## 3 Results
### Mechanisms for Enhanced Lycopene Accumulation by Glutamate and Proline  
No reports were found to be available on enhanced growth and lycopene accumulation in PNSB by glutamate and proline in the literature. To elucidate the mechanisms, differential expression analysis and differential metabolomic analysis were performed in the present study. The results show that, compared with the control, the addition of glutamate to the culture medium led to the upregulation of 1506 genes and the downregulation of 473 genes in the bacterial cells. Similarly, the addition of proline resulted in the upregulation of 1290 genes and the downregulation of 511 genes (Figure 4A). The differentially expressed genes were involved in energy metabolism, the TCA cycle, amino acid metabolism, fatty acid metabolism, and carotenoid metabolism, among others. Notably, genes closely related to lycopene synthesis were predominantly downregulated rather than upregulated. For instance, the expression levels of genes related to the conversion of lycopene, such as _crtC_, _crtD_, and _crtF_, were significantly lower than those in the control group. In contrast, no significant changes were observed in the expression levels of genes related to lycopene synthesis, such as _crtI_, _crtB_, and _crtE_ (Figure 4C-E). This suggests that reducing the conversion of lycopene represents the first mechanism by which glutamate or proline promotes lycopene accumulation in PNSB strain DT.1  
On the other hand, the expression of five genes involved in fatty acid synthesis--_fabB_, _fabF_, _fabG_, _fabl_, and _fabZ_--was also significantly downregulated (Figures 4F,G and 5). These genes encode enzymes responsible for synthesizing long-chain fatty acids using acetyl-CoA as the substrate. The downregulation of expression of these genes implies a reduction in the consumption of acetyl-CoA within the cells, which is evidently conducive to increasing intracellular acetyl-CoA levels. Concurrently, the expression of genes in the TCA cycle, such as _glutA_ and _acnA_, as well as the gene _glIB_ in amino acid metabolism, was also significantly downregulated. These genes encode enzymes that catalyze the stepwise conversion of  
Figure 4: Transcriptome and metabolome analysis and RT-qPCR validation. (**A**) Number of differentially expressed genes. (**B**) Number of differential metabolites (**C–H**). RT-qPCR validation of differentially expressed genes, where \({}^{*}\) denotes \(p<0.05\) and \({}^{**}\) denotes \(p<0.01\). acetyl-CoA, ultimately leading to the synthesis of glutamate. Therefore, the downregulation of these genes also contributes to an increase in intracellular acetyl-CoA levels [28; 29]. The accumulation of acetyl-CoA can inhibit the activity of the pyruvate dehydrogenase complex, leading to the buildup of pyruvate, thereby providing more precursors for lycopene synthesis. Metabolomic results confirm that acetyl-CoA and pyruvate were significantly upregulated in treatments supplemented with glutamate or proline. Based on the above results, a second mechanism is proposed in which the downregulation of genes encoding enzymes that consume acetyl-CoA results in increased intracellular levels of acetyl-CoA and pyruvate, which, in turn, enhances the availability of the precursor substances required for lycopene synthesis. Differential metabolomic analysis showed, that compared with the control, 83 metabolites were upregulated and 154 metabolites were downregulated (POS), 70 metabolites were upregulated and 116 metabolites were downregulated (NEG) in the DT.1 cells cultured in the medium supplemented with glutamate culture. Additionally, 74 metabolites were upregulated and 151 metabolites were downregulated for POS and 88 metabolites were upregulated and 105 were downregulated for NEG in the DT.1 cells cultured in the medium supplemented with proline (Figure 4B).