Joural of Agricultural and Food Chemistry Article This ■ASSOCIATED CONTENT K-2 was success y engin here upporting Info tio isavailable free of chargeon the ower than that of so dent strain the optimizations including metabolic engineering strat D0:10.1021/acs jafe.9%01755. d fo the fermentation process are used for PCR in this study Effects of Glutamate Addition on y-PGA ated and de y-PCA the diffe nentat entrati of glu dded to the m a to es (DEC EGs),GO enrichm nd KEG ut the optim. -PGA prod nd on the NADPH acct to 50 g/L the -PGA yield in sed co and the maximum-PGA yield the y-PC with the ■AUTHOR INFORMATION wth and the th 58139433;e-mail,xuh @njtech.edu.cn ded 40 40 a y-PGA production of 35. This ng rk was funded by the National Natural Scie 0f63. of C na (No 218 52 and 2177633 oy R vince Pol ce Program (N able 7-PGA e In of the (No.) -PG NX-24 was not the athors declare no competing financial interest addition,the and yield on total suga ■REFERENCES of th 0.44 1)H Y.H:Huang K.Y,Kunl within a relatively short time (48 h).When NX-24 8mL2017,182264 i were at a persp eg o th 3 of Nh synth with gl tha ate c could facilitate the utilization of (4)Tang B.Xu.H.:Xu. es in B. is cons d)pro sults.B.subtilis NX-24 is a promisine producer for X. of poly glntamic acid cient y-PGA pro th mo 3- ate der PGA (6)Ko y H. ducing ferm ntation.Genes involved the PPP mic 1998,37%450437 sts,an eo Y-PG ion of halo strains cultivated with and without added to 1600 133 nthesis has a ke e in the of r-PGA r uctior lutamate-depe ndent strain This study offer poter 20 ngA:Hong Y.;Hung produced via glutamate-intensive pathways of po-uta acid)with organic acid addition in 627 This approach is a new and efficient genetic engineering method for economical γ-PGA production. Although B. subtilis NX-2 was successfully engineered here to synthesize γ-PGA without exogenous glutamate addition, the yield of γ-PGA is still lower than that of some glutamate-independent strains. Further optimizations including metabolic engineering strat￾egies and improvement in the fermentation process are required for desirable γ-PGA production.43 3.5. Effects of Glutamate Addition on γ-PGA Production. To further analyze the influence of glutamate addition on γ-PGA production by strain NX-24, different concentrations of glutamate were added to the media to screen out the optimal content for γ-PGA production. As shown in Figure 5, when the glutamate concentration increased from 10 to 50 g/L, the γ-PGA yield increased continuously and the maximum γ-PGA yield reached 38.46 ± 0.36 g/L with 50 g/L glutamate addition. Although the γ-PGA production was enhanced with the increased glutamate concentration, the cell growth was inhibited and the apparent conversion rate of glutamate to γ-PGA decreased gradually as the glutamate exceeded 40 g/L. The addition of 40 g/L glutamate resulted in a γ-PGA production of 35.52 ± 0.26 g/L with a maximum apparent conversion rate of glutamate to γ-PGA of 63.27%. Thus, 40 g/L glutamate is suitable for high γ-PGA production by B. subtilis NX-24. Table 3 compares the γ-PGA titers and productivities reported in the literature for glutamate-dependent and glutamate-independent production and the data of the present study. Generally, the γ-PGA yield of NX-24 was not the highest, but the productivity and yield on total sugar were relatively higher than those of the other strains. With glutamate addition, the productivity and yield on total sugar of the strain NX-24 were 0.74 g/(L h) and 0.44 g/g, respectively, indicating that most of the carbon source could be converted to γ-PGA within a relatively short time (48 h). When NX-24 was cultured without glutamate addition, the yield and productivity of γ-PGA were at a moderate level in comparison to the other glutamate-independent strains. This result may be due to the enhancement of intracellular glutamate synthesis. Interestingly, the yield on total sugar of NX-24 with glutamate addition was higher than that of the strain without glutamate addition, which indicated that glutamate could facilitate the utilization of carbon sources in B. subtilis. This phenomenon is consistent with the result of transcriptome assay. On the basis of the above results, B. subtilis NX-24 is a promising producer for efficient γ-PGA production. In conclusion, our results provide the first insights into the glutamate dependence mechanism in B. subtilis during γ-PGA￾producing fermentation. Genes involved in glycolysis, the PPP, TCA cycle, glutamate synthesis, and the γ-PGA synthesis pathway were identified by comparing the transcriptome data of strains cultivated with and without added glutamate. Overexpression of genes gltA, gltB, putM, and rocA led to γ- PGA accumulation, suggesting that intracellular glutamate synthesis has a key role in the regulation of γ-PGA production in glutamate-dependent strains. This study offers potential metabolic targets for further improvement of γ-PGA production and is applicable to a wide range of compounds produced via glutamate-intensive pathways. ■ ASSOCIATED CONTENT *S Supporting Information The Supporting Information is available free of charge on the ACS Publications Web site. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b01755. Primers and their sequences used for PCR in this study, primer sequences used for qRT-PCR, list of genes upregulated and downregulated by glutamate addition, effects of putM and rocA deletion and complementation on γ-PGA production, analysis of the differentially expressed genes (DEGs), GO enrichment and KEGG pathway, effects of the overexpression of zwf, pgl, and gnd on the NADPH accumulation in Bacillus subtilis, and expression level of candidate genes determined using quantitative PCR (qRT-PCR) (PDF) ■ AUTHOR INFORMATION Corresponding Author * H.X.: tel/fax, +86-25-58139433; e-mail, xuh@njtech.edu.cn. Funding This work was funded by the National Natural Science Foundation of China (Nos. 21878152 and 21776133), the Natural Science Foundation of Jiangsu Province (No. 55129017), Jiangsu Province Policy Guidance Program (No. BZ2018025), Nanjing Science and Technology Program (No. 201818026), and the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (No. XTB1804). Notes The authors declare no competing financial interest. ■ REFERENCES (1) Hsueh, Y. H.; Huang, K. Y.; Kunene, S. C.; Lee, T. Y. Poly-γ- glutamic acid synthesis, gene regulation, phylogenetic relationships, and role in fermentation. Int. J. Mol. Sci. 2017, 18 (12), 2644. (2) Luo, Z.; Guo, Y.; Liu, J.; Qiu, H.; Zhao, M.; Zou, W.; Li, S. Microbial synthesis of poly-γ-glutamic acid: current progress, challenges, and future perspectives. Biotechnol. Biofuels 2016, 9 (1), 134. (3) Chettri, R.; Bhutia, M. O.; Tamang, J. P. 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