Constitutive Expression of Chimeric Transcription Factors Enables Cellulase Synthesis under Non‐Inducing Conditions in Penicillium oxalicum

Abstract

Industrial production of cellulase by filamentous fungi is largely dependent on cellulose, which serves as a natural inducer of cellulase expression. However, insoluble cellulose is unfavorable to submerged fermentation and thus limits the production level of cellulase. The possibility of cellulase production under non-inducing conditions is explored in Penicillium oxalicum by overexpressing two chimeric transcription factors. The chimeric transcription factors contain the DNA binding domain of cellulase transcriptional activator ClrB linked to the C-terminal sequences of XlnR , a constitutively active mutant of hemicellulase transcriptional activator. The obtained recombinant mutants exhibited dramatically improved basal production of cellulase, which was not observed with the overexpression of intact ClrB. When cultivated in a complex cellulosic medium, one of these mutants, OE-CX -S-1, displayed a 7.3-fold increase in cellulase production (2.8 U mL ) relative to the parent strain. The results demonstrate that the dependence of cellulase synthesis on cellulose could be reduced by the overexpression of artificially designed chimeric transcription factors, and offers a potential strategy to engineer fungal strains for improving cellulase production.

Mutation of a Conserved Alanine Residue in Transcription Factor AraR Leads to Hyperproduction of α‐l‐Arabinofuranosidases in Penicillium oxalicum

“…The counterpart of alanine residue 824 of T. reesei XYR1 is identical in all the seven AraR/XlnR homologs (Figure S1, Supporting Information), suggesting the possibility of engineering AraR for constitutive transcription activation through mutation of this site. Therefore, an analogous mutant AraR A731V was constructed and expressed under the control of A. nidulans gpdA promoter in CX C , a strain exhibiting cellulose‐independent cellulase production . The resulting strain was named CX C ‐gAraR A731V .…”

“…The AraR A731V overexpression cassette containing the same promoter and marker genes was constructed by introducing the A731V mutation with overlapping PCR. The overexpression cassettes were transformed to strain CX C , which was constructed by overexpressing a chimeric transcription factor CX C ‐S in strain M12 using the selection marker gene AnpyrG from A. nidulans . The transformants were purified and identified via PCR and DNA sequencing.…”

Modulating Transcriptional Regulation of Plant Biomass Degrading Enzyme Networks for Rational Design of Industrial Fungal Strains

“…Another example of synthetic TFs (CX C -S) was shown in P. oxalicum , where the DNA-binding domain of the constitutively active XlnR A871V was replaced with that of ClrB. This synthetic TF was overexpressed using the A. nidulans gpdA promoter, yielding to inducer-independent, but still glucose-repressed, expression of cellulases and xylanases (Gao et al, 2017a , b ).…”

Protein hyperproduction in fungi by design

“…Initially, traditional mutagenesis and screening methods were used to developed high-protein production strains. Initial strains developed have largely manipulated transcriptional regulators, including creA , and in subsequent work have generated an activated Xylanase regulator 1 (called xlnR (A871V) as well as a chimeric clrB-xlnR (A871B) (Derntl et al 2013 ; Gao et al 2017a ; Gao et al 2017b ; Li et al 2015 ; Yao et al 2015 ). Similar combinatorial genetics have been developed in Myceliophthora thermophile , a thermophilic ascomycete.…”