Journal of Agricultural and Food Chemistry Article Table 2.Comparison of y-PGA Fermentation Proces ong Recombinants and Wild-Type Strain strain s(/L) dual glucose(g/L】 -PGA (g/L) e(mg/gpcw) 421±035 Nx22 4.48±03 383±036 763±036 2236±1.8 Nx.24 456034 331±038 102042 2532士192 and rocA were ow explained by adapt to the changes distri also increased, rocA tructed two the ant ere de ted a NX-2-AputM and tigate the eff hese gene tor y the argin prolin the mut metabolites in NX-2 and NX-2(Glutan de with those of the pd- in These mpanson purposes.As 138 the uption 81 NX2-gB(1239 ±179mg say.the by rely,at the to str NX-2 3 were co pparableto thos of NX-and ere was a e e levels. in B.iNK-2 si th ot pr ate were nat int of the NX-2.This is is is the io 8 is pa nate-dep ent s rains In this s th strain was PCA-producing rate tation by strengthening the synthesis of intrac n glut .of th 3.5.Enhancement of the y-PGA Synthe sis Module.In E to ds.Ths the s an rol ove d in tha th fate and ucos verexpression and ed that DegS-DegU activate s bio ng no ne th rate. n ast to sthase (GS-GOGAT)P and the up u and theg upregul the n of y-PG in Bo ll th rielded the tilis.Furthe t the nla ep of glut -PGA ctive oenhpeeeievewnton to n oth put （encoding prolin hanism or gluta dence in strains and rocA were overexpressed, the synthesis of intracellular glutamate was enhanced. To adapt to the changes of intracellular glutamate concentration, the transcription of DegQ and DegU was also increased, which further activated the expression of γ-PGA synthase and resulted in high levels of γ-PGA production. To further investigate the effects of these genes over￾expression on the intracellular metabolites, the concentrations of the intracellular glutamate, arginine, and proline were measured in this study. The concentrations of these metabolites in NX-2 and NX-2(Glutamate) were also detected as a control for comparison purposes. As shown in Figure 3E, the intracellular glutamate concentrations of NX-2-gltA (13.85 ± 1.81 mg/gDCW), NX-2-gltB (12.39 ± 1.79 mg/gDCW), NX-2- putM (18.32 ± 1.92 mg/gDCW), and NX-2-rocA (16.84 ± 1.85 mg/gDCW) increased by 50.21%, 34.38%, 98.70%, and 82.65% in comparison to strain NX-2 (9.22 ± 1.82 mg/gDCW). The arginine and proline concentrations of NX-2-gltA and NX-2- gltB were comparable to those of NX-2, and there was a slight decrease in the arginine and proline concentrations in NX-2- putM and NX-2-rocA. Moreover, the arginine and proline levels in the strain NX-2(Glutamate) were even slightly higher than those of the NX-2. This is contrary to the expectation that the downregulation of the arginine and proline synthesis pathways and the upregulation of the proline degradation pathway might decrease the concentrations of arginine and proline greatly. Previous studies have demonstrated that there are several routes for the biosynthesis of arginine and proline proceeding from glutamate.38 Therefore, even though one of the synthesis pathways is inhibited, the other synthesis pathways of arginine and proline will provide supplements continuously to maintain a reasonable concentration for bacterial growth and other metabolic needs. This might be one reason for the small differences in arginine and proline concentrations in B. subtilis NX-2, which supported the cell growth to high density and maintained the cellular vitality. These results proved that overexpression of genes gltA, gltB, putM, and rocA for glutamate generation is an effective way to improve γ-PGA production during no-glutamate cultivation. In B. subtilis, genes gltA and gltB are involved in the glutamine synthesis− glutamate synthase (GS-GOGAT) pathway, and the upregu￾lation of gltA would push more carbon flux distribution toward the glutamic acid point, thereby further increasing the production of γ-PGA.39 Notably, among all the genes expressed, the gene putM yielded the greatest improvement in γ-PGA production. Li et al. revealed that the overexpression of the gene ycgM (encoding proline dehydrogenase) and ycgN (encoding Δ1 -pyrroline-5- carboxylate dehydrogenase) decreases the production of γ- PGA by B. licheniformis WX-02 by disturbing the intrinsic reactive oxygen species level.40 In contrast to B. licheniformis WX-02, overexpression of both putM (encoding proline dehydrogenase) and rocA (encoding Δ1 -pyrroline-5-carbox￾ylate dehydrogenase) increased γ-PGA production in B. subtilis. This difference might be partially explained by differences in the distribution of reactive oxygen species between B. licheniformis and B. subtilis. To analyze the role of putM and rocA in more detail, we constructed two gene disruption mutants of the putM and rocA genes, respectively. Then, the mutant strains were designated as NX-2-ΔputM and NX-2-ΔrocA and were investigated for γ-PGA production in the fermentation medium. As shown in Table S4, the concentration of intracellular glutamate of the mutants was decreased, while the intracellular proline level was increased in comparison with those of the wild-type strain. These results indicated that the disruption of putM and rocA genes has a negative effect on the glutamate accumulation in B. subtilis. In addition, for the complementation assay, the single deletion strain was further complemented by chromosomal reintegra￾tion of putM and rocA genes, respectively, at the original locus to validate the function of the deleted genes. Complementation of the putM and rocA gene in mutant strains restored the intracellular glutamate and proline levels. In conclusion, the genes putM and rocA play key roles in the catabolism of proline into glutamate in B. subtilis NX-2. Collectively, these results strongly indicated that intracellular glutamate synthesis is the key metabolic node limiting γ-PGA production in glutamate-dependent strains. In this study, the glutamate-dependent strain was first engineered into a glutamate-independent strain during γ-PGA-producing fer￾mentation by strengthening the synthesis of intracellular glutamate. 3.3.5. Enhancement of the γ-PGA Synthesis Module. In B. subtilis, the response regulators (DegQ, DegU, and DegS) activate the γ-PGA synthetase genes (pgsB, pgsC, and pgsA) for γ-PGA production.41 To verify the effects of regulators and pgsBCA operon on γ-PGA production without the addition of glutamate, these genes were overexpressed in strain NX-2. As illustrated in Figure 3F, the overexpression of DegU and DegS upregulated the growth rate and glucose consumption. Stanley et al. revealed that DegS-DegU activates biofilm formation for increased resistance to environmental stress, which might be the reason for upregulation of the growth rate.42 In contrast to our speculation, overexpression of the pgsBCA operon decreased glucose consumption and the growth rate remarkably (Figure 3F). According to Feng et al., upregulation of pgsBCA expression may disrupt the cell balance or other membrane-associated metabolic activity in Bacillus sp.12 Thus, pgsBCA overexpression posed a great threat to the growth of B. subtilis. Furthermore, none of these manipulations had active effects on γ-PGA production, suggesting that the γ-PGA synthesis pathway does not play a key role in the dependence of glutamate in B. subtilis for γ-PGA production. In this study, the five metabolic modules related to γ-PGA production were strengthened individually to reveal the mechanism of glutamate dependence in B. subtilis strains. The overexpression of the significantly upregulated genes involved in glycolysis, the PPP, TCA cycle, and the γ-PGA Table 2. Comparison of γ-PGA Fermentation Process among Recombinants and Wild-Type Strain strain biomass (g/L) residual glucose (g/L) γ-PGA (g/L) intracellular glutamate (mg/gDCW) NX-2 4.03 ± 0.32 7.21 ± 0.41 0 9.22 ± 1.82 NX-2-putM 4.38 ± 0.35 4.41 ± 0.39 4.21 ± 0.35 18.32 ± 1.88 NX-22 4.48 ± 0.35 3.83 ± 0.36 7.63 ± 0.36 22.36 ± 1.84 NX-23 4.50 ± 0.38 3.62 ± 0.37 8.25 ± 0.38 23.45 ± 1.86 NX-24 4.56 ± 0.34 3.31 ± 0.38 10.21 ± 0.42 25.32 ± 1.92 Journal of Agricultural and Food Chemistry Article DOI: 10.1021/acs.jafc.9b01755 J. Agric. Food Chem. 2019, 67, 6263−6274 6270