关键词: 地衣芽孢杆菌；细胞生长；补料优化；转录组分析；代谢组分析

Abstract: To study the limiting factors of cell growth and sporulation during the late stage of Bacillus licheniformis HW fermentation in a 30 L bioreactor, we analyzed the differences in the transcriptome and metabolome of strain HW at the middle (25 h) and late (32 h) logarithmic growth stages. It was found that the expression levels of the glycolysis genes gapA, pfkA, and pyK decreased by 65.1% to 80.5% at the late logarithmic growth stage; the tricarboxylic acid (TCA) cycle genes sucC, sdhB, and fumC decreased by 56.8% to 80.2%, and the abundance of the corresponding metabolites citric acid, isocitric acid, cofactor NAD+, and NADH decreased by 32.2% to 96.3%. The gluconeogenesis pathway genes pckA and gapB increased by 1.21 and 1.29 times, respectively. Additionally, the phospholipid synthesis genes tagB, tagD, and tagO decreased by 54.6% to 64.0%, and the abundance of the related metabolites N-acetylglucosamine, 6-phosphoadenosylglucosamine, and UDP-N-acetylglucosamine significantly decreased by 35.4% to 95.6%. The amino acid metabolism-related genes gltA, gltB, gdhA, avtA, and serC decreased by 71.3% to 98.8%, while aspB expression increased by 1.849 times. The expression levels of pdxT and pdxK decreased by 64.8% and 51.4%, respectively; the abundance of the corresponding metabolites glutamic acid, glutamine, valine, and aspartic acid decreased by 20% to 56%. Additionally, the genes related to spore synthesis spo0A, spo0F, kinA, and kinE were downregulated by 51% to 81%. Meanwhile, the Spo0A–Spo0P inhibiting genes abrB and rapH were upregulated by 1.439 to 4.653 times, while the spore coat synthesis genes cotE, cotF, cotA, cotY, cotZ, and yhcN were downregulated by 81.3% to 91.2%. Furthermore, the abundance of tetrapeptide subunits in the spore cortex (such as lysyl-aspartyl-glutamyl-leucine) and tripeptide subunits in the spore coat (such as leucyl-prolyl-isoleucine) was reduced by 48.6% to 77.1%. This indicates that during the logarithmic phase of strain HW fermentation, there are bottlenecks including insufficient energy and phosphate supply as well as low amino acid metabolic efficiency. Based on this, by uniformly supplementing glucose to a final concentration of 3% from 20 to 26 h of fermentation and phosphates to 0.1% and VB6 to 50 mg/L from 20 to 22 h, we found that the biomass reached a peak of 5.11 × 1010 CFU/mL after 33 h and the spore count reached 4.89 × 1010 CFU/mL, which increased by 44.7% and 60.3% when compared with the control without supplementation, respectively. This feeding strategy significantly enhanced both cell growth and sporulation efficiency, providing theoretical and technical support for high-yield spore production of B. licheniformis on an industrial scale.

Key words: Bacillus licheniformis; cell growth; feeding optimization; transcriptome analysis; metabolome analysis