Metabolic Regulation Of Acetobacter Pasteurianus To Improve Acetic Acid Productivity

Acetic acid bacteria are widely used in the vinegar fermentation industry due to their unique ability to oxidize ethanol to produce acetic acid and high acetic acid tolerance.However,in industrial production,the continuous accumulation of acetic acid causes serious feedback inhibition on cell growth and product synthesis.This leads to the decrease of metabolic activity of cells,which is the bottleneck of acetic acid production and production intensity.The use of synthetic biology to control and modify the key metabolic pathways of cell acid production and acid resistance is considered to be an effective means to increase the production of acetic acid.Therefore,this study uses a strain of Acetobacter pasteurianus CICIM B7003（Shanghai Brew 1.01）as a model strain for the vinegar brewing industry.From the perspective of metabolic engineering,molecular biology methods are applied.Explore a series of constitutive promoters in A.pasteurianus CICIM B7003.Then,for the coenzyme PQQ,an electron receptor for redox reactions,the adaptive regulation relationship between acetate synthesis pathway enzymes and cofactors was explored,and the law of action between them and cell specific growth rate,acid production rate and acid stress resistance was revealed;On this basis,the study of static and dynamic regulation of tolerance pathways involved in the TCA cycle is further combined to achieve rapid cell growth under initial acetic acid stress.The main research results are as follows:（1）Using RNA-Seq technology,a whole-genome transcription library of A.pasteurianus CICIM B7003 in deep liquid acetic acid fermentation was constructed at logarithmic initial stage,mid-logarithmic stage,stable stage,stable intermediate stage,and stable late stage;built to enhance A.pasteurianus CICIM B7003 promoter screening platform with type green fluorescent protein as the reporter gene;a constitutive promoter P_tuf with high expression intensity during acetic acid fermentation was screened,and its activation activity was 8 times that of the universal alcohol dehydrogenase promoter P_{adh A}.（2）Through the high-strength constitutive and coordinated regulation of the key dehydrogenases ADH and ALDH of the alcohol respiratory chain and the cofactor PQQ,the optimal adaptation relationship of the combined metabolic regulation of dehydrogenase and PQQ is clarified;not only can it effectively alleviate the increase in acetic acid production Conflict with impaired cell adaptability,and can greatly improve the tolerance of Acetobacter pasteurianus to 30 g·L^-1 high initial acetic acid.Obtained an engineered strain A.pasteurianus/p T-adh A-pqq ABCDE with excellent traits,which produces 70 g·L^-1 acetic acid.The start-up time of semi-continuous fermentation can be shortened from 116 h to 99 h from the original strain,and the fermentation cycle is also shortened to34 h～35 h,the average acidification rate of the whole process reached 0.99 g·L^-1·h^-1,which was 32%higher than the original strain.（3）Through the overexpression study of the constitutive strong promoter P_tuf,the key enzymes of the acetic acid tolerance mechanism mediated by the carbon center metabolism TCA cycle were determined,which are encoding acetyl Co A synthetase ACS1 and encoding succinyl coenzyme A:acetyl coenzyme,respectively A transferase Aar C;compared with the original bacteria A.pasteurianus CICIM B7003,the specific growth rate increased by 16.2%and 38.7%,respectively;the acetic acid production increased by 42.0%and 23.5%.Under the initial acetic acid stress containing 20 g·L^-1,compared with the static regulation of the constitutive promoter P_tuf,regulating the expression of ACS and Aar C through the acid pressure-driven promoter P_{gro ESL} can further increase the cell growth rate by 56.4%and 89.2%,and at the same time The acetic acid output of the engineered strain AP/p AR-P_{gro ESL}-acs1 was increased by 12.1%compared with the original strain,reaching 35.86 g·L^-1.