| N-acetylglucosamine is synthesized in all organisms, including bacteria, yeast, fungi, plants and animals. In humans, GlcNAc is the precursor of the disaccharide units in glycosaminoglycans, such as hyaluronic acid, chondroitin sulfate and keratan sulfate. GlcNAc is necessary to repair and maintain healthy articular cartilage and joint function. GlcNAc can also effectively increase the production of hyaluronic acid in the skin. Otherwise, GlcNAc is the precursor for biosynthesizing N-acetylneuraminic acid. Owing to its applications in pharmaceutical, cosmetics and chemical industry, large-scale production of GlcNAc is required. At present, GlcNAc is prepared based on chitin hydrolysis. Chemical methods for GlcNAc production are not widely commercialized, because the process includes large quantities of chemical waste, which is not environmentally friendly. The yields of enzymes that specifically hydrolyze chitin are very low, whether they are purified from the mass production of microbes or from genetically engineered microorganisms, limiting the industrial application of enzymatic hydrolysis of chitin. In addition, the source of chitin is seasonal, which might lead to unpredictable limitations on production capacity. Because of its advantages, such as short fermentation period and high yield, non-seasonal raw materials and environmentally friendly procedures, microbial fermentation has drawn much attention for GlcNAc production.Firstly, we produced GlcNAc in engineered Escherichia coli with recombinant plasmids containing different promoters and copy numbers with glycerol as carbon source. We compared the content of GlcNAc, the expression level of glmSM and GNA1and the plasmid stability. As a result, we found that the yield of GlcNAc in strains harboring low copy plasmid pCLTrcGM was the highest. Secondly, we studied the function of mannose transporter EIIMan, encoded by manXYZ genes. After deletion of manXYZ genes in DN1, the yield of GlcNAc was improved, which means manXYZ-encoded PTS transporter absorbed GlcNAc and converted into GlcNAc-6P in these conditions. What is interesting, deletion of manXYZ genes accelerated the metabolism of glycerol and greatly reduced the fermentation period. Finally, we compared the yield of GlcNAc with glycerol or glucose as carbon source. Glucose produced7.42g/1GlcNAc,48%higher than glycerol did.Sialic acids are a highly diverse family of nine-carbon amino that are widespread in the nature. N-acetylneuraminic acid (NeuAc), the most ubiquitous sialic acid and the precursor for all other sialic acids, generally located at the terminal of glycoprotein and glycolipid on the surface of cell membrane. There are several important physiological functions for NeuAc:maintainment of the conformation and function of biomacromolecules; information transfer between cells and molecules; recognition of specified glycoconjugates and cells based on specific bio-activities. Based on these roles, NeuAc is used as the starting material for pharmaceutical drugs, such as potential anti-cancer, antiviral, anti-adhesion, and anti-inflammatory agents. Zanamivir, a derivative of NeuAc, which inhibits neuraminidases of influenza virus of both types A and B, has been considered the most useful anti-influenza virus agent. Sialic acids could promote infant brain development, so they have been used as additives in new types of milk powders.NeuAc is traditionally extracted from natural products, such as bird’s nest, egg yolk membrane and milk whey. But the low content of NeuAc in these materials limits its large-scale production. For chemical synthesis, the complex protection and de-protection processes would not be suitable for mass production. Otherwise, NeuAc produced by chemical method is racemal, which can not meet the practical needs. In the last decades, enzymatic synthesis and whole-cell biocatalysis are major tools for NeuAc production.Enzymatic synthesis of NeuAc from N-acetyl-mannosamine (ManNAc) and pyruvate using NeuAc aldolase as catalyst has been reported many years ago. Afterwards, researchers found ManNAc can be epimerized by N-acetylglucosamine. Replacing ManNAc with GlcNAc as synthetic substrate greatly reduced the substrate cost during the enzymatic production.Meanwhile, whole-cell biocatalysis is considered as an alternative tool for NeuAc production since it eliminates the tedious manipulation of protein purification. For whole-cell biocatalysis, normally necessary genes are over-expressed in microorganisms. After cultivation and collection of microbial cells, NeuAc is synthesized without purification of the catalytic enzymes or additional supplementation of ATP. But precursors, pyruvate and GlcNAc, have to be added in the reaction buffer.With the development of metabolic engineering and synthetic biology, analysis and regulation of the metabolic pathway in microorganism towards efficient accumulation of target compound became realistic and easy to operate. Metabolic engineering has been used to produce terpenoids, polyhydroxyalkanoate, amino acids, biofuels, and other useful compounds. Direct microbial fermentative NeuAc production from glucose or other unrelated carbon sources is an attractive alternative since it can synthesize NeuAc in one step without adding any direct precursors. Few reports described the fermentative NeuAc production.In this study, by rational design, we constructed a NeuAc fermentative pathway in engineered E. coli, which provides a new way for NeuAc production. Introducing NeuAc aldolase (nanA), N-acetylglucosamine2-epimerase (slr1975) and glucosamine-6-phosphate acetyltransferase (GNA1) into E. coli, we got0.12g/1NeuAc. By analyzing this pathway, we found that the first enzyme glucosamine-6-phosphate synthase (GlmS), which is involved in the formation of hexosamine, is tightly regulated both at transcriptional and post-transcriptional level. In order to eliminate the transcription effect of the gene, glmS was over-expressed under an IPTG induced artificial promoter. Meanwhile, to derepress the feedback inhibition caused by GlcN-6P, the wild type GlmS was mutated to GlmSM. Additional expression of glmS and glmSM gene improved NeuAc concentration. By blocking in vivo N-acetylglucosamine catabolism, we determined6.48and1.55g/l GlcNAc and ManNAc, respectively. But the recombinant strain only produced0.30g/l NeuAc. After deletion of ackA, poxB and ldhA genes, another precursor pyruvate was accumulated, and1.36g/1NeuAc was produced. By preventing NeuAc retro-transportation and catabolism, NeuAc concentration increased to1.62g/1.To explore the NeuAc production potential, we performed fed-batch fermentation under indicated cultivation condition. NeuAc production reached7.85g/1with10%glucose was consumed. In addition,4.81g/l pyruvate,3.49g/1ManNAc and15.51g/1GlcNAc were also detected. The remaining pyruvate, ManNAc and GlcNAc was found to continuously synthesize NeuAc after glucose was consumed.We took measures to increase the conversion of GlcNAc to NeuAc, or decrease the production of GlcNAc, yet we found that reduced GlcNAc yield accompanied by decline of NeuAc content. Addition of surfactant decreased the metabolism of glucose and the cell mass, and eventually resulted in the decline of NeuAc accumulation. Through overexpression of NeuAc synthase from E. coli EV36, we found its catalytic efficiency was lower than NeuAc aldolase. In addition, we overexpressed NeuAc biosynthetic pathway of E. coli itself, while no NeuAc was detected. |
