CRISPR-Cas9-mediated seb1 disruption in Talaromyces pinophilus EMU for its enhanced cellulase production

https://doi.org/10.1016/j.enzmictec.2020.109646Get rights and content

Highlights

  • CRISPR Cas9 platform was established in Talaromyces pinophilus EMU.

  • Gene seb1 was cloned and disrupted by this CRISPR-Cas9 platform.

  • Disruption of seb1 in EMU caused more hyphal branching and reduced cell growth.

  • Disruption of seb1 in EMU enhanced protein secretion and cellulase production.

  • Disruption of seb1 affected transcription of rhoA and ras1, but not cbh1 and bgl1.

Abstract

Filamentous fungi are working horses for industrial enzyme production. Combinatory approaches, such as random mutagenesis and rational genetic engineering, were adopted to improve their enzyme productivity. The filamentous fungus Talaromyces pinophilus EMU is a hyper cellulase-producing filamentous fungus obtained through random mutagenesis. This study further enhanced its cellulase production through the disruption of seb1 gene, which encodes Seb1, a transcription factor that binds to the stress response element (STRE) and regulates a variety of cellular processes. Gene seb1 was cloned from strain T. pinophilus EMU and disrupted using CRISPR-Cas9 technology. The seb1-disruptants (TpΔseb1 strains) showed distinct morphology from its parent strain. They presented a hyphal branching phenotype with decreased transcription levels of rhoA and ras1 genes involved in hyphal branching. Furthermore, TpΔseb1 strains displayed lower cell biomass, higher specific protein content, and 20%–40% enhancement in filter paper cellulase (FPase) activity, however, insignificant changes in the transcription levels of cbh1 and bgl1 genes involved in cellulase production. Through this study, we confirmed that seb1 gene disruption in T. pinophilus EMU caused more hyphal branching, reduced cell growth, increased protein secretion, and enhanced cellulase production. In addition, we successfully established the CRISPR-Cas9 genome-editing platform in T. pinophilus EMU.

Introduction

The high cost of cellulolytic enzymes is one of the limiting factors in biorefinery industries [1,2]. There is an increased interest in novel fungal strain isolation and development for high-yield cellulase production. Strain improvement of filamentous fungi is tedious and labor-intensive; it often takes decades to improve a strain to obtain an enzyme productivity that can meet the industrial requirements [3]. Combinatory approaches, such as random mutagenesis and rational genetic engineering were adopted for strain improvement [[3], [4], [5], [6], [7]]. The fungus Talaromyces pinophilus EMM was a hyper cellulase-producer derived from a wild-type T. pinophilus strain OPC4−1 through consecutive random mutagenesis [7] and strain EMU is a uracil auxotrophic mutant strain derived from strain EMM. It secretes large amount of proteins and can potentially be a cell factory for cellulolytic enzyme production. The objective of this study is to further enhance its cellulase production through rational genetic engineering.

It has been suggested that hyphal morphology plays an important role in its performance [8]. Short and highly branched hypha are favorable to protein secretion and enzyme production [4]. Seb1 is a C2H2-type transcription factor, which binds to the stress response element (STRE) and regulates various cellular processes. Recently, Seb1 was shown to play an important role in cell growth, asexual development, oxidative stress response, cell wall integrity maintenance, and virulence in Valsa mali [9]. In Neurospora crassa, seb1 gene deletion caused strain morphological defects; the mutant exhibited cauliflower-like growth and a hyper-hyphal-branching phenotype [10]. In an earlier patent publication, Seb1-disrupted variants of a Pezizomycotina strain- displayed varied cell growth characteristics and viscosity phenotype; Such filamentous fungus variant strains were suitable for submerged fermentation, in particular, large-scale enzyme production [11]. In view of the above, this study aims to disrupt seb1 gene in T. pinophilus EMU to improve its enzyme produciton.

Research on filamentous fungi has recently targeted towards development of molecular biology tools for strain genetic modification. This not only assists in genetic engineering of fungal strains, but also helps better understand their cellular processes. The current molecular biology tools are underdeveloped, or they are mostly limited to model strains. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 is a bacterial immune system [12] and has been successfully applied in the modification of a broad range of microorganisms [13]. The CRISPR-Cas 9 system requires two main components, the Cas 9 enzyme and the single guide RNA (sgRNA). The Cas9 enzyme catalyses a double stranded break in the target DNA and the sgRNA, which consists of a 20-base-pair (bp) protospacer region, guides the Cas9 enzyme to the target site in the target DNA. Recently, this technology has been successfully acquired in a few prominent filamentous fungi such as A. aculeatus [14], N. crassa [15], T. reesei [16], T. atroroseus [17] and Penicillium strains [18,19]. This opens a new frontier in fungal genetic engineering. Here, in this study, the CRISPR Cas9 genome-editing platform for fungus T. pinophilus strain EMU was established and gene seb1 was disrupted using this platform. Its effects on strain morphology, hyphal branching, protein secretion, and cellulase production were investigated. To the best of our knowledge, this is the first study on seb1 gene disruption for enhanced enzyme production in a T. pinophilus strain. It is also the first demonstration on the establishment of the CRISPR-Cas9 platform in a T. pinophilus strain for gene disruption.

Section snippets

Microorganisms, media and cultivation conditions

Strain T. pinophilus EMM is a mutant strain derived from T. pinophilus strain OPC 4-1 [7] and strain EMU is a uracil auxotrophic mutant strain derived from strain EMM. Strain EMU was used for genetic engineering and strain improvement. Escherichia coli strain JM109 obtained from Promega (Promega Corporation, Madison, WI, USA) was used for cloning and plasmid propagation.

Strain EMU and its mutant strains were maintained on potato dextrose agar (PDA) plates or broth (PDB) (Merck, Kenilworth, NJ.

CRISPR-Cas9-mediated seb1 gene disruption in strain EMU

The seb1 gene was amplified from the genomic DNA of strain EMU using primers Seb1.Seq.F and Seb1.Seq.R. It was subsequently sequenced. The seb1 gene sequence contained 1808 bp and it was deposited at NCBI GenBank with an accession number of MN548885. It showed 90%, 83% and 75% identity to that of T. marneffei, T. stipitatus and A. niger, respectively. The sequence analysis using Clustal W and N-J tree showed that the seb1 gene in strain EMU was grouped with the Talaromyces seb1 genes (data not

CRISPR-Cas9-mediated seb1 gene disruption in strain EMU

In this study, for the first time, we report the seb1 gene in a T. pinophilus strain, which showed high identity to that in other Talaromyces strains. Further, we disrupted the seb1 gene in strain EMU using CRISPR-Cas9 technology. Plasmid pFCΔseb1, which contained the Aspergillus codon-optimized Cas9 gene expression cassette and the sgRNA expression cassette for seb1 gene, was transformed to strain EMU without any donor DNA using the method outlined in Session 2.3. This resulted in T. pinophilus

Conclusion

In this study, we report the cloning of the seb1 gene in a T. pinophilus strain and its disruption by using the CRISPR-Cas9 technology. The disruption of the seb1 gene in strain EMU resulted in morphology defects and more hyphal branching in seb1 mutant strains. The transcription levels of rhoA and ras1 genes were reduced. This subsequently affected the fungal hyphae polarity and branching. Although TpΔseb1 strains did not present much increase in transcription levels of cbh1 and bgl1 genes,

Funding

The research was supported by Ministry of Education Singapore (Grant no. MOE2015-TIF-2-G-024).

CRediT authorship contribution statement

Rupali Rahul Manglekar: Conceptualization, Methodology, Investigation, Validation, Writing - original draft. Anli Geng: Conceptualization, Project administration, Supervision, Writing - review & editing.

Declaration of Competing Interest

The authors declare no financial or commercial conflict of interest.

Acknowledgement

Authors are thankful for the research platform provided by Ngee Ann Polytechnic, Singapore.

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