Modification Of L-threonine-producing Strain And Optimization Of Fermentation Process

L-threonine is one of the eight essential amino acids for human health.It has unique physiological function and has been widely used in many industries,including food,pharmaceutical,cosmetics,feed and so on.In this paper,the effects of pep C gene over-expression on L-threonine fermentation was tested.Then,the fermentation process of L-threonine was optimized using Escherichia coli JLTHR p JL225-9,and the effects of betaine and B-vitamin on the fermentation of L-threonine were investigated.And the metabolic flux analysis of the addition of betaine during L-threonine fermentation was carried out.Finally,the influence of cell cycle fermentation on L-threonine fermentation was studied.The fermentation experiments in this paper were carried out in a 5L fermentor.The main research contents and results are as follows:（1）The plasmid p JL225-9 containing pep C was transformed into L-threonine producing strain E.coli JLTHR.The L-threonine production of E.coli JLTHR p JL225-9 was 111.2g/L,which was 2.8% higher than that of E.coli JLTHR.The metabolic flow model of L-threonine fermentation was constructed and the metabolic flux distribution of E.coli JLTHR p JL225-9 and E.coli JLTHR were compared.The results showed that GLC6 P,PEP and α-KG were the key nodes.By metabolic flow analysis,at the GLC6 P node,the metabolic flux of HMP pathway was increased by 22%,the metabolic flux of EMP was reduced by 7.5%.And the metabolic flux of E.coli JLTHR p JL225-9 flowed into the replenishment pathway of oxalacetic acid was increased by 4% at the PEP node.（）（2）The effects of betaine on the fermentation of L-threonine were studied by adding three different types of anhydrous betaine,including Sigma anhydrous betaine,the domestic anhydrous betaine with calcium stearate and the domestic anhydrous betaine without calcium stearate.The results showed that domestic anhydrous betaine without calcium stearateon was the most significant.It enhanced the production of L-threonine to 119.3g/L,which was increased by 7.3% compared with the control.In addition,the effects of betaine salt type and concentration on the fermentation of L-threonine were studied by adding different concentrations of betaine hydrochloride or betaine phosphate.It was found that the production of L-threonine reached 127.3 g/L fed with 2 g/L of betaine hydrochloride,which was increased by 14.5% compared with the control.And the highest OD_600nm of 63.4 was obtained fed with 2g/L of betaine phosphate,which was increased by 15.3% compared with the control.Betaine contained three active methyl groups,choline chloride and methionine also contained active methyl groups,which could promote the synthesis of L-threonine.The results showed that L-threonine production were 119.3g/L and 118.4g/L by addition of choline chloride and methionine,respectively,which was similar to that of adding anhydrous betaine.（3）The effect of betaine on the metabolic flux distribution of L-threonine was investigated.Based on the metabolic flux analysis theory,the metabolic network of L-threonine strain was constructed and the MATLAB software linear program was used to obtain the metabolic flux distribution of L-threonine producing strains in the middle and late fermentation stage.The metabolic flux distribution of carbon in EMP,HMP and TCA cycles and the key nodes of GLC6 P,PEP and α-KG were obtained.The results showed that at the node GLC6 P,the metabolic flux of carbon to the HMP pathway with betaine addition was 57.3% higher than that of without betaine addition.It was found that at the node PEP,the metabolic flux to oxaloacetic acid（OAA）was increased by 10.1% and the metabolic flux to TCA pathway was decreased by 6.9% when betaine was added in the fermented broth.（4）The effects of B-vitamins,including calcium pantothenate（VB₅）,cobalamin(VB₁₂),choline chloride（VB₄）and nicotinamide（VB₃）,on L-threonine fermentation by fed-batch culture of E.coli JLTHR p JL225-9 in a 5L fermentor were investigated.The results showed that VB₅ and VB₁₂ had no obvious effects on L-threonine fermentation.The production of L-threonine reached 133.4 g/L,which was increased by 7.8% when supplemented with 1 g/L VB₄.The production of L-threonine reached 130.6 g/L,which was increased by 5.6% when supplemented with 10mg/L VB₃.When a combination of 1 g/L VB₄ and 10 mg/L VB₃ together with 2 g/L betaine hydrochloride was added to the glucose solution,138.4 g/L L-threonine was accumulated,which was increased by 11.9%.（5）The results of cell recycle fermentation showed that the production of centrifugal separation fermentation was 141.4 g/L,which was higher than that of ceramic membrane separation fermentation.By analyzing the cell cycle and cycle fermentation strategies,the highest L-threonine production of 148.4 g/L was obtained when the cycling time was 16 h,the cycle strategy was V（supernatant）: V（concentrated solution）= 1: 2,and the L-threonine production was 13.1% higher than that of without cell recycle.