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N-Acetylglucosamine Fermentation Process

 

GNA1 genes from the yeasts Saccharomyces cerevisiae and Candida albicans, and the higher plant, Arabidopsis, were evaluated for NAG production. Significant amounts of NAG were produced with all three genes; however, the gene from Saccharomyces performed the best. The NAG production process was improved by optimizing lactose induction and medium compositions.

The established process conditions, summarized in Table 2, consist of an aerobic fed-batch fermentation in a defined, simple mineral medium supplemented with glucose. No antibiotic is required, since expression cassettes are stably integrated into the chromosome. Timing of lactose induction is critical. Although NAG added to the growth medium is not inhibitory to the host cells, amino sugar phosphates generated internally could potentially inhibit a number of enzymes of the central metabolism.

When NAG production was induced from the start of the fermentation, cell growth was totally inhibited and little or no NAG was produced. Therefore, a two-phase protocol was developed: first, grow the culture to a relatively high density; then, add lactose to induce NAG production. NAG titer reached 110 g/L at 72 hours in one-liter fermentors (Figure 8). The process was scaled up to 14- and 250-liter fermentors.
  

Table 2. Process Parameters for N-Acetylglucosamine Fermentation

  

Figure 8. Lactose-Induced N-Acetylglucosamine Production

NAG Production

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