Optimization Of Production Process Of Pyruvic Acid And Its Derivatives By Microbial Methods

Pyruvic acid is an important organic acid and industrial raw material.In recent years,the production of pyruvic acid through microbial fermentation has attracted much attention due to its advantages such as high product purity and environmental friendliness.However,there are problems in the process of microbial production of pyruvic acid,such as the inhibition of metabolic products,the high production cost of raw materials,and limited research on high-value conversion applications.Therefore,how to eliminate or alleviate the acid stress inhibition caused by the accumulation of pyruvic acid and improve the economy and application value of microbial production of pyruvic acid is the key to solving the problem.In this study,Candida glabrata capable of accumulating pyruvic acid extracellularly was used as the starting strain.The strain was screened using atmospheric and room temperature plasma（ARTP）mutation technology,a fully automated high-throughput microbial microdroplet culture（MMC）system,and adaptive evolution technology.The fermentation performance of the selected strains was verified.Nitrogen source optimization and fermentation process optimization were carried out to reduce the production cost of pyruvic acid.Finally,high-value L-tyrosine and levodopa were derived from pyruvic acid using whole-cell catalysis to explore the application of high-value conversion of pyruvic acid.The main research results are as follows:（1）The combination of ARTP mutation and MMC adaptive evolution was used to screen acid-resistant and high pyruvic acid-producing mutant strains.Firstly,an acid-tolerant strain Cg-B2 with high pyruvic acid production was successfully screened through ARTP mutagenesis and MMC adaptive acid-tolerance evolution.Under normal p H fermentation conditions and p H 4.5 acid fermentation conditions,the cell concentration of strain Cg-B2was 11%and 21%higher than that of the original strain,and the acetonic acid production was9%and 19%higher than that of the original strain,respectively.Subsequently,fermentation validation and process optimization were carried out in a 5 L fermenter,and the pyruvic acid production was 61.69 g?L^-1.（2）Optimization of nitrogen sources reduces the cost of producing pyruvic acid by C.glabrata fermentation.Using spent yeast cells to prepare recyclable of spent yeast cells dry powder instead of soybean peptone as a seed nitrogen source to produce pyruvic acid.The effects of different nitrogen sources,different drying process methods,and different concentrations of spent yeast cells dry powder on pyruvic acid fermentation in seed culture medium were compared at the shaker.The vitamin combination and Mg²⁺addition amount were optimized at the 5 L fermenter.The production of pyruvic acid was scaled up by batch fermentation in a 15 L and 50 L fermenter,with titers of 63.74 g?L^-1 and 65.10 g?L^-1,respectively.It was estimated that using spent yeast cell dry powder as the seed nitrogen source for pyruvic acid fermentation could reduce the production cost by 2688 yuan per ton of pyruvic acid.（3）Whole-cell catalytic synthesis of L-tyrosine and levodopa from pyruvate,expanding the application value of pyruvic acid.Firstly,the expression conditions of tyrosine phenol lyase（TPL）and key factors for whole-cell catalytic reactions were optimized at the shakers.Then,using pyruvic acid fermentation broth and phenol as substrates,the effects of different feeding methods on the catalytic synthesis of L-tyrosine were compared,and an optimal feeding strategy was determined:at 1,2,and 4 hours of the reaction,pyruvic acid and phenol were added until the final concentrations were 12 g?L^-1 and 6 g?L^-1,respectively,three times.Under this condition,the titer of L-tyrosine reached 50.13 g?L^-1,an increase of 83%compared to no supplementation.Finally,optimized the process of whole-cell catalytic synthesis of levodopa from pyruvic acid.Based on the bottom flow addition strategy,the pyruvic acid was fed with 12 g?L^-1 for the entire reaction process,and catechol was fed with 9 g?L^-1 for 0.5～2,2～4,and 4～5 hours,respectively.Under these conditions,the highest titer of levodopa reached 55.37 g?L^-1,with a conversion rate of 76%.