Aanlysis Of The Acid-tolerant Mechanism And Metabolic Regulation Of Propionibacterium Acidipropionici

Propionic acid（PA） is an important platform chemical used in the agriculture, food, and pharmaceutical industries. Achieving high PA titers and productivity is difficult due to end-product inhibition of cell growth and PA synthesis during the biosynthesis of PA by propionibacteria. A strategy that rational regulation of metabolic pathways through genetic modifications based on the systematic understanding of acid-tolerant mechanism of strains may be effective for the further improvement of the acid tolerance as well as PA production. In this study, the wild type of Propionibacterium acidipropioniciand its acid-tolerant mutants obtained by genome shuffling were used as the research models, and the acid-tolerant mechanism of P. acidipropionici at levels of gene transcription and protein expression, metabolic regulation and intracellular microenvironment were investigated in combination with the research methods of genomics, transcriptonmics, proteomics, metabolomics, and so on. On this basis, the metabolic pathway was directional controlled by metabolic engineering. As a result, the acid tolerance and PA production of P. acidipropionici were improved significantly. It is of guiding significance for the study of stress-tolerant mechanism of microbial cells and the optimization of fermentation process to synthesize organic acids. Major results achieved with this research are highlighted below:（1） Three acid-tolerant mutants were obtained through genome shufflingfrom the wild type Propionibacterium acidipropionici CGMCC 1.2230. The genomes of P. acidipropioniciCGMCC 1.2230 and the shuffled strain P. acidipropionici WSH1105 were sequenced, and the comparative analysis showed that amino acids metabolism and transport systems have a great impact on the acid tolerance of P. acidipropionici.Transcriptomics was then used to compare the genes expression of the two strains between low pH and neutral pH via RNA-seq. It is revealed that the metabolism of CoA in PA biosynthetic pathway of P. acidipropionici was closely related to acid tolerance of cells.（2） A comparative proteomics study of P. acidipropionici CGMCC 1.2230 and P. acidipropionici WSH1105 was performed. MALDI-TOF/MS identified 24 proteins that significantly differed between the parental and shuffled strains. The differentially expressed proteins were mainly categorized as key components of crucial biological processes and the acid stress response. Membrane-associated proteins control the transmembrane transport of substances by generating energy; cell growth and metabolic activity are regulated by enzymes of central carbon metabolism, which maintains the supply of energy and precursors and redox balance. Proteins involved in regulating gene transcription, translation and posttranslational modification constitute the DNA repair and protection mechanisms of P. acidipropionici response to acid. Quantitative reverse transcriptase polymerase chain reaction（qRT-PCR） was used to confirm differential expression of nine key proteins. Overexpression of the secretory protein glyceraldehyde-3-phosphate dehydrogenase and ATP synthase subunit α in Escherichia coli BL21 improved PA and acetic acid tolerance; overexpression of NADH dehydrogenase and methylmalonyl-CoA epimerase improved PA tolerance.（3） Metabolomics analysis of P. acidipropionici CGMCC 1.2230 and P. acidipropionici WSH1105 was undertaken to find the key metabolic nodes influencing PA production, and reveal the regulation mechanism of PA sunthesis at the levels of metabolites and metabolic pathways. In total, 142 intracellular metabolites were identified, of which those produced in amounts of greater than twofold difference between the two strains were further investigated with principal components analysis. The regulatory functions of key metabolites involved in the PA biosynthetic pathway were also forecast and analyzed according to their potential impact on metabolism. The results indicated that the amounts of metabolic intermediates of glycolysis, the Wood–Werkman cycle, and amino acid metabolism differed markedly between parental P. acidipropionici and its mutants. Based on the results of comparative metabolomics analysis, exogenous addition of key metabolites（precursors and amino acids） and gene deletion to weaken the by-product synthesis were performed to improve PA production. Under optimized conditions, the PA titer increased by 55.0% and 18.6% respectively.（4） The acid-tolerant mechanism of P. acidipropionici was unraveled by comparing the physiological changes between P. acidipropionici and three mutants.The parameters used for comparison included intracellular pH（pHi）, NAD+/NADH ratio, H+-ATPase activity and the intracellular amino acids concentrations. It was indicated that the acid tolerance of P. acidipropionici was systematically regulated. Specifically, low pHi promoted the P. acidipropionici to biosynthesize more H+-ATPase to pump the protons out of the cells, and as a result, the NAD+/NADH ratio increased due to the decreased protons concentration.The increased arginine, aspartic acid and glutamic acid concentrations helped to resist the acidic environment by consuming more H+ and generating more ATP and NH3. Based on what analyzed above, 20 mM arginine and aspartic acid were added during the shaker culture of P. acidipropionici, and the maximal PA titer reached 14.38 g?L^-1, which was increased by 39.9% compared with the control.（5） The acid resistance and PA production of Propionibacterium jensenii ATCC 4868 were improved by engineering acid-tolerant elements. Specifically, five genes（arcA, arcC, gadB, gdh, and ybaS） encoding the arginine deaminase and glutamate-based acid-tolerant systems were overexpressed in P. jensenii. The activities of the five enzymes in the corresponding engineered strains were 26.7% to 489.0% higher than those in wild-type P. jensenii. Compared with that of the wild-type, the growth rate of the engineered strains decreased, whereas specific PA production significantly increased. Among the overexpressed genes,gadB（encoding glutamate decarboxylase） increased PA resistance and yield most effectively; the PA resistance of P. jensenii-gadB was more than 10-fold that of the wild type, and the titer, yield, and conversion ratio of PA reached 10.81 g?L^-1, 5.92 g?g^-1 cell, and 0.56 g?g^-1 glycerol, increases of 22.0%, 23.8%, and 21.7%, respectively. The effects of introducing these acid resistance elements were also investigated at the transcriptional level of related enzymes in the engineered pathways and in the intracellular pools of amino acids.