 Number of giant African snails attracted by different dough at different time durations.
Fig.2 Three fermented doughs mixed with different fruits are placed to attract the giant African snails in the yard.
In order to increase the production of isoamyl acetate, we are determined to increase the accumulation of isopentanol by overexpression of ARO10 and ADH7. (Fig.3A) The overexpression of ATF1 enables the conversion from the alcohol to isoamyl acetate. The endogenous DNA strands were extracted and amplified from CEN.PK2-1C genome and integrated simultaneously to site 106 of CEN.PK2-1C using lithium acetate transformation, in which CRISPR-Cas9 is utilized to cut the site and the donor DNA strands, which were our coding sequences, would be inserted through homologous recombination. (Fig.3B.1) The construction was then verified by conducting yeast colony PCR followed by gel electrophoresis, which shows the target strands were integrated into the genome successfully (Fig.3C). The constructed strain was named SCIE L1. The fermentation was carried out and lasted for 48 hours using YPD+2% glucose medium and dodecane as solvent. We collected the product, and detected by GC-MS analysis. The result of the analysis demonstrates that we have successfully produced isoamyl acetate through our engineered S. cerevisiae using isoamyl acetate as the control. (Fig.4)
For further improvement in the production of isoamyl acetate, it is necessary to enhance the supply of precursor 2-ketoisocaproate (KIC) in our modified SCIE L1. This can be achieved by overexpression of endogenous LEU4, LEU2 and LEU1 (Fig.3A). Since the presence of leucine can result in a decrease in the activity of LEU4, the 547th amino acid of the LEU4 gene is deleted (LEU4S547∆) to diminish its sensitivity to leucine while maintaining its function. Furthermore, to maximize the supply of KIC and minimize the formation of by-products, LEU4S547∆, LEU2 and LEU1 were targeted and overexpressed in mitochondria, reaching a higher regional enzyme concentration. It can be achieved by appending the first 26, 47, 41 amino acids from COX4, CDC9, COX6 to the N-terminus of LEU4S547∆, LEU2, and LEU1 coding sequences, respectively. The strands are simultaneously integrated into SCIE L1 at site His3 (Fig.3B.2). Yeast colony PCR and gel electrophoresis were carried out, and it successfully proved that the DNA strands have been integrated into the genome (Fig.3C). The constructed strain was named SCIE L2. We conducted fermentation of the yeast and tested our product through GC-MS using isoamyl acetate as the control. The results show an increase in the overall expression level of isoamyl acetate (Fig.4).
Fig.3 Expression of isoamyl acetate in S. cerevisiae. (A) Genetic pathway of producing isoamyl acetate. LEU4^S547∆, encoding for 2-isopropylmalate synthase (2-IPMS), in which the codon of the 547th amino acid S is deleted. LEU1, encoding isopropylmalate isomerase. LEU2, encoding 3-IPM dehydrogenase. ARO10, encoding 2-oxoacid decarboxylase. ADH7, encoding for alcohol dehydrogenase 7. ATF1, encoding for alcohol acetyl-coenzyme A (acetyl-CoA) transferase (AATase) (B) Genetic circuit construction for producing isoamyl acetate. ARO10, ADH7, and ATF1 are transformed into site 106 through homologous recombination using 106 upstream (106 US) and 106 downstream (106 DS). CDC9(first 47 amino acids)-LEU1, COX6(first 41 amino acids)-LEU2, COX4(first 26 amino acids)-LEU4S547∆ are transformed into site His3 through homologous recombination using His3 upstream (His3 US) and His3 downstream (His3 DS) (C) Gel electrophoresis analysis of integrated sequence ARO10-ADH7-ATF1 and CDC9-LEU1-COX6-LEU2-COX4-LEU4S547∆
Fig.4 GC-MS analysis of the product isoamyl acetate of SCIE L1 and SCIE L2 fermentation, using Standard isoamyl acetaet as control
According to GC-MS analysis, the production of isoamyl acetate of SCIE L1 is 22.5, and SCIE L2 is 32.7 mg/L (Fig.5), which shows an increase in the yield.
Fig.5 Testing the production of isoamyl acetate (A) standard curve of isoamyl acetate (B) the production of isoamyl acetate of SCIE L1 and SCIE L2.
In order to produce the odor of papaya oil, we engineered S.cerevisiae to produce alpha-pinene. The endogenous genes tHMG1 and IDi were overexpressed in the pathway to increase the supply of precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (Fig.6A). The strands were simultaneously integrated into CEN.PK2-1C at the His3 site (Fig.6B). The transformation was proven to be successful by colony PCR and gel electrophoresis (Fig.7A). The accuracy of the transformation was proved by the result of sequencing (Fig.7B.1). The modified strain was named SCIE L3. We carried out fermentation, and sent the product for GC-MS analysis using CEN.PK2-1C as the control (Fig.8).
To achieve a high expression level of alpha-pinene, it is indispensable to increase the supply of geranyl pyrophosphate (GPP) in our strain SCIE L3 and minimize the supply of farnesyl diphosphate (FPP) which would result in the production of by-products. Thus, ERG20 was overexpressed with the introduction of the mutations F96W and N127W (ERG20ww), mitigating the formation of FPP while maintaining its activity for producing GPP. The heterologous gene derived from Pinus taeda encoding for alpha-pinene synthase (PS) is required to convert GPP to alpha-pinene. In order to maximize the production of alpha-pinene, a N-terminus truncation of 48 amino acids was introduced (t48PS) (Fig.6A). In addition, we decided to express ERG20ww and t48PS in two possible ways. First, ERG20ww and t48PS were simultaneously transformed into SCIE L3 (Fig.6B.2). Second, we are determined to express ERG20ww linked to the t48PS through a flexible linker (FL) GGGGSGGGGS, which would increase the amount of GPP converted to alpha-pinene (Fig.6B.3). The result of the transformation was verified successfully by gel electrophoresis (Fig.7A) and sequencing (Fig.7B.2). The strain was named SCIE L4. Fermentation was conducted using the same procedure, and the product was collected and sent for GC-MS analysis using CEN.PK2-1C as the control (Fig.8). Although the result of the analysis was not alpha-pinene, the odor of our product resembled the smell of papaya.
Fig.6 Expression of alpha-pinene in S. cerevisiae. (A) Genetic synthesis pathway of producing alpha-pinene. tHMG1, endocing truncated hydroxymethylglutaryl-CoA reductase. IDi, encoding Isopentenyl-diphosphate delta-isomerase. ERG20ww, encoding farnesyl pyrophosphate synthetase (FPP synthase), with mutations F96W and N127W. t48PS, encoding alpha-pinene synthase, with a truncation of N-terminus 48 amino acids. (B) Genetic circuit construction for producing alpha-pinene. 1. tHMG1 and IDi are transformed into site His3, through homologous recombination using His3 upstream (His3 US) and His3 downstream (His3 DS). 2. ERG20ww and t48PS are transformed into site 106 through homologous recombination using 106 upstream (106 US) and 106 downstream (106 DS). 3. Another method of construction suggests ERG20ww links to t48PS through a flexible linker (FL), then transformed into site 106 of the modified strain SCIE L3.
Fig.7 (A) Gel electrophoresis analysis of transformed tHMG1-IDi, ERG20ww-t48PS and ERG20ww-FL-t48PS. (B) Results of sequencing of transformed 1. tHMG1-IDi and 2. ERG20ww-t48PS
Fig.8 GC-MS analysis for the product of CEN.PK2-1C, SCIE L3 and SCIE L4 fermentation.
After successful attraction of snails, the elimination of parasitic nematode A. cantonensis in the snails are completed by Cry1518-35 proteins. Cry1518-35 is a crystal protein originally produced in Bacillus thuringiensis(Fig.9A). The Cry protein was first expressed in E.coli(Fig.9B) in large amounts for testing on nematodes which is under active progression. In the construction, we fused codon-optimised Cry coding sequence with 6x His tag and PET28a vector(Fig.9B). SDS-page was performed and the result shows that Cry protein was successfully expressed with induction(Fig.9C). The result was confirmed in Western Blot analysis(Fig.9D).
Fig.9 Cry1518-35 protein expression in E.Coli ; (A) Cry protein from B.thuringiensis. (B) Construction of Cry protein with 6x His tag. (C) SDS-page analysis of Cry protein with His tag. (D) Western Blot analysis of Cry protein with His tag. M:marker; 1:PET28a-His-Cry; 2:PET28a-Cry-His; C:control, 1&2 without induction; W:whole cell; S:supernate; P:precipitate. (E) Toxicity assay of Cry protein with C. elegans.
For continuous secretion expression in modified Saccharomyces cerevisiae, an effective signal peptide is necessary. The different signal peptides, JFm, JF, ScMα were primarily constructed with RFP to compared their functionality(Fig.10A). After comparison, Cry was also connected with signal peptides(Fig.10A,Fig.10B).
Fig.10 Construction of different signal peptides with Cry and RFP proteins in S.Cerevisiae with 021 vector. (A) Genetic circuits for SP-Cry/RFP. (B) 3D Structure of Cry proteins with His tag and with different signal peptides.
SP: different signal peptides, JF, JFm, ScMα, OPT.
SDS-page analysis showed that JFm was the most effective one in secretion RFP to supernate(Fig.11A). The fluorescence seen in JFm construction and absent in control CEN.Pk2(Fig.11D) and intensity quantitatively detected in supernates from different signal peptides construction further verified JFm was the most efficient one(Fig.11C). SDS-page analysis for Cry construction also showed that JFm was efficient which was consistent with the previous outcome in RFP construction(Fig.11B).
Fig.11 Verification of SP-RFP/Cry (A)SDS-page analysis of SP-Cry expression. 1:JF-His-Cry, 2:JFm-His-Cry; 3:ScMα-His-Cry; 4:OPT-His-Cry; 5:JF-Cry-His; 6:JFm-Cry-His; 7:ScMα-Cry-His; 8:OPT-Cry-His.(B) SDS-page analysis of SP-RFP expression. 1:JF-RFP; 2:JFm-RFP; 3:ScMα-RFP; 4:OPT-RFP. (C) Fluorescence intensity of RFP in supernates from samples with different signal peptides. (D) The fluorescence indication of different signal peptides.
M:marker; C:control, CENPK2-1C; S:supernate; P:precipitate.
In attraction and elimination steps, genetically modified Saccharomyces cerevisiae is involved. For safety consideration, the biocontainment strategy is necessary. Mammalian derived pro-aproptotic protein BAX functions to promote cell death and BAX inhibitor-1, or BI-1 counteract the effect of BAX(Fig.13). Our biocontainment strategy, kill switch system is based on their combination. We designed synthetic arabinose promoters and tried to use them in the yeast(Fig.12). However, the characterisation was not obivous. Therefore, we utilised existing inducible promoters(Fig.13).
Fig.12 Synthetic arabinose promoter design. (A) Inducible arabinose promoter circuit. (B) NOT gate arabinose promoter circuit.
Fig.13 BAX and BI-1 circuit. (A) BI-1 expresses with inducers and inhibits BAX. (B) Only BAX expresses without inducers.
Detection monitors the efficiency of elimination and ensures the safety of operators using GAStroPurifier hardware. SHERLOCK is a precise technology to detect expected DNA or RNA. Parasitic nematode Angiostrongylus cantonensis on the snail releases cfDNA to the mucus of snail. Therefore, we envision using SHERLOCK to detect the mucus extracted from snail in real time to conduct the detection.
After consideration of the practical application of our project, we decided to design and build a hardware that could seamlessly integrate our synthetic biology products, including genetically modified S.cerevisiae expressing isoamyl acetate, alpha-pinene and Cry1518-35, into daily use while prioritizing biosafety with all our GMOs inserted a Bax kill switch to prevent bio release (Fig.14). We have successfully constructed our hardware (Fig.15). We look forward to not only contributing iGEM community, but also serving to create inspirational effects for the community workers and synbio scientists in the future to confront invasive species.
Fig.14 The design of hardware
Fig.15 The hardware we constructed
