Our previous study demonstrated that an Escherichia coli heterologous secretion expression system, mediated by superfolder green fluorescent protein (sfGFP) mutants, significantly enhances recombinant lipase yield and reduces large-scale production costs. In this study, we identified mScarlet3, a fast-folding fluorescent protein, as another effective mediator of secretion expression in E. coli. A novel lipolytic enzyme, named LipHu6, was identified through sequence alignment. Secretion expression of LipHu6 was achieved by fusing mScarlet3 to either its N- or C-terminus. The specific activity of mScarlet3-LipHu6 reached 669,151.75 U/mmol, slightly surpassing that of LipHu6 alone (646,682.69 U/mmol) and markedly exceeding that of sfGFP(-15)-LipHu6 (492,432.39 U/mmol). Notably, N-terminal mScarlet3 fusion had no impact on LipHu6 hydrolytic activity toward short-chain p-nitrophenyl fatty acyl esters (C2-C8). In contrast, mScarlet3-LipHu6 exhibited approximately 1.5- and 1.7-fold increases in hydrolytic activity toward p-nitrophenyl palmitate (p-NPP, C16) and p-nitrophenyl stearate (p-NPS, C18), respectively. In conclusion, this study establishes a novel E. coli heterologous secretion expression system mediated by mScarlet3, offering a highly efficient and cost-effective strategy for the large-scale production of lipolytic enzymes.

Previous reports indicated that self-assembling amphipathic peptide S1v1 (AEAEAHAH)2 significantly enhances the soluble expression, thermostability, and activity of the target proteins when fused to them. In order to obtain high-efficiency enzymes for the large-scale degradation of polyethylene terephthalate (PET), this multifunctional peptide was fused to the N- and C-terminus of FAST-PETase, a variant of Ideonella sakaiensis PETase (IsPETase), with a PT-linker (TTVTTPQTS) harbored between the target protein and the multifunctional peptide. Consistent with previous reports, S1v1 increased the solubility of FAST-PETase slightly. Moreover, it increased the activity of FAST-PETase dramatically. The amount of terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalic acid (MHET) released from PET substrate after 24 h of digestion at 50°C by fusion enzymes bearing N- and C-terminal S1v1 tag was approximately 2.9- and 4.6-fold that of FAST-PETase, respectively. Furthermore, the optimal temperature and thermostability of the fusion proteins increased in comparison with FAST-PETase. The present study provides a novel strategy to improve the depolymerization efficiency of FAST-PETase.

Pneumonia remains a prevalent childhood illness and is the foremost cause of mortality due to infectious diseases among children under the age of five. Mycoplasma pneumoniae (M. pneumoniae) causes the most frequent type of atypical pneumonia in this age group, has raised global health concern. Therefore, there is a pressing need for a rapid, low cost, and user-friendly method for the early diagnosis of M. pneumoniae pneumonia. Herein, we develop a CRISPR sensing platform for on-site M. pneumoniae detection, termed CRAFT (Crispr-based rapid assay device for field testing). The CRAFT provides "sample in-result out" functionality. It completed sample processing and nucleic acid extraction within 5 min at room temperature, with efficiency comparable to commercial kits. RPA and CRISPR/Cas12a reagents were isolated in a closed tube using a movable magnetic bead valve, and the RPA product was then mixed with the CRISPR reagent. The limit of detection for M. pneumoniae using CRAFT was 100 copies/μL, and the method exhibited no cross-reactivity with other respiratory pathogens. CRAFT was utilized to validate 50 clinical samples, and the results demonstrated 100 % consistency with those obtained by qPCR. This versatile platform holds significant potential for point-of-care testing of M. pneumoniae, particularly in resource-limited settings.

Abstract Background Rhizomucor miehei (RM) lipase is a regioselective lipase widely used in food, pharmaceutical and biofuel industries. However, the high cost and low purity of the commercial RM lipase limit its industrial applications. Therefore, it is necessary to develop cost-effective strategies for large-scale preparation of this lipase. The present study explored the high-level expression of RM lipase using superfolder green fluorescent protein (sfGFP)-mediated Escherichia coli secretion system. Results The sfGFP(−15) mutant was fused to the C-terminus of RM lipase to mediate its secretion expression. The yield of the fusion protein reached approximately 5.1 g/L with high-density fermentation in 5-L fermentors. Unlike conventional secretion expression methods, only a small portion of the target protein was secreted into the cell culture while majority of the fusion protein was still remained in the cytoplasm. However, in contrast to intracellular expression, the target protein in the cytoplasm could be transported efficiently to the supernatant through a simple washing step with equal volume of phosphate saline (PBS), without causing cell disruption. Hence, the approach facilitated the downstream purification step of the recombinant RM lipase. Moreover, contamination or decline of the engineered strain and degradation or deactivation of the target enzyme can be detected efficiently because they exhibited bright green fluorescence. Next, the target protein was immobilized with anion-exchange and macropore resins. Diethylaminoethyl sepharose (DEAE), a weak-basic anion-exchange resin, exhibited the highest bind capacity but inhibited the activity of RM lipase dramatically. On the contrary, RM lipase fixed with macropore resin D101 demonstrated the highest specific activity. Although immobilization with D101 didn’t improve the activity of the enzyme, the thermostability of the immobilized enzyme elevated significantly. The immobilized RM lipase retained approximately 90% of its activity after 3-h incubation at 80 °C. Therefore, D101 was chosen as the supporting material of the target protein. Conclusion The present study established a highly efficient strategy for large-scale preparation of RM lipase. This innovative technique not only provides high-purity RM lipase at a low cost but also has great potential as a platform for the preparation of lipases in the future.

The polymeric chain reaction (PCR) has come under fire for being time-consuming, requiring expensive equipments, and requiring the extraction and purification of nucleic acids. Here, an ultra-fast and sensitive detection platform without nucleic acid extraction solved the above problems. Firstly, the RoomTemp Sample Lysis Kit released the nucleic acid in 3 min and removed the inhibition to facilitate the amplification reaction. What's more, ultra-fast PCR (UF-PCR) can complete 40 cycles in just 15 min and 50 s. To improve the sensitivity and provide more convenient reading modes, CRISPR/Cas12a was mediated to detect Lumpy skin disease virus

Foot-and-mouth disease virus (FMDV) causes Foot-and-mouth disease, which was listed as a reported disease by Office international des e ' pizooties. It has caused great economic losses to the meat industry. Here, we developed a sensitive, rapid, and visible method based Reverse Transcriptase Enzyme-assisted recombinant isothermal amplification technique (RT-ERA) and CRISPR/Cpf1 for detecting FMDV. RT-ERA achieved simultaneous reverse transcription and amplification in a short time at 37 celcius. Moreover, CRISPR/Cpf1 specifically recognized the products and amplified the signals through the trans-cleavage activity, which converted the DNA signals into fluorescence signals and lateral flow assay strip. Within 30 min, the method detected FMDV as low as 10 copies center dot mu L-1, making it 103 times more sensitive than gel electrophoresis and 10 times more sensitive than reverse transcription quantitative real-time PCR. Moreover, the method had good performance in actual samples. Therefore, this method realized visual detection without the large instruments and showed significant potential for FMDV assay.

The spread of African swine fever virus (ASFV) caused huge economic costs, so early detection is particularly important. Here, we established a fluorescence biosensor based on carbon nanodots (CNDs) and loop-mediated isothermal amplification (LAMP) to ultra-sensitively detect ASFV. LAMP with high efficiency produced a large amount of pyro phosphoric acid and caused pH change in a short time. CNDs with strong light stability had a large fluorescence response at the emission wavelength of 585.5 nm to small pH change by the excitation wavelength of 550 nm. The biosensor realized “turn-off–on” mode for ASFV detection with the detection limit as low as 15.21 copies μL−1. In addition, the biosensor had high accuracy in the actual sample assay. Therefore, the biosensor achieved rapid, sensitive, low-cost, and simple detection for ASFV. Moreover, the biosensor broadened the detection pathway of LAMP as a tool with great development prospect.

The livestock industry has been deeply affected by African swine fever virus (ASFV) and Capripoxvirus (CaPV), which caused an enormous economic damage. It is emergent to develop a reliable detection method. Here, we developed a rapid, ultra-sensitive, and one-pot DNA detection method combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a for ASFV and CaPV, named one-pot-RPA-Cas12a (OpRCas) platform. It had the virtue of both RPA and CRISPR/Cas12a, such as high amplification efficiency, constant temperature reaction, and strict target selectivity, which made diagnosis simplified, accurate and easy to be operated without expensive equipment. Meanwhile, the reagents of RPA and CRISPR/Cas12a were added to the lid and bottom of tube in one go, which overcame the incompatibility of two reactions and aerosol contamination. To save cost, we only need a quarter of the amount of regular RPA per reaction which is enough to achieve clinical diagnosis. The OpRCas platform was 10 to 100 times more sensitive than qPCR; the limit of detection (LOD) was as low as 1.2 × 10−6 ng/µL (3.07 copies/µL by ddPCR) of ASFV and 7.7 × 10−5 ng/µL (1.02 copies/µL by ddPCR) of CaPV with the portable fluorometer in 40 min. In addition, the OpRCas platform combined with the lateral flow assay (LFA) strip to suit for point-of-care (POC) testing. It showed 93.3% consistency with qPCR for clinical sample analysis. Results prove that OpRCas platform is an easy-handling, ultra-sensitive, and rapid to achieve ASFV and CaPV POC testing. • The platform realizes one-pot reaction of RPA and Cas12a. • Sensitivity is 100 times more than qPCR. • Three output modes are suitable to be used to quantitative test or POC testing.

ABSTRACT Argonaute proteins are widespread in prokaryotes and eukaryotes. Most prokaryotic Argonaute proteins (pAgos) use 5’P-gDNA to target complementary DNA. However, more and more studies on the properties of pAgos make their functions more diversified. Previously reported pAgos only possess several forms of high activity in all eight cleavage patterns, which limits their practical applications. Here, we described a unique pAgo from Marinitoga hydrogenitolerans (MhAgo) with eight cleavage activities. MhAgo can utilize all four types of guides (5’OH-gDNA, 5’P-gDNA, 5’OH-gRNA, and 5’P-gRNA) for ssDNA and RNA cleavage. Further studies demonstrated that MhAgo had high activities with 16-21 nt guides and no obvious preferences for the 5’-end nucleotides of 5’OH-guides. Unexpectedly, MhAgo had different preferences for the 5’-end nucleotides of 5’P-guides depending on the types of targets. Although the specificity of MhAgo was related to the types of guides, single mismatches in the central and 3’-supplementary regions of guides greatly reduced the cleavage efficiency. Additionally, the electrophoretic mobility shift assay (EMSA) demonstrated MhAgo had the weakest affinity for 5’P-gRNA:tRNA duplex, which was consistent with its cleavage efficiency. In conclusion, MhAgo is highly active under a wide range of conditions and can be used for programmable endonucleolytic cleavage of both ssDNA and RNA substrates. The abundant biochemical characteristics of MhAgo broaden our understanding of pAgos and expand the potential application in nucleic acids manipulations.

CP4-EPSPS (Agrobacterium sp. strain CP4 5-enolpyruvylshikimate-3-phosphate synthase) protein showed remarkable thermostability and was highly resistant to proteases, such as trypsin. In order to eliminate the pollution of CP4-EPSPS from the accumulated straws to the surrounding environment during the winter, the present study investigated the extracellular proteases of 21 psychrophilic strains isolated from the south polar region. The results indicated that Stenotrophomonas maltophilia 780 was able to degrade CP4-EPSPS at 18 °C efficiently. Further study indicated that it was able to grow in the extract of Roundup Ready soybean at 18 °C, with CP4-EPSPS degraded to an undetectable level within 72 h. The extracellular proteases of Stenotrophomonas maltophilia 780 are thermo-sensitive, with an optimal temperature of 65 °C. The genomic sequencing result indicated that this strain had more than a hundred putative protease and peptidase coding genes, which may explain its high capability in decomposing CP4-EPSPS.

The binding affinities between E-group colicin (CE) and cognate immunity proteins (Im) are among the strongest interactions in nature. Aiming at mining protein pairs with ultrahigh affinity for artificial multienzyme complexes, DNase domains of CE2, 8, and 9 were engineered to generate CL2, 8, and 9 variants without DNA binding and catalytic activity but retaining Im binding activity, respectively. Further study discovered that all these variants, in addition to a CE7 variant (CL7) from the previous report, and their cognate Im proteins are highly thermostable. Taking advantage of the specific and ultrahigh affinity of these hyperthermostable affinity pairs, a hyperthermostable artificial scaffold was established and applied for cellulose degradation. Four hyperthermostable cellulolytic enzymes were integrated into the system through specific pairing of CL and cognate Im proteins. This complex with four enzymes assembled sequentially exhibited an obvious synergistic effect in hydrolyzing cellulosic substrates at elevated temperature. In comparison with free enzymes, the catalytic activity of the complex enhanced more than half-fold at 70 degrees C with phosphoric acidswollen cellulose as the substrate, while it increased almost onefold at 75 degrees C with Avicel as the substrate. The present study provided four affinity pairs with ultrahigh affinity, hyperthermostability, high specificity, a small and unified structure, and high assembly efficiency. More importantly, it provided an unconventional idea of mining hyperthermostable affinity pairs from mesophilic microorganisms instead of the less-abundant thermophiles, which are the main source pool of the thermostable affinity pairs for the time being.

In the present study, we upgraded Pyrococcus furiosus Argonaute (PfAgo) mediated nucleic acid detection method and established a highly sensitive and accurate molecular diagnosis platform for the large-scale screening of COVID-19 infection. Briefly, RT-PCR was performed with the viral RNA extracted from nasopharyngeal or oropharyngeal swabs as template to amplify conserved regions in the viral genome. Next, PfAgo, guide DNAs and molecular beacons in appropriate buffer were added to the PCR products, followed by incubating at 95 degrees C for 20-30 min. Subsequently, the fluorescence signal was detected. This method was named as SARS-CoV-2 PAND. The whole procedure is accomplished in approximately an hour with the using time of the Real-time fluorescence quantitative PCR instrument shortened from >1 h to only 3-5 min per batch in comparison with RT-qPCR, hence the shortage of the expensive Real-time PCR instrument is alleviated. Moreover, this platform was also applied to identify SARS-CoV-2 D614G mutant due to its single-nucleotide specificity. The diagnostic results of clinic samples with SARS-CoV-2 PAND displayed 100% consistence with RT-qPCR test.

Infectious diseases caused by viruses such as SARS-CoV-2 and HPV have greatly endangered human health. The nucleic acid detection is essential for the early diagnosis of diseases. Here, we propose a method called PLCR (PfAgo coupled with modified Ligase Chain Reaction for nucleic acid detection) which utilizes PfAgo to only use DNA guides longer than 14-mer to specifically cleave DNA and LCR to precisely distinguish single-base mismatch. PLCR can detect DNA or RNA without PCR at attomolar sensitivities, distinguish single base mutation between the genome of wild type SARS-CoV-2 and its mutant spike D614G, effectively distinguish the novel coronavirus from other coronaviruses and finally achieve multiplexed detection in 70 min. Additionally, LCR products can be directly used as DNA guides without additional input guides to simplify primer design. With desirable sensitivity, specificity and simplicity, the method can be extended for detecting other pathogenic microorganisms.

Argonaute (Ago) proteins are conserved nucleic acid-guided proteins present in all domains of life. Eukaryotic Argonaute proteins (eAgos) are key players in RNA interference pathways and function as RNA-guided RNA endonucleases at physiological temperatures. Although eAgos are considered to evolve from prokaryotic Argonaute proteins (pAgos), previously studied pAgos were unable to catalyze RNA-guided RNA cleavage at physiological temperatures. Here, we describe a distinctive pAgo from mesophilic bacteria Kurthia massiliensis (KmAgo). KmAgo utilizes DNA guides to cleave single-stranded DNA (ssDNA) and RNA targets with high activity. KmAgo also utilizes RNA guides to cleave ssDNA and RNA targets at moderate temperatures. We show that KmAgo can use 5′ phosphorylated DNA guides as small as 9-mers to cut ssDNA and RNA, like Clostridium butyricum Ago. Small DNA binding confers remarkable thermostability on KmAgo, and we can suppress the guide-independent plasmid processing activity of empty KmAgo by elevating the DNA guide loaded temperature. Moreover, KmAgo performs programmable cleavage of double-stranded DNA and highly structured RNA at 37◦C. Therefore, KmAgo can be regarded as a DNA-guided programmable omnipotent nuclease for cleaving most types of nucleic acids efficiently. This study broadens our understanding of Ago proteins and could expand the pAgo-based DNA and RNA ma-

A simple and user-friendly nucleic acid sensing platform with 10 aM sensitivity, named USPCRP (combines ultrashort PCR with Pyrococcus furiosus Argonaute cleavage for nuleic acids detection) is reported. The product of this ultrashort PCR could be directly used as a DNA guide to mediate PfAgo cleavage of molecular beacons.

Abstract Argonaute (Ago) proteins are conserved nucleic acid-guided proteins present in all domains of life. Eukaryotic Argonaute proteins (eAgos) are key players in RNA interference pathways and function as RNA-guided RNA endonucleases at physiological temperatures. Although eAgos are considered to evolve from prokaryotic Argonaute proteins (pAgos), previously studied pAgos were unable to catalyze RNA-guided RNA cleavage at physiological temperatures. Here, we describe a distinctive pAgo from mesophilic bacteria Kurthia massiliensis (KmAgo). KmAgo utilizes DNA guides to cleave single-stranded DNA (ssDNA) and RNA targets with high activity. KmAgo also utilizes RNA guides to cleave ssDNA and RNA targets at moderate temperatures. We show that KmAgo can use 5′ phosphorylated DNA guides as small as 9-mers to cut ssDNA and RNA, like Clostridium butyricum Ago. Small DNA binding confers remarkable thermostability on KmAgo, and we can suppress the guide-independent plasmid processing activity of empty KmAgo by elevating the DNA guide loaded temperature. Moreover, KmAgo performs programmable cleavage of double-stranded DNA and highly structured RNA at 37°C. Therefore, KmAgo can be regarded as a DNA-guided programmable omnipotent nuclease for cleaving most types of nucleic acids efficiently. This study broadens our understanding of Ago proteins and could expand the pAgo-based DNA and RNA manipulation toolbox.

Adenosine triphosphate (ATP) and S-adenosyl-L-methionine (SAM) are important intermediates that are widely present in living organisms. Large-scale preparation and application of ATP or SAM is limited by expensive raw materials. To lower the production costs for ATP/SAM, in this study we used strategies applying engineered multidomain scaffold proteins to synthesize ATP and SAM. An artificial scaffold protein containing CBM3 domain, IM proteins and CL-labeled proteins was assembled to form complex 1 for catalytic reactions to increase ATP production. The ATP synthesis system produced approximately 25 g/L of ATP with approximately 15 g/L of ADP and 5 g/L of AMP using 12.5 g/L of adenosine and 40 g/L of sodium hexametaphosphate reaction at 35 °C and a pH of 8.5 for 6 h. Based on the above ATP synthesis system, two CL-labeled methionine adenosyltransferases (CL9-MAT4 and CL9-MAT5) were applied to construct scaffold protein complex 2 to achieve SAM synthesis. Approximately 25 μg of MAT4 in a reaction system with 0.3 M MgCl2 catalyzed at 20 °C and a pH of 8 catalyzed 0.5 g/L of l-Met to produce approximately 0.9 g/L of SAM. Approximately 25 μg of MAT5 in a reaction system with 0.7 M MgCl2 catalyzed at 35 °C and a pH of 8 catalyzed 0.5 g/L of l-Met to produce approximately 1.2 g/L of SAM. Here, we showed that low-cost substrates can be efficiently converted into high-value additional ATP and SAM via multi-enzyme catalytic reactions by engineered multidomain scaffold proteins.

In order to establish a high-throughput identification technique that simultaneously detects six major pathogens including APP, HPS, PRRSV, Mhp, PCV-2 and CSFV, six pairs of primers and probes were designed based on the specific conservative sequences of the pathogens, a multiplex PCR system was developed, hybrid parameters were optimized, and evaluation of the technology was performed. The results showed that the present detection method had a sensitivity of 5.8 × 102copies/μL for APP, 7.8 × 103 copies/μL for HPS, 6.8 × 103 copies/μL for Mhp, 6.3 × 102 copies/μL for PCV-2, 4.8 × 103 copies/μL for PRRSV, and 5.5 × 102 copies/μL for CSFV, respectively; and it produced no cross reaction against the other nine pathogens like swine-origin pseudorabies virus, porcine parvovirus, Japanese B encephalitis virus, swine vesicular disease virus, vesicular stomatitis virus, foot-and-mouth disease virus, bluetongue virus, peste des petits ruminants virus and salmonella. Application of the multiplex oligonucleotide microarray established here to testing 285 clinical blood samples indicated a single infection rate of 18.2 % (52/285) and a mixed infection rate of 6.3 % (18/285) which were consistent with the results of the sequencing verification. This technique might serve as a rapid and high-throughput method of detection for epidemic investigation and clinical diagnosis of multiple pathogens.

Aliphatic α,ω‐dicarboxylic acids (DCAs) are a class of useful chemicals that are currently produced by energy-intensive, multistage chemical oxidations that are hazardous to the environment. Therefore, the development of environmentally friendly, safe, neutral routes to DCAs is important. We report an in vivo artificially designed biocatalytic cascade process for biotransformation of cycloalkanes to DCAs. To reduce protein expression burden and redox constraints caused by multi-enzyme expression in a single microbe, the biocatalytic pathway is divided into three basic Escherichia coli cell modules. The modules possess either redox-neutral or redox-regeneration systems and are combined to form E. coli consortia for use in biotransformations. The designed consortia of E. coli containing the modules efficiently convert cycloalkanes or cycloalkanols to DCAs without addition of exogenous coenzymes. Thus, this developed biocatalytic process provides a promising alternative to the current industrial process for manufacturing DCAs.

BACKGROUND Chlamydiae are spread globally and cause infectious diseases in both humans and animals. The existing detection methods for this disease have numerous shortcomings, including low sensitivity, time consuming procedures, and high contamination vulnerability. MATERIAL AND METHODS To overcome shortcomings for detecting animal chlamydiosis, a multiplex quantitative polymerase chain reaction (PCR) assay was established for simultaneously detecting and differentiating 3 Chlamydia species (C. pecorum, C. abortus, and C. psittaci) by real time PCR based on TaqMan-MGB technology. RESULTS The limit of detection was 20.2 copies/µL for Chlamydophila (Cp.) abortus, 30.8 copies/µL for Cp. pecorum, and 16 copies/µL for Cp. psittaci. This method has good repeatability and stability as coefficients of variation range from 0.04% to 1.38%. Furthermore, compared with OIE (World Organization for Animal Health) recommended PCR assay and previously reported animal chlamydia shell PCR, this multiplex PCR assay demonstrated 99% concordance in detecting clinical samples of porcine nasal swabs and vaginal swabs. CONCLUSIONS The novel established method in this study was able to detect 3 types of Chlamydia species simultaneously, and had high sensitivity, strong specificity, and good stability. It provided a rapid, reliable, and convenient method for epidemiological and clinical diagnosis of chlamydiosis in animals.