Adaptive Evolution And High-throughput Screening Of Acid Alpha-amylase Producing Bacillus Licheniformis

Alpha-amylase(EC 3.2.1.1)is an industrially important enzyme,which cuts the α-1,4-glycosidic bonds in starch and glycogen molecules.It is widely used in starch processing,textile,paper,and feed industries.Bacillus licheniformis is the principal producing strain of acid α-amylase.It has the characteristics of heat resistance,strong protein secretion ability and food safety.However,B.licheniformis strains still cannot fully meet the industry needs due to their problems such as insufficient α-amylase-producing capacity and poor acid resistance of α-amylase.To improve the performance of acid α-amylase producing B.licheniformis,we developed the growth coupled adaptive evolution to enrich α-amylase high-yielding strains,and established a droplet microfluidic high-throughput screening method to screen high-yielding B.licheniformis strains.Finally,these evolved strains were analyzed by genome resequencing.The main results are as follows:(1)Acid α-amylase producing strains B.licheniformis BLL and BLH were screened out from soil.The BLH was selected as the original strain for adaptive evolution based on enzyme activity-growth coupling due to its better performance.The adaptive evolution was carried out in the selective medium with starch as the sole carbon source.After continuous subculture under low p H conditions,different mutant libraries with improved enzyme producing performance were finally obtained.A preliminary evaluation of the S200 generation library revealed that its fermentation enzyme activity increased by 35.45%(2)A high-throughput screening method was established through optimization of single cell preparation,droplet embedding with fluorescent starch substrate,and microfluidic sorting.The specific process includes: 1)prepare highly active B.licheniformis single cells via two minutes’ ultrasonic treatment;2)embed single cell droplets with 2% Pico-Surf in Novec 7500 as oil phase;3)incubate single cell droplets at 37°C under dark for 3 hours before screening.4)The deflection voltage was set to 300 V for droplet microfluidic screening.Finally,the droplet microfluidic high-throughput screening method was established for selection of acid α-amylase high-yielding B.licheniformis.(3)Compared with the original strain BLH,the evolved high-yielding strains showed that enzyme producing capacity of S200-3-3,S367-49 and S427-5-17 per cell increased by 43.30%,93.48% and 88.52%,and fermentation enzyme activity increased by 66.20%,36.82% and 41.04%respectively.Besides,the starch hydrolysis capacity under acidic conditions of S367-49 and S427-5-17 was also improved.Combined with the resequencing analysis,it is preliminarily speculated that mutations of genes related to bacterial growth,membrane protein quality control and morphology,such as fts W,glp G and rod Z,may affect the enzyme producing performance,which could guide the efficiently rational engineering of producing strains.