Redesigning the regulatory pathway to enhance cellulase production in Penicillium oxalicum

Abstract

BackgroundIn 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.ResultsIn 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0253-8) contains supplementary material, which is available to authorized users.

Effect of different terminators on transcription regulatory factor ClrB and XlnR in Penicillium oxalicum 114-2

“…By changing the terminator region of the regulatory factor, cellulase activity was significantly increased and the highest FPA enzyme activity reached 1.4 U/mL (c-d15-2-1), although there was a gap between this and the high-yielding cellulase strain RE-8, RE-10 [14,15] however, it provided a new method to transform strains, terminator in the transformation of strains still haved a great potential. In order to obtain high yield cellulase strains, these efforts were clearly not enough.…”

Genetic modification: a tool for enhancing cellulase secretion

“…over expression while gene CreA was deleted by using gene bar as the marker, obtaining a trigenic recombinant strain 'RE 10'. Several screenings were performed for comparative analysis of 'RE 10', wild type, and JU-A10-T, showing considerably improved cellulolytic ability and higher cellulase expression and secretion of 'RE 10' vs. the wild type (Yao et al, 2015). Interestingly, the cellulolytic ability of the mutant was comparable with that of the industrial strain P. oxalicum mutant (JU-A10-T).…”

“…Furthermore, a single-gene deletion library has been established for 470 transcription factors as well as 15 novel and 4 major transcriptional regulators. Their roles in the cellulase expression regulatory network have been identified and characterized (Li et al, 2015). The Reconstruction of Expression Regulatory Network (REXRN) technology has been developed as a new strategy to engineer fungi that enhance cellulase and protein production.…”

“…Such synergisms exist between endo and exoglucanases (exo/endo synergism) and between exoglucanases. BGL has been regarded as the rate-limiting enzyme as it completes the hydrolysis by hydrolyzing cellobiose which otherwise if remains in the medium inhibits CBHs production (Yao et al, 2015). BGL itself gets feedback-inhibited if glucose accumulates, thus emphasizing on the need to have glucose tolerant BGLs so as to drive reaction forward even in the presence of glucose (Singhania et al, 2013).…”

A Newly Isolated Penicillium oxalicum 16 Cellulase with High Efficient Synergism and High Tolerance of Monosaccharide

“…Furthermore, P. oxalicum 16 secreted 584.6 mg/L of protein almost with an equivalent amount of protein from T. reesei RUT-C30. P. oxalicum 16 produced a lower protein expression level in comparison to the other reports [24,25], because it was not mutated and engineered, and was cultured under nonoptimized conditions in 250-mL Erlenmeyer flasks with the basal medium which did not contain corncob residue, bagasse, been cake, Tween 80, lactose, peptone, and yeast extract. P. oxalicum 16 had a higher cellulase activity than P. oxalicum reported by Saini et al [7], Penicillium echinulatum [26], Penicillium citrinum [27], and so on.…”