Gene Mining And Expression Optimization Of Tobacco-specific Alpha-amylase

The tobacco industry occupies the important position in national economy,while some problems were also exposed in the development process,such as the tobacco quality problem.The type,ratio and content of macromolecular substances in tobacco are important for the quality of tobacco.As a major macromolecular substance in tobacco,starch has a great impact on the quality of tobacco.On the one hand,the starch content affects the sensory properties of tobacco combustion,such as aroma,irritation,alcohol and so on.On the other hand,the harmful substances such as polycyclic aromatic hydrocarbons,tar,benzo alpha pyrene were produced by starch and other macromolecular substances after combustion,which will affect human health.Therefore,reducing the content of tobacco starch has become one of the breakthroughs to improve the quality of tobacco and reduce its harm.In recent years,enzymatic treatment technology has provided a new means for the degradation of macromolecular substances in tobacco.However,the commercial food enzymes currently used are not specific to tobacco starch,and it is urgent to develop specific amylase for tobacco starch.In this study,tobacco-specific amylase producing bacteria and their genetic resources were screened using tobacco starch as substrate,and food-grade engineered Bacillus strains were constructed,which significantly improved the production of tobacco specific amylase.The results are as follows:Firstly,tobacco-related samples,fermented foods and existing strains in the laboratory were selected as the screening objects.Using tobacco starch as the only carbon source in medium,18 natural strains with efficient degradation of tobacco starch were obtained by the primary and secondary screening.Based on the analysis of amylase fermentation activity,6 strains with high fermentation activities were selected for gene mining.The corresponding gene fragments were amplified by PCR,and five potentialα-amylase gene fragments were obtained by gene sequence and amino acid sequence analysis.Secondly,the optimal host strain forα-amylase gene was provided by optimizing the host strain.Five gene knockout mutant strains BAX-5,BAX-6,BAX-7,BAX-8,and BAX-9 preserved in the laboratory were used as alternative host strains.The nativeα-amylase gene of Bacillus amyloliquefaciens HZ-12 was used as the target gene to construct a free expression vector,and five recombinant expression strains were finally constructed.The enzyme activity data ofα-amylase were compared and analyzed,and BAX-5 was selected as the host strain for the subsequent expression of tobacco-specificα-amylase gene.The five tobacco-specificα-amylase genes obtained by mining were used as the target genes,and the BAX-5 was used as the host strain to construct the free expression strain to select the optimalα-amylase gene.The engineering strain BAX-5/p HY300Pamy A（LC）showed the strongestα-amylase activity（47.36 U/m L）,which was significantly higher than that of other recombinant strains.Therefore,the gene amy A（LC）was identified as the optimal gene for the expression of tobacco specific amylase.The signal peptide of amy A（LC）was further optimized.Three potential strong signal peptides obtained from signal peptide library were used to replace the original signal peptide coding region of amy A（LC）,and three recombinant expression engineering strains containing different signal peptides were constructed.The fermentation analysis showed that the strain BAX-5/PT-17SP₀₀₃amy A（LC）had the highestα-amylase fermentation activity of 67.20 U/m L,increased by 41.89%compared with the original signal peptide.In this study,the tobacco-specificα-amylase gene and the efficient signal peptide SP₀₀₃were obtained,and an engineered strain BAX-5/PT-17SP₀₀₃amy A（LC）which can efficiently express tobacco-specificα-amylase was constructed.This study laid a good foundation for the fermentation production and application of tobacco specific amylase,and it has potential application value.