This project belongs to the field of animal husbandry feed processing technology. In recent years, feed enzyme preparations have been widely used in the animal husbandry industry, and the global annual output value of feed enzymes has reached 10 billion yuan. Phytase, xylanase and beta;-glucanase are the most widely used feed enzymes, which have the effect of eliminating anti-nutritional factors in feed and improving feed utilization. Phytase can also degrade phytic acid in plant feeds, release available phosphorus, reduce the use of inorganic phosphorus, and reduce phosphorus pollution in the environment. In view of the problems such as poor effectiveness and unsatisfactory enzyme properties that often occur in the application of the above three feed enzymes, this project has transformed feed enzyme production from three aspects: gene, expression and fermentation. 1) Innovation in genetic modification methods ① Innovation in genetic optimization methods. This project integrates many factors such as gene transcription and translation efficiency, RNA and protein stability, and innovates the theory of optimal design of foreign genes. Based on this theory, thousands of gene optimization and efficient expression in heterologous hosts have been completed. Compared with similar methods, the optimized gene structure is stable and the expression level is high. The expression level of the modified phytase gene in high-density fermentation in engineering bacteria was as high as 40g/L, nearly three times that reported at home and abroad.② Improved DNA rearrangement technology. DNA rearrangement technology won the 2018 Nobel Prize. Our team has been tracking and improving this technology, taking the lead in organically combining gene synthesis, inferential design, family rearrangement and other technologies to establish an in vitro directed molecular evolution system for genes based on semi-inferential design; created a DSN enzyme homogeneous mutation library construction system, which improves the problem of low mutation rate; applied the ClonExpress in vitro recombination system to efficiently build mutant DNA libraries, increasing the capacity of the mutant gene library by more than 10 times. The improved method was successfully applied to the performance modification of multiple genes. It was invited by Human Press to write a monograph and was invited to publish a review in authoritative journals in this field.③ Optimize the feed enzyme gene screening platform. This project combines bioinformatics, gene synthesis technology and membrane carrier enzyme activity analysis technology to establish a high-throughput feed enzyme gene screening platform. 2) Construction and improvement of feed enzyme engineering strain This project has created an engineering strain that can utilize glucose fermentation through modification of the promoter, reducing fermentation costs by 30-40%; through the structural modification of the signal peptide, the secretion and expression efficiency of feed enzyme has been improved. 20-30%; genomic rearrangement has been used to improve the stability of the strain and the expression of feed enzymes. 3) Innovation in feed enzyme fermentation technology This project starts from biochemical engineering technology and develops an automatic batch fermentation control process for high-density fermentation of Pichia pastoris engineered bacteria, forming a domestic technology that combines fermentation process and equipment, and establishing the world's largest production line. This project uses a high-throughput feed enzyme gene screening platform to mine multiple feed enzyme genes with high specific activity from different microorganisms. Gene optimization and DNA rearrangement technology are used to modify phytase genes derived from Escherichia coli, Citrobacter and Yersinia to obtain various phytase with excellent properties such as high specific activity, pepsin resistance, high temperature resistance to 90 ° C and high activity at room temperature; The xylanase gene is modified to obtain feed xylanases that are thermally stable and suitable for animal gastrointestinal pH; The beta;-glucanase gene is modified to obtain beta;-glucanase with high specific activity and wide pH. This project has published 1 foreign monograph and 27 papers, including 17 SCI papers (2 IF gt;5.0); 15 invention patents have been applied, and 8 of them have been granted patent authorization; the created feed enzyme has been produced in Qingdao Weilan Biological Group Co., Ltd. In the past three years, the added output value has been nearly 250 million yuan. The feed enzyme has been cumulatively used in 48 million tons of feed, creating social benefits of 1.776 billion yuan. The commercial application of the above green feed enzyme preparations has achieved good ecological benefits. After novelty search and consultation, the project as a whole has reached the international advanced level.