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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. Key points • 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.

Increasing evidence suggests that the intestinal microbiota is closely correlated with the host’s health status. Thus, a serious disturbance that disrupts the stability of the intestinal microecosystem could cause host disease. Shrimps are one of the most important products among fishery trading commodities. However, digestive system diseases, such as white feces syndrome (WFS), frequently occur in shrimp culture and have led to enormous economic losses across the world. The WFS occurrences are unclear. Here, we compared intestinal bacterial communities of WFS shrimp and healthy shrimp. Intestinal bacterial communities of WFS shrimp exhibited less diversity but were more heterogeneous than those of healthy shrimp. The intestinal bacterial communities were significantly different between WFS shrimp and healthy shrimp; compared with healthy shrimp, in WFS shrimp, Candidatus Bacilloplasma and Phascolarctobacterium were overrepresented, whereas Paracoccus and Lactococcus were underrepresented. PICRUSt functional predictions indicated that the relative abundances of genes involved in energy metabolism and genetic information processing were significantly greater in WFS shrimp. Collectively, we found that the composition and predicted functions of the intestinal bacterial community were markedly shifted by WFS. Significant increases in Candidatus Bacilloplasma and Phascolarctobacterium and decreases in Paracoccus and Lactococcus may contribute to WFS in shrimp.

The genetic manipulation of basidiomycete mushrooms is notoriously difficult and immature, and there is a lack of research reports on clustered regularly interspaced short palindromic repeat (CRISPR) based gene editing of functional genes in mushrooms. In this work, Ganoderma lucidum, a famous traditional medicinal basidiomycete mushroom, which produces a type of unique triterpenoid-anti-tumor ganoderic acids (GAs), was used, and a CRISPR/CRISPR-associated protein-9 nuclease (Cas9) editing system for functional genes of GA biosynthesis was constructed in the mushroom. As proof of concept, the effect of different gRNA constructs with endogenous u6 promoter and self-cleaving ribozyme HDV on ura3 disruption efficiency was investigated at first. The established system was applied to edit a cytochrome P450 monooxygenase (CYP450) gene cyp5150l8, which is responsible for a three-step biotransformation of lanosterol at C-26 to ganoderic acid 3-hydroxy-lanosta-8, 24-dien-26 oic acid. As a result, precisely edited cyp5150l8 disruptants were obtained after sequencing confirmation. The fermentation products of the wild type (WT) and cyp5150l8 disruptant were analyzed, and a significant decrease in the titer of four identified GAs was found in the mutant compared to WT. Another CYP gene involved in the biosynthesis of squalene-type triterpenoid 2, 3; 22, 23-squalene dioxide, cyp505d13, was also disrupted using the established CRISPR-Cas9 based gene editing platform of G. lucidum. The work will be helpful to strain molecular breeding and biotechnological applications of G. lucidum and other basidiomycete mushrooms.

Late blight caused by Phytophthora infestans is an economically important disease of potato and tomato worldwide. In Canada, an increase in late blight incidence and severity coincided with changes in genetic composition of P. infestans . We monitored late blight incidence on tomato and potato in Pacific western and eastern Canada between 2019 and 2022, identified genotypes of P . infestans , and examined their population genetic diversity. We identified four major existing genotypes US11, US17, US8, and US23 as well as 25 new genotypes. The US11 genotype was dominant in Pacific western Canada, accounting for 59% of the total population. We discovered the US17 genotype for the first time in Canada. We revealed a higher incidence of late blight and quite diverse genotypes of P . infestans in Pacific western Canada than in eastern Canada. We found high genetic diversity of P. infestans population from Pacific western Canada, as evidenced by the high number of multilocus genotypes, high values of genetic diversity indices, and emergence of 25 new genotypes. Considering the number of disease incidence, the detection of diverse known genotypes, the emergence of novel genotypes, and the high number of isolates resistant to metalaxyl-m (95%) from Pacific western Canada, the region could play a role in establishing sexual recombination and diverse populations, which could ultimately pose challenges for late blight management. Therefore, continuous monitoring of P. infestans populations in Pacific western region and across Canada is warranted. Key points • Genotypes of P. infestans in Pacific western were quite diverse than in eastern Canada. • We discovered US17 genotype for the first time in Canada and identified 26 novel genotypes. • Approximately 95% of P. infestans isolates were resistant to metalaxyl-m.

The oleaginous yeast Yarrowia lipolytica represents a potential microbial cell factory for the recombinant production of various valuable products. Currently, the commonly used selection markers for transformation in Y. lipolytica are limited, and successive genetic manipulations are often restricted by the number of available selection markers. In our study, we developed a dominant marker, dsdA , which encodes a D-serine deaminase for genetic manipulation in Y. lipolytica . In Y. lipolytica , this marker confers the ability to use D-serine as a nitrogen source. In addition, the selection conditions of several infrequently used dominant markers including bleoR (zeocin resistance), kan MX (G418 resistance), and guaB (mycophenolic acid resistance) were also analyzed. Our results demonstrated that these selection markers can be used for the genetic manipulation of Y. lipolytica and their selection conditions were different for various strains. Ultimately, the selection markers tested here will be useful to expand the genetic toolbox of Y. lipolytica . Key points • The dsdA from Escherichia coli was developed as a dominant marker. • The applicability of several resistance markers in Y. lipolytica was determined. • We introduced the Cre/mutant lox system for marker recycling.

As a kind of biosurfactants, iturin A has attracted people’s wide attentions due to their features of biodegradability, environmentally friendly, etc.; however, high production cost limited its extensive application, and the aim of this research wants to improve iturin A production in Bacillus amyloliquefaciens . Firstly, dual promoter was applied to strengthen iturin A synthetase expression, and its yield was increased to 1.25 g/L. Subsequently, original 5′-UTRs of downstream genes ( ituA , ituB , and ituC ) in iturin A synthetase cluster were optimized, which significantly increased mRNA secondary stability, and iturin A yield produced by resultant strain HZ-T3 reached 2.32 g/L. Secondly, synthetic pathway of α-glucosidase inhibitor 1-deoxynojirimycin was blocked to improve substrate corn starch utilization, and iturin A yield was increased by 34.91% to 3.13 g/L. Thirdly, efficient precursor (fatty acids, Ser, and Pro) supplies were proven as the critical role in iturin A synthesis, and 5.52 g/L iturin A was attained by resultant strain, through overexpressing yngH , serC , and introducing ocD . Meanwhile, genes responsible for poly-γ-glutamic acid, extracellular polysaccharide, and surfactin syntheses were deleted, which led to a 30.98% increase of iturin A yield. Finally, lipopeptide transporters were screened, and iturin A yield was increased by 17.98% in SwrC overexpression strain, reached 8.53 g/L, which is the highest yield of iturin A ever reported. This study laid a foundation for industrial production and application development of iturin A, and provided the guidance of metabolic engineering breeding for efficient production of other metabolites synthesized by non-ribosomal peptide synthetase. Key points • Optimizing 5′-UTR is an effective tactics to regulate synthetase cluster expression. • Blocking 1-DNJ synthesis benefited corn starch utilization and iturin A production. • The iturin A yield attained in this work was the highest yield reported so far.

Bacillus subtilis as an important host has been widely used in synthetic biology, metabolic engineering, and production of industrial enzymes. To fully take advantage of this organism, 114 endogenous putative promoters were measured with a green fluorescent protein reporter and four classes of phase-dependent promoters (class I: exponential phase; class II: middle-log and early stationary phases; class III: lag-log and stationary phases; class IV: stationary phase) with different strengths were identified. The transcriptional strengths ranged from 0.03 to 2.03-fold of that of the commonly used strong promoter P 43 . On this basis, the temperature (for instance P bltD , P ydaD , and P gerBC ) and pH (such as P abrB , P ydjO , and P opuE ) inducible phase-dependent promoters were further identified and characterized. In comparison, P abrB (class I), P spoVG (class II), and P lytR (class III) achieved the highest expression levels of esterase, keratinase, and alkaline polygalacturonate lyase, respectively. The constructed phase-dependent promoter library should have great application potentials for enzyme production, metabolic engineering, and synthetic biology.

Species of the genera Bacteroides and Phocaeicola play an important role in the human colon. The organisms contribute to the degradation of complex heteropolysaccharides to small chain fatty acids, which are in part utilized by the human body. Furthermore, these organisms are involved in the synthesis of vitamins and other bioactive compounds. Of special interest is Phocaeicola vulgatus , originally classified as a Bacteroides species, due to its abundance in the human intestinal tract and its ability to degrade many plant-derived heteropolysaccharides. We analyzed different tools for the genetic modification of this microorganism, with respect to homologous gene expression of the ldh gene encoding a D-lactate dehydrogenase (LDH). Therefore, the ldh gene was cloned into the integration vector pMM656 and the shuttle vector pG106 for homologous gene expression in P. vulgatus . We determined the ldh copy number, transcript abundance, and the enzyme activity of the wild type and the mutants. The strain containing the shuttle vector showed an approx. 1500-fold increase in the ldh transcript concentration and an enhanced LDH activity that was about 200-fold higher compared to the parental strain. Overall, the proportion of lactate in the general catabolic carbon flow increased from 2.9% (wild type) to 28.5% in the LDH-overproducing mutant. This approach is a proof of concept, verifying the genetic accessibility of P. vulgatus and could form the basis for targeted genetic optimization. Key points • A lactate dehydrogenase was overexpressed in Phocaeicola (Bacteroides) vulgatus. • The ldh transcript abundance and the LDH activity increased sharply in the mutant. • The proportion of lactate in the catabolic carbon flow increased to about 30%.