The Argentine shortfin squid Illex argentinus is an economically important short-lived species widely distributed in the southwest Atlantic Ocean.The abundance and distribution of I.argentinus are associated with clim...The Argentine shortfin squid Illex argentinus is an economically important short-lived species widely distributed in the southwest Atlantic Ocean.The abundance and distribution of I.argentinus are associated with climate change and environmental fluctuations.The potential distribution of I.argentinus was modeled with various environmental variables including sea surface temperature(SST),sea surface height(SSH),chlorophyll a,sea surface salinity(SSS),net primary productivity(NPP),mixed layer depth(MLD),eddy kinetic energy(EKE),and photosynthetically active radiation(PAR)using the maximum entropy(MaxEnt)approach during the peak fishing seasons(January–April).The habitat suitability index(HSI)was defined as the probability of species emergence from the MaxEnt model and the area of HSI≥0.6 was regarded as suitable.Results indicate that the predicted habitat correlated with the actual fishing position,with similar trends in the percentages of suitable habitats and catch per unit effort(CPUE)of I.argentinus from January to April.Moreover,SST,SSH,PAR,and MLD were identified critical environmental variables for the distribution of I.argentinus.In addition,the median of preferred ranges of the critical environmental variables were concentrated within the suitable habitats of I.argentinus.The Area under the Receiver Operating Characteristic Curve(AUC)was greater than 0.96 for all four months.Variations in latitudinal and longitudinal gravity centers(LATG and LONG)of fishing effort were consistent with latitudinal and longitudinal gravity centers(LATG_H and LONG_H)of the HSI.Our findings suggest that the MaxEnt model is an effective tool to predict the potential distribution of I.argentinus.Meanwhile,SST,SSH,PAR,and MLD should be given with more extensive attention in predicting the potential distribution of I.argentinus,as they are important environmental indicators that can help decision-makers search for the fishing ground of I.argentinus in the Southwest Atlantic.展开更多
Efficient gene cluster editing tools are one of the key techniques for discovering novel compounds encoded by silent natural product(NP)biosynthetic gene clusters(BGCs)in microbial genomes.Currently,in vivo BGC editin...Efficient gene cluster editing tools are one of the key techniques for discovering novel compounds encoded by silent natural product(NP)biosynthetic gene clusters(BGCs)in microbial genomes.Currently,in vivo BGC editing tools developed in E.coli is the most widely used,but they often introduces DNA scars into gene clusters,which may affect the function of target NP BGCs.Herein,a genome-integrated Cas9/λRed system-based in vivo scarless gene cluster editing tool(iCASRED)was established in E.coli BL23,which was constructed on the basis of BL21/DE3 with recA deletion and simultaneous integration of an inducible sgRNA targeting the editing plasmid(an all-in-one plasmid with the BGC-targeting sgRNAs and repair templates).iCASRED achieved scarless editing of single targets in three tested gene clusters(44.2,72.0,and 76.2 kb)cloned in either a single-copy BAC plasmid or a high-copy plasmid pCAP01 with the efficiencies of 28.8%±3.9%-100%±0%.Furthermore,this tool could enable convenient,high-efficiency iterative editing.Finally,we achieved 24.4%±3.8%efficiency for simultaneous double-target editing by replacing Cas9 by nCas9(Cas9D10A).Collectively,iCASRED provides a simple,convenient,and cost-effective approach for engineering gene clusters,which may facilitate the discovery of novel NPs and strain improvements for high-yield of target compounds.展开更多
Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inac...Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inaccessibility and lack of effective genome engineering tools.In this work, we characterized the activity of an endogenous type I-B CRISPR-Cas system as well as its recognized protospacer adjacent motifs(PAMs) based on bioinformatics analysis combined with a plasmid interference assay in S. spinosa. By delivering editing plasmids containing a designed mini CRISPR array(repeat+self-targeting spacer+repeat) and repair templates, we achieved 100% editing efficiency for gene deletion. Using this tool, the genetic barrier composed of the restriction-modification(RM) systems was systematically disarmed. We showed that by disarming one type I RM system(encoded by A8926_1903/1904/1905) and two type Ⅱ RM systems(encoded by A8926_1725/1726 and A8926_2652/2653) simultaneously, the transformation efficiency of the replicative and integrative plasmids(pSP01 and pSI01) was increased by approximately 3.9-fold and 4.2-fold, respectively. Using the engineered strain with simultaneous knock-out of these three RM genes as the starting strain, we achieved the deletion of 75-kb spinosyns biosynthetic gene cluster(BGC) as well as gene insertion at high efficiency. Collectively, we developed a reliable and highly efficient genome editing tool based on the endogenous type I CRISPR-Cas system combined with the disarmament of the RM systems in S. spinosa. This is the first time to establish an endogenous CRISPR-Cas-based genome editing tool in the non-model industrial actinomycetes.展开更多
基金Supported by the Natural Science Foundation of Shanghai(No.23ZR1427100)the National Key R&D Program of China(No.2023YFD2401303)the Shanghai Talent Development Funding for the Project(No.2021078)。
文摘The Argentine shortfin squid Illex argentinus is an economically important short-lived species widely distributed in the southwest Atlantic Ocean.The abundance and distribution of I.argentinus are associated with climate change and environmental fluctuations.The potential distribution of I.argentinus was modeled with various environmental variables including sea surface temperature(SST),sea surface height(SSH),chlorophyll a,sea surface salinity(SSS),net primary productivity(NPP),mixed layer depth(MLD),eddy kinetic energy(EKE),and photosynthetically active radiation(PAR)using the maximum entropy(MaxEnt)approach during the peak fishing seasons(January–April).The habitat suitability index(HSI)was defined as the probability of species emergence from the MaxEnt model and the area of HSI≥0.6 was regarded as suitable.Results indicate that the predicted habitat correlated with the actual fishing position,with similar trends in the percentages of suitable habitats and catch per unit effort(CPUE)of I.argentinus from January to April.Moreover,SST,SSH,PAR,and MLD were identified critical environmental variables for the distribution of I.argentinus.In addition,the median of preferred ranges of the critical environmental variables were concentrated within the suitable habitats of I.argentinus.The Area under the Receiver Operating Characteristic Curve(AUC)was greater than 0.96 for all four months.Variations in latitudinal and longitudinal gravity centers(LATG and LONG)of fishing effort were consistent with latitudinal and longitudinal gravity centers(LATG_H and LONG_H)of the HSI.Our findings suggest that the MaxEnt model is an effective tool to predict the potential distribution of I.argentinus.Meanwhile,SST,SSH,PAR,and MLD should be given with more extensive attention in predicting the potential distribution of I.argentinus,as they are important environmental indicators that can help decision-makers search for the fishing ground of I.argentinus in the Southwest Atlantic.
基金supported by the National Key Research and Devel-opment Program(2019YFA0905400 to Y.L.)the National Natural Science Foundation of China(32270095 to Y.L.).
文摘Efficient gene cluster editing tools are one of the key techniques for discovering novel compounds encoded by silent natural product(NP)biosynthetic gene clusters(BGCs)in microbial genomes.Currently,in vivo BGC editing tools developed in E.coli is the most widely used,but they often introduces DNA scars into gene clusters,which may affect the function of target NP BGCs.Herein,a genome-integrated Cas9/λRed system-based in vivo scarless gene cluster editing tool(iCASRED)was established in E.coli BL23,which was constructed on the basis of BL21/DE3 with recA deletion and simultaneous integration of an inducible sgRNA targeting the editing plasmid(an all-in-one plasmid with the BGC-targeting sgRNAs and repair templates).iCASRED achieved scarless editing of single targets in three tested gene clusters(44.2,72.0,and 76.2 kb)cloned in either a single-copy BAC plasmid or a high-copy plasmid pCAP01 with the efficiencies of 28.8%±3.9%-100%±0%.Furthermore,this tool could enable convenient,high-efficiency iterative editing.Finally,we achieved 24.4%±3.8%efficiency for simultaneous double-target editing by replacing Cas9 by nCas9(Cas9D10A).Collectively,iCASRED provides a simple,convenient,and cost-effective approach for engineering gene clusters,which may facilitate the discovery of novel NPs and strain improvements for high-yield of target compounds.
基金supported by the National Key Research and Development Program of China(2019YFA0905400)the National Natural Science Foundation of China(32270095)the Open Funding Project(MMLKF23-08)of State Key Laboratory of Microbial Metabolism.
文摘Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inaccessibility and lack of effective genome engineering tools.In this work, we characterized the activity of an endogenous type I-B CRISPR-Cas system as well as its recognized protospacer adjacent motifs(PAMs) based on bioinformatics analysis combined with a plasmid interference assay in S. spinosa. By delivering editing plasmids containing a designed mini CRISPR array(repeat+self-targeting spacer+repeat) and repair templates, we achieved 100% editing efficiency for gene deletion. Using this tool, the genetic barrier composed of the restriction-modification(RM) systems was systematically disarmed. We showed that by disarming one type I RM system(encoded by A8926_1903/1904/1905) and two type Ⅱ RM systems(encoded by A8926_1725/1726 and A8926_2652/2653) simultaneously, the transformation efficiency of the replicative and integrative plasmids(pSP01 and pSI01) was increased by approximately 3.9-fold and 4.2-fold, respectively. Using the engineered strain with simultaneous knock-out of these three RM genes as the starting strain, we achieved the deletion of 75-kb spinosyns biosynthetic gene cluster(BGC) as well as gene insertion at high efficiency. Collectively, we developed a reliable and highly efficient genome editing tool based on the endogenous type I CRISPR-Cas system combined with the disarmament of the RM systems in S. spinosa. This is the first time to establish an endogenous CRISPR-Cas-based genome editing tool in the non-model industrial actinomycetes.
