Metabolic Engineering Of Klebsiella Oxytoca To Produce Pyruvate

With the increasingly serious problems of energy demand and environmental pollution in the world,more and more attention has been paid to the renewable energy materials and chemicals.Pyruvate can be used as precursors of some high value products such as alanine,phenylalanine derivatives,isobutanol and carotenoids directly or indirectly.Pyruvate is also widely used as a raw material for drugs,pesticides,chemical and food.In cells,it is the key point of metabolism of EMP and TCA cycle,the intermediate product of carbohydrate and lipids metabolism,and a synthetic precursor of several macromolecules.It plays an important role in the process of cell metabolism.A variety of microbes,such as Escherichia coli,Torulopsis glabrata,Corynebacterium glutamicum,and Saccharomyces cerevisiae,can produce pyruvate from glucose.Klebsiella oxytoca is widely used in industry,which mainly used to produce 2,3-butanediol,1,3-propanediol,ethanol and lactate.In this study,we chose K.oxytoca to be a host to accumulate pyruvate.Firstly,we investigated the influence of lactate dehydrogenase(ldh)on the production of pyruvate in several K.oxytoca mutants.We reported lactate formation in K.oxytoca PDL-0 strain knoking out 2,3-butanediol,ethanol,acetate,succinate and other products to obatain PDL-5,PDL-6,PDL-7.Since these strains can product lactate by glycerol,the activity of ldh is generally high.So it is unfavorable to the production of pyruic acid by K.oxytoca.We detected the activity of lactate dehydrogenase in PDL-5,PDL-6 and PDL-7 and found that if lactic dehydrogenase activity was high in the process of fermentation,strains will accumulate a lot of lactate.And there was no such phenomenon in the condition of low activity.In addition,after knocking out pyruvate formate-lyase encoding gene pflB,the accumulation of acetate reduced and the yield of pyruvate increased.Thus,PDL-7?pflB?ldh was constructed by metabolic engineering.However,the redox state was unbalanced in the cells after the byproduct synthesis pathways were blocked.Thus,we chose NADH oxidase to regenerate NAD+,which is beneficial to the growth of bacteria and the accumulation of pyruvate.Finally,we got the strain PDL-7?pflB?ldhD::nox,which produced 54.99 g/L pyruvate with a yield of 0.859 g/g pyruvate/glucose.In order to enhance the production of pyruvate and reduce the costs,the fermentation conditions of PDL-7 ?pflB?ldhD::nox were optimized including screening fermentative medium,aeration rate,agitation speed and neutralizer.Fed-batch fermentation was carried out under optimized conditons:No.2 medium(the medium without yeast extract),stirrer speed of 600 rpm,airflow rate of 1.6 vvm,and pH 6.8 regulated by KOH.PDL-7 ?pflB?ldhD::nox produced 61.57 g/L pyruvate from 121 g/L glucose in 54 h with a yield of 0.509 g/g pyruvate/glucose.To further cut the production costs,mixed saccharides(mainly glucose and galactose)were used to produce pyruvate.Methylglyoxal is an inhibitor of multiple sugars metabolism and it was reported that deletion of gene mgsA encoding methylglyoxal synthase could improve the efficiency of carbohydrate utilization.Thus,we constructed PDL-7 ?pflB?ldhD::noxAmgs to improve the efficiency of mixed saccharides utilization.In order to promote the transport of galactose,which is inhibited by carbon catabolite repression,the galactose permease gene(galP)was cloned and transferred into the strain,generating PDL-7 ?pflB?ldhD::nox?mgs pDK7-galp.Fed-batch fermentation showed that the strain consumed 17.3 g/L glucose and 5.32 g/L galactose in 20 h,indicating that the expression of galactose permease encoding gene galP could promote the co-utilization of glucose and galactose.