Study On The Biological Reduction Strategy Of P-Cresol Produced By Clostridium Fermenticellae

P-cresol（PC）is the main compound producing mud odor in strong flavor Baijiu.At present,the methods of p-cresol reduction mainly focus on biodegradation,however,few research has been done on biodegradation of p-cresol produced in Baijiu brewing.Previously,Clostridium fermenticellae JNCS001 that can produce p-cresol was screened from high-quality100-year pit mud of Luzhou-type liquor in our laboratory.In this study,C.fermenticellae JNCS001 was taken as an example,and food safety level Sacchromyces cerevisiae was chose as the chassis to explore the reduction strategy of p-cresol.This study firstly evaluated the ability of C.fermenticellae JNCS001 to synthesize p-cresol and analyzed the mode of using the precursor tyrosine（Tyr）to produce p-cresol.On this basis,the strategies that include competitive utilization of precursor Tyr through the heterologous expression of TAL and direct degradation of p-cresol through the heterologous expression of Cre IH were explored,respectively.Through the exploration of the above strategies,this study achieved effective degradation of p-cresol,which provides theoretical support and strategical reference for reduction of p-cresol,and is of great significance for solving the off-odor problem caused by p-cresol.The main research content and results of this article are as follows:（1）This study firstly evaluated the ability of C.fermenticellae JNCS001 to synthesize p-cresol,and the results showed that it can synthesize 7.5 mg·L^-1 p-cresol.Then,the mode of using the precursor Tyr by C.fermenticellae JNCS001 was analyzed,the concentration of p-cresol was increased by adding Tyr externally,which proved that the precursor of p-cresol produced by C.fermenticellae JNCS001 was Tyr.Meanwhile,C.fermenticellae JNCS001could utilize both internal and external Tyr to synthesize p-cresol.In order to further verify that Tyr is the precursor for the synthesis of p-cresol by C.fermenticellae JNCS001,this study successfully identified the hpd gene in the synthetic pathway of p-cresol,then RT-q PCR was used to analyze the expression level of hpd during the fermentation process.When adding 0.25,0.5 and 1.0 mmol·L^-1 Tyr,it was found that the expression level of hpd was 1.6,2,and 1.4 times higher than that without adding Tyr,respectively.The results indicated that external addition of Tyr can increase the expression level of hpd which can lead to overproduction of p-cresol.Based on the above research results,this study proposes to explore the methods of p-cresol biodegradation from two aspects,which are the competitive utilization precursor Tyr and the direct degradation of p-cresol.（2）In order to explore the strategy of competing with the precursor Tyr to reduce p-cresol,this study used Saccharomyces cerevisiae CEN.PK 2-1C as the host and introduced the TAL enzyme from Rhodotorula glutinis.The recombinant S.cerevisiae 2-1C-p Y15-TAL strain was constructed,and evaluation its ability to utilize external Tyr was performed by adding 0.25,0.5,and 1.0 mmol·L^-1 Tyr.Subsequently,the recombinant S.cerevisiae 2-1C-p Y15-TAL was co-cultured with C.fermenticellae JNCS001 anaerobically adding 0.25,0.5,and 1.0 mmol·L^-1 Tyr.The production of p-cresol by co-culture of C.fermenticellae JNCS001 and 2-1C-p Y15-TAL was reduced by 12%,21%,and 9%,respectively,compared to that produced by mono-culture of C.fermenticellae JNCS001.The experimental results showed that 2-1C-p Y15-TAL could utilize Tyr in the culture medium to reduce the precursor of p-cresol synthesized by C.fermenticellae JNCS001 decreasing the content of p-cresol to a certain extent.（3）To explore the strategy of directly degrading p-cresol produced by C.fermenticellae JNCS001,S.cerevisiae CEN.PK 2-1C was used as the host for the first time,and the key enzyme 4-methylphenyl phosphate synthase Cre IH derived from Corynebacterium glutamicum ATCC 13032 was heterologously expressed for degrading p-cresol.When adding 10 mg·L^-1 of p-cresol to RCM medium,the recombinant S.cerevisiae was able to degrade up to 44%of p-cresol.On this basis,by increasing the copy numbers of the cre IH gene,the ability of recombinant S.cerevisiae to degrade p-cresol was gradually improved.When the copy number of the cre IH gene was increased to three,recombinant S.cerevisiae had the strongest ability to degrade p-cresol.After 36 hours of aerobic cultivation,87%of p-cresol can be degraded,while65%of p-cresol can be degraded after 48 hours of anaerobic cultivation.Finally,50%of p-cresol can be degraded when co-cultured with C.fermenticellae JNCS001,which is more effective than the former strategy of competing precursor Tyr to reduce p-cresol.