Study On Metabolic Mechanism And Application Of Ethyl Acetate Production By Nakazawaea Ishiwadae GDMCC 60786

Currently,commercial yeast is used for the wine industry in China,which can better control the fermentation process and product quality,but leads to homogenization of wine quality,low complexity,and a lack of characteristic style.Esters are important flavour substances that provide fruit aroma and increase complexity in wine.Ethyl acetate is an important ester that directly affects wine quality.Therefore,selecting yeast strains with good fermentation performance and strong ester production ability is particularly important for wine industry applications.In this paper,a novel non-saccharomyces yeast,Nakazawaea ishiwadae GDMCC 60786,was used as the starting strain.Through previous studies,it was found that it has the ability to produce ethyl acetate,but it produces low levels of ethanol and cannot complete alcohol fermentation.We deeply analysed the metabolic response mechanism of the strain’s multiple pathways of ester production by fermentation performance analysis and omics technology,explored key genes of ester metabolic pathways,and analysed ester content and fermentation potential.It is of great significance to broaden the fermentation field and enrich the microbial database of fermented foods.Firstly,the fermentation characteristics of the starting strain under different carbon sources and initial sugar concentrations showed that glucose and fructose produce esters,reaching 380.32 mg/L and 555.21 mg/L,respectively.While ethanol was 3.24%（v/v）and3.35%（v/v）,respectively,and not reaching 6%（v/v）.Thus,this strain could not independently complete alcohol fermentation.Therefore,the yeast is non-Saccharomyces yeast.Maltose and sucrose produced esters at relatively low rates and almost no ethanol.Ethanol and acetic acid were used as supplemental carbon sources;ethanol increased ethyl acetate to 1212.82 mg/m L in 4%ethanol.However,acetic acid was used as a supplemental carbon source,resulting in a loss of the ability to produce esters.Cell growth rate was analysed under different tolerance conditions and was determined to be 6%Na Cl,200 ppm SO_2,p H=7,and 4%ethanol.Finally,PCR amplification and sequence comparison of 26S r RNA,ITS r DNA,and 18S r RNA regions revealed that the starting strain belonged to the genus Nakazawaea and was named N.ishiwadae GDMCC 60786.Based on genome sequencing analysis and KEGG functional annotation,pyruvate metabolism and fatty acid degradation are important metabolic pathways involved in acetyl-Co A synthesis.In order to enhance the starting strain’s ability to produce ethyl acetate,mutant strains were screened through physical and chemical mutagenesis.Mutants with high esterase activity were screened using transparent rings of tributyl glycerol.Then,secondary screening was performed for shake flask fermentation.Finally,ethanol and acetic acid were added to determine tolerance and ester production capacity.After mutagenic treatment of the starting strain through esterase activity,shake flask fermentation and screening,and substrate tolerance evaluation,a mutant strain N5 was finally obtained with a high ester yield and good genetic stability.After five consecutive passages,the average value of ethyl acetate was 764.52 mg/L.At the same time,after adding ethanol,it reached 1426.81 mg/L;acetic acid inhibited cell growth and lost its ability to produce esters,indicating that the strain was highly resistant to ethanol.At the same time,the activities of intracellular esterase,acetyl-Co A,and alcohol acyltransferase of the mutant strain were higher than those of the original strain,consistent with the high yield of ethyl acetate.Finally,through hemolysis and drug resistance analysis,it has good characteristics for in vitro safety.Transcriptomic analysis was performed on the starting strain and mutant strain,and the differentially expressed genes obtained under FC≥2 and FC≤0.5 were combined with functional annotation and enrichment of the GO and KEGG databases.The up-regulation of key genes（PDC,ALDH,ACS1/2,PDHA,PDHB,and ACAA1）in pyruvate metabolism and fatty acid degradation enables efficient accumulation of acetyl-Co A.The key genes of the cell membrane component ergosterol（ERG6,ERG2,ERG3）were down-regulated,and all of them could accumulate acetyl-Co A,the precursor of ethyl acetate.Based on the detection of esterase activity mentioned above,it has been clarified that the use of pyruvate decarboxylase,acetaldehyde dehydrogenase,acetyl-Co A synthase,and pyruvate dehydrogenase in pyruvate metabolism promotes the synthesis of acetyl-Co A.At the same time,the up-regulated gene ACAA1 of fatty acid degradation and the down-regulated genes ERG6,ERG2 and ERG3 of ergosterol were analysed,which can accumulate acetyl-Co A.Based on the above detection of esterase activity,it is preliminarily determined that pyruvate decarboxylase,acetaldehyde dehydrogenase,acetyl-Co A synthetase,and pyruvate dehydrogenase positively regulate the synthesis of ethyl acetate in pyruvate metabolism.The accumulation of acetyl-Co A through three pathways is the response mechanism for the high yield of ethyl acetate in this strain.In addition,key genes for flavour substances such as higher alcohols,amino acids,and organic acids were analysed as FAS1/2,ACSL,BDH,and ARO8.In order to analyse the changes in the production of ethyl acetate by the starting strain and the mutant strain under different fermentation conditions and different medium components.The optimal fermentation conditions were 1×10⁸ CFU/m L,p H=4,20%dissolved oxygen with the starting strain and mutagenic strain.But the temperature was inconsistent,with the starting strain at 20℃and the mutagenic strain at 30℃.Based on the fermentation conditions of wine,response surface optimization experiments were conducted to obtain the optimal fermentation conditions for producing ethyl acetate for the starting strain and mutant strain.From the culture medium components,glucose and fructose were utilised to form esters.Ethanol can be used to enhance ethyl acetate,but adding ethanol has no effect on ester production during the cell stabilisation phase.The ester production effects of organic nitrogen sources were better than those of inorganic nitrogen sources,among which yeast extract had the highest ester production and NH₄H₂PO₄ had the highest ester production from inorganic nitrogen sources.The metal ions that inhibit ester generation were Zn²⁺,Fe²⁺,Fe³⁺,Cu²⁺,and Mn²⁺.Among them,iron ion and copper ion are components of complex I and complex IV in electron transport chain,respectively.Iron and copper can increase the efficiency of respiratory chain,improve the flux of Acetyl-Co A into TCA cycle,further restrict the flux of Acetyl-Co A into the synthesis of ethyl acetate,indicating that the precursor of producing ethyl acetate is Acetyl-Co A with the strain.Amino acids and vitamins had no significant effect on improving ester production.Starting strains and mutagenic strains were mixed with Saccharomyces cerevisiae to brew wine,and co-inoculation and sequential inoculation were used to analyse the complexity of wine style.The physical and chemical indicators had reached national standards in all samples.The detection and analysis of volatile substances,organic acids,and amino acids showed that the highest total amount was organic acids,followed by amino acids,and the lowest was volatile substances.But volatile substances had the greatest contribution to aroma,mainly divided into esters and higher alcohols.The analysis of 19aroma substances with OAV>0.1 showed that sequential inoculation could significantly enhance the content of acetic esters such as ethyl acetate,isobutyl acetate,and phenylethyl acetate.At the same time,co-inoculation could significantly enhance the content of medium long chain esters,including ethyl phenylacetate,ethyl hexanoate,ethyl caproate,and ethyl lauric acid.Co-inoculation of SC+NI and SC+NI5 was 82.61 mg/L and 98.95 mg/L with ethyl acetate,respectively;sequential inoculation of NI+SC and NI5+SC was 207.13 mg/L and 441.14 mg/L,respectively.The results showed that three days before fermentation,the mutant strain could use glucose and fructose in grape to synthesize ethyl acetate more efficiently than the original strain.Co-inoculation and sequential inoculation enhances lactic acid,acetic acid,pyruvic acid,and tartaric acid.Sequential inoculation increased the content of arginine and alanine,but amino acids were not significantly increased by co-inoculation.Through sensory evaluation and analysis,mixed fermentation can enhance the fruit aroma,sweet aroma,harmonious taste and high flavour complexity of wine.