Systematic Metabolic Engineering And Fermenatation Optimization Of Corynebacterium Glutamicum For The Production Of L-phenylalanine

L-phenylalanine (L-Phe), an essential amino acid for humans and other animals, hasbeen widely used in food, feed additives and pharmaceutical industries. In particular, L-Phe isused as the precursor for the production of aspartame in food industry. Over the past years,microbial production of L-Phe has attracted much attention and most of the studies weremainly concentrated on Escherichia coli. However, E. coli belongs to the pathogenic strainand is tend to be infected by phage, which confine it application in the industrial production.Consequently, it is significant to construct a food grade recombinant strain for efficientproduction of L-Phe.In this thesis, metabolic engineering and biotechnology engineering strategies were usedto regulate L-Phe biosynthesis pathway, reconstruct the transport pathway of glucose andL-Phe, inactivate the byproducts biosynthesis pathway and optimize the fermentationconditions in Corynebacterium glutamicum ATCC13032to improve the production of L-Phe.The main contents and results are shown as follows:(1) Construction a C. glutamicum platform strain for L-Phe production. Two key genesaroH(E)(3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, DAHPS) from E. coli andpheAfbr(feedback-inhibition resistance chorsmate mutase/prephenate dehydratase, CM-PDT)from plasmid (pAP-B03) were individually expressed with pXMJ19in C. glutamicum ATCC13032. As a result, the production of L-Phe was increased to0.99±0.06g L-1and1.34±0.02g L-1, respectively. More impressively, the titers of L-Phe and shikimate were improved to4.64±0.09g L-1and3.20±0.05g L-1by co-overexpression of aroH(E) and pheAfbrwhich were29.0and11.0-fold of that of the wild-type C. glutamicum ATCC13032, respectively.(2) Modification of the N-terminal of AroF and construction of a feedback inhibitionresistant mutant. On the analysis the effect of overexpression of aroF, an AroF*(withN-terminal Ile11deficiency) muant was unexpectedly generated with a2.41-fold of L-Phethan the control, indicated that the N-terminus of AroF plays an important role regulation ofits activity and feedback inhibition. As a consequence, the N-terminal fragment wassystematically truncated and the variant AroFΔ(1-11)with the deletion of11residues led to thehighest prodcution of L-Phe (1.23±0.03g L-1). When adding1.0g L-1L-Tyr, L-Phe wasfurther increased to1.54±0.12g L-1. The results confirmed that feedback inhibition resultedfrom L-Tyr was completely relieved. Further cultivation results in3L fermentor showed thatC. glutamicum (pXM-pheAfbr-aroFfbr) produced much higher titer of L-Phe (5.93±0.17g L-1)than strain C. glutamicum (pXM-pheAfbr-aroH) with the increase of24.6%. In this regard, C.glutamicum (pXM-pheAfbr-aroFfbr) was chosen for further studies.(3) Identification of key enzymes of L-Phe biosynthesis pathway: In addition to PheAfbrand AroFfbr, another six key enzymes TktA, PpsA, AroE, AroL, AroA and TyrB in L-Phebiosynthesis pathway from E. coli and C. glutamicum were identified. On this base, theseeight key enzymes were divided into two modules with shikimate as a node (upstream anddownstream). Plasmids pEC-XK99E, pXMJ19and three promoters were used for furtherreseach. As a result, the recombinant strain harboring pSUTL (pEC-XK99E-Ptac-aroFfbr-aroE-Plac-ppsA-tktA) and pSDTL (pXMJ19-Ptac-pheAfbr-aroA-Plac-tyrB-aroL) produced more L-Phe (5.59±0.11g L-1) and less shikimate (0.31±0.11g L-1). Furthermore, the production of L-Phewas increased to7.42±0.21g L-1in a3L fermentor with a25.1%increase compared with C.glutamicum (pXM-pheAfbr-aroFfbr).(4) Genome engineering and modification to enhance L-Phe production: C. glutamicumΔptsI:: iolT2-ppgK (pSUTL, pSDTL), deletion of ptsI in PTS system and reconstructionnon-PTS pathway, accumulated higher intracellular PEP and L-Phe with62.3%and12.0%increase, respectively. Furthermore, to modify the L-Phe transport pathway, aroP gene wasdeleted, yielding recombinant strain C. glutamicum ΔptsI:: iolT2-ppgK ΔaroP (pSUTL,pSDTL). Correspondingly, the intracelluar L-Phe was decreased42.0%coupled withextracellular L-Phe increased6.9%compared with C. glutamicum ΔptsI:: iolT2-ppgK(pSUTL, pSDTL). Moreover, the recombinant strain C. glutamicum ΔptsI:: iolT2-ppgKΔaroP ΔaceE Δldh (pSUTL, pSDTL) with deletion of aceE and ldh accumulated higher titerof L-Phe (7.46±0.16g L-1) and less acetatic acid (0.74±0.093g L-1). As expected, no lacticacid was detected. The production of L-Phe was further increased to10.13±0.22g L-1in3Lfermentor with an increased ratio of L-Phe to glucose (0.073g g-1).(5) Fermentation optimization of recombinant strain C. glutamicum ΔptsI:: iolT2-ppgKΔaroP ΔaceE Δldh (pSUTL, pSDTL) to enhance the production of L-Phe: Key factors of theseed medium (glucose, corn steep liquor, KH2PO4and (NH4)2SO4), fermentation medium(glucose, corn steep liquor and (NH4)2SO4), and fermentation condition (loaded liquid,concentration of IPTG, initial pH) were confirmed through orthogonal experiment, responsesurface methodology and single factor experiment. The production of L-Phe was9.35±0.02g L-1with the increase of25.3%under the optimum fermentation conditions. In a3Lfermentor, two-stage DO control strategy and glucose feeding strategy was applied for theL-Phe production. The control strategy was that0-20h DO was controlled at20%and20-80h at10%,30-80h control glucose concentration was controlled at10g L-1. Finally, theproduction of L-Phe, the ratio of L-Phe to glucose and productivity was improved to17.26±0.08g L-1,0.121g g-1and0.240g L-1h-1with the increase of70.4%,65.8%and70.4%,respectively.