Optimization Of The Conditions And Molecular Mechanism For The Reduction Of Acetic Acid Formation By Bisulfite In Succinic Acid Fermentation Of Actinobacillus Succinogenes

Actinobacillus succinogenes is a natural succinic acid（SA）producing strain,but its by-product acetic acid（AC）is relatively high（about one-third of the total succinic acid production）in the fermentation process,which restricts its industrial development.Previous studies found that the addition of small amounts of NaHSO₃ significantly reduced the yield of by-product AC and had no significant effect on the yield of SA,but the targets and related mechanisms of action of NaHSO₃ are not yet clear.Therefore,in this study,A.succinogenes GXAS137 was used as the research object.Firstly,the medium and culture conditions of reducing AC generation by adding NaHSO₃ were optimized by single factor,and then the factors with great influence were optimized by orthogonal test.Next,multi-omics methods（transcriptomics,proteomics and metabolomics）were used to explore the differences of A.succinogenes GXAS137 in m RNA and protein metabolism levels after the addition of NaHSO₃,and their biological functions and metabolic pathways were analyzed to initially explore the targets and internal molecular mechanisms of NaHSO₃.Finally,the effect of SA fermentation with sweet potato powder as substrate was explored,and the effect of reducing by-product AC in the process of succinic acid fermentation with cheap carbon source（sweet potato powder and xylose mother liquor）was explored,and the application value of NaHSO₃ in industrial production of SA fermentation was discussed.The main findings were as follows:（1）Through single factor experiment,the process of SA fermentation was obtained as follows:NaHSO₃0.25%（w/v）,and addition time of NaHSO₃ was 6 h after fermentation,glucose 50 g/L,Mg CO₃ 60 g/L.Further orthogonal experiments were carried out to optimize the factors with great influence on SA,and the optimal fermentation process of SA was determined as follows:glucose 60 g/L,Mg CO₃ 60 g/L,NaHSO₃ 0.15%（w/v）,and addition time of NaHSO₃ was 8 h after fermentation.After optimization,the yield of SA and AC were 44.42±0.18 g/L and5.73±0.12 g/L,respectively,and the ratio of SA to AC was 7.74.Compared with before optimization,SA increased by 100.72%,AC decreased by 21.18%,and the ratio of SA/AC increased by 1.55 times.The complex of sulfonated hydroxyacetaldehyde was detected in the fermentation liquid supplemented with NaHSO₃,but not in the control.It was speculated that the possible mechanism of NaHSO₃ was to react with acetaldehyde to form sulfonated hydroxyacetaldehyde,which led to acetaldehyde couldn’t be oxidized as a hydrogen donor.At the same time,the results of enzyme activity assay showed that the enzyme activity of acetate kinase was reduced,which led to the decrease of AC production.（2）Transcriptome sequencing（RNA-seq）technology was used to study the transcriptional expression difference of A.succinogenes under the two treatments of with 0.15%（w/v）NaHSO₃（treatment group）and without NaHSO₃（control group）,to explore the reduction mechanism of AC at the gene expression level.A total of 210 differentially expressed genes（DEGs）were screened by transcriptome analysis,among which 83 were up-regulated and 127 were down-regulated.COG,GO and KEGG functional annotation and enrichment analysis of DEGs were performed,and it was found that these genes were mainly involved in the transport and metabolism of carbohydrates,inorganic ions and amino acids,and the generation and transformation of energy.The reduction mechanism of AC production in A.succinogenes might be related to two aspects:（a）the genes encoding lipoyl transferase（Lip B）and lipoyl synthase（Lip A）in the lipoic acid synthesis pathway were significantly down-regulated,which might inhibit the synthesis of lipoic acid,and then inhibited the lipoylation of pyruvate dehydrogenase complex（PDHc）,thus inhibiting the pathway of PDHc to synthesize acetyl-COA by pyruvate;（b）The gene encoding acetaldehyde/alcohol dehydrogenase（Adh E）was significantly up-regulated,which promoted the synthesis of ethanol from acetyl-COA.However,the reaction of NaHSO₃ with the intermediate metabolite acetaldehyde blocked ethanol production and consumed acetyl-COA,resulting in decreasing AC production.（3）The proteomic sequencing technology,tandem mass tag（TMT）,was used to study the protein expression difference of A.succinogenes under the two treatments of with 0.15%（w/v）NaHSO₃（treatment group）and without NaHSO₃（control group）,to explore the reduction mechanism of AC.A total of 54differentially expressed proteins（DEPs）were screened,among which 35 were up-regulated and 17 were down-regulated.The decrease mechanism of AC production in A.succinogenes might be related to three aspects:（a）the significant up-regulation of phosphoenolpyruvate carboxykinase（PEPCK）would promote the carbon flow to C4 pathway and decrease the carbon flow into C3 pathway;（b）Down-regulation of acetyltransferase component in PDHc inhibited the binding of acetyl group to COA,thus inhibiting the production of acetyl-COA;（c）The significant increase in the expression of alcohol dehydrogenase and Adh E promoted the entry of acetyl-COA into the ethanol synthesis pathway and consumed acetyl-COA,resulting in a decrease in AC production.（4）The metabolomics sequencing technology,liquid chromatography-mass spectrometry（LC-MS）,was used to study the difference of extracellular metabolites of A.succinogenes under the two treatments of with 0.15%（w/v）NaHSO₃ and without NaHSO₃,to explore the reduction mechanism of AC.In the positive/negative ion mode,128 and 29 differential metabolites were screened,among which 66 and 16 metabolites were up-regulated and 62 and 13 metabolites were down-regulated,respectively.The mechanism of the decrease of AC production might be that the addition of NaHSO₃ inhibited the synthesis of riboflavin,thus reducing the content of riboflavin,and the production of flavin adenine dinucletide（FAD）from riboflavin was an important coenzyme of PDHc.The decrease of riboflavin content would affect the production of acetyl-COA from pyruvate,resulting in the accumulation of pyruvate and the decrease of AC production.Integrating results of multi-omics analysis,the possible mechanisms of NaHSO₃ decreasing the production of by-product AC in A.succinogenes mainly included the following:（a）After the addition of NaHSO₃,the expression and function of PDHc were inhibited,and the conversion pathway from pyruvate to acetyl-COA was hindered,thus reducing the content of acetyl-COA;（b）The significant up-regulation of the expression of alcohol dehydrogenase and Adh E promoted the entry of acetyl-COA into the ethanol synthesis pathway,while the reaction of NaHSO₃ with the intermediate metabolite acetaldehyde to form sulfonated hydroxyacetaldehyde consumed acetyl-COA,thus decreasing the production of AC;（c）In response to the stress of NaHSO₃,amino acid metabolism and energy metabolism pathways in A.succinogenes were promoted,thus increasing the consumption of carbon flow.（5）Single factor and orthogonal experiments were used to optimize the fermentation conditions of SA from sweet potato powder.The optimal culture parameters were:sweet potato powder 115 g/L,mixed nitrogen source（yeast powder:corn steep liquor powder=1:2）33 g/L,liquifying enzyme 0.008KUN-S/g substrate,glucoamylase 3.09 AGU/g substrate,Mg CO₃ 60 g/L.Under the optimal condition,the concentration of SA in the anaerobic bottle could reach69.89±0.59 g/L,which was 64.6%higher than the initial condition（42.46±0.24g/L）.In the process of SA fermentation with sweet potato powder and xylose mother liquor as substrate,the AC yield after adding NaHSO₃ was significantly decreased compared with the blank group（without NaHSO₃）,which decreased by16.52%and 16.28%respectively.The results show that NaHSO₃ can be used as a metabolic regulator to decrease the production of by-product AC in the fermentation process of A.succinogenes,which has a good industrial application prospect.