Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228

Abstract

Background Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus. Results Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome. Conclusions In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.

Profiling of the β-glucosidases identified in the genome ofPenicillium funiculosum: Insights from genomics, transcriptomics, proteomics and homology modelling studies

“…The β-glucosidase deletion cassettes for Pf Bgl3A, Pf Bgl3B and Pf Bgl1A were constructed by replacing 2090bp, 1779bp and 2178bp region respectively at the middle of the ORF with a 1.9 kb hygromycin gene by restriction digestion and ligation. The hygromycin insert previously obtained in the laboratory (21) was amplified from pARI vector through PCR using Hyg primers (Table 1).…”

“…Transformants were selected with 50 μg/ml kanamycin. Plasmids isolated from positive transformants were used to transform Agrobacterium tumefaciens LBA4404 through the heat shock method as described by Randhawa et al (21).…”

“…Recently, Ogunmolu et al (16) identified a strain of the filamentous fungus Penicillium funiculosum (NCIM1228) which has outstanding biomass hydrolyzing potential comparable to Trichoderma reesei. The organism was found to be a hypercellulolytic fungus and in order to develop NCIM1228 into an industrial workhorse, several genetic interventions have been carried out to deregulate and overexpress the cellulolytic genes to improve cellulase production, activity and stability of the enzymes, resulting in increased catalytic activity, expression and secretion of both exo-and endo-cellulases (17)(18)(19)(20)(21). However, little has been studied for the enhancement of the activity and thermostability of P. funiculosum β-glucosidase.…”

Genome editing in Penicillium funiculosum using in vitro assembled CRISPR-Cas9 ribonucleoprotein complexes

“…This technique is beneficial for researchers interested in functional analysis of genes as it improves reproducibility and replicability of the experiment. For complete details on the use and execution of this protocol, please refer to Randhawa et al. (2021) .…”

“…For complete details on the use and execution of this protocol, please refer to Randhawa et al. (2021) .…”