Acremonium species are prolific producers of therapeutic molecules which include the widely used beta-lactam antibiotic,cephalosporin.In light of their significant medical value,an efficient gene disruption method is ...Acremonium species are prolific producers of therapeutic molecules which include the widely used beta-lactam antibiotic,cephalosporin.In light of their significant medical value,an efficient gene disruption method is required for the physiological and biochemical studies on this genus of fungi.However,the number of selection markers that can be used for gene targeting is limited,which constrain the genetic analysis of multiple functional genes.In this study,we established a uridine auxotrophy based marker recycling system which achieves scarless gene deletion,and allows the use of the same selection marker in successive transformations in a deep seaderived fungus Acremonium sp.HDN16-126.We identified one homologue of Acremonium chrysogenum pyrG(also as a homologous gene of the yeast URA3)from HDN16-126,designated as pyrG-A1,which can be used as a selection marker on uridine free medium.We then removed pyrG-A1 from HDN16-126 genome via homologous recombination(HR)on MM medium with 5-fluoroortic acid(5-FOA),a chemical that can be converted into a toxin of 5-flurouracil by pyrG-A1 activity,thus generating the HDN16-126-△pyrG mutant strain which showed auxotrophy for uridine but insensitivity to 5-FOA and enabled the use of exogenous pyrG gene as both positive and negative selection marker to achieve the scarless deletion of target DNA fragments.We further applied this marker recycling system to successfully disrupt two target genes pepL(encodes a putative 2OG-Fe(Ⅱ)dioxygenase)and pepM(encodes a putative aldolase)identified from HDN16-126 genome,which are proposed to be functional genes related to 2-aminoisobutyric acid metabolism in fungi.This work is the first application of uridine auxotrophy based scarless gene deletion method in Acremonium species and shows promising potential in assisting sequential genetic analysis of filamentous fungi.展开更多
1836年,Schmidberger首次发现蛀木质部小蠹是以坑道壁上排列的栅栏状物为食,由于弄不明白这些栅栏状物为何物,便将它们称为"ambrosia",意为"神的食物"(Fisher et al.,1953;1954;Schmidberger,1836)。1844年,Hartig...1836年,Schmidberger首次发现蛀木质部小蠹是以坑道壁上排列的栅栏状物为食,由于弄不明白这些栅栏状物为何物,便将它们称为"ambrosia",意为"神的食物"(Fisher et al.,1953;1954;Schmidberger,1836)。1844年,Hartig明确了被北方材小蠹(Xyleborus dispar)幼虫取食的栅栏状物是丛梗孢属(Monilia)的一种真菌(Batra,1967)。此后,"ambrosia fungi",即"虫道真菌",便被广泛用于对生长在坑道中、为蠹虫所取食的真菌之称。展开更多
基金funded by the National Key R&D Program of China(2019YFC0312504)the National Natural Science Foundation of China(41976105,81991522)+5 种基金Major Basic Research Programs of Natural Science Foundation of Shandong Province(ZR2019ZD18)the Fundamental Research Funds for the Central Universities(201941001)the NSFC-Shandong Joint Fund(U1906212)the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology(Qingdao)(No.2018SDKJ0401-2)the Youth Innovation Plan of Shandong province(2019KJM004)the Taishan Scholar Youth Expert Program in Shandong Province(tsqn201812021).
文摘Acremonium species are prolific producers of therapeutic molecules which include the widely used beta-lactam antibiotic,cephalosporin.In light of their significant medical value,an efficient gene disruption method is required for the physiological and biochemical studies on this genus of fungi.However,the number of selection markers that can be used for gene targeting is limited,which constrain the genetic analysis of multiple functional genes.In this study,we established a uridine auxotrophy based marker recycling system which achieves scarless gene deletion,and allows the use of the same selection marker in successive transformations in a deep seaderived fungus Acremonium sp.HDN16-126.We identified one homologue of Acremonium chrysogenum pyrG(also as a homologous gene of the yeast URA3)from HDN16-126,designated as pyrG-A1,which can be used as a selection marker on uridine free medium.We then removed pyrG-A1 from HDN16-126 genome via homologous recombination(HR)on MM medium with 5-fluoroortic acid(5-FOA),a chemical that can be converted into a toxin of 5-flurouracil by pyrG-A1 activity,thus generating the HDN16-126-△pyrG mutant strain which showed auxotrophy for uridine but insensitivity to 5-FOA and enabled the use of exogenous pyrG gene as both positive and negative selection marker to achieve the scarless deletion of target DNA fragments.We further applied this marker recycling system to successfully disrupt two target genes pepL(encodes a putative 2OG-Fe(Ⅱ)dioxygenase)and pepM(encodes a putative aldolase)identified from HDN16-126 genome,which are proposed to be functional genes related to 2-aminoisobutyric acid metabolism in fungi.This work is the first application of uridine auxotrophy based scarless gene deletion method in Acremonium species and shows promising potential in assisting sequential genetic analysis of filamentous fungi.
文摘1836年,Schmidberger首次发现蛀木质部小蠹是以坑道壁上排列的栅栏状物为食,由于弄不明白这些栅栏状物为何物,便将它们称为"ambrosia",意为"神的食物"(Fisher et al.,1953;1954;Schmidberger,1836)。1844年,Hartig明确了被北方材小蠹(Xyleborus dispar)幼虫取食的栅栏状物是丛梗孢属(Monilia)的一种真菌(Batra,1967)。此后,"ambrosia fungi",即"虫道真菌",便被广泛用于对生长在坑道中、为蠹虫所取食的真菌之称。
