Study On The All Fermentable Sugars Metabolism Capability And Inhibitor Tolerance Of Pediococcus Acidilactici

Hexose derived from cellulose in the composition of lignocellulose is mainly glucose.Hexose derived from hemicellulose is galactose and mannose,and pentose is mainly xylose and arabinose.Among them,xylose and arabinose account for about 30-40%of total carbohydrates,and galactose and mannose account for about 1%of total carbohydrates in lignocellulose.One of the key issues in current biorefinery fermentation technology is the efficient utilization of sugars.The problem of many fermentation strains is glucose inhibition.When glucose is present,only glucose can be used preferentially,and other sugars are used after it is consumed.As a result,the utilization of non-glucose by fermentation strains is low.In recent research work on lactic acid fermentation of lignocellulose,no effective solution has been explored to alleviate the inhibitory effect of glucose.On the other hand,more inhibitors such as furans,phenols,and weak acids are generated during the pretreatment process of biorefinery.Furan and weak acid inhibitors can be quickly removed through detoxification of biological,but it is difficult to effectively remove phenolic inhibitors.If the detoxification time is prolonged,glucose and xylose will be lost,which will affect the fermentation index in the later stage.In addition,the metal ions enriched during the pretreatment process are also a kind of major inhibitors,among which sodium ions are typical metal ions enriched by pH adjustment of the biorefining technology.It will seriously inhibit the fermentation performance of the strain in a certain concentration range.This article explores the three key issues mentioned above.We selected P.acidilactici ZY271 and P.acidilactici ZY15,which have constructed the xylose metabolism pathway in the early stage of the laboratory.Investigating the utilization ability of all fermentable sugars of two constructing strains in different systems.And the analysis of the fermentation phenomenon was conducted from the transcription levels of sugar metabolism and sugar transport.Secondly,enhancing inhibitor tolerance of phenolic inhibitors and sodium ions for P.acidilactici was achieved from genetic engineering.In the first part,we evaluated the fermentation of P.acidilactici ZY271 and P.acidilactici ZY15 in different systems.In order to improve the utilization of sugar and the rate of metabolizing for the constructed strain,we carried out the domestication strategy of mixed sugar.It could be obtained from the fermentation phenomenon that the two constructed strains can realize the utilization of all fermentable sugars without glucose inhibition.And the construction of the xylose metabolic pathway increased the strain’s metabolic rate of mannose.Aiming at these fermentation phenomena,the transcription level analysis of related genes was conducted from two aspects of sugar metabolism and sugar transporter.It was found that the gene cluster of manXYZ in EIIMan played a key role in the efficient utilization of sugars.Further exploration of related genes would provide gene sources for efficient utilization of sugars in other strains.In the second part,enhancing the inhibitor tolerance for P.acidilactici ZY15 of phenolic inhibitors from genetic engineering.The tolerance of P.acidilactici ZY15 on 4-hydroxybenzaldehyde,vanillin,syringaldehyde and BQ were assayed,respectively.Vanillin showed the most inhibition on P.acidilactici ZY15.And in the previous work,the laboratory has discovered that BQ has a strong inhibitory effect on P.acidilactici ZY15,and the strain is more sensitive to quinone compounds.The selected effective gene CGS9114 RS09725 of phenolic tolerance and effective gene ZM01116 of benzoquinone degradation were integrated into the P.acidilactici ZY15 genome.The resulting strain P.acidilactici ZY15-AackA2::CGS9114_RS09725 performed better fermentation than P.acidilactici ZY15 under the stress of vanillin.It indicated that CGS9114_RS09725 integration strengthened the vanillin tolerance of P.acidilactici ZY15.P.acidilactici ZY15-ΔackA2::ZM01116 had a pathway of BQ degradation,and the final fermentation performance was also improved.High solids loading SSCF using corn stover was conducted.Compared with the original strain of P.acidilactici ZY15,the fermentation performance of the two integrated strains was better than the original strain.In the third part,enhancing the inhibitor tolerance for P.acidilactici ZY271 of sodium ions from genetic engineering.The tolerance of P.acidilactici ZY271 on sodium ions was assayed and we found that a certain concentration(about 25 g/L)of sodium ions could completely inhibit the growth activity of P.acidilactici ZY271.The gene of Na+/H+antiporter_RS02775 was overexpressed in P.acidilactici ZY271.The recombinant strain of P.acidilactici ZY271(pZY36e-RS02775)performed better fermentation than P.acidilactici ZY271 under the stress of sodium ions.High solids loading SSCF using wheat straw was conducted and the pH neutralizer was NaOH solution of 13.5 M.Compared with the original strain of P.acidilactici ZY271,the fermentation index of sodium lactate was improved.Conclusively,this study investigated the utilization capacity of all fermentable sugars for P.acidilactici which have established a xylose metabolism pathway,and conducted a preliminary molecular mechanism analysis on the fermentation phenomenon of all sugars utilization.Enhancing inhibitor tolerance of phenolic inhibitors and sodium ions for P.acidilactici by genetic engineering.This study provided genes with reference value for the efficient utilization of fermentable sugars,as well as effective strains for the fermentation of high-quality lactic acid and sodium lactate from lignocellulose.