title: Enhanced production of Aspergillus niger laccase-like multicopper oxidases through mRNA optimization of the glucoamylase expression system.
abstract: In filamentous fungi, most of the strategies used for the improvement of protein yields have been based on an increase in the transcript levels of a target gene. Strategies focusing at the translational level have been also described, but are far less explored. Here the 5' untranslated sequence of the glaA mRNA, a widely used expression system for the expression of recombinant proteins, was modified by the introduction of different nucleotide elements that have positive role in the translation process. Five Aspergillus niger laccase-like multicopper oxidases (MCOs) coding genes were fused to the native glaA 5'UTR and the three synthetic versions (sUTR1, sUTR2, and sUTR3) as well, and placed under the control of the glucoamylase gene promoter. Afterwards, a total of 20 fungal transformations were done using A. niger N593 as a recipient strain and 50 transformants per transformation were isolated and analyzed. The result of the incorporation of the synthetic 5'UTRs on the overall productivity of the transformants was assessed, on one hand by monitoring the laccase activity of all the isolated transformants, and on the other hand by quantifying and comparing the activity of those secreting the highest level of each MCO. For this purpose, a high-throughput method for the screening and selection of the best producers was developed. Once the best transformants producing the highest yield of McoA, McoB, McoC, McoD, and McoJ laccases were selected, their production level was quantified in supernatants of liquid cultures. The results obtained in this work indicate that modifications in the native glaA 5'UTR can lead to improvements in protein yields.
authors.affiliation: Microbial Systems Biology, Laboratory of Systems and Synthetic Biology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands.
authors.forename: Juan Antonio
authors.initials: JA
authors.lastname: Tamayo-Ramos
authors.forename: Sharief
authors.initials: S
authors.lastname: Barends
authors.forename: Dennis
authors.initials: D
authors.lastname: de Lange
authors.forename: Annemarie
authors.initials: A
authors.lastname: de Jel
authors.forename: Raymond
authors.initials: R
authors.lastname: Verhaert
authors.forename: Leo
authors.initials: L
authors.lastname: de Graaff
mesh_terms: D020121:5' Untranslated Regions; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D010088:Oxidoreductases / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; D005656:Fungal Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D010088:Oxidoreductases; C024748:copper oxidase; D005087:Glucan 1,4-alpha-Glucosidase