Effects Of Overexpression Of LaeA And VeA Genes On Aspergillus Niger Growth And Metabolism

As an important industrial production strain,Aspergillus niger can not only produce a variety of organic acids and enzymes,but also produce a variety of physiologically active secondary metabolites.This article intended to study the effects of global regulatory factors laeA and veA on the growth and development of Aspergillus niger,primary metabolism(taking citric acid as an example)and secondary metabolism(taking ochratoxin A as an example).From these three aspects,the global regulation of laeA and veA was comprehensively analyzed on Aspergillus niger CICC₄1702.In this experiment,an agrobacterium-mediated transformation method was used to integrate and express a copy of the laeA or veA gene at the ku70 site of Aspergillus niger CICC₄1702 to construct overexpressing strains of laeA or veA gene.Two laeA overexpressed strains OE::laeA-PgpdA and OE::laeA-PlaeA,which were regulated by the glyceraldehyde 3-phosphate dehydrogenase promoter(PgpdA)and laeA gene self-promoter(PlaeA),and two veA overexpressed strains OE::veA-PgpdA and OE::veA-PveA regulated by PgpdA and veA gene self-promoter(PveA),were screened.Tability experiment verified that four overexpressed strains all had good genetic stability.Real-time quantitative PCR analysis was performed to test gene expression level of laeA gene expression about laeA overexpressed strains OE::laeA-PgpdA and OE::laeA-PlaeA and veA gene expression about veA overexpressed strains OE::veA-PgpdA and OE::veA-PveA.The strain OE::laeA-PgpdA with higher laeA gene expression level and the OE::veA-PgpdA with higher veA gene expression level were screened for subsequent experiments.Compared with the starting strain Aspergillus niger CICC₄1702,the color of the edge mycelium of the OE::laeA-PgpdA strain was significantly darker,the diameter of the colony growth was significantly reduced,mycelia were slender and straight,and fewer hypha branches;the citric acid content was significantly increased;The content ofochratoxin ?(OT?)and ochratoxin a(OTa)decreased,and the early synthesis of ochratoxin A(OTA)slowed down,but the final content increased.OE::veA-PgpdA strain had relatively lighter mycelium at the edge of the colony,no obvious change in colony diameter,shorter mycelium length,reduced branches,overall evacuation,and more conidial heads;no significant change in citric acid synthesis.The synthesis of OT?,OT? and OTA was significantly reduced.Transcriptome data showed that laeA and veA were involved in regulating multiple genes related to growth and development,mainly involved in genes related to the growth of spores,hyphae and vegetative growth.In addition,in the 85 secondary metabolite biosynthesis gene clusters,overexpression of laeA and veA regulated 32 of them respectively,of which laeA participated in the up-regulation of OTA biosynthesis core gene ANI₁₁836134,and caused increased OTA content in the OE::laeA-PgpdA strain.It was proved that laeA and veA were widely involved in regulating secondary metabolites of Aspergillus niger,and might play an important role in improving target products and activating silent secondary metabolic gene clusters.