Process Research For L-malic Acid Production By Engineering Corynebacterium Glutamicum M5

L-malic acid is an important natural organic acid,playing an important role in the TCA cycle.It is widely used in food,medicine and chemical industry,with a huge market prospect.Microbial production of L-malic acid by fermentation has many advantages and good prospects,therefore it becomes one of the hotesttopics in recent years.There are a large number of by-products besides L-malic acid among the fermentation products by wild-type strains,but L-malic acid producing strain constructed by genetic engineering method can eliminate the influence of by-products and lead the distributed carbon to flow to the target product as much as possible.Because of the well-known genetic background and simple operation,C.glutamicum constructed by genetic engineering method as a L-malic acid producing strain is feasible in theory.Firstly,a simple rough fermentation detection technology and intuitive liquid chromatography detection technology was established in this paper.The liquid chromatography conditions:United States Agilent liquid chromatograph,BDS HYPERSIL C₁₈ column?4.6 mm×200 mm?with a mobile phase containing acetonitrile and 20 mmol/L KH₂PO₄ buffer?pH 2.8??v:v=1:99?.The column temperature was 29?,the flow rate was 0.5 mL/min,the detection wavelength was 210 nm,and the sample volume was 20?L.This method is rapid,accurate and reproducible.The C.glutamicum ATCC13032 was taken as the original strain.Based on the known metabolic flux analysis,the strategy of gene knock-out and SacB genetic screening was designed.The gene pqo?pdh?lldh?malE?mqo was deleted from chromosome,and a recombinanted strain named M5?C.glutamicum ATCC13032 M5?was obtained finally,by the combination of gene knock operation and molecular verification.Based on the molecular verification,the extracellular accumulation of L-malic acid was found in the fermented broth by HPLC.The result proved that the engineering strain M5 designed by rational strategy was reasonable.Secondly,in order to improve the yield of L-malic acid,the different nutritional conditions were studied in this paper,such as:carbon source,nitrogen source,acetate,sodium bicarbonate,etc.The optimal medium compoents were ultimately determined as follows?g/L?:glucose 50?sodium acetate 5??NH₄?₂SO₄ 5?KH₂PO₄ 1.5?MgSO₄·7H₂O0.5?CaCl₂ 0.2,pH was adjusted to 7.0 by NaHCO₃;Added vitamin:B₂,B₅,B₆,B₁₂,NA,Fa,Biotin;the yield of L-malic acid could reach 23.5 g/L in shake flask culture within the72 h.The sugar acid conversion was 66.4%.The optimal fermentation conditions for engineering strain M5 were ultimately determined as follows:2.5 per cent of inoculation,fermentation temperature 30?,the initial pH 7.0,aerobic fermentation for 36 h was converted into limited oxygen fermentation for 72 h with stable pH at 7.5.At the end of the fermentation,the final production of L-malic acid was 16.2 g in the 505 mL fermentation broth by engineering strain M5.The L-malic acid quality yield was 69.8%,and the molar conversion rate of L-malic acid was 94.5%.Based on the continuous multi-batch fermentation experiments by 2 L fermentor,the average L-malic acid yield and the average conversion rate of L-malic acid were 32.16 g/L and 69.38%.Finally,the L-malic acid crystallization process has preliminarily explored.The best crystallization process for high purity and yield of L-malic acid were determined as follows:decolored 2 h with 4%actived carbon,decompression filtration,concentrated at70?,gradient freezing crystallization.The L-malic acid purity could reach more than83%,and the L-malic acid yield could reach 76.7%with the above process.