| Bacillus subtilis,as a common probiotic,can improve the body’s growth performance and repair the intestinal barrier.Bacillus subtilis has clear genetic characteristics and can secrete a variety of proteases,so it has become one of the important industrial strains.Synthetic biology technology can integrate the synthetic path of target substances into Bacillus subtilis,and realize the efficient expression of heterologous enzymes through its powerful secretory ability.As an endogenous hormone,melatonin has the functions of regulating biological rhythm,antioxidant,anti-aging and anti-inflammatory.In order to achieve sustainable and efficient delivery of melatonin in animals,this study intended to use CRISPR-Cas9 gene editing technology to integrate melatonin synthetase AANAT and HIOMT into Bacillus subtilis,construct melatonin producing Bacillus,and carry out preliminary application in laying hens at the end of egg production.The main test results are as follows:1.The biological characteristics of Bacillus subtilis MY016 were analyzed.MY016 began to enter a stable period at 10 h of culture,with the highest OD600 value reaching 4.599.At 12 h of culture,the number of vegetative bacteria reached the highest,about 5.320×109CFU/m L.MY016 began to form spores 12-16 h after inoculation,and fully formed spores at 72 h.The highest number of spores was about 1.900×109 CFU/m L.The enzyme-producing characteristics of MY016 showed that the highest levels of lipase,amylase and cellulase produced by MY016 were about 241.880 mg/L,28.667mg/m L and 80.444 mg/L,respectively.The highest enzyme activity was 281.883 U/L,94.473 U/m L and 72.153 U/m L.When MY016 was cultured,melatonin could not be secreted by basic medium,addition of tryptophan,change of temperature and p H,and MY016 would decompose 18-27.8% of melatonin in the environment during its growth and survival.A search of the genome of MY016 found that it did not contain key enzymes for melatonin synthesis,so this study planned to insert exogenous melatonin synthetase into the lipase site to synthesize melatonin.2.Construction of melatonin producing Bacillus.Based on the codon preference of Bacillus subtilis,the chicken melatonin synthetase encoding genes AANAT and HIOMT were optimized and inserted into the p JOE8999 plasmid,and sg RNA sequence and lipase encoding gene homologous fragments were inserted into it.The recombinant plasmid was introduced into MY016 and eliminated,and the engineering bacterium MY017 successfully recombined into the genome by AANAT-HIOMT was screened.The analysis of MY017’s melatonin production capacity showed that the highest melatonin production concentration was about 0.131 mg/L at 48 h of growth.This indicates that this experiment has initially successfully constructed melatonin producing Bacillus subtilis.3.Application of Bacillus melatonin-producing MY017 in laying hens at the end of laying period.Strain MY017 was cultured in the common feed of laying hens,and the change of melatonin content in the culture medium was detected.The experimental results showed that the melatonin content in the culture medium reached the maximum at 48 h,about 0.087 mg/L.MY017 was added to the feed at 1.579×1012 CFU/kg and fed to 490-day-old Hyland-white laying hens.The results showed that feeding MY017 could maintain and improve the egg production rate at the end of egg laying.After two weeks of feeding,melatonin content in duodenum of experimental group was significantly increased by about 1.561 pg/g compared with control group.After four weeks of feeding,compared with the control group,the melatonin content in the duodenum of the experimental group was significantly increased by about 3.193 pg/g,and the melatonin content in the blood was significantly increased by 1.848 pg/g.The analysis of intestinal flora of laying hens after feeding for four weeks showed that the feeding of melatonin-producing Bacillus subtilis could improve the intestinal flora of laying hens,and was conducive to the colonization of beneficial bacteria,such as Barnessiaceae,Comamonadaceae,Oxalobacteriaceae,Acetobacteriaceae and Sporichthyaceae.These results indicated that the recombinant strain was beneficial to the maintenance of laying performance of laying hens,and could significantly improve the content of melatonin in intestinal tract and blood of laying hens at the end of laying period.In conclusion,this study successfully achieved the heterologous expression of the key enzyme of chicken melatonin synthesis in the genome of Bacillus subtilis,and the secretion synthesis of melatonin was realized.The content of melatonin in the culure medium could reach 0.131 mg/L.The application of recombinant bacteria in practical production showed that it could maintain the laying performance of laying hens at the late stage of laying,and was beneficial to improve the intestinal flora of laying hens.The development of this study provides a new realization path for the improvement of comprehensive performance of laying hens,a new idea for the application of melatonin in laying hens,and a theoretical support for the application of synthetic biology technology in the livestock industry. |
