Study On Mutation Breeding Of Candida Util Is For High Single-cell Protein Production

With the rapid development of the animal husbandry industry and the arrival of the feed industry anti-ban era,the demand for protein feed in China is also increasing.The shortage of protein feed has seriously affected the development of China’s animal husbandry.Single cell protein(SCP)has been proven to be a high-quality protein substitute.Candida utilis is a better industrial strain for SCP production.It has fast growth and reproduction,high single cell protein content,and is rich in a variety of nutrients,such as nucleotides,enzymes,vitamins B and oligosaccharide.It has been certified by the US FDA as Generally Regarded as Safe,GRAS,and can be used as feed additives.In this study,firstly,three highly productive starting strains were screened from nine strains of C.utilis by the weighted average method.Then,12C6+ion beam irradiation was used for mutation screening,and the ammonia nitrogen assimilation ability of mutant strains with high protein content was compared.Finally,the mechanism of improving the ammonia nitrogen utilization ability of mutant strains was comprehensively analyzed from the aspects of phenotype,DNA and mRNA,and the following conclusions were drawn.C.utilis CGMCC 2.2878,C.utilis CGMCC 2.2951,and C.utilis CICC 31856 were selected as the starting strains using the weighted average method.The SCP content of single cell protein producing strains Y-3-2,J-4-1 and A-3-1 were 63.06%,64.74%and 60.12%respectively by heavy ion mutagenesis.Compared with the original strains,the SCP content were increased by 20.60%,11.91%and 11.19%respectively.The experimental results of ammonia assimilation ability showed that the mutant strain Y-3-2 had the greatest increase in ammonia nitrogen utilization rate and the strongest ammonia assimilation ability at 24 hours,with an ammonia nitrogen utilization rate of 94.27%,which was 9.86%higher than the ammonia nitrogen utilization rate of 85.81%for Y-0-0.The glutamate dehydrogenase(GDH)enzyme in the ammonia assimilation pathway reached its maximum activity at 4.51 U/mL after 18 hours of fermentation.The enzyme activities of glutamate synthase(GOGAT)and glutamine synthase(GS)reached their maximum values at 12 hours of fermentation,reaching 4.80 U/mL and 1.10 U/mL,respectively.Analyze the reasons for the increased utilization of ammonia nitrogen in C.utilis Y-32 through a comprehensive analysis of phenotype,DNA,and mRNA.The surface of Y-3-2 cells becomes rough and wrinkled.The expression level of the CYBJADRAFT-161299 gene encoding the ammonium transporter protein of Y-3-2 was significantly downregulated.The expression level of the ammonium transporter encoding gene CYBJADRAFT-161303 was significantly upregulated.The expression level of the ammonium transporter encoding gene CYBJADRAFT-188980 was significantly downregulated.Thus,the increase in ammonium transporter gene expression was an important reason for the increased ammonia nitrogen utilization by C.utilis Y-3-2.In this study,we obtained mutant strains with high protein and efficient utilization of ammonium sulfate through screening,mutagenesis breeding and comparison of ammonia assimilation ability of C.utilis.It is intended to solve the current problem of lack of protein resources and to provide new ideas and technical means for selecting and breeding high protein strains.At the same time,it will lay the theoretical and application foundation for the production of protein feed by C.utilis as a probiotic additive.