A new high-performance heterologous fungal expression system based on regulatory elements from the Aspergillus terreus terrein gene cluster

Abstract

Recently, the Aspergillus terreus terrein gene cluster was identified and selected for development of a new heterologous expression system. The cluster encodes the specific transcription factor TerR that is indispensable for terrein cluster induction. To identify TerR binding sites, different recombinant versions of the TerR DNA-binding domain were analyzed for specific motif recognition. The high affinity consensus motif TCGGHHWYHCGGH was identified from genes required for terrein production and binding site mutations confirmed their essential contribution to gene expression in A. terreus. A combination of TerR with its terA target promoter was tested as recombinant expression system in the heterologous host Aspergillus niger. TerR mediated target promoter activation was directly dependent on its transcription level. Therefore, terR was expressed under control of the regulatable amylase promoter PamyB and the resulting activation of the terA target promoter was compared with activation levels obtained from direct expression of reporters from the strong gpdA control promoter. Here, the coupled system outcompeted the direct expression system. When the coupled system was used for heterologous polyketide synthase expression high metabolite levels were produced. Additionally, expression of the Aspergillus nidulans polyketide synthase gene orsA revealed lecanoric acid rather than orsellinic acid as major polyketide synthase product. Domain swapping experiments assigned this depside formation from orsellinic acid to the OrsA thioesterase domain. These experiments confirm the suitability of the expression system especially for high-level metabolite production in heterologous hosts.

Strategies and Challenges for the Development of Industrial Enzymes Using Fungal Cell Factories

“…High-throughput screening enables large numbers of mutants to be screened in search for the desired phenotype, i.e., higher enzyme yields. The increase in copy number typically leads to an increase in product formation, but it also reaches a maximum when transcription is not limiting and other cellular processes like secretion become a bottleneck (Gressler et al 2015). Overview of a generic recovery process for enzyme production.…”

Induced Chemical Defense of a Mushroom by a Double‐Bond‐Shifting Polyene Synthase

“…Transgene expression was elicited when 100 mm d-glucose and 20 mm l-glutamine were present in the medium, while 1 % casamino acids as the sole carbon and nitrogen source repress transgene expression. [19] A yellow coloration was already visible by the naked eye after 24 h when A. niger tPB01 was grown under inducing conditions ( Figure S3), while it did not change its color in non-inducing medium.…”

“…The 8211 bp (base pair) cDNA of PPS1 was isolated from injured BY1 mycelium and inserted into the plasmid SM-Xpress [19] to place this PKS gene behind the inducible terA promoter. The resulting plasmid pPB25 (Figure S2) was used to transform Aspergillus niger P2 and create strain tPB01.…”

Induzierte chemische Verteidigung eines Ständerpilzes durch eine doppelbindungsverschiebende Polyensynthase

“…Die 8211 bp lange cDNA von PPS1 wurde aus verletztem BY1-Mycel isoliert und in das Plasmid SMXpress eingefügt, [19] um dieses PKS-Gen unter die Kontrolle des induzierbaren terA-Promotors zu stellen. Das resultierende Plasmid pPB25 (Abbildung S2) wurde verwendet, um Aspergillus niger P2 zu transformieren und den Stamm tPB01 zu erzeugen.…”

“…Die Expression des Transgens wird angeregt, wenn 100 mm d-Glucose und 20 mm l-Glutamin im Medium vorhanden sind, wohingegen 1 % Casaminosäuren als einzige Kohlenstoff-und Stickstoffquelle die Expression des Transgens unterdrücken. [19] Nach 24 h war eine Gelbfärbung bereits mit bloßem Auge sichtbar, wenn A. niger tPB01 unter induzierenden Bedingungen angezogen wurde (Abbildung S3), während der Pilz seine Farbe im nichtinduzierenden Medium nicht änderte.…”