Increasing The Fermentation Level Of L-lysine Via Enhancing Glucose Metabolism Pathways

Phosphoenolpyruvate-dependent glucose phosphorylation via the phosphoenolpyruvatedependent glucose phosphotransferasesystem(PTS^Glc)is the major pathway of glucose uptake in Corynebacterium glutamicum（C.glutamicum）.However,the phosphoenolpyruvate（PEP）is used as phosphate donor during glucose phosphorylation,which will lead to the imbalance of carbon source distribution,thus accumulating a large number of by-products（e.g.,acetic acid,pyruvic acid,etc.）and affectting the growth of bacteria.It is well documented that C.glutamicum can also uptake glucose by PTS^Glc-independent glucose transport route which consist of the inositol permease（IolT1/IolT2）and glucokinase（PPGK/GLK）.In this study,C.glutamicum ZL-8 is an L-lysine producing strain via classical mutagenesis,which shows the low rate of glucose uptake and phosphorylation.Here,we focused on the metabolic engineering of C.glutamicum ZL-8 to improve the consumption of glucose and the production of L-lysine.Moreover,we optimized the fermentation medium and conditions in shake-flask fermentation.And different agitation speeds and the concentration of glucose and nitrogen of fermentation medium in batch fermentation were optimized.The main results are as follows:（1）In order to improve the consumption rate of glucose and the production of L-lysine in the original strain C.glutamicum ZL-8,the overexpressions of PTS^Glc system genes in C.glutamicum ZL-8 were studied.The results showed that the consumption rate of glucose（increased by 16.7%、27.3%、27.3%、33.2% and 33.2%,respectively）、the L-lysine yield（increased by 13.6%、16.9%、16.4%、17.5% and 18.8%,respectively）and the activity of PTS^Glc system（increased by 38.9%、69.8%、62.7%、102.9% and 107.9%,respectively）of C.glutamicum ZL-8/pXMJ^-19-ptsI 、 C.glutamicum ZL-8/pXMJ^-19-ptsG 、 C.glutamicum ZL-8/pDXW-8-ptsG、C.glutamicum ZL-8/pXMJ^-19-ptsIG、C.glutamicum ZL-8/pXMJ^-19-ptsIHG were increased compared with C.glutamicum ZL-8.However,the production of by-products（e.g.,acetic acid,lactic acid and valine,etc.）was also increased.（2）In order to improve the consumption rate of glucose and the production of L-lysine in the original strain C.glutamicum ZL-8 and to decrease the production of by-product in fermentation liquid,the overexpressions of gene iolT1 encoding IolT1 and gene ppgK encoding glucokinase（PPGK）in C.glutamicum ZL-8 were studied.The results showed that the consumption rate、the L-lysine yield and the glucokinase activity of C.glutamicum ZL-8/pECXK-99E-ppgK-iolT1（increased by 21.6%、25.6% and 768%,respectively）were increased compared with C.glutamicum ZL-8.In addition,the production of by-product（e.g.,acetic acid,lactic acid and valine,etc.）was decreased in some extent.Therefore,it is concluded that the recombinant strain C.glutamicum ZL-8/pECXK-99E-ppgK-iolT1 has the advantages in the cell growth and the L-lysine biosynthesis.（3）Based on the results of single factor experiments in shake-flask,corn steep liquor,（NH₄）2SO₄ and MgSO₄·7H₂O were selected as independent variables to optimize fermentation medium from 8 factors by Plackett-Burman design.The optimal value of medium was determined by Box-Behnkne design,which is(g·L^-1): glucose 120,（NH₄）2SO₄ 47.85,corn steep liquor 17.32,Molasses 12,KH₂PO₄ 1,MgSO₄·7H₂O 1.57,betaine 0.05,FeSO₄·7H₂O 2×10-2,MnSO₄ 2×10-2,thiamine 4.5×10-4,biotin 8.5×10-4,nicotinamide 8×10-3.Under the above-mentioned conditions,L-lysine production reached to 58.73±0.67 g·L^-1,which is 9.34% higher than that before optimization.In addition,the fermentation conditions were optimize by single experiment,and the optimal fermentation conditions were initial pH 7.0,10% inoculum size seed at 24 h,liquid medium volume 25 m L/500 m L,CaCO3 concentration 35 g·L^-1,0.6 mmol·L^-1 inducer was at 12 h,the temperature was at 30℃ in fermentation(0^-16 h)and increased to 33℃ after 16 h.Under the above-mentioned conditions,L-lysine production reached to 62.87±1.02 g·L^-1,which is 7.04% higher than that before optimization.（4）The agitation speed and feeding（e.g.,glucose and（NH₄）2SO₄）were optimized in the batch fermentation.The results showed that the dissolved oxygen levels were changed in different fermentation stages of L-lysine fermentation.Therefore,controlling the dissolved oxygen level via different agitation speeds has important role in the biosynthesis of L-lysine.In addition,the osmotic pressure of the fermentation medium was decreased by maintain appropriate concentration of glucose(5^-10 g·L^-1)and nitrogen(1-2 g·L^-1)in fed-batch,and the L-lysine production reached to 86.52±1.16 g·L^-1,which is 21.9% higher than that before optimization.