Combination of genetic engineering and random mutagenesis for improving production of raw-starch-degrading enzymes in Penicillium oxalicum

Abstract

Background Raw starch-degrading enzyme (RSDE) is applied in biorefining of starch to produce biofuels efficiently and economically. At present, RSDE is obtained via secretion by filamentous fungi such as Penicillium oxalicum. However, high production cost is a barrier to large-scale industrial application. Genetic engineering is a potentially efficient approach for improving production of RSDE. In this study, we combined genetic engineering and random mutagenesis of P. oxalicum to enhance RSDE production. Results A total of 3619 mutated P. oxalicum colonies were isolated after six rounds of ethyl methanesulfonate and Co60-γ-ray mutagenesis with the strain A2-13 as the parent strain. Mutant TE4-10 achieved the highest RSDE production of 218.6 ± 3.8 U/mL with raw cassava flour as substrate, a 23.2% compared with A2-13. Simultaneous deletion of transcription repressor gene PoxCxrC and overexpression of activator gene PoxAmyR in TE4-10 resulted in engineered strain GXUR001 with an RSDE yield of 252.6 U/mL, an increase of 15.6% relative to TE4-10. Comparative transcriptomics and real-time quantitative reverse transcription PCR revealed that transcriptional levels of major amylase genes, including raw starch-degrading glucoamylase gene PoxGA15A, were markedly increased in GXUR001. The hydrolysis efficiency of raw flour from cassava and corn by crude RSDE of GXUR001 reached 93.0% and 100%, respectively, after 120 h and 84 h with loading of 150 g/L of corresponding substrate. Conclusions Combining genetic engineering and random mutagenesis efficiently enhanced production of RSDE by P. oxalicum. The RSDE-hyperproducing mutant GXUR001 was generated, and its crude RSDE could efficiently degrade raw starch. This strain has great potential for enzyme preparation and further genetic engineering.

Creating a novel genetic diversity of Trichoderma afroharzianum by γ-radiation for xylanase-cellulase production

“…In most cases, it influences the overall economics of enzyme production because it allows an organism to carry out a biotechnological process more efficiently. For instance, a large number of the high-secreting mutants offer suitable strains for specific industrial objectives [ 56 ], such as the overproduction of antibiotics [ 57 , 58 ], and enhanced production of cellulase enzymes [ [59] , [60] , [61] ], lipases [ 62 ], citric acid [ 63 ] and bioethanol [ 64 , 65 ].…”

Strategies for the Development of Industrial Fungal Producing Strains

“…γ irradiation (Co 60 ) Chemical mutagenesis (EMS) Genetic engineering (TF-based) [29] Penicillium oxalicum JU-A10-T Cellulase production (36%) UV mutagenesis, Chemical mutagenesis (NTG) [40] Pichia stipitis Ethanol production (70%) and tolerance UV mutagenesis [50] Pleurotus ostreatus Laccase activity (77%) UV mutagenesis [39] Saccharomyces cerevisiae Ethanol production (13.2-25%) and tolerance UV mutagenesis [51,52] S. cerevisiae Ethanol production (81.02%) and tolerance Atmospheric and room temperature plasma (ARTP) [6] S. cerevisiae Amylase activity (250%) UV mutagenesis [53] Talaromyces pinophilus Cellulase production (28%) UV mutagenesis Chemical mutagenesis (NTG and EMS) [54] Trichoderma reesei Cellulase production (250%) UV mutagenesis Chemical mutagenesis (NTG) [9] 2.1.2. Adaptive Evolution Another classical technique for strain improvement is adaptive evolution, also known as adaptive laboratory evolution, evolutionary engineering or whole-cell directed evolution.…”

“…Random (physical or chemical) mutagenesis and screening have been successfully performed in several filamentous fungi. As a result, many of the high-secreting mutants provide suitable strains for specific industrial goals [ 20 ], such as the overproduction of penicillin [ 21 , 22 ], and increased production of lignocellulolytic enzymes [ 23 , 24 , 25 ], lipases [ 26 ], citric acid [ 27 ] and bioethanol [ 28 , 29 ].…”

“…Fusarium oxysporum treated with UV followed by NTG also improved CMCase production [ 38 ], and exposure of Pleurotus ostreatus to UV caused an increase in laccase activity [ 39 ]. Random mutagenesis was also successfully applied on Penicillium oxalicum to enhance starch-degrading enzyme production [ 29 ] and for the generation of improved cellulase-producing strains. One of the cellulase-producing mutants, JU-A10-T, has been utilized for industrial-scale cellulase production since 1996 in China with a productivity of 160 U/L/h [ 40 , 41 ].…”