Research On Metabolic Engineering Of Corynebacterium Glutamicum Using Xylose To Synthesize L-Ornithine

L-Ornithine is a basic amino acid,which has the functions of protecting the liver,protecting the liver,treating liver diseases,anti-cancer,and stimulating the secretion of growth hormone.The industrial chain synthesis of L-ornithine is usually carried out by means of microbial fermentation.Among them,Corynebacterium glutamicum is widely used as a dominant strain for the fermentation and breeding of various amino acids,and is also a mainstream strain for the production of L-ornithine.However,the synthesis of L-ornithine by microbial fermentation requires the consumption of a large amount of substrate glucose,the extraction pressure increases,and the negative impact of this problem is increasing day by day under the environment of declining cultivated area and increasing population.Therefore,the search for alternative carbon sources of glucose has become the focus of current research.Lignocellulose is one of the wide-ranging and reproducible natural resources.It can be converted into reducing sugars by acidolysis or enzymolysis,with xylose and glucose occupying the largest proportion of all reducing sugars.In this study,based on the existing recombinant C.glutamicum SO26,the use of metabolic engineering strategies to build a strain that can simultaneously ferment and synthesize L-ornithine using glucose and xylose.The main research contents include:(1)The xylAB coding genes of xylose isomerase and xylulose kinase from Xanthomonas campestris were cloned into the shuttle expression vector pXMJ19 and introducing it into recombinant C.glutamicum SO26 by electrotransform,the optimal addition time and concentration of IPTG were optimized.The results of the study showed that the initial concentration of IPTG added at a final concentration of 0.5 mM yielded the largest L-ornithine production.Xylose consumption 47.9%and the L-ornithine production reached 21.6 ± 0.19 g/L.(2)To investigate the effects knock-in of the gene encoding pentose transporter(AraE)and overexpression of phosphoenolpyruvate carboxylase(PEPC)and site-directed mutation modification on L-ornithine production in C.glutamicum.After modification by genetic engineering tools,L-omithine production can reach 27.1 ± 0.32 g/L.(3)To investigate whether adding biotin and thiamine hydrochloride as coenzymes of various carboxylase can improve the biosynthesis of L-ornithine.The fermentation results showed that promoting the growth rate of C.glutamicum when adding 0.2 mg/L biotin and 5 mg/L thiamine hydrochloride,the L-ornithine production increased to 33.4±0.49 g/L.The components and conditions of the fermentation medium were optimized.The most suitable pH was mainly investigated.The fermentation results showed that when the pH of the fermentation medium was 6.7,37.5± 0.63 g/L of L-ornithine was obtained.Cultivation of recombinant strains through a mixed ratio of glucose and xylose at a concentration ratio of 2:1 and simulated use of lignocellulose in the laboratory,the results show that mixed sugar has a significant advantage over glucose and xylose when cultured separately.Furthermore,the L-ornithine production can reach 41.5± 0.02 g/L,which is 7%higher than that of the starting bacteria,and the average yield is 0.576 g/(L·h).