| Bacitracin, a broad spectrum antibiotic peptide, is synthesized non-ribosomally bystrains of Bacillus licheniformis. In this study, strategies and metabolic regulationmethods was designed to promote bacitracin synthesis efficiency in10L and2000Lscale fermenter based on the bottleneck in the fermentation process. The results were asfollows:(1) Base on the time courses of precursor amino acids, an inefficient supply ofglutamate was a metabolic bottleneck for bacitracin synthesis. Glutamate addition (0.03g/L) at20h enhanced bacitracin yield by13.4%in shaker flasks. In a10-L fermenter,glutamate addition at the rate of3mg/(L·h) during16-26h was conducted, and thebacitracin production was1020U/mL that was9.6%higher than control.(2) The acetaldehyde addition (0.05g/L) at16h in a10-L bioreacror promotedbacitracin yield by5.7%, up to952U/mL, whereas, biomass was decreased by21.4%-30.8%from18to34h relative to the control. The acetaldehyde addition (0.048g/L) from12h to22h in a2000-L bioreacror promoted bacitracin synthesisefficiencyby15.5%, corresponding to57.2U（/mL·h）, and bacitracin production at32h was1153U/mL that was4.3%higher than control. Meanwhile, the content of2,3-butanediol andacetate was lower than control after addition, which suggested that NADH oxidation byethanol formation reduced formations of acetate and2,3-butanediol and maintained ahigher bacitracin synthesis efficiency. Meanwhile, the content of pyruvate,α-ketoglutarate, citrate and succinate was21.3～45.4mg/L,13.1～26mg/L,109.3～138.5mg/L and82.5～175.8mg/L, which was increased by23.5～101.7%,38.5～122.0%,9.5～55.8%and14.8～44.5%from16h to28h relative to the control,respectively. The results suggested that acetaldehyde addition eased redox imbalance,increased TCA cycle flux, thus promoting the bacitracin synthesis.(3) The BaCl2addition (0.3mg/L) at0h in a10-L bioreacror promoted bacitracinyield by3.1%, up to939U/mL, whereas, biomass was nearly no difference with thecontrol. The bacitracin production with BaCl2addition (0.3mg/L) at0h in a2000-Lbioreacror was enhanced by3.0%corresponding to1138U/mL. However, the acetionutilization rate and2,3-butanediol synthesis from24h to28h was3.53times and3.65 times of the control, which suggested that addition accelerated the consumption ofacetoin and2,3-butanediol formation by promoting acetoin reductase activity.Meanwhile, the volatile fatty acid (VFA) content was higher than control, which wasrelative with the higher biomass of addition.(4) Glucose addition with the rate of2g/L·h from24h to28h, agitation controlsand aerate control in a2000-L bioreactor was conducted, respectively. The bacitracinsynthesis during the course of glucose addition was halted, which suggested thatglucose addition at the middle culture disturbed bacitracin synthesis severely. Throughthe controls of agitation and aerate, maintaining pH at7.4or so and the dissolvedoxygen at2or so, the cell autolysis time delayed16h, VFA content from12h to32hwas varied from4.5g/L to7.5g/L that was40-152.2%higher than the control, and thebacitracin synthesis rate from28h to36h was17U/mL·h that was2.79-time that of thecontrol. The results suggested that a higher VFA content at the later stage of culturecould facilitate bacitracin synthesis efficiency. Whereas, owe to the lower bacitracinsynthesis efficiency before28h, the production was only increased by2.6%relative tothe control in the end, which also reflected that some bottlenecks in the earlyfermentation process was still uncertain. |
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