Journal of Agricultural and Food Chemistry thus inhibit 251 g ese th the glycolyti tho e of strain NX-2 the PPP flu n.Prev studie the s(w pgl,and gnd)on y not highest NADPH titer the t thesi (-PGA- r Dee an and 4A.the of was an A d srupt the balanceof NaDPH/naDe e r depe he TCA cycle i mediate amic acid is the p for the svnthesi of y-PGA an 易pw for the b10 hA.and f )As p d NX-2-icd 0.21,0.42±0.22 y.As th ther acce ate the of a-k oglut ate to had ca no signi ts ot the relative tr citA,citE A rate,af sis:(B)genes related to regulatio 34n t of t of h ndicated that ther this success h f the fo gre the expression ous study has rev 3D the r roduction of y-PGA 626thus inhibit the conversion of acetyl-CoA in the TCA cycle.31 As a result, glucose metabolism was found to be reduced in pdhA-, pdhB-, and pdhC-overexpressing strains. Additionally, overexpression of all these genes had no significant effect on γ- PGA production, which reflected that the glycolytic pathway is not the key regulator of glutamate dependence in B. subtilis. 3.3.2. Enhancement of the PPP. Strengthening the PPP flux increases the NADPH supply, which may be beneficial for product biosynthesis.32 In this study, the effects of over￾expression of PPP related genes (zwf, pgl, and gnd) on γ-PGA biosynthesis were investigated. As shown in Figure S2, the highest NADPH titer of 25.21 ± 0.85 μmol/gDCW was obtained by overexpression of zwf; this NADPH titer was 2.59-fold higher than that of the wild-type strain. The NADPH productions of the other strains, NX-2-pgl and NX-2-gnd, were 14.43 ± 0.83 and 18.56 ± 0.82 μmol/gDCW, respectively. These results indicated that overexpression of zwf was an effective way to improve NADPH production. However, glucose consumption, cell growth, and γ-PGA biosynthesis did not increase obviously in these recombinant strains (Figure 3B). The reason may be that overexpression of zwf weakens the glycolytic pathway, which further affects the overflow metabolism.33 Moreover, overaccumulation of NADPH may disrupt the balance of the NADPH/NADP+ cofactor pair, which is a prerequisite for both catabolism and anabolism.34 Thus, there was no noticeable influence of zwf, pgl, and gnd overexpression on γ-PGA production. These results confirmed that the PPP is not the key factor for glutamate dependence in B. subtilis. 3.3.3. Enhancement of the TCA Cycle. The TCA cycle is responsible for the complete oxidation of acetyl-CoA and formation of intermediates required for ATP production and other anabolic pathways, such as amino acid biosynthesis.35 Glutamic acid is the precursor for the synthesis of γ-PGA and is mainly generated from α-ketoglutarate from the TCA cycle when no glutamic acid is added into the medium.36 Thus, an adequate supply of α-ketoglutarate for the biosynthesis of glutamate was expectedly achieved after the overexpression of these genes (citA, citB, icd, sdhA, and fumC). As presented in Figure 3C, the γ-PGA yields of strains NX-2-citA, NX-2-citB, and NX-2-icd were slightly higher: 0.51 ± 0.21, 0.42 ± 0.22, and 0.34 ± 0.24 g/L, respectively. As the first and key enzyme in the TCA cycle, CitA converts acetyl-CoA to citrate, which further accelerates the conversion of α-ketoglutarate to glutamate and eventually improves the γ-PGA production. Nonetheless, the overexpression of sdhA and fumC had almost no influence on γ-PGA synthesis. The reason may be that the overexpression of sdhA and fumC had negative effects on α- ketoglutarate accumulation, thereby causing no significant increase in the γ-PGA synthesis. Collectively, these results suggested that the overexpression of citA, citB, and icd further shifted the carbon flux to α-ketoglutarate, affording a slight increase in γ-PGA production. 3.3.4. Enhancement of the Glutamate Synthesis Pathway. The production of γ-PGA was slightly improved by modification of the TCA cycle, likely by increasing the accumulation of α-ketoglutarate. In addition, this result indicated that there was an increase in the pool of intracellular glutamate, but greater flux was needed for shifting to the synthesis of glutamate. For this purpose, we overexpressed genes gltA, gltB, putM, rocA, and racE, thus generating strains NX-2-gltA, NX-2-gltB, NX-2-putM, NX-2-rocA, and NX-2-racE, respectively. As shown in Figure 3D, the production of γ-PGA was significantly (p < 0.05) increased from 0 to 2.51 ± 0.31, 2.03 ± 0.32, 4.21 ± 0.35, and 3.52 ± 0.32 g/L by the overexpression of gltA, gltB, putM, and rocA, respectively. The glucose consumption and growth rates of the four strains accordingly increased in comparison to those of strain NX-2. However, the overexpression of racE did not have a significant effect on the γ-PGA production. Previous studies have revealed that racE is responsible for the conversation of L-glutamate to D-glutamate and is not necessary for γ-PGA synthesis in B. subtilis. 37 Therefore, enhanced expression of racE did not increase the γ-PGA yield. In addition, the transcription of the key enzymes involved in glutamate synthesis (GltA, GltB, PutM, and RocA) and γ-PGA synthesis (γ-PGA-synthesis regulator DegQ and DegU and γ- PGA synthase PgsB and PgsC) was investigated in different B. subtilis derivatives. As shown in Figure 4A, the transcription levels of gltA, gltB, putM, and rocA were increased by 3.7-, 3-, 4-, and 3.5-fold, respectively, indicating that gltA, gltB, putM, and rocA were overexpressed successfully in B. subtilis NX-2. As shown in Figure 4B, overexpression of the four genes upregulated the expression of DegQ, DegU, pgsB, and pgsC remarkably in comparison with those in the control strain. A previous study has revealed that DegQ and DegU are important response regulators of γ-PGA synthase and their expression is regulated by glutamate concentration.18 When gltA, gltB, putM, Figure 4. Effects of overexpression of genes involved in glutamate synthesis pathway on the relative transcriptional levels of regulatory genes and γ-PGA biosynthesis genes: (A) genes related to glutamate synthesis; (B) genes related to regulation and biosynthesis of γ-PGA. Journal of Agricultural and Food Chemistry Article DOI: 10.1021/acs.jafc.9b01755 J. Agric. Food Chem. 2019, 67, 6263−6274 6269