title: Redesigning the regulatory pathway to enhance cellulase production in Penicillium oxalicum.
abstract: BACKGROUND
  In cellulolytic fungi, induction and repression mechanisms synchronously regulate the synthesis of cellulolytic enzymes for accurate responses to carbon sources in the environment. Many proteins, particularly transcription regulatory factors involved in these processes, were identified and genetically engineered in Penicillium oxalicum and other cellulolytic fungi. Despite such great efforts, its effect of modifying a single target to improve the production of cellulase is highly limited.
  
  
  RESULTS
  In this study, we developed a systematic strategy for the genetic engineering of P. oxalicum to enhance cellulase yields, by enhancing induction (by blocking intracellular inducer hydrolysis and increasing the activator level) and relieving the repression. We obtained a trigenic recombinant strain named 'RE-10' by deleting bgl2 and creA, along with over-expressing the gene clrB. The cellulolytic ability of RE-10 was significantly improved; the filter paper activity and extracellular protein concentration increased by up to over 20- and 10-fold, respectively, higher than those of the wild-type (WT) strain 114-2 both on pure cellulose and complex wheat bran media. Most strikingly, the cellulolytic ability of RE-10 was comparable with that of the industrial P. oxalicum strain JU-A10-T obtained by random mutagenesis. Comparative proteomics analysis provided further insights into the differential secretomes between RE-10 and WT strains. In particular, the enzymes and accessory proteins involved in lignocellulose degradation were elevated specifically and dramatically in the recombinant, thereby confirming the importance of them in biomass deconstruction and implying a possible co-regulatory mechanism.
  
  
  CONCLUSIONS
  We established a novel route to substantially improve cellulolytic enzyme production up to the industrial level in P. oxalicum by combinational manipulation of three key genes to amplify the induction along with derepression, representing a milestone in strain engineering of filamentous fungi. Given the conservation in the mode of cellulose expression regulation among filamentous fungi, this strategy could be compatible with other cellulase-producing fungi.
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan City, Shandong Province 250100 China.
authors.forename: Guangshan
authors.initials: G
authors.lastname: Yao
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan City, Shandong Province 250100 China ; Qingdao Vland Biotech Group Co. Ltd., Shandong Expressway Mansion, Miaoling Road, Qingdao, Shandong China.
authors.forename: Zhonghai
authors.initials: Z
authors.lastname: Li
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan City, Shandong Province 250100 China.
authors.forename: Liwei
authors.initials: L
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan City, Shandong Province 250100 China.
authors.forename: Ruimei
authors.initials: R
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan City, Shandong Province 250100 China.
authors.forename: Qinbiao
authors.initials: Q
authors.lastname: Kan
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan City, Shandong Province 250100 China.
authors.forename: Guodong
authors.initials: G
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan City, Shandong Province 250100 China ; National Glycoengineering Research Center, Shandong University, Jinan City, Shandong Province 250100 China.
authors.forename: Yinbo
authors.initials: Y
authors.lastname: Qu
keywords: Beta-glucosidase; Cellulase; Genetic engineering; Penicillium oxalicum; Secretome; Transcription factor