| In the process of feed utilization of crop straw,lignin can be closely cross-linked with hemicellulose molecules to form a hydrophobic network structure,and the whole cell wall becomes a tight network,which increases the mechanical strength of the cell wall and its resistance to microorganisms.At the same time,it also hinders the contact between rumen microbial hydrolase and cellulose and hemicellulose in the cell wall of straws,thus reducing the degradation efficiency of cellulose polysaccharides.Therefore,lignin is considered to be the main limiting factor to inhibit the utilization rate of straw.At present,the production of lignin degrading enzyme was too lower to industrial production,and high cost of lignin-degrading enzymes are the main bottlenecks of biological lignin degradation.In order to increase the yield of lignin-degrading enzymes and promote the digestion and utilization of straw feed,lignin peroxidase gene and laccase gene from white rot fungi Phanerochaete Chrysosporium and Trametes Versicolor were cloned and expressed in Candida utilis.The genetic stability,protease properties,lignin degradation ability,Transcriptome sequencing data and phenotypic characteristics of recombinant Candida utilis were also studied.The main results are as follows:(1)In this study,lignin peroxidase gene and laccase gene were cloned from non-toxic and harmless Phanerochaete Chrysosporium and Trametes Versicolor,and the full-length sequence of cDNA was 1239 bp and 1571 bp,respectively.pGMLR and pGQLR 2 expression vectors were constructed and these two genes were also effectively expressed in Candida utilis.(2)All genetic elements of vectors come from probiotic Candida utilis and non-toxic and harmless fungi,do not carry antibiotic resistance genes,do not carry other toxic protein genes,and do not bring harm to the environment or human and animal biosafety.It has reached the food grade and can be directly added to food and feed.(3)The Engineering bacteria constructed in this experiment still contained target bands in 100 passages,and maintained high genetic stability in the process of subculture.The Enzyme activities of recombinant bacteria ZHMX4 and ZHQX1 reached the highest values on the 96h of culture,which were 65.28 U/L and 1078.9 U/L respectively,which were 5.3 times and 4.8 times higher than those of starter culture.(4)The properties of enzyme activity results showed that the optimum reaction temperature of ZHMX4 was 30℃,and the optimum reaction pH was 3.0.When pH was higher than 6.5,ZHMX4 was basically inactivated.The optimum reaction temperature of ZHQX1 was 30℃,and the optimum reaction pH was 4.5.when pH was higher than 6.0,the enzyme activity of ZHQX1 decreased gradually.(5)The ability of two kinds of engineering bacteria to degrade straw lignin was tested.The results showed that when ZHMX4 and ZHQX1 were mixed with equal 2 mL and fermented,the lignin content decreased by 4.14%(the degradation rate was 50.12%).When the dose is increased to 5 mL,the ability of lignin degradation did not change significantly.The experimental results also showed that the addition of cellulase and hemicellulase had an impedance effect on the degradation of straw lignin by engineering bacteria.(6)Transcriptome sequencing data analysis showed that compared with Candida utilis strains,It was found that a total of 6024 genes were identified in ZHMX4 compared with Candida utilis,of which 583 genes were differentially expressed.There were 291 up-regulated genes and 292 down-regulated differentially expressed genes;A total of 6024 genes were identified in ZHQX1 compared with Candida utilis,of which 748 genes were differentially expressed.The number of up-regulated genes and down-regulated genes was 418 and 330,respectively.(7)The results of Biolog identification and phenotypic analysis showed that the material metabolism of the engineering bacteria changed obviously.From the experimental results,we can find the culture characteristics and nutrient requirements of the two engineering bacteria,which can be used to optimize the culture medium of the engineering bacteria.In this study,two engineering bacteria with intellectual property rights were constructed,and their genetic stability,enzyme activity,lignin degradation ability,transcriptional differences and phenotypic characteristics were studied.It laid a good foundation for the development of lignin anti-nutritional factors Culture for the straw degradation,the industrial production of lignin-degrading enzymes and the construction of artificial white rot fungi. |
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