| Polysialic acid(PSA), a polyanion-type linear polymer of N-acetylneuraminic acid, is a promising biomaterial that can be used as scaffold material in tissue engineering and control-release agent for protein drugs as a result of good biocompatibility, hydrophilicity, non-immunogenicity and biodegradability. In this work, using Escherichia coli K235 as the starting strain for production of PSA, different strategies were used to perturb the intracellular energy metabolism and redox state, to investigate the relationship between biosynthesis of PSA and intracellular energy metabolism.Different carbon sources with different redox state, including sorbitol, glucose, xylose and sodium gluconate, were used to investigate the relationship between biosynthesis of PSA and intracellular nucleotides in single carbon source fermentation. It was found that different nucleotides levels were achieved with different carbon sources, which had a significant influence on PSA and organic acid biosynthesis. Studies indicated the importance of sorbitol’s higher reducing state in enhancing PSA production. The effect of 10 g·L-1 different carbon sources presented in the sorbitol fermentation medium on PSA biosynthesis was investigated. The addition of different carbon sources had great influence on energy metabolism and redox state in E. coli, which resulted in lower PSA production. At the same time, ethanol that was not found in single carbon source fermentation can be synthesized in fermentation with mixing carbon sources.In addition, the effect of different carbon sources on the expression of genes related to PSA synthesis was investigated using RT-PCR. The transcriptional levels of kdsD, kpsF, kdsA, kdsC, kdsB, waaA that governed synthesis and polymerization of KDO were enhanced under glucose, xylose or sodium gluconate fermentation compared to sorbitol fermentation, indicating that LPS biosynthesis was the major metabolic route of KDO. The transcriptional level of neuS was decreased under glucose, sodium gluconate fermentation and was increased under xylose fermentation compared to sorbitol fermentation.Metabolic flux distribution of E. coli K235 was investigated under different carbon sources(sorbitol, glucose and xylose). The result suggested that metabolic flux of Ac Co A to acetic acid was increased and the competition between PSA and acetic acid biosynthesis was enhanced with glucose or xylose as single carbon source compared to sorbitol as single carbon source. Meanwhile, the flux of PPP pathway was decreased, resulting in low flux of cell growth and PSA biosynthesis with glucose or xylose as single carbon source. Transport of sorbitol needs phosphoenolpyruvate and pyruvate is generated. Subsequently, inhibition effect of energy charge to pyruvate kinase was weakened, leading to farvorable condition for cell growth and PSA biosynthesis.Nicotinic acid(NA), the precursor of NAD+, had a significant effect on synthesis of PSA. PSA and organic acid, synthesized by E. coli K235, were lower with addition of NA to the fermentation medium compared to the control. When the concentration of NA was 10 mg·L-1, PSA yield was only 0.37 g·L-1. Higher NAD+ titer was achieved with addition of NA to the fermentation medium compared to the control, which led to the raise of cellular oxidative stress, sequentially the decrease of the reducing metabolites such as polysialic acid, succinate and lactate, and even the vanish of them.Orotic acid(OA), the precursor of UMP, affected biosynthesis of PSA and organic acid. PSA fermentation by Escherichia coli K235 with the addition of OA caused an increased ethanol concentration but a decreased lactate concentration. The addition of 1.5 g·L-1 OA to the fermentation medium led to a significant increase in PSA production(2.08 g·L-1) because of increasing the availability of UTP and UDP. |
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