Metabolic Engineering Of Bacillus Subtilis For Efficient Production Of N-acetylglucosamine

Glucosamine(GlcN) and its derivate N-acetylglucosamine(Glc NAc) have been widely used as nutraceutical and pharmaceutical for maintenance of joint health and treatment of osteoarthritis. Newly discovered function of GlcN and GlcNAc for lifespan further widen their application fields. However, current method of GlcN and GlcNAc production has several severe drawbacks, such as variable raw material supply, heavy pollution during production, and potential allergic reaction for the consumers. Therefore, a sustainable and environmentally-friendly method for food and pharmaceutical grade GlcN and GlcNAc production is needed to be developed.In this work, Bacillus subtilis 168 was used as the initial host for metabolic engineering for microbial GlcN and GlcNAc production, which is a wild type strain of B. subtilis and meets the requirement for generally regarded as safe(GRAS) grade for biochemical production. Synthetic biology tools and systems biology methods were combined with metabolic engineering to optimize the cellular properties of engineered B. subtilis. The main results of this work are summarized as follows:(1) Rational metabolic engineering was first used to construct GlcNAc synthetic pathway. Specifically, GlcN-6-P synthase(GlmS) and GlcN-6-P N-acetyltransferase(Gna1) were co-expressed, which realized for the first time GlcNAc overproduction in B. subtilis(240mg/L). Next, encoding gene of NagP, the GlcNAc-specific enzyme of phosphotransferase system, was knocked out to block extracellular GlcNAc transport into the cell, which further facilitated extracellular GlcNAc accumulation(615 mg/L). Finally, knocking out of encoding gene of GlcNAc-6-P deacetylase(NagA), GlcN-6-P deaminase(GamA and NagB) entirely block intracellular GlcNAc catabolism, which enhanced GlcNAc titer to 1.85 g/L in shake flask culture.(2) On the basis of the constructed recombinant GlcNAc-producing strain, DNA-guided scaffold was employed to co-localize the key pathway enzymes GlmS and Gna1. When GlmS and Gna1 were co-localized on the DNA-guided scaffold at the ratio of 1:2, Glc NAc titer further increased to 4.55 g/L, which is 2.5-fold of that without enzyme co-localization. Next, to reduce the maintenance metabolism of the engineered strain for enhancing GlcNAc production, encoding genes of cytochrome bd oxidase(subunit II, CydB) and ATP-binding protein for the expression of cytochrome bd(CydC) were knocked out to block the less efficient respiration chain. The maintenance coefficient of engineered strain reduced 19.0 %, with 39.6 % increase of GlcNAc titer(6.15 g/L).(3) Modular pathway engineering strategy was further used to optimize GlcNAc synthetic network, including GlcNAc synthesis module, glycolysis module, and peptidoglycan synthesis module. Firstly, two promoter system was used to fine tune GlcNAc synthesis module. GlcNAc yield on dry cell weight(DCW) reached 0.42 g GlcNAc/g DCW in shake flask culture when the expression of GlmS was under the control of tightly inducible promoter PxylA and Gna1 was under the control of constitutive promoter P43. Secondly, synthetic small regulatory RNAs(sRNAs) were used to modulate the activities of glycolysis module and peptidoglycan module. When the activities of glycolysis module and peptidoglycan module were controlled at low activities by expressing anti-pfk sRNA, anti-glmM sRNA, and Hfq protein, GlcNAc yield on cell reached 2.00 g GlcNAc/g DCW. Finally, GlcNAc production was further enhanced by batch fermentation and fed-batch fermentation in 3-L bioreactor. In batch culture, GlcNAc titer reached 9.41 g/L, which is 13.4 % higher than shake flask culture. In fed-batch culture, GlcNAc titer reached 31.65 g/L.(4) Targeted metabolomics was implemented to investigate metabolite pool sizes in central metabolism and GlcNAc synthetic pathway in steady state. In engineered B. subtilis, concentration of metabolites in central carbon metabolism, central nitrogen metabolism, and amino acid synthetic pathway were significantly lower than those of wild type strain B. subtilis 168. Overconsumption of glutamine was identified as the reason for lower metabolite concentration in central metabolism. In GlcNAc synthetic pathway, GlcNAc-6-P was accumulated, reaching 33.71 mM, which indicated that there was a certain bottleneck in GlcNAc synthetic pathway, and further investigation was needed.(5) Kinetic modeling and dynamic metabolomics were combined to investigate GlcNAc synthetic pathway. Possible scenarios of GlcNAc synthetic pathway with various rate-limiting step were firstly simulated, which showed distinct qualitative features. Next, dynamic metabolomics during the start-up phase of GlcNAc synthesis was monitored, which showed kinetics feature of GlcNAc pathway with a futile cycle between GlcNAc-6-P and intracellular GlcNAc. Dynamic labeling using [U-13C]glucose was implemented to verify the existence of futile cycle between GlcNAc-6-P and intracellular GlcNAc. Finally, we identified glucokinase(GlcK) as the responsible enzyme and a deletion strain restored intracellular GlcNAc-6-P concentration and 2.32-fold GlcNAc productivity.