Fermentation Process Optimization And Metabolic Mechanism Of β-Phenylethanol Production By Saccharomyces Cerevisiae Based On Parallel Bioreactors

β-Phenylethanol(2-PE)is widely used in food,medicine,cosmetics and other fields because of its soft rose smell and antibacterial effect.Saccharomyces cerevisiae possesses a natural metabolic pathway to synthesize 2-PE but the yield is low.In order to increase the 2-PE production in S.cerevisiae and analyze the metabolic mechanism of high yield,this study first established a parallel culture platform based on a self-made 500 mL quadruple bioreactors system to increase the culture throughput.Then the fully synthetic medium fermentation was optimized,and the production of 2-PE was greatly improved through fed-batch mode.Finally,from the perspective of the combination of macro and micro metabolism,the metabolic mechanism of high-yielding 2-PE was analyzed.It provides a reference for the subsequent metabolic engineering strategy of 2-PE production in S.cerevisiae and the optimization of the fermentation process.The bioreactor used in this research is a 500 mL quadruple parallel bioreactors system.Firstly,cold model experiments were conducted to investigate the parallelism of the bioreactor,and secondly,a common culture of S.cerevisiae was conducted to study whether the bioreactor could meet the needs of culture.Based on the culture results and the simulation of computational fluid dynamics(CFD),the 500 mL parallel bioreactor was improved and the culture conditions were optimized to enhance the oxygen supply capacity.The KLa value in the optimized bioreactor during the cultivation of S.cerevisiae was improved from 243.36 h-1 to 2151.13 h-1,which could not only realize the parallel culture of S.cerevisiae,but also characterize the macro-metabolic parameters well.The fully synthetic medium with clear components is the basis for the subsequent analysis of the metabolic mechanism.Herein,S.cerevisiae S288C,W303,and W101 were used as the culture objects to optimize concentrations of inorganic organic nitrogen sources in the fully synthetic medium.The results demonstrated that the optimal nitrogen source concentration is 5.0 g/L L-phenylalanine(L-Phe)and 2.5 g/L ammonium sulfate.The cell growth and metabolic characteristics of the three strains further showed that the W303 is a dominant 2-PE producer with a shorter fermentation time and higher substrate utilization yield.Thus,the W303 strain was selected for subsequent glucose concentration optimization,and consequently the 2-PE concentration was 2.83 g/L with the optimized synthetic medium,which was 1.75 times that of the initial medium.Moreover,the 2-PE concentration further reached to 4.23 g/L through the feed strategy in fed-batch fermentation.Finally,the 2-PE high-yielding strain S.cerevisiae W303 was used as the research object,and the model strain S.cerevisiae S288C was used as the control.The differences of macro-and micro-metabolism in two strains were compared.In terms of macro-characterization parameters,the fermentation time of W303 was about 12 hours shorter than that of S288C,and the oxygen uptake rate(OUR),which characterizes the intensity of respiratory metabolism,was 1.7 times that of S288C.In the microscopic aspect,the chemostat culture at different specific growth rates was carried out,and the two strains in different culture conditions were subjected to quantitative metabonomic analysis,metabolic flux calculation and non-targeted metabolome analysis.Metabonomics results showed that the intermediates pools of central carbon metabolism such as the pentose phosphate pathway(PPP)and the tricarboxylic acid cycle pathway(TCA)of the W303 were significantly higher than those of the S288C.Metabolic flux results indicated that the difference in the flux of pyruvate(PYR)node to TCAis the key factor leading to the difference in 2-PE product concentration of the two strains.The TCA flux of the W303 was 3.2 times that of the S288C,which could provide sufficient supply of precursors,cofactors and energy for cell maintenance and 2-PE product synthesis.In addition,the results of non-targeted analysis showed that a variety of metabolites related to 2-PE toxicity tolerance in the W303 were significantly up-regulated.