Adaptive Evolution And Mechanism Analysis Of Lysine-Producing Escherichia Coli

Lysine is one of the essential amino acids,which has been broadly used in feed,food additives,chemistry industry,the global annual production reached 2.77 million tons in 2017.Lysine is mainly produced via microbial fermentation,with the production capacity of above 136 g/L by Escherichia coli as reported.Most industrial strains were obtained through numerous mutations or metabolic engineered by rational design on known pathways or cofactors,wherein further rational design always needs new targets.Moreover,hyperproducer strains generally face complex pressureful environment due to accumulated high-concentration products and various unknown by-products or intermediates in the late stage of fermentation,whether they can resist to the severe stress condition and maintain high producing ability is the key issue to be concerned.Although a lot of work has been devoted to improving the tolerance of strains to a single stress factor,the achieved good candidates may sometimes unsatisfy industrial application,since the evolution condition did not reproduce the real complex fermentation conditions.To address these problems,the following work were carried out in this study:(1)The GREACE technology was improved.The expression of DnaQ was controlled by replacing the original promoter with an inducible promoter.The plasmid was readily cured by replacing with a temperature-sensitive replicon to stabilize the genomic phenotype of the evolved strains.(2)The real end fermentation liquor containing increasing lysine concentration was used to mimic the real complicated stress condition.Combining with the GREACE technology to enhance the mutation percentage,the parent strain MU-11,an Escherichia coli lysine high-producer,was applied for adaptive evolution in an endpoint fermentation broth containing increasing lysine concentration.Finally,six mutant strains with increased tolerance and lysine titer were achieved in microplate culture system.Of which,the best mutant strain RS3 showed that the proportion of undamaged cell increased by 40%in comparison to the parent strain MU-11,the lysine titer was 155 g/L in a 42-hour fed-batch fermentention in a 5 L reactor,with an increase of 14.8%,and the yield increased at a value of 9.2%.The metabolomic analysis revealed that the metabolic flow in the mutant favors for lysine production.(3)The whole genome sequencing and assembly were carried out for the parent strain MU-11 and the mutant RS3,further comparative genomic analysis between both strains showed the point mutation on the gene speB,atpB,secY.Overexpression of speB and atpB can increase lysine production by 12.2%-14.4%in 24-30 h fermentation in shake flasks.This work provides new targets for engineering lysine industrial strain.Meanwhile,the present study developed an adaptive evolution method under a simulated real complex stress condition,which might benefit other stress tolerce property improvement for strain development.