Insight Into The Phenotype And Secondary Metabolites Of Aspergillus Niger CBS 513.88 Under The Control Of Global Regulator McrA

Fungi are a type of microorganism commonly found in nature and are famous for their ability to synthesize a variety of small active molecules.Since the discovery of penicillin,more active products have been discovered from fungi.Aspergillus niger is a common filamentous fungus that has received much attention due to the industrial fermentation of citric acid.Gluconic acid and fumaric acid have also been identified in Aspergillus niger.The genome of Aspergillus niger CBS 513.88 was analyzed and found to contain dozens of secondary metabolic gene clusters,but the compounds identified therefrom were far less than expected,indicating that a large number of silent gene clusters occur in Aspergillus niger CBS 513.88,which produced a variety of natural products.Studies have found that changing the regulatory mechanism of transcription factors can activate the expression of silent gene clusters or increase the yield of corresponding compounds,which also provides an important idea for exploration of new active natural products.There is a class of global regulator McrA in the fungus.Knockout of mcrA activate gene clusters in Aspergillus nidulans,Penicillium and Aspergillus terreus.In the research we used the global regulator McrA as the starting point,and activated gene clusters or increase the yield of corresponding compounds by knocking out mcrA.First,the hygromycin resistance gene hph was used as a screening marker to successfully construct a mcrA knockout strain of Aspergillus niger CBS 513.88.Second,in order to verify the sensitivity of knockout and wild-type strains to high salt hyperosmotic pressure and heavy metal ions,a series of media were selected to cultivate the strains.At the same time,the products in the mutant strain were separated,purified and identified by HPLC,HRMS and NMR.Several compounds in the mutant and wild type were quantitatively analyzed.The results showed that the knockout mcrA strain increased sensitivity to high salt hyperosmotic pressure,decreased sensitivity to heavy metal ions,and the colony turned yellow.Using the wild type as a negative control,we successfully isolated and identified three compounds in the mutant.Compounds 1,2 and 3 were(3S,8R)-8-hydroxy-3-carboxy-2-methylenenonanoic acid,(3S)-9-hydroxy-3-carboxy-2-methylenenonanoate,asperitaconic acid A.The quantitative analysis of the compounds showed that the compound 1 yield increased by 29.9% and the compound 2 yield was increased by nearly 6.47 times compared with the wild type,but yield of 3 was significantly reduced.The knockout of mcrA in this study may stimulate the transcription of multiple genes in Aspergillus niger,thereby positively regulating the synthesis of 1 and 2.Our research successfully constructed the knockout strain of mcrA for the first time in Aspergillus niger and improved the production of some metabolites,which provided a new direction for the research the strain Aspergillus niger.