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Enzyme Production Potential of Penicillium oxalicum M1816 and Its Application in Ferulic Acid Production.

ABSTRACT: The present study focused on isolating an efficient enzyme production microorganism for ferulic acid (FA) production from wheat bran. A wild-type cellulase-, xylanase-, and feruloyl esterase-producing strain was isolated and identified as Penicillium oxalicum M1816. The genome was sequenced and assembled into 30.5 Mb containing 8301 predicted protein-coding genes. In total, 553 genes were associated with carbohydrate metabolism. Genomic CAZymes analysis indicated that P. oxalicum M1816, comprising 39 cellulolytic enzymes and 111 hemicellulases (including 5 feruloyl esterase genes), may play a vital role in wheat bran degradation and FA production. The crude enzyme of strain M1816 could release 1.85 ± 0.08 mg·g^-1 FA from de-starched wheat bran (DSWB) at 12 h, which was significantly higher than other commercial enzymes. Meanwhile, when the strain M1816 was cultured in medium supplemented with DSWB, up to 92.89% of the total alkali-extractable FA was released. The process parameters of solid-state fermentation were optimized to enhance enzyme production. The optimized wheat bran Qu of P. oxalicum M1816 was applied to huangjiu fermentation, and the FA content was increased 12.4-fold compared to the control group. These results suggest that P. oxalicum M1816 is a good candidate for the development of fermented foods bio-fortified with FA.

SUBMITTER: Zhang J

PROVIDER: S-EPMC8621443 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:Cellulolytic enzyme hydrolysis of lignocellulose biomass to release fermentable sugars is one of the key steps in biofuel refining. Gene expression of fungal cellulolytic enzymes is tightly controlled at the transcriptional level. Key transcription factors such as activator ClrB/CLR2 and XlnR/XYR1, as well as repressor CreA/CRE1 play crucial roles in this process. The putative protein methyltransferase LaeA/LAE1 has also been reported to regulate the gene expression of the cellulolytic enzyme. The formation and gene expression of the cellulolytic enzyme was compared among Penicillium oxalicum wild type (WT) and seven mutants, including ?laeA (deletion of laeA), OEclrB (clrB overexpression), OEclrB?laeA (clrB overexpression with deletion of laeA), OExlnR (xlnR overexpression), OExlnR?laeA (xlnR overexpression with deletion of laeA), ?creA (deletion of creA), and ?creA?laeA (double deletion of creA and laeA). Results revealed that LaeA extensively affected the expression of glycoside hydrolase genes. The expression of genes that encoded the top 10 glycoside hydrolases assayed in secretome was remarkably downregulated especially in later phases of prolonged batch cultures by the deletion of laeA. Cellulase synthesis of four mutants ?laeA, OEclrB?laeA, OExlnR?laeA, and ?creA?laeA was repressed remarkably compared with their parent strains WT, OEclrB, OExlnR, and ?creA, respectively. The overexpression of clrB or xlnR could not rescue the impairment of cellulolytic enzyme gene expression and cellulase synthesis when LaeA was absent, suggesting that LaeA was necessary for the expression of cellulolytic enzyme gene activated by ClrB or XlnR. In contrast to LaeA positive roles in regulating prominent cellulase and hemicellulase, the extracellular ?-xylosidase formation was negatively regulated by LaeA. The extracellular ?-xylosidase activities improved over 5-fold in the OExlnR?laeA mutant compared with that of WT, and the expression of prominent ?-xylosidase gene xyl3A was activated remarkably. The cumulative effect of LaeA and transcription factor XlnR has potential applications in the production of more ?-xylosidase.

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Enzyme Production Potential of Penicillium oxalicum M1816 and Its Application in Ferulic Acid Production.

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