| Sialic acid, also known as neuraminic acid, is an amino sugar with nine carbons and pyranose structures. It is ubiquitous in nature, and more than 60 categories of sialic acids have been identified. Among these categories, N-acetylneuraminic acid(Neu5Ac) is the most important because it is the precursor for the synthesis of other sialic acids and has a close relationship with human health. Neu5 Ac is used to synthesize several antiviral drugs and cure H1N1 and H5N1. In addition, Neu5 Ac has great nutritional value and can promote brain development in infants. Neu5 Ac is commonly produced through enzyme catalysis. Although enzyme catalysis has high output of Neu5 Ac, adding excessive pyruvate to drive the reaction equilibrium to Neu5 Ac synthesis, but it also leads to substrate waste, high costs, and considerable environmental pressure. This study attempts to develop a Neu5 Ac synthesis strategy without extracellular pyruvic acid. This strategy utilizes N-Acetyl glucosamine(GlcNAc) as the substrate to synthesize N-acetylmannosamine(ManNAc) by GlcNAc 2-epimerase(AGE). ManNAc and intracellular PEP then undergo an irreversible reaction catalyzed by N-acetylneuraminic acid synthetase(NeuB) to synthesize Neu5 Ac. The synthesis pathway was constructed in Escherichia coli and strengthened by metabolic and fermentation engineering. The developed synthesis strategy is summarized below:(1) First, the activity and enzymatic property of AGE and NeuB from different sources were investigated. Results demonstrated that pAGE and bAGE had similar stability, temperature preference, and pH preference. Their optimal pH and temperature were 7.0 and 50°C, respectively. These enzymes maintained high activity at 35–55°C, and higher than 90% of activity at 50°C. At 37°C in Tris-HCl(pH 7.0), pAGE and bAGE activities were 392.1 U?mg-1 and 2411.5 U?mg-1, respectively. The optimal pH of eNeu B and cNeuB was neutral and their optimal temperature was 45°C. They maintained more than 60% activity at 30–47.5°C. At 37°C in Tris-HCl(pH 7.0) eNeuB and cNeuB activities were 0.037 U?mg-1 and 6.380 U?mg-1 total proteins, respectively. However, cNeuB had poor stability and retained only 50% catalytic activity after 2 h. When coexpressed with pGro7, cNeuB activity was 17.8% higher compared with the control group. Inducing the expression of the GroEL/Gro ES composite inhibited the erratic agglomeration of proteins and accelerated protein folding, thus increasing the soluble expression of cNeuB in E. coli. Subsequent experiments constructed the Neu5 Ac synthesis pathway based on bAGE and cNeuB.(2) The Neu5 Ac synthesis pathway was constructed based on bAGE and cNeuB. The eliminating of host bacterial genes related to the Neu5 Ac degradation pathway and GlcNAc transmembrane transport pathway promoted Neu5 Ac synthesis. Given that cNeuB had relatively lower activity than bAGE, we explored strategies to increase cNeuB activity. The exchange sequence of the two genes and pDTrc-AB and pDTrc-BA coexpression carriers was investigated. The results showed that cNeuB in pDTrc-AB was highly active, and that Neu5 Ac did not accumulate when the Neu5 Ac degradation pathway in E. coli MG1655 was retained. E. coli SA-01 was constructed by eliminating the nanATEK gene clusters of the related enzymes in the Neu5 Ac degradation pathway via the ? Red recombinant system. pDTrc-AB and pDTrc-BA were transformed into E. coli SA-01, and the transformed bacteria was used to synthesize Neu5 Ac. The Neu5 Ac outputs were 2.61 and 2.03 g?L-1, respectively. The transformation of the GlcNAc transmembrane transport system not only prevented GlcNAc phosphorylation and saved intracellular PEP, but also increased intracellular GlcNAc concentration and enhanced Neu5 Ac synthesis. Therefore, Neu5 Ac production increased by 38.3% to 3.61 g?L-1.(3) In E. coli, Neu5 Ac synthesis was further improved as the intracellular PEP supply increases via the independent overexpression or co-overexpression of genes related to PEP synthesis. Genes related to PEP synthesis(pck and ppsA) were cloned from the E. coli MG1655 genome. The Duet series carrier induced the independent overexpression or co-overexpression of these genes, and the host E. coli was DE3 lysogenized. Neu5 Ac yield reached 5.80 g?L-1 and 7.09 g?L-1 for independent pck and ppsA overexpression, respectively. However, the transcriptional level of another gene was inhibited. When pck and ppsA were co-overexpressed, their transcriptional levels increased simultaneously and were positively correlated with the copy number of carriers. The optimal Neu5 Ac output was contributed by the PEP supply system, which was composed of the copy carrier, pCDF-pck-ppsA. The Neu5 Ac yield reached 8.63 g?L-1, which was higher than that of the original strain by 139%. The intracellular PEP supply was increased by optimizing pck and ppsA overexpression levels in E. coli, resulting in high Neu5 Ac production.(4) The nutrition for Neu5 Ac synthesis in the biotransformation stage, as well as the effect of surfactants on Neu5 Ac synthesis were investigated. In a culture medium with nitrogen sources, the host E. coli cells grow exponentially, but Neu5 Ac synthesis is inhibited. In nitrogen-free glucose culture medium, host bacterial biomass was slightly lower than the initial value, and the inhibition of Neu5 Ac synthesis was disrupted. Neu5 Ac yield was 8.61 g?L-1. The exponential growth of the host E. coli consumed PEP and competed with Neu5 Ac synthesis for intracellular PEP, and was therefore disadvantageous to Neu5 Ac synthesis. When glycerol was used as the carbon source, transmembrane transport was no longer dependent on PTS and thereby more PEP molecules were available for Neu5 Ac synthesis, Neu5 Ac yield increased to 10.43 g?L-1. Adding Triton X-100 to the transformation culture medium promotes Neu5 Ac synthesis by approximately 6.1%. Finally, Neu5 Ac biotransformation was implemented in a bioreactor. Neu5 Ac biotransformation was carried out under low phosphate concentration and the osmotic pressure of the reaction system. Meanwhile, the fermenter facilitated mass transfer condition for Neu5 Ac synthesis. At 65 h, the Neu5 Ac yield in the fermenter reached 16.02 g?L-1, which was 53.6% higher than that in a shake flask under same conditions. Therefore, the constructed Neu5 Ac synthesis pathway attained highly efficient Neu5 Ac synthesis. |
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