Metabolic Engineering Of Corynebacterium Glutamicum For Efficient Biosynthesis Of L-glutamine

L-glutamine,a conditionally essential amino acid,is widely used in medicine,food,feed and other industries because of its dual physiological functions of immunity and nutrition.In this study,Corynebacterium glutamicum N01 with L-glutamine titer of 25.7±2.7 g·L^-1was used as the starting strain,and combined strategies involved in rational metabolic engineering and fermentation process optimization were implemented to improve L-glutamine production.The main contents are as follows:（1）Increasing the carbon flux towards L-glutamine.L-glutamate and L-proline were found the main by-products by determining the amino acid spectrum of the N01 fermentation broth.The pro B gene encoding gamma-glutamyl kinase that catalyzes the first reaction in the biosynthetic pathway of L-proline was first deleted in N01 strain to block the competitive branch pathway.The resulting strain CGQ01 could produce 27.2±2.5 g·L^-1L-glutamine.Then,NCgl1221 encoding channel protein mainly responsible for modulating glutamate export L-glutamate was knocked out to construct CGQ02.Compared with CGQ01,the intracellular supply of L-glutamate was improved and intracellular accumulation was 40.3%lower than that of CGQ01 to 9.2 g·L^-1,and L-glutamine production increased to 31.3±2.4 g·L^-1.（2）Increasing catalytic efficiency of the key enzyme glutamine synthase（GS）.The gln E gene encoding glutamine synthase adenylyltransferase was knocked out to relieve GS adenylation,which lead to a 128.6%increase of GS enzyme activity compared to the control and L-glutamine production was increased to 36.2±2.5 g·L^-1.Subsequently,Sc GS derived from Saccharomyces cerevisiae with high catalytic activity,which was screened out from 8different microorganisms,was overexpressed to further increase GS activity.The GS activity of recombinant strain increased by 8.3 times,and L-glutamine production increased by 26.8%to 45.9±1.4 g·L^-1,illustrating the key importance of increasing GS activity for L-glutamine accumulation.（3）Enhancing intracellular ATP supply.The polyphosphate kinase gene ppk derived from Escherichia coli was overexpressed in CGQ03 to construct an ATP regeneration system.Compared with CGQ03,the intracellular ATP content and L-glutamine production of recombinant strain CGQ03/p XMJ19-ppk ^Ecwas increased by 22.6%and 13.8%(41.2±2.7g·L^-1),respectively.Afterwards,gln A^Sc（encoding Sc GS）and ppk ^Ecwas co-expressed in CGQ03 leading to a 49.5±3.9 g·L^-1L-glutamine production.A strong RBS5 was obtained to further improve gene expression level by ribosome binding site engineering,and the yield of L-glutamine was improved to 52.8±4.1 g·L^-1.（4）The optimization of two-stage p H control strategy.According to the enzymatic properties of Cg GS,the influences of different p H on L-glutamine fermentation was explored,and the optimal p H for amine production period was determined to be 6.2.The two-stage p H control strategy was optimized for resolving the imbalance between cell growth and L-glutamine production.The final engineered strain CGQ03/p XMJ19-R5-gln A^Sc-ppk ^Ecproduced 73.5±3.1 g·L^-1L-glutamine with a sugar-acid conversion rate of 36.8%at 66 h,using the optimized two-stage p H control strategy in a 5 L fermenter.（5）The effects of dissolved oxygen（DO）control on L-glutamine fermentation.Two different ways of DO control which included uncontrolled DO（constant speed and ventilation）and controlled DO levels at 30%-40%（rotating speed coupled with dissolved oxygen and manually increasing the ventilation rate）were studied for the influences on L-glutamine fermentation.It was found that controlling the DO level more conducive to cell growth and L-glutamine fermentation.Under the best conditions,the final engineered strain CGQ03/p XMJ19-R5-gln A^Sc-ppk ^Ecwas fed-batch fermented in a 5 L fermenter for 66 h.The maximum titer of L-glutamine was 89.2±1.7 g·L^-1with a yield and productivity of 33.5%and1.35 g·L^-1·h^-1.