关键词: 地衣芽孢杆菌；羟基酪醇；转录组学；代谢组学；发酵优化；柠檬酸；豆油

Abstract: To enhance the hydroxytyrosol (HT) biosynthesis efficiency of Bacillus licheniformis, transcriptomic and metabolomic differential analyses were performed on samples collected at three fermentation stages: 14 h (biosynthetic phase), 16 h (stagnant phase), and 18 h (secondary biosynthetic phase). In the stagnant phase, the expression levels of Embden-Meyerhof-Parnas (EMP) pathway-related genes including pgi, fbaA and pdhC were down-regulated by 42.4%–80.9%, accompanied by a 14%–16% reduction in the intracellular abundances of glucose and pyruvate. The expression levels of the zwf, tkt and gndA genes were down-regulated by 64.1%–95.6%, and the abundances of ribose 5-phosphate and sedoheptulose 7-phosphate were increased by 97% and 68%, respectively. The expression levels of the citA, icd, sdhA and mdh genes were down-regulated by 75.0%–92.1%, leading to a 37%–47% decrease in the levels of isocitrate, succinate, fumarate, and malate. The expression levels of the organic acid transporter genes yhfQ, maeN, and cimH were down-regulated by 71.9%–73.0%. The expression levels of the oxidative phosphorylation genes atpA, ctaE and sdhB were down-regulated by 89.5%–92.2%, and those of the HT biosynthetic genes aroCDK, yugJ and hpaC by 53.3%–96.6%. These results demonstrated that cellular carbon metabolism and energy metabolism were severely restricted during the stagnant phase. After oxygen supply was enhanced at 17 h, the expression levels of pgi and fbaA were increased by 356%–994%, those of zwf, tkt and gndA by 301%–4 084%, those of pdhC, citA, icd, sdhA, lpdV, nadB, and rocG by 204%–2 325%, those of atpA, ctaE and sdhE by 443%–1 245%, and those of aroA, aroC, tyrA and hpaC by 244%–736%; the expression level of the citrate transporter gene cimH was up-regulated by 10-fold, while that of the β-oxidation gene fadABDN was down-regulated by 94.1%–99.1%. The intracellular abundances of pyruvate, glyceraldehyde 3-phosphate, citrate, oxalosuccinate, fumarate, malate and ribose 5-phosphate were increased by 10%–125% compared with those at 16 h (stagnant phase). Collectively, elevated aeration effectively boosted the efficiency of glycolysis and the pentose phosphate pathway (HMP), and revealed the potential of citrate and fatty acids to participate in carbon metabolism. On this basis, an optimized fermentation strategy integrating pH-controlled citrate supplementation and soybean oil addition was established. Using this strategy an HT yield of 7.21 g/L was achieved, which was 106.6% higher than that of the control group (3.49 g/L). This study provides a valuable reference for the industrial fermentative production of HT.

Key words: Bacillus licheniformis; hydroxytyrosol; transcriptomics; metabolomics; fermentation optimization; citric acid; soybean oil