Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides

Expression of Drosophila melanogaster acetylcholinesterase ( DmAChE ) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides 果蝇乙酰胆碱酯酶基因剪接变种在毕赤酵母中的表达及其对有机磷农药敏感性的评价 1 2 first-author https://orcid.org/0000-0001-5124-7845 1 2 1 2 1 2 corresp Shoufeng WANG, sfwang@zju.edu.cn Shoufeng WANG, sfwang@zju.edu.cn sfwang@zju.edu.cn https://orcid.org/0000-0003-0896-5440 Institute of Pharmaceutical Biotechnology, School of Medicine, Zhejiang University, Hangzhou310058, China Institute of Pharmaceutical Biotechnology, School of Medicine, Zhejiang University, Hangzhou310058, China Zhejiang Provincial Key Laboratory for Microbial Biochemistry and Metabolic Engineering, Hangzhou310058, China Zhejiang Provincial Key Laboratory for Microbial Biochemistry and Metabolic Engineering, Hangzhou310058, China Acetylcholinesterase (AChE) is a key enzyme used to detect organophosphorus pesticide residues by the enzyme inhibition method. An accidental discovery of a mutant strain with AChE activity was made in our laboratory during the process of AChE expression by Pichia pastoris . The pPIC9K- Drosophila melanogaster acetylcholinesterase ( DmAChE ) -like expression vector was constructed by codon optimization of this mutant strain, which was transformed into P. pastoris GS115, and positive clones were selected on yeast peptone dextrose (YPD) plate with G418 at 4.0 mg/mL. The GS115-pPIC9K- DmAChE-like strain was subjected to 0.5% methanol induction expression for 120 h, with a protein band at 4.3 kDa found by the tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern of the fermentation supernatant. After preliminary purification by ammonium sulfate precipitation, the enzyme activity was detected to be 76.9 U/(mL⋅min). In addition, the pesticide sensitivity test proved that DmAChE-like is selective and sensitive to organophosphorus pesticides. 目的 2 通过比较果蝇乙酰胆碱酯酶基因剪接变种（DmAChE-like）与果蝇乙酰胆碱酯酶（DmAChE）之间的酶活差异以及对有机磷农药的敏感性差异，为酶抑制法检测农药残留提供新的酶源。 创新点 2 DmAChE-like 是果蝇乙酰胆碱酯酶基因发生剪接突变产生的，在此之前并未被报道过。本文通过检测证明了DmAChE-like具有乙酰胆碱酯酶活性，并且对大多数有机磷农药敏感。这个发现为酶抑制法检测农药残留提供了新的酶源。 方法 2 在本研究中，我们通过构建毕赤酵母重组表达菌株GS115-pPIC9K- DmAChE-like 并进行诱导表达，利用Ellman法检测其酶活，并进行酶学性质研究。我们选取了5种常见农药进行农药敏感性试验，比较其与DmAChE之间的差异性。 结论 2 在本研究中，我们构建了基因工程的GS115-pPIC9K- DmAChE-like 菌株，并在实验室水平诱导了基因表达。我们测试了各个发酵上清液的酶活性，证明了DmAChE-like具有乙酰胆碱酯酶活性。通过Tricine-SDS-PAGE，我们发现特异的蛋白条带为4.3 kDa，与预期结果一致。农药敏感性试验发现DmAChE-like对大多数选择的有机磷农药敏感，表明它可以用作有机磷农药检测酶抑制方法的新酶源。 Acetylcholinesterase (AChE) Pichia pastoris Enzyme activity determination Pesticide sensitivity 乙酰胆碱酯酶 巴斯德毕赤酵母 酶活性测定 农药敏感性 1 Introduction 1 Organophosphorus compounds are the most widely used pesticides due to their broad insecticidal spectrum and relatively low residue ( Chen et al., 2010 Chen et al., 2010 ). They can inhibit acetylcholinesterase (AChE, EC 3.1.1.7) activity and cause excessive nerve excitement, thereby achieving the purpose of killing insects ( Jokanović, 2009 Jokanović, 2009 ; Hossain and Sikder, 2015 Hossain and Sikder, 2015 ; Liang et al., 2019 Liang et al., 2019 ). With the widespread use of organophosphorus pesticides, chronic poisoning in agricultural products has become an issue of increasing concern ( van Dyk and Pletschke, 2011 van Dyk and Pletschke, 2011 ; Koureas et al., 2012 Koureas et al., 2012 ). The main hazard of organophosphorus pesticides pose to the human body is acute toxicity, which usually occurs after large doses or repeated exposure, manifesting in a series of neurotoxic symptoms. In severe cases, respiratory paralysis and even death may occur ( Sakata, 1993 Sakata, 1993 ; Yang et al., 2018 Yang et al., 2018 ). Therefore, finding a method for the rapid detection of pesticide residues is of great significance to ensure food safety. Current conventional methods for determining pesticide residues include gas chromatography (GC) or high-performance liquid chromatography (HPLC) combined with mass spectrometry (MS) ( Ponomarev and Shtykov, 2000 Ponomarev and Shtykov, 2000 ; Albero et al., 2003 Albero et al., 2003 ; Viñas et al., 2003 Viñas et al., 2003 ; Apilux et al., 2015 Apilux et al., 2015 ), which are complicated and time-consuming protocols requiring skilled technicians and expensive instruments ( Villatte et al., 1998 Villatte et al., 1998 ; Duford et al., 2013 Duford et al., 2013 ). Thereby, the enzyme inhibition method is currently the routine technology for detecting organophosphorus pesticide residues. It has the characteristics of simple operation and fast detection, and does not require large instruments or advanced professional knowledge ( Amine et al., 2006 Amine et al., 2006 ), and thus it is suitable for rapid on-site detection ( Mai et al., 2017 Mai et al., 2017 ). Its detection principle is detailed below. Under normal physiological conditions, AChE can hydrolyze acetylcholine into choline and acetic acid. Once the organophosphorus pesticides have entered the human body through food intake, they will combine with the active site of AChE to make it phosphorylate, resulting in phosphorylated AChE that is not easily hydrolyzed, thus losing its catalytic activity ( Aldridge and Davison, 1953 Aldridge and Davison, 1953 ; Fournier and Mutero, 1994 Fournier and Mutero, 1994 ; Pope, 1999 Pope, 1999 ; Tougu, 2001 Tougu, 2001 ; Worek et al., 2012 Worek et al., 2012 ; Li et al., 2013 Li et al., 2013 ). The enzyme inhibition method employs this reaction characteristic, using filter paper or electrode as a carrier to adsorb AChE, and judging the residual amount of pesticides in the sample by the color change of the developer ( Yang et al., 2019 Yang et al., 2019 ). Enzymes used for the enzyme inhibition method include AChE, butyrylcholinesterase, as well as animal and plant esterases. Among these, AChE is the most sensitive to organophosphorus pesticides while being selective, resulting in its wide application. At present, AChE is mainly extracted from insect heads or animal brain tissues ( van Dyk and Pletschke, 2011 van Dyk and Pletschke, 2011 ), which sources are relatively limited, preventing the general use of such enzyme inhibition methods. Choosing a suitable and stable enzyme source is the key to rapid and accurate detection. Compared with other sources of AChE genes, insect-derived AChE is more sensitive to organophosphorus pesticides ( Schulze et al., 2005 Schulze et al., 2005 ). We have previously discovered a naturally spliced gene mutant ( Drosophila melanogaster acetylcholinesterase ( DmAChE )- like ) by chance during the in vitro recombinant expression of DmAChE . That was the first time DmAChE-like was reported. The DmAChE-like enzyme has a smaller molecular weight compared with DmAChE, and is easy to work with. This study planned to demonstrate the AChE activity of DmAChE-like by expressing DmAChE-like in Pichia pastoris , and compare its differences from DmAChE. We performed pesticide susceptibility experiments to compare the sensitivity and selectivity of DmAChE-like and DmAChE to pesticides, so as to provide a new source of enzymes for routine monitoring of organophosphorus pesticides. 2 Materials and methods 1 2.1 Strains and media 2 The pUC57- DmAChE-like plasmid was synthesized by Shanghai Sangon Biotechnology (Shanghai, China). The Escherichia coli BL21 and P. pastoris GS115 strains, as well as the GS115-pPIC9K- DmAChE and the integrative expression vector (pPIC9K-top10) were all stored in our laboratory. Colonies of E . coli were grown in Luria-Bertani (LB) medium (0.01 g/mL peptone, 5 g/L yeast extract, and 0.01 g/mL sodium chloride), while P. pastoris was cultured in yeast peptone dextrose (YPD) medium (0.01 g/mL yeast extract, 0.02 g/mL peptone, and 0.02 g/mL glucose). Transformants of P . pastoris were selected on minimal dextrose (MD) medium plates (0.02 g/mL glucose, 0.4 mg/L biotin, 0.013 g/mL yeast nitrogen base (YNB), and 0.015 g/mL agarose), whereas P . pastoris positive transformants were fermented in buffered glycerol-complex medium (BMGY) or buffered methanol-complex medium (BMMY) (0.01 g/mL yeast extract, 0.02 g/mL peptone, 100 mmol/L phosphate-buffered saline (PBS, pH 6.0), 0.4 mg/L biotin, 0.013 g/mL YNB, and 0.015 g/mL agarose). 2.2 Reagents 2 High-fidelity DNA polymerase (PrimeSTAR HS DNA polymerase), restriction endonucleases, and DNA polymerases were acquired from TaKaRa Biotechnology Co., Ltd. (Dalian, China). Kits for polymerase chain reaction (PCR) product purification were purchased from Shanghai Sangon Biotechnology (Shanghai, China). Plasmid small extraction kits, agarose gel DNA purification kit, and column purification kits were obtained from Axygen (Hangzhou, China). Organophosphate insecticides (purity of ≥98.4%) were purchased from Beijing Century Aoke Biotechnology Co., Ltd. (Beijing, China), and AChE standard products were obtained from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). 2.3 Plasmid construction 2 After codon optimization of the original sequence of DmAChE-like , the pUC57- DmAChE-like plasmid containing the DmAChE-like gene was constructed by Shanghai Sangon Biotechnology (Shanghai, China). Using pUC57- DmAChE-like as a template, the DmAChE-like fragment was amplified by PCR using DmAChE-like -up and DmAChE-like -down primers ( Table 1 Table 1 ), and the PCR amplification products were purified by agarose gel DNA purification kit. The DmAChE-like and pPIC9K fragments were digested by the restriction enzymes Sna BI and Not I, and the digested fragments were ligated using the In-Fusion cloning kit for the construction of pPIC9K- DmAChE-like . * Underlining indicates sequences of restriction sites. AOX: alcohol oxidase. Primer by function Sequence (5'→3') Amplification DmAChE-like -up * GAGGCTGAAGCT TACGTA ATGGCTATCTCTTGTAG DmAChE-like -down * GAATTAATTC GCGGCCGC TTACAACAAAGATTC Verification 5'-AOX GACTGGTTCCAATTGACAAGC 3'-AOX GCAAATGGCATTCTGACATCC 2.4 Bioinformatics analysis 2 Based on the sequencing results, DmAChE-like was analyzed by bioinformatics. The DNAMAN V6 (LynnonBiosoft, USA) was applied to analyze the molecular mass, base composition, and distribution of nucleic acid sequences; ExPASy ( https://web.expasy.org/compute_pi https://web.expasy.org/compute_pi ) was used to calculate its isoelectric point and relative molecular mass; the ProtParam online tool ( https://web.expasy.org/protparam https://web.expasy.org/protparam ) was utilized to predict the protein properties of the protein to be expressed; SignalP software Version 2.0 ( http://www.cbs.dtu.dk/services/SignalP-2.0 http://www.cbs.dtu.dk/services/SignalP-2.0 ) was used for signal peptide analysis ( Nielsen et al., 1997 Nielsen et al., 1997 ; Goujon et al., 2010 Goujon et al., 2010 ; Petersen et al., 2011 Petersen et al., 2011 ). 2.5 P . pastoris transformation and selection 2 The pPIC9K- DmAChE-like plasmid linearized by Sal I was transformed into the host strain GS115 by electric shock, and His + recombinant transformants were selected by MD and G418 plates. Colony PCR was performed using universal primers 5'-alcohol oxidase (5'-AOX) and 3'-AOX ( Table 1 Table 1 ) to obtain positive GS115-pPIC9K- DmAChE-like transformants. 2.6 Induced expression 2 The recombinant GS115-pPIC9K- DmAChE-like strain was shake-cultured in 50 mL BMGY medium at 30 °C to get optical density at 600 nm (OD 600 ) of 2.0–6.0. The bacterial solution was centrifuged at 4000 g for 5 min at room temperature, and the precipitate was collected. The OD 600 value was adjusted to 1.0 with a fresh BMMY medium. Strain culturing was continued at 30 °C, with 5 mL of sample taken every 12 h before adding anhydrous methanol to a final volume fraction of 0.5%. After 120 h of induced expression, the fermentation supernatant was collected by centrifugation at 10 000 r/min for 10 min. 2.7 Enzyme activity assay 2 AChE activity was measured by ultraviolet (UV) spectrophotometry according to Ellman et al. (1961) Ellman et al. (1961) . In brief, the reaction solution contained 100 mmol/L PBS (pH 7.4), 10 mmol/L 5,5'-dithiol-bis-2-nitrobenzoic acid (DTNB), 75 mmol/L acetylthiocholine iodide (ATC), and fermentation supernatant. The mixture was incubated at 37 °C for 10 min, and the change in absorbance of the solution was measured within 1 min at 412 nm. Based on the results for the commercially pure AChE enzyme, the relative enzyme activity was obtained from the standard curve ( Li et al., 2013 Li et al., 2013 ). The GS115-pPIC9K- DmAChE strain expression product was used as a control. 2.8 Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 2 The process of discontinuous electrophoresis included a protein gel, which consists of 16.5% separation gel, 10.0% sandwich gel, and 4.0% concentrated gel (all volume fraction). During electrophoresis, cathode buffer was added to the inner tank and anode buffer was added to the outer tank. The fermented protein supernatant was concentrated with trichloroacetic acid (TCA). Firstly, electrophoresis was performed at 30 V for 1‒2 h. When the leading edge reached the upper edge of the separation gel, the voltage was adjusted to 130 V until the end of electrophoresis ( Pardo and Natalucci, 2002 Pardo and Natalucci, 2002 ; Schägger, 2006 Schägger, 2006 ). Following the electrophoresis, the gel was stained for 4 h and then decolorized for another hour for imaging. 2.9 Effect of pH and temperature 2 AChE enzyme activity was measured in PBS with pH values of 5.0, 6.0, 7.0, 7.4, 8.0, and 9.0, respectively. Changes in absorbance were recorded at 412 nm, and the relative enzyme activity was calculated to get the optimal pH. Under different temperature conditions ranging from 25 to 75 °C, the best reaction temperature was obtained by measuring the enzyme activity of DmAChE-like. The GS115-pPIC9K- DmAChE strain expression product was used as a control. 2.10 Detection of organophosphorus and carbamate pesticides using the DmAChE-like expression product 2 The fermentation broth was centrifuged to remove the cells, and the supernatant was subjected to ammonium sulfate fractionation. The target product was packed in a dialysis bag and desalted in a low-salt buffer solution, and then concentrated with polyethylene glycol to obtain the crude enzyme solution. Five kinds of pesticide standard products (methamidophos, trichlorfon, carbaryl, chlorpyrifos, and carbofuran) were prepared in dilutions of 10 -4 ‒10 -1 mg/mL with distilled water. Volumes of 140 μL of the crude enzyme solutions were mixed with equal volumes of pesticide and left for the reaction to take place at 37 °C for 15 min. At the same time, PBS was used as blank control instead of pesticide. Finally, 560 μL Ellman reagent was added to each reaction mixture to measure DmAChE-like activity for the calculation of the activity inhibition rate (IR): IR=( E 1 - E 2 )/ E 1 ×100%, where E 1 indicates the enzyme activity of the reaction system without pesticide control and E 2 is the enzyme activity of the reaction system with pesticide inhibition. Taking the inhibition rate of enzyme activity as the ordinate and the logarithmic value of the pesticide concentration as the abscissa, the pesticide sensitivity curves were drawn. 3 Results 1 3.1 Identification of clones 2 The mutant strain was a result of spontaneous mutation introduced during the construction of the expression vector pPIC9K- DmAChE , and the complete sequence was obtained by sequencing, with the sequencing results shown in Table 2 Table 2 . The original gene sequence is DmAChE-like (original), and DmAChE-like is the gene sequence after codon optimization ( Fig. 1 Fig. 1 ). A 237-bp DmAChE-like gene fragment was amplified from the plasmid pUC57- DmAChE-like ( Fig. 2 Fig. 2 a). The DmAChE-like was cloned into the expression vector pPIC9K, with the colony PCR results shown in Fig. 2 Fig. 2 b. Sequencing results showed that the recombinant plasmid pPIC9K- DmAChE-like was successfully constructed. Results of electric shock conversion are shown in Fig. 3 Fig. 3 . These results demonstrated that the number of transformants gradually decreased with the gradual rise of G418 concentration. Gene Sequence (5'→3') DmAChE-like (original) ATGGCCATCTCCTGTCGGCAGAGCAGAGTCCTGCCCATGTCCTTGCCCCTGCCTCTGACCATCCCGCTGCCCCTGGTGCTGGTGCTATCACTGCACCTGTCCGGCGTCTGCGGCGTCATCGATCGCCTGGTCGTGCAGACATCCTCCGGACCTGTACGCGGTCGCTCCGTGACGGTGCAGGGCAGGGAGGTGCATCCATATCCCCGAGGCTCCAGCTCCTTGGAATCGCTGCTCTGA DmAChE-like (after codon optimization) ATGGCTATCTCTTGTAGACAATCTAGAGTTTTGCCAATGTCTTTGCCATTGCCATTGACTATCCCATTGCCATTGGTTTTGGTTTTGTCTTTGCACTTGTCTGGTGTTTGTGGTGTTATCGACAGATTGGTTGTTCAAACTTCTTCTGGTCCAGTTAGAGGTAGATCTGTTACTGTTCAAGGTAGAGAAGTTCACCCATACCCAAGAGGTTCTTCTTCTTTGGAATCTTTGTTGTAA A single colony on a G418 plate was picked for PCR using the universal primer (5'-AOX and 3'-AOX) to obtain GS115-pPIC9K- DmAChE-like positive transformants. As shown in Fig. 2 Fig. 2 c, a band of 718 bp appeared (the expected size of the band obtained from the universal primer PCR was 718 bp, showing that the constructed vector has been integrated into the genome of P. pastoris GS115). 3.2 Bioinformatics analysis of DmAChE-like 2 Due to splicing mutations in the gene, the intermediate sequence of DmAChE is missing, resulting in the connection of base pairs 1‒194 of the gene sequence with the 1867‒1909 bp gene fragment, thus forming DmAChE-like . Among them, the open reading frame of DmAChE-like is 237 bp long. Through the bioinformatics analysis of DmAChE-like , the protein sequence identity between DmAChE-like and DmAChE was found to be 79.5%. The result of protein sequence alignment is shown in Fig. 4 Fig. 4 . The first 38 amino acids of DmAChE-like are signal peptides, and the last 40 amino acids are mature peptides. The relative molecular weight of DmAChE-like is 4.3 kDa and the theoretical isoelectric point (pI) is 10.6 according to the analysis, with its main physical and chemical properties as shown in Table 3 Table 3 . The specific mechanism of the reaction between DmAChE-like and organophosphorus pesticides is yet unclear and needs to be further investigated in subsequent work. Physical and chemical properties Predicted result Formula C 186 H 313 N 59 O 59 Molecular weight 4319.9 Number of amino acids 40.0 Theoretical isoelectric points (pI) 10.6 Absorbance 0.1% (1 g/L) 0.3 Total number of negatively charged residues (Asp+Glu) 3.0 Total number of positively charged residues (Arg+Lys) 5.0 Grand average of hydropathicity (GRAVY) -0.2 Instability index 37.0 Aliphatic index 99.5 3.3 Expression and enzyme activity of recombinant protein in the GS115-pPIC9K- DmAChE-like strain 2 A single GS115-pPIC9K- DmAChE-like colony was selected to induce expression with methanol at 30 °C for 120 h. A volume of 1 mL fermentation supernatant was taken at each time point, and after desalting and concentrating by TCA, this was used for tricine-SDS-PAGE gel electrophoresis for qualitative analysis. The target band can be observed at 4.3 kDa, as shown in Fig. 5 Fig. 5 . The enzyme activity of the sampled fermentation supernatant was detected according to the Ellman method. Based on the time curve as shown in Fig. 6 Fig. 6 , the enzyme activity of DmAChE-like gradually increased with fermentation time, and began to stabilize at 96 h. Its value for DmAChE was 14.7 U/(mL⋅min) at 120 h, while that of DmAChE-like also reached 12.3 U/(mL⋅min), with little difference observed. This demonstrates that DmAChE-like does have AChE activity, the level of which is not much different from that of DmAChE enzyme activity. 3.4 Characteristics of DmAChE-like 2 Under the above conditions, pH=8.0 and temperature ( T )=37 °C were optimal for DmAChE-like. As shown in Fig. 7 Fig. 7 , DmAChE-like reached the highest enzyme activity at an optimum of pH=8.0. Between pH 5.0–8.0, this activity for DmAChE-like gradually increased with the rise of pH, but declined significantly when pH > 8.0. Compared with DmAChE, DmAChE-like is less tolerant to acidic environments, while its ability to tolerate alkaline environments is improved, and has an increased optimum pH of 8.0 instead of 7.4. When the temperature was changed from 25 to 75 °C, the activity of DmAChE-like showed a trend of rising first and then decreasing ( Fig. 8 Fig. 8 ). It reached its peak value when the temperature was 37 °C. At T > 50 ℃, the rate of decline of enzyme activity became immediately clear. Similarly to DmAChE, DmAChE-like was basically inactive at temperatures above 75 ℃. 3.5 Detection of organophosphorus and carbamate pesticides using DmAChE-like 2 With the aim to study the sensitivity of DmAChE-like to organophosphorus pesticides, we compared the sensitivity of DmAChE with that of DmAChE-like to five organophosphorus pesticides. The enzyme activity determination and inhibition analyses were performed as described in the experimental part. As shown in Fig. 9 Fig. 9 , DmAChE-like was more easily inhibited than DmAChE under the action of 1.0 mg/mL pesticide. Most pesticides inhibited DmAChE-like by over 50% at 0.1 mg/mL. In addition, DmAChE and DmAChE-like showed different sensitivities to organophosphorus pesticides. As the concentration of carbofuran decreased, the sensitivities of both became more consistent. For the remaining four organophosphorus pesticides, the new splice variant was more sensitive than DmAChE along a decreasing concentration gradient. At the same time, the selectivity between the two enzymes to different organophosphorus pesticides was also different. The order of DmAChE sensitivity to five pesticides was: carbofuran > chlorpyrifos > methamidophos > carbaryl > trichlorfon. The order of DmAChE-like sensitivity to the five pesticides was: methamidophos > chlorpyrifos > carbaryl > trichlorfon > carbofuran. This difference may be attributed to the altered structure of DmAChE-like, leading to changes in the mechanism of its interaction with organophosphorus pesticides. In summary, DmAChE-like showed good sensitivity for the determination of most selected organophosphorus pesticides. This also showed that a further experimental optimization of DmAChE-like expressed by P. pastoris is expected to meet the requirements of pesticide detection and provide a new source of enzyme for routine organophosphorus pesticide monitoring. Currently, only the activity of DmAChE-like has been verified by the present work; however, subsequent studies are planned to focus on the molecular structure of this splice variant and the interaction mechanism between DmAChE-like and organophosphorus pesticides, thereby laying the foundations of methods to improve enzyme activity. 4 Discussion 1 Detection methods used for organophosphorus pesticides currently include chromatography, biosensing, and enzyme inhibition. Among these, chromatography and biosensing have higher sensitivity. As shown in Table 4 Table 4 , the use of biosensors in the detection of low concentration organophosphorus pesticides features a good linear relationship, can achieve lower detection limits, and has high sensitivity and selectivity. The enzyme inhibition method mainly uses spectrophotometry to quantify organophosphorus pesticides. This approach has a wider detection range and can meet the organophosphorus pesticide detection requirements ( Table 4 Table 4 ). In addition, it also has the advantages of low detection cost and fast detection speed, providing the advantage of practicality for on-site pesticide testing. Detection method Substrate Linear range Limit of detection Reference 3 Spectrophotometric Paraoxon 0.5‒150.0 μmol/L 0.2 μmol/L 3 Tang et al., 2014a Tang et al., 2014a Parathion 1.0‒200.0 μmol/L 0.4 μmol/L Methyl parathion 2.5‒200.0 μmol/L 1.0 μmol/L 3 Electrochemical microbial biosensor Paraoxon 0.05‒25.00 μmol/L 9.0 nmol/L 3 Tang et al., 2014b Tang et al., 2014b Parathion 0.05‒25.00 μmol/L 10.0 nmol/L Methyl parathion 0.08‒30.00 μmol/L 15.0 nmol/L 2 Amperometric acetylcholinesterase biosensor Paraoxon 0.001‒5.000 μmol/L 0.7 nmol/L 2 Lang et al., 2016 Lang et al., 2016 Dimethoate 0.005‒1.000 μmol/L 3.9 nmol/L 2 Spectrophotometric Paraoxon 0.5‒10.0 μg/mL 0.136 ng/mL 2 Liang et al., 2019 Liang et al., 2019 Parathion 5.0‒10.0 μg/mL 3.720 ng/mL Furthermore, we used the P. pastoris expression system to induce GS115-pPIC9K- DmAChE-like expression. After detecting the enzyme activity of the expressed product, we confirmed that DmAChE-like indeed presents AChE activity. In the follow-up pesticide susceptibility experiments, we selected five commonly used pesticides for pesticide susceptibility testing, which proved the sensitivity and selectivity of DmAChE-like to these pesticides. Although its measured sensitivity was not very high, DmAChE-like can be tentatively used as a new enzyme source for pesticide residue detection upon further improving its activity and optimizing detection methods. The yeast expression system selected for this study is an eukaryotic expression system, which has the advantages of fast reproduction, simple nutritional requirements, and easy industrial large-scale fermentation production ( Zhou et al., 2015 Zhou et al., 2015 ). In addition, it is capable of a variety of post-translational modifications to proteins in order to help maintain the activity and stability of biological products. In this paper, the original sequence was optimized by codons to make it more suitable for expression in P. pastoris . Nevertheless, the expression level of DmAChE-like is still low, and ways to effectively increase this level remain a challenge. The expression level of DmAChE-like obtained in P. pastoris can be improved by increasing the number of gene copies and optimizing fermentation conditions in subsequent work. In addition, we only tested and verified the enzyme activity of DmAChE-like in this experiment. Further work will focus on the molecular structure of this splice variant and study the interaction mechanism between DmAChE-like and organophosphorus pesticides, to lay the foundations of molecular transformation for the improvement of enzyme activity. 5 Conclusions 1 The genetically engineered GS115-pPIC9K- DmAChE-like strain was constructed in the present study with gene expression induced at the laboratory level. The enzyme activity of the respective fermentation supernatant was tested, which proved that DmAChE-like has AChE activity. A specific protein band was found to be 4.3 kDa by tricine-SDS-PAGE, which was consistent with the expected result. The pesticide susceptibility test found that DmAChE-like was sensitive to most selected organophosphorus pesticides, indicating that it can be used as a new enzyme source for the enzyme inhibition method for organophosphorus detection. References Albero B , Sánchez-Brunete C , Tadeo JL , 2003 . Determination of organophosphorus pesticides in fruit juices by matrix solid-phase dispersion and gas chromatography . J Agric Food Chem , 51 ( 24 ): 6915 - 6921 . https://doi.org/10.1021/jf030414m https://doi.org/10.1021/jf030414m Determination of organophosphorus pesticides in fruit juices by matrix solid-phase dispersion and gas chromatography Aldridge WN , Davison AN , 1953 . The mechanism of inhibition of cholinesterases by organophosphorus compounds . Biochem J , 55 ( 5 ): 763 - 766 . https://doi.org/10.1042/bj0550763 https://doi.org/10.1042/bj0550763 The mechanism of inhibition of cholinesterases by organophosphorus compounds Amine A , Mohammadi H , Bourais I , et al. , 2006 . Enzyme inhibition-based biosensors for food safety and environmental monitoring . Biosens Bioelectron , 21 ( 8 ): 1405 - 1423 . https://doi.org/10.1016/j.bios.2005.07.012 https://doi.org/10.1016/j.bios.2005.07.012 Enzyme inhibition-based biosensors for food safety and environmental monitoring Apilux A , Isarankura-Na-Ayudhya C , Tantimongcolwat T , et al. , 2015 . Paper-based acetylcholinesterase inhibition assay combining a wet system for organophosphate and carbamate pesticides detection . EXCLI J , 14 : 307 - 319 . https://doi.org/10.17179/excli2014-684 https://doi.org/10.17179/excli2014-684 Paper-based acetylcholinesterase inhibition assay combining a wet system for organophosphate and carbamate pesticides detection Chen DQ , Chen CHZ , Du D , 2010 . Detection of organophosphate pesticide using polyaniline and carbon nanotubes composite based on acetylcholinesterase inhibition . J Nanosci Nanotechnol , 10 ( 9 ): 5662 - 5666 . https://doi.org/10.1166/jnn.2010.2477 https://doi.org/10.1166/jnn.2010.2477 Detection of organophosphate pesticide using polyaniline and carbon nanotubes composite based on acetylcholinesterase inhibition Duford DA , Xi YQ , Salin ED , 2013 . Enzyme inhibition-based determination of pesticide residues in vegetable and soil in centrifugal microfluidic devices . Anal Chem , 85 ( 16 ): 7834 - 7841 . https://doi.org/10.1021/ac401416w https://doi.org/10.1021/ac401416w Enzyme inhibition-based determination of pesticide residues in vegetable and soil in centrifugal microfluidic devices Ellman GL , Courtney KD , Andres V , et al. , 1961 . A new and rapid colorimetric determination of acetylcholinesterase activity . Biochem Pharmacol , 7 ( 2 ): 88 - 95 . https://doi.org/10.1016/0006-2952(61)90145-9 https://doi.org/10.1016/0006-2952(61)90145-9 A new and rapid colorimetric determination of acetylcholinesterase activity Fournier D , Mutero A , 1994 . Modification of acetylcholinesterase as a mechanism of resistance to insecticides . Comp Biochem Physiol C Pharmacol Toxicol Pharmacol Endocrinol , 108 ( 1 ): 19 - 31 . https://doi.org/10.1016/1367-8280(94)90084-1 https://doi.org/10.1016/1367-8280(94)90084-1 Modification of acetylcholinesterase as a mechanism of resistance to insecticides Goujon M , McWilliam H , Li WZ , et al. , 2010 . A new bioinformatics analysis tools framework at EMBL‒EBI . Nucleic Acids Res , 38 ( suppl_2 ): W695 - W699 . https://doi.org/10.1093/nar/gkq313 https://doi.org/10.1093/nar/gkq313 A new bioinformatics analysis tools framework at EMBL‒EBI Hossain MS , Sikder MT , 2015 . Potential human health impacts and medical treatment of acute poisoning with organophosphorus pesticides (OPs): a review . Int J Environ Prot Policy , 3 ( 2 ): 6 - 13 . https://doi.org/10.11648/j.ijepp.s.2015030201.12 https://doi.org/10.11648/j.ijepp.s.2015030201.12 Potential human health impacts and medical treatment of acute poisoning with organophosphorus pesticides (OPs): a review Jokanović M , 2009 . Medical treatment of acute poisoning with organophosphorus and carbamate pesticides . Toxicol Lett , 190 ( 2 ): 107 - 115 . https://doi.org/10.1016/j.toxlet.2009.07.025 https://doi.org/10.1016/j.toxlet.2009.07.025 Medical treatment of acute poisoning with organophosphorus and carbamate pesticides Koureas M , Tsakalof A , Tsatsakis A , et al. , 2012 . Systematic review of biomonitoring studies to determine the association between exposure to organophosphorus and pyrethroid insecticides and human health outcomes . Toxicol Lett , 210 ( 2 ): 155 - 168 . https://doi.org/10.1016/j.toxlet.2011.10.007 https://doi.org/10.1016/j.toxlet.2011.10.007 Systematic review of biomonitoring studies to determine the association between exposure to organophosphorus and pyrethroid insecticides and human health outcomes Lang QL , Han L , Hou CT , et al. , 2016 . A sensitive acetylcholinesterase biosensor based on gold nanorods modified electrode for detection of organophosphate pesticide . Talanta , 156 - 157 : 34 - 41 . https://doi.org/10.1016/j.talanta.2016.05.002 https://doi.org/10.1016/j.talanta.2016.05.002 A sensitive acetylcholinesterase biosensor based on gold nanorods modified electrode for detection of organophosphate pesticide Li JQ , Ba Q , Yin J , et al. , 2013 . Surface display of recombinant Drosophila melanogaster acetylcholinesterase for detection of organic phosphorus and carbamate pesticides . PLoS ONE , 8 ( 9 ): e72986 . https://doi.org/10.1371/annotation/5c68b3d3-240f-4e87-b394-ca5301479cef https://doi.org/10.1371/annotation/5c68b3d3-240f-4e87-b394-ca5301479cef Surface display of recombinant Drosophila melanogaster acetylcholinesterase for detection of organic phosphorus and carbamate pesticides Liang B , Wang G , Yan L , 2019 . Functional cell surface displaying of acetylcholinesterase for spectrophotometric sensing organophosphate pesticide . Sens Actuators B Chem , 279 : 483 - 489 . https://doi.org/10.1016/j.snb.2018.09.119 https://doi.org/10.1016/j.snb.2018.09.119 Functional cell surface displaying of acetylcholinesterase for spectrophotometric sensing organophosphate pesticide Mai CQ , Chen S , Chen Y , 2017 . Enzyme inhibition rate method for rapid detection of organophosphorus and carbamate pesticides in cowpea . Plant Dis Pests , 8 ( 4 ): 30 - 32 . https://doi.org/10.19579/j.cnki.plant-d.p.2017.04.008 https://doi.org/10.19579/j.cnki.plant-d.p.2017.04.008 Enzyme inhibition rate method for rapid detection of organophosphorus and carbamate pesticides in cowpea Nielsen H , Engelbrecht J , Brunak S , et al. , 1997 . Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites . Protein Eng , 10 ( 1 ): 1 - 6 . https://doi.org/10.1093/protein/10.1.1 https://doi.org/10.1093/protein/10.1.1 Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites Pardo MF , Natalucci CL , 2002 . Electrophoretic analysis (tricine-SDS-PAGE) of bovine caseins . Acta Farm Bonaerense , 21 ( 1 ): 57 - 60 . Electrophoretic analysis (tricine-SDS-PAGE) of bovine caseins Petersen TN , Brunak S , von Heijne G , et al. , 2011 . SignalP 4.0: discriminating signal peptides from transmembrane regions . Nat Methods , 8 ( 10 ): 785 - 786 . https://doi.org/10.1038/nmeth.1701 https://doi.org/10.1038/nmeth.1701 SignalP 4.0: discriminating signal peptides from transmembrane regions Ponomarev AS , Shtykov SN , 2000 . Determination of pesticides and other physiologically active compounds by capillary gas chromatography with an atomic-emission detector without using standard reference materials . J Anal Chem , 55 ( 1 ): 47 - 51 . https://doi.org/10.1007/BF02757632 https://doi.org/10.1007/BF02757632 Determination of pesticides and other physiologically active compounds by capillary gas chromatography with an atomic-emission detector without using standard reference materials Pope CN , 1999 . Organophosphorus pesticides: do they all have the same mechanism of toxicity? J Toxicol Environ Health B Crit Rev , 2 ( 2 ): 161 - 181 . https://doi.org/10.1080/109374099281205 https://doi.org/10.1080/109374099281205 Organophosphorus pesticides: do they all have the same mechanism of toxicity? Sakata M , 1993 . Organophosphorus pesticides . In: Suzuki O, Watanabe K (Eds.), Drugs and Poisons in Humans . Springer , Berlin, Heidelberg. https://doi.org/10.1007/3-540-27579-7_60 https://doi.org/10.1007/3-540-27579-7_60 Organophosphorus pesticides Schägger H , 2006 . Tricine-SDS-PAGE . Nat Protoc , 1 ( 1 ): 16 - 22 . https://doi.org/10.1038/nprot.2006.4 https://doi.org/10.1038/nprot.2006.4 Tricine-SDS-PAGE Schulze H , Muench SB , Villatte F , et al. , 2005 . Insecticide detection through protein engineering of Nippostrongylus brasiliensis acetylcholinesterase B . Anal Chem , 77 ( 18 ): 5823 - 5830 . https://doi.org/10.1021/ac050383p https://doi.org/10.1021/ac050383p Insecticide detection through protein engineering of Nippostrongylus brasiliensis acetylcholinesterase B Tang XJ , Liang B , Yi TY , et al. , 2014a . Cell surface display of organophosphorus hydrolase for sensitive spectrophotometric detection of p -nitrophenol substituted organophosphates . Enzyme Microb Technol , 55 : 107 - 112 . https://doi.org/10.1016/j.enzmictec.2013.10.006 https://doi.org/10.1016/j.enzmictec.2013.10.006 Cell surface display of organophosphorus hydrolase for sensitive spectrophotometric detection of p-nitrophenol substituted organophosphates Tang XJ , Zhang TT , Liang B , et al. , 2014b . Sensitive electrochemical microbial biosensor for p -nitrophenylorganophosphates based on electrode modified with cell surface-displayed organophosphorus hydrolase and ordered mesopore carbons . Biosens Bioelectron , 60 : 137 - 142 . https://doi.org/10.1016/j.bios.2014.04.001 https://doi.org/10.1016/j.bios.2014.04.001 Sensitive electrochemical microbial biosensor for p-nitrophenylorganophosphates based on electrode modified with cell surface-displayed organophosphorus hydrolase and ordered mesopore carbons Tougu V , 2001 . Acetylcholinesterase: mechanism of catalysis and inhibition . Curr Med Chem Cent Nerv Syst Agents , 1 ( 2 ): 155 - 170 . https://doi.org/10.2174/1568015013358536 https://doi.org/10.2174/1568015013358536 Acetylcholinesterase: mechanism of catalysis and inhibition van Dyk JS , Pletschke B , 2011 . Review on the use of enzymes for the detection of organochlorine, organophosphate and carbamate pesticides in the environment . Chemosphere , 82 ( 3 ): 291 - 307 . https://doi.org/10.1016/j.chemosphere.2010.10.033 https://doi.org/10.1016/j.chemosphere.2010.10.033 Review on the use of enzymes for the detection of organochlorine, organophosphate and carbamate pesticides in the environment Villatte F , Marcel V , Estrada-Mondaca S , et al. , 1998 . Engineering sensitive acetylcholinesterase for detection of organophosphate and carbamate insecticides . Biosens Bioelectron , 13 ( 2 ): 157 - 164 . https://doi.org/10.1016/S0956-5663(97)00108-5 https://doi.org/10.1016/S0956-5663(97)00108-5 Engineering sensitive acetylcholinesterase for detection of organophosphate and carbamate insecticides Viñas P , Campillo N , López-García I , et al. , 2003 . Capillary gas chromatography with atomic emission detection for pesticide analysis in soil samples . J Agric Food Chem , 51 ( 13 ): 3704 - 3708 . https://doi.org/10.1021/jf021106b https://doi.org/10.1021/jf021106b Capillary gas chromatography with atomic emission detection for pesticide analysis in soil samples Worek F , Eyer P , Thiermann H , 2012 . Determination of acetylcholinesterase activity by the ellman assay: a versatile tool for in vitro research on medical countermeasures against organophosphate poisoning . Drug Test Anal , 4 ( 3-4 ): 282 - 291 . https://doi.org/10.1002/dta.337 https://doi.org/10.1002/dta.337 Determination of acetylcholinesterase activity by the ellman assay: a versatile tool for in vitro research on medical countermeasures against organophosphate poisoning Yang X , Dai J , Yang L , et al. , 2018 . Oxidation pretreatment by calcium hypochlorite to improve the sensitivity of enzyme inhibition-based detection of organophosphorus pesticides . J Sci Food Agric , 98 ( 7 ): 2624 - 2631 . https://doi.org/10.1002/jsfa.8755 https://doi.org/10.1002/jsfa.8755 Oxidation pretreatment by calcium hypochlorite to improve the sensitivity of enzyme inhibition-based detection of organophosphorus pesticides Yang XM , Gu YP , Wu SJ , et al. , 2019 . Research on a rapid detection method of pesticide residues in milk by enzyme inhibition . E3S Web Conf , 79 : 03013 . https://doi.org/10.1051/e3sconf/20197903013 https://doi.org/10.1051/e3sconf/20197903013 Research on a rapid detection method of pesticide residues in milk by enzyme inhibition Zhou WJ , Yang JK , Mao L , et al. , 2015 . Codon optimization, promoter and expression system selection that achieved high-level production of Yarrowia lipolytica lipase in Pichia pastoris . Enzyme Microb Technol , 71 : 66 - 72 . https://doi.org/10.1016/j.enzmictec.2014.10.007 https://doi.org/10.1016/j.enzmictec.2014.10.007 Codon optimization, promoter and expression system selection that achieved high-level production of Yarrowia lipolytica lipase in Pichiapastoris Liujia SHI performed the experimental research and data analysis, wrote and edited the manuscript. Fangfang YANG discovered the DmAChE-like gene and verified its function. Yanyan XU provided assistance for the experiment. Shoufeng WANG contributed to the study design, data analysis, and writing and editing of the manuscript. All authors have read and approved the final manuscript and, and therefore have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the National Development and Reform Commission of China (No. 20111158). We thank Wentao JING Engineer (Biological Experiment Teaching Center, College of Life Sciences, Zhejiang University, Hangzhou, China) for providing the RNA samples from the head of Drosophila melanogaster. 10.1631/jzus.B2000525 1673-1581（2021）03-0204-10 2020-09-04 2020-11-16 2021-01-29 2021-03-15 2022-05-09 © Zhejiang University Press 2021 Liujia SHI, Fangfang YANG, Yanyan XU, and Shoufeng WANG declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 3 22