Breeding Of High-yielding L-Proline Strains And Optimization Of Fermentation Conditions

L-Proline belongs to heterocyclic amino acid and has certain physiological activities.It is widely used in industry,agriculture,medicine food and other fields.Since the mid-1990s,the production process of L-Proline fermentation has not changed greatly.The further improvement and stabilization of the fermentation production level and the reduction of production costs have become the main means to improve the market competitiveness of L-Proline.In this study,a laboratory-preserved strain,Corynebacterium glutamicum P1,was used as the original strain.The mutant strain SG-56 was obtained by stepwise mutagenesis of diethyl sulfate（DES）and atmospheric and room temperature plasma（ARTP）.Then,the fermentation medium and conditions of the mutant were optimized at the shake flask level by single factor experiment,response surface design and other methods.Then,the shake fermentation conditions of L-Proline were determined and some parameters in the fed-batch fermentation were optimized in 5 L fermenter.Finally,the intracellular metabolic flow of the original strain P1 and mutant strain SG-56 were compared and analyzed.The main findings are as follows:（1）The ability of C.glutamicum P1 to produce L-Proline was enhanced by stepwise mutagenesis.In this study,C.glutamicum P1 was used as the original strain,which was mutated by DES and ARTP,and then the strain was screened by using sulfaguanidine（SG）resistant plate and succinic acid plate as the sole carbon source.Finally,a mutant SG-56 with high L-Proline yield was screened.Under the condition of not optimizing the fermentation conditions,it could accumulate 48.71 g·L^-1 L-Proline,which was 19.4%higher than the original strain.（2）The composition of fermentation medium and culture conditions for the synthesis of L-Proline by C.glutamicum SG-56 were optimized.In this study,the concentration gradient experiments of carbon source,nitrogen source,inorganic salt and biotin in the fermentation medium were carried out,and the components of the fermentation medium were further optimized by response surface design.Then,based on the optimization of culture medium,the effects of fermentation conditions such as p H,inoculation amount,liquid volume and shake flask type on the acid production ability of the strain were investigated.The optimal medium composition are as follows:glucose 161.6 g·L^-1,peptone 7.92 g·L^-1,corn steep liquor 4.0g·L^-1,（NH₄）₂SO₄ 50.4 g·L^-1,KH₂PO₄ 0.94 g·L^-1,Mg SO₄·7H₂O 0.5 g·L^-1,Fe SO₄·7H₂O 0.01g·L^-1,Mn SO₄·4H₂O 0.01 g·L^-1,biotin 2.0×10^-4 g·L^-1,thiamine 1.0×10^-4 g·L^-1,Ca CO₃ 40.0g·L^-1.The conditions of optimal culture are as follows:initial p H of 7.5,baffle shake flask,4%inoculation volume and filling volume of 25 m L/250 m L.Under the above optimum process,the concentration of L-Proline reached 55.18±0.74 g·L^-1 for 48 h,which was 14.8%higher than before optimization.Finally,the effect of different surfactants on the fermentation was explored.The results showed that adding 2 g·L^-1 of Tween 80 at 15 h could improve the L-Proline production capacity of the strain.（3）The fed-batch fermentation parameters of C.glutamicum SG-56 were optimized at 5L fermenter.In this study,parameters such as corn pulp dry powder,initial glucose,residual glucose and dissolved oxygen level were optimized.The optimal medium composition as follows:corn pulp dry powder 2 g·L^-1,initial sugar 60 g·L^-1,residual sugar 10 g·L^-1 and dissolved oxygen 35%,which is more favorable for the accumulation of L-Proline.Under these conditions,the concentration of L-Proline yield reached 75.84 g·L^-1,and the conversion rate was 0.318 g·g^-1,which were 15.9%and 34.1%higher than that before optimization,respectively.（4）Metabolic flow analysis of L-Proline biosynthesis.Based on the metabolic flow analysis theory and the metabolic pathway of L-Proline biosynthesis,the metabolic network model of L-Proline was constructed,and the metabolic flow of original strain and mutant strain were compared and analyzed.The results showed that the flow distribution at the pyruvate node changed greatly.The metabolic flow from pyruvate to L-Proline increased from 32.96 mmol·L^-1·h^-1 of the starting strain to 43.21 mmol·L^-1·h^-1 of the mutant strain,The metabolic flux from pyruvate to"miscellaneous acid"and"by-product"decreased from 25.14mmol·L^-1·h^-1 and 41.90 mmol·L^-1·h^-1 of the starting strain to 17.26 mmol·L^-1·h^-1 and 39.53mmol·L^-1·h^-1 of the mutant strain,respectively.