Metabolic Engineering Of Escherichia Coli And Process Optimization For Microbial Production Of Glucosamine

Glucosamine (GlcN), also called amino sugar, is a compound derived from the substitution of ahydroxyl group of glucose molecule with an amino group. GlcN finds a wide-range of applications inhealthy food, food and pharmaceutical industries. In this dissertation, Escherichia coli ATCC25947(DE3)was used as an initial strain for the construction of GlcN and N-acetylglucosamine (GlcNAc) producer bymetabolic engineering. The genes fragments of glucosamine synthase encoding gene (glmS) andglucosamine-6-P N-acetyltransferase encoding gene (gna1), amplified from Escherichia coli BL21genomeand Saccharomyces cerevisiae S288C genome respectively, were inserted between the restriction sites ofthe plasmid pET-28a, yielding the recombined plasmid pET-28a-glmS-gna1. Then a recombinant E. coli-glmS-gna1was constructed by transforming the recombined plasmid into E. coli ATCC25947(DE3). Theeffects of glucose feeding strategies and dissolved oxygen (DO) levels on the GlcN and GlcNAc wereinvestigated and a multi-stage glucose supplying approach and a step-wise DO control strategy wereproposed respectively. To alleviate or block the transportation process of GlcN from the culture broth tothe inside of cells, GlcNAc-specific transporter encoding gene (nagE), mannose transporter encodinggene (manX) and glucose transporter encoding gene (ptsG) were knocked out with Red homologousrecombination method, and the engineered strains, E. coli-glmS-gna1-ΔnagE (with nagE genedeletion), E. coli-glmS-gna1-ΔnagE-ΔmanX (with nagE and manX genes deletion) and E. coli-glmS-gna1-galP-glk-ΔnagE-ΔmanX-ΔptsG (harboring the plasmids of pETDuet-1-gna1-glk and pRSFDuet-1-glms-galP) were successfully formed. For the further enhancement of GlcN and GlcNAc production, thenode of pyruvate kinase encoding genes (pykA/F) as the switch point for carbon flux distribution in E. coli-glmS-gna1-galP-glk-ΔnagE-ΔmanX-ΔptsG was selected based on the results of transcriptional levelanalysis and pykA and pykF were knocked out. The main results were described as follows:1) Escherichia coli ATCC25947(DE3) was used as an initial strain for the construction of GlcN andGlcNAc producer by metabolic engineering. The genes fragments of glmS and gna1, amplified fromE. coli BL21genome and S. cerevisiae S288C genome respectively, were inserted between therestriction sites of Sac I, Hind III, and Not I and Xho I of the plasmid pET-28a, yielding therecombined plasmid pET-28a-glmS-gna1. Then a recombinant E. coli-glmS-gna1was constructed bytransforming the plasmid pET-28a-glmS-gna1into E. coli ATCC25947(DE3). The fermentationresults showed that the GlcN yield reached24.15g/L at18h and was enhanced by1.97-fold incomparison with that of Escherichia coli ATCC25947(DE3).2) For the further enhancement of GlcN and GlcNAc production by E. coli-glmS-gna1, effects ofdifferent glucose feeding strategies including constant-rate feeding, interval feeding, and exponentialfeeding on GlcN and GlcNAc production were investigated in a3-L fermentor. The results indicatedthat exponential feeding resulted in relatively high cell growth rate and low acetate formation rate,while the constant feeding contributed to the highest specific GlcN and GlcNAc production rate. Basedon this, a multi-stage glucose supplying approach was proposed to enhance GlcN and GlcNAcproduction. In the first stage (0-2h), a batch culture with an initial glucose concentration of27g/L wasconduc-1ted, and the second culture stage (2-10h) was performed with an exponential feeding at μset=0.20h, which was followed by feeding a concentrated glucose (300g/L) at a constant rate of32mL/hin the third stage (10-16h). With this time-variant glucose feeding strategy, the total GlcN and GlcNAcyield reached69.66g/L, which was enhanced by1.59-fold in comparison with that of batch culture(43.80g/L) with the same glucose supplyment.3) Influence of DO levels (10,20,30and40%) on the GlcN and GlcNAc production by E. coli-glmS-gna1was investigated in a3-L fermentor. It was found that the highest specific GlcN and GlcNAcproduction rates were obtained at different DO levels and different culture stages. Namely, the highestspecific GlcN and GlcNAc production rates were obtained at20%during0-2h,30%during2-8h,40%during8-12h, and30%during12-18h. Accordingly, a step-wise DO control strategy wasproposed, namely DO was controlled at20%during0-2h,30%during2-8h,40%during8-12h and30%during12-18h. With this DO control approach, the total production of GlcN and GlcNAc reached72.89g/L, which was1.37times that without DO control (53.31g/L).4) To alleviate or block the transportation process of GlcN from the culture broth to the inside of cells,nagE gene and manX gene were knocked out with Red homologous recombination method, and twoengineered strains, E. coli-glmS-gna1-ΔnagE (with nagE gene deletion) and E. coli-glmS-gna1-ΔnagE- ΔmanX (with nagE and manX genes deletion), were successfully constructed. The two strains werecultured in a3-L fermentor for the production of GlcN and GlcNAc. The maximal GlcN concentrationof control strain E. coli-glmS-gna1reached4.06g/L, and the maximal GlcNAc concentration reached41.46g/L. The maximal GlcN and GlcNAc concentration of E. coli-glmS-gna1-nagE reached4.38g/L and71.80g/L, which were1.08-fold and1.70-fold of those of E. coli-glmS-gna1, respectively. Themaximal GlcN and GlcNAc concentration of E. coli-glmS-gna1-nagE-manX reached4.82g/L and118.78g/L, which were1.20-fold and2.86-fold of those of E. coli-glmS-gna1, respectively. Theseresults suggested that the deletion of nagE and manX could significantly increase the extracellularaccumulation of GlcN and GlcNAc.5) To understand the effects of ptsG gene knockout on GlcN and GlcNAc production, an engineered strain,E. coli-glmS-gna1-galP-glk-ΔnagE-ΔmanX-ΔptsG with ptsG gene deletion and harboring the plasmidsof pETDuet-1-gna1-glk and pRSFDuet-1-glms-galP was constructed, The fermentation resultssuggested that the decrease of glucose titer and the rise of GlcN titer in the culture broth ofE. coli-glmS-gna1-galP-glk-nagE-manX-pykF-ptsG changed more slowly in comparison withthat of the control strain; the deletion of ptsG gene also helped to prevent the drop of GlcN titer inthe later stages of fermentation; in comparison with that of the control strain E. coli-glmS-gna1, thetiter of GlcN and GlcNAc increased by97%and60%respectively and the concentration of aceticacid decreased by71%, the total titer of GlcN and GlcNAc in the culture broth increased by65.4%and reached128.75g/L.6) For the further enhancement of GlcN and GlcNAc production, the node of pykA/F as the switch pointfor carbon flux distribution in E. coli-glmS-gna1-galP-glk-nagE-manX-ptsG was selected based onthe results of transcriptional level analysis. Then, pykA gene and pykF gene were knocked out and thefermentation results showed that the maximum GlcN and GlcNAc total titer of the pykA gene deletionstrain and the pykF gene deletion strain increased in comparison with that of the control strain andreached130.19g/L and132.43g/L, respectively, however, the maximum GlcN and GlcNAc total titerof the pykA/F genes deletion strain decreased by61%in comparison with that of the control strain.