Fusion Breeding And Rational Transformation Of Cellulose-degrading Filamentous Fungi

As a kind of major agricultural country,a large amount of agricultural waste biomass is produced every year in China.In which,the amount of cellulose is up to 40%-50%,which is the main product of carbon fixation in crops through photosynthesis.Efficient cellulose degradation technology is essential for the sustainable utilization of waste biomass.Compared with the traditional chemical treatment methods,the strategy of biocatalysis,carried out by microbial fermentation,owning the advantages of high efficiency and cleanliness.Among which,reasonable fermentation strategies and efficient engineering strains are critical to degrade cellulose in fermentation process.Especially,the regarding of waste biomass as a type of solidstate fermentation substrate and the acquisition of heat-resistant microorganisms for open solidstate fermentation can not only improve the degradation efficiency of waste biomass significantly,but also inhibit bacterial contamination in open fermentation systems effectively under the circumstance of high-temperature.This process is of great practical significance for accelerating global carbon cycling.However,there are no cellulose-degrading model strains that simultaneously owning the properties of high cellulose degradation comprehensive ability and high-temperature tolerance.The rapid development of industrial microbial breeding technology provides rich strategies for the construction of strains with excellent characteristics.Among which,protoplast fusion technology is a meaningful strategy for the mergence of excellent performance between different species,thereby obtaining evolved strains that simultaneously possess excellent parental properties.In this study,a fusion strain with strong heat resistance was obtained with the strategy of protoplast fusion technology and was used as a chassis for genetic engineering.Finally,an engineered strain,which can degrade cellulose under high-temperature conditions,was constructed.The main results can be listed into three parts:(1)Construction and screening of fusion strains.This study comprehensively evaluated several cellulose-degrading fungi,including Aspergillus niger,Aspergillus oryzae,Neurospora crassa,Trichoderma longibrachiatum,and Myceliophthora thermophila.Finally,A.niger with good growth performance and M.thermophila with good heat resistance were recruited as parental strains for protoplast fusion.Subsequently,the strategy for protoplast preparation was optimized systematically.In addition,the polyethylene glycol mediated fusion method,a type of chemical method,was applied and then optimized so that generated a fusion strain with the properties of superior growth performance and higher cellulase activity at 45℃.(2)Optimization of solid-state fermentation process for fusion strains.The solid-state fermentation process for the fusion strain was optimized and established the optimal fermentation strategy: ammonia concentration of 12.95% for corn stalks pretreatment,initial fermentation p H of 6.46,liquid-solid ratio of 2.03,and fermentation period of 5.68 days.Finally,the cellulase activity of the fusion strain was measured: filter paper enzyme activity of 8.64 U/g,β-glucosidase activity of 18.66 U/g,endo-cellulase activity of 3.67 U/g,and exo-cellulase activity of 2.45 U/g.(3)Construction of an engineered strain for efficient cellulose degradation.Firstly,the genes eg7 a and cel6 a encoding efficient endo-cellulase and exo-cellulase were heterologously expressed in the fusion strain.Next,the cellulase activity was measured,and the endo-cellulase activity of the engineered strain An Mt31-eg7 a was increased by 1.23-fold,while the exocellulase activity of the engineered strain An Mt31-cel6 a was increased by 1.89-fold.Furthermore,both engineered strains owning the property of heat resistance.In this study,a fusion strain with good heat resistance and cellulose degradation performance was obtained by the strategy of protoplast fusion,and the solid-state fermentation strategy for efficient cellulose degradation was established.Subsequently,rational strategy was applied by heterologously expressing the eg7 a and cel6 a genes in the fusion strain,so that further improved the comprehensive performance of cellulases.This study provides a novel strategy for improving the comprehensive performance of cellulases and provides guidance for future research.