Metabolic Engineering Modification Of An Escherichia Coli L-threonine Production Strain

L-threonine is the second limiting amino acid?behind L-lysine?in pig feed and the third limiting amino acid?behind L-methionine and L-lysine?in poultry feed.It can improve the immune function,fat metabolism of the body and the meat quality,promote the growth and the secretion of intestinal mucin,maintain the intestinal health,etc.it has important physiological functions.L-threonine is also widely used in food,medicine,cosmetics and other fields.At present,L-threonine is the third amino acid in terms of yield,next to L-glutamic acid and L-lysine,and mainly produced by the fermentation of Escherichia coli in the industry.Although the level of L-threonine produced by E.coli is very high,there is still some distance from the theoretical production and the conversion rate.So,it is of great significance to further improve the ability of E.coli to produce L-threonine and develop some new genetic engineering methods.In this study,the high-yielding L-threonine E.coli strain TWF001 was taken as the main research object,and a set of non-resistant expression vector system and dynamic regulation components were developed.The main work was described as follows:?1?The growth and the amino acid production of E.coli TWF001 were studied by shake flask fermentation and fed-batch fermentation.The growth of TWF001 was lower than wild type strain E.coli K-12 W3110.In shaker fermentation,TWF001 could produce L-threonine without affecting the metabolism of other amino acids.In fed-batch fermentation,TWF001could produce 30.35 g/L L-threonine in 23 h,with 1.32 g/L/h production efficiency,and the main by-products were L-glycine?0.22 g/L?and L-alanine?0.46 g/L?.?2?The mechanism about high-yield L-threonine of TWF001 was analyzed by using W3110 as control in logarithmic and stable phase?growth and L-threonine production phase?.The results of transcriptome analysis showed that the transcription levels of 1739 genes?249up-regulated,1490 down-regulated?in logarithmic stage and 2361 genes?319 up-regulated,2042 down regulated?in stable stage were significantly changed.Combining the transcriptome analysis results and metabolic pathways revealed that the genes associated with L-threonine biosynthesis?aspC,thrABC,and asd?were significantly up-regulated.The genes associated with NAD?P?H regeneration?uxaB,tyrA,pntAB,wrbA,ndh,and gabD?were significantly up-regulated.The genes associated with the TCA cycle?sucCD,sdhC,acnA,sucB,aceB,mdh,sdhACD?were significantly down-regulated.The key genes for L-threonine degradation?tdh,tdcD,tdcE,tynA,mhpF,tdcB and pta?were significantly down-regulated.The L-threonine output gene rhtA was up-regulated,while the L-threonine input genes sstT and tdcC were down-regulated.An amino supply ring was formed by multiple up-regulation genes to provide an amino donor for L-threonine synthesis.Many genes encoding the 30S and 50S subunits of ribosomes are significantly up-regulated.?3?To meet the requirements of countries and enterprises for efficient,environmentally friendly and safe production,an expression plasmid system for no antibiotics and no antibiotic genes was constructed.eight plasmids were constructed in the early stage to determine the optimal scheme.The results showed the bases in PAM were mutanted at least two sites could effectively avoid being recognized by Cas9 protein,and the more the number of silenced mutants in PAM,the higher the knockout efficiency for lpxA in chromosome.A new E.coli non-resistant expression system based on the plasmids p Cas9Cre,pTF-A-UD and pRSFCmlpxA was constructed.The plasmid pCas9Cre could express the enzymes Cas9,?-Red and Cre,removed at 42?;pTF-A-UD contained the DNA fragments used to knock out the gene lpxA in chromosome,removed by adding IPTG;pRSFCmlpxA contained the mutant gene lpxA123 and the chloramphenicol resistance gene CamR removed by adding IPTG.The results of application in wild-type E.coli MG1655 and L-threonine producing E.coli TWF006 showed that the system could be well applied to improve the production of L-threonine in E.coli by overexpressing genes thr ABC and rhtA,and the overexpression of lxpA would not affect the synthesis of L-threonine.?4?A set of more reasonable regulatory expression elements than knockout(inhibit element thrR and promote element P_cysH)were developed.Applying the threonine operon lead control element thrL to control the expression of genes arcA,cpxR,gadE,fadR and pykF,the result shown that all the recombinant strains had more L-threonine synthesis than the original strain TWF001.Different parts of thrL was inserted into the regulatory regions of gene iclR in TWF001.The optimal sequence of regulatory elements?thrR?was determined,and the strain TWF063 was screened out which could produce 16.34 g/L L-threonine with the initial 40 g/L glucose in 30 h.Threonine activation promoters P_cysH,P_cysJ and P_cysD were studied to regulate the expression of gene aspC,and finally the optimal combination sequence of P_cysH was determined,which could further improve the L-threonine production of TWF063?TWF066?.Using TWF066 as the initial strain,the optimal strain of TWF083 was obtained by regulating the expression of arcA,cpxR,gadE,pykF and fad R genes.?5?All high-yielding L-threonine strains?TWF001,TWF058,TWF063,TWF066,TWF070,TWF077,TWF078 and TWF083?were cultured in the same time.The fermentation results showed that TWF083 had the advantages of the largest dry weight?30.87 g/L?,the highest yield of L-threonine?26.95 g/L?,the highest conversion rate?67.37%?,the lowest acetic acid concentration and the highest concentration of pyruvate and oxaloacetate.Fermentation of TWF001 and TWF083 at different glucose concentrations showed that TWF083 had the advantage of high yield of L-threonine at each glucose concentration,and the optimal initial glucose concentration was 40 g/L.TWF001 and TWF083 were studied in fed-batch fermentation.After 48 h,strain TWF083 produced 116.62 g/L L-threonine,with the conversion rate of 48.6%and the production efficiency of 2.43 g/L/h,and the acetic acid concentration?1.14 g/L?was reduced by 89%,pyruvate?0.04 g/L?was basically exhausted,while NADPH?4.97 g/L?and acetyl-coenzyme A?0.21 g/L?remained.