Assessment of the ptxD gene as a growth and selective marker in Trichoderma atroviride using Pccg6, a novel constitutive promoter

Abstract

Background: Trichoderma species are among the most effective cell factories to produce recombinant proteins, whose productivity relies on the molecular toolkit and promoters available for the expression of the target protein. Although inducible promoter systems have been developed for producing recombinant proteins in Trichoderma, constitutive promoters are often a desirable alternative. Constitutive promoters are simple to use, do not require external stimuli or chemical inducers to be activated, and lead to purer enzyme preparations. Moreover, most of the promoters for homologous and heterologous expression reported in Trichoderma have been commonly evaluated by directly assessing production of industrial enzymes, requiring optimization of laborious protocols. Results: Here we report the identification of Pccg6, a novel Trichoderma atroviride constitutive promoter, that has similar transcriptional strength as that of the commonly used pki1 promoter. Pccg6 displayed conserved arrangements of transcription factor binding sites between promoter sequences of Trichoderma ccg6 orthologues genes, potentially involved in their regulatory properties. The predicted ccg6-encoded protein potentially belongs to the SPE1/SPI1 protein family and shares high identity with CCG6 orthologue sequences from other fungal species including Trichoderma reesei, Trichoderma virens, Trichoderma asperellum, and to a lesser extent to that of Neurospora crassa. We also report the use of the Pccg6 promoter to drive the expression of PTXD, a phosphite oxidoreductase of bacterial origin, which allowed T. atroviride to utilize phosphite as a sole source of phosphorus. We propose ptxD as a growth reporter gene that allows real-time comparison of the functionality of different promoters by monitoring growth of Trichoderma transgenic lines and enzymatic activity of PTXD. Finally, we show that constitutive expression of ptxD provided T. atroviride a competitive advantage to outgrow bacterial contaminants when supplied with phosphite as a sole source of phosphorus. Conclusions: A new constitutive promoter, ccg6, for expression of homologous and heterologous proteins has been identified and tested in T. atroviride to express PTXD, which resulted in an effective and visible phenotype to evaluate transcriptional activity of sequence promoters. Use of PTXD as a growth marker holds great potential for assessing activity of other promoters and for biotechnological applications as a contamination control system.

Dual RNA-Seq Profiling Unveils Mycoparasitic Activities of Trichoderma atroviride against Haploid Armillaria ostoyae in Antagonistic Interaction Assays

“…The reaction was realized using the following conditions: denaturation at 95°C for 3 min, followed by 40 cycles of amplification (95°C for 5 s, 60°C for 30 s, and 72°C for 30 s). The relative quantification of gene expression was executed with the 2 −ΔΔCt method ( 92 ) using the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase ( gpd , ARMOST_14637) or actin (ARMOST_03733) for A. ostoyae and the gpd (ID 297741) or elF-4 (ID 301614) genes for T. atroviride ( 93 ). For each sample, 2 technical replicates of the qRT-PCR assay were used with a minimum of 3 biological replicates.…”

Dual RNA-Seq profiling unveils mycoparasitic activities ofTrichoderma atrovirideagainst haploidArmillaria ostoyaein antagonistic interaction assays

“…The reaction was realized using the following conditions: denaturation at 95 °C for 3 min, followed by 40 cycles of amplification (95 °C for 5 s, 60 °C for 30 s and 72 °C for 30 s). The relative quantification of gene expression was executed with the 2-ΔΔCt method (93) using the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase ( gpd , ARMOST_14637) or actin (ARMOST_03733) for A. ostoyae and the gpd (ID 297741) or elF-4 (ID 301614) genes for T. atroviride (94). For each sample, 2 technical replicates of the qRT-PCR assay were used with a minimum of 3 biological replicates (except for samples for the mycoparasite stage which only contained 2 biological replicates).…”

“…or actin (ARMOST_03733) for A. ostoyae and the gpd (ID 297741) or elF-4 (ID 301614) genes for T. atroviride (94). For each sample, 2 technical replicates of the qRT-PCR assay…”

A novel dominant selection system for plant transgenics based on phosphite metabolism catalyzed by bacterial alkaline phosphatase

“…Moreover, it has also been extensively used to genetically engineer important microorganisms (e.g. yeast [33], Trichoderma fungi [34]) and microalgae (e.g. Chlamydomonas [35][36][37][38], Cyanophyta alga [20,21]) as cost-effective bio-production platforms under non-axenic conditions, owing to Phi-directed control of biological contaminants.…”