Journal of Agricultural and Food Chemistry Table 1.continued HY HY.putM P43,h DR-phest-ArocA-A rms for the 14L 2.MATERIALS AND METHODS PCA in B etheles the depend ce on exogenous glutamat the ing in the of ction of -PGA it is ar to ed the GXG-5 (a dent Y-PGA-pro ce at h re mutated in GXG C fo 48h validat the in thi ce at the de HY K02174 lent s ed by e ge y,was n s we I 937 NX-2 High-th e ofrNA (RNA (so used for st dan :C0 it is sible to ide PCR IN ion ve pro typ F/pDR-put -R an pDR tain more the mechanism dep mploye NX. NX (wit ther (ATP.NADPH PGA Y-PGA this PGA PBS to a Th stud us te ydles of 3 by).The b ent 626straw14 have been developed for environmentally friendly and economical production of γ-PGA in B. subtilis NX-2. Nonetheless, the dependence on exogenous glutamate resulting in the addition of large quantities of exogenous L￾glutamate remains the major obstacle limiting the large-scale production of γ-PGA. Thus, it is necessary to reveal the mechanisms underlying glutamate dependence in glutamate￾dependent strains. Zeng et al. analyzed the difference between GXA-28 (a glutamate-dependent γ-PGA-producing strain) and GXG-5 (a glutamate-independent γ-PGA-producing strain) in terms of glutamate dependence at the genomic level, and the results showed that only 13 genes related to γ-PGA synthesis are mutated in GXG-5.15 No further validation was conducted to reveal the connection between the mutation in these genes and glutamate dependence of the strains. Furthermore, the difference at the genomic level between the dependent and independent strains, as revealed by this comparative genomics study, was not as substantial as we had expected, indicating the importance of transcriptional regulation for glutamate depend￾ence. High-throughput sequencing of RNA (RNA-Seq) is one of the most useful next-generation sequencing methods to fully elucidate the landscape of a transcriptome and has been successfully used for studying the adaptation of B. subtilis C01 to alumina nanoparticles.16 Therefore, it is feasible to identify the glutamate dependence mechanism in B. subtilis by transcriptome analysis, which might suggest new directions for improving the efficiency of γ-PGA production. B. subtilis NX-2 has been proven to be a typical efficient glutamate-dependent γ-PGA producer.4 In the present study, to gain more insights into the molecular mechanisms underlying glutamate dependence in B. subtilis, we employed global transcriptome analysis to assess the differences in gene expression between two groups: NX-2 (without glutamate addition) and NX-2(Glutamate) (with glutamate addition). Then, the significantly upregulated and downregulated genes were catalogued and analyzed to identify the key genes. The selected genes were then systematically overexpressed in B. subtilis NX-2 to characterize their functions during fermenta￾tive production of γ-PGA. Finally, the identified genes increasing γ-PGA production were artificially overexpressed in combination to obtain an increased γ-PGA yield. To the best of our knowledge, this is the first report that reveals the mechanisms behind glutamate dependence of a γ-PGA￾producing strain. The findings of this study will allow us to understand the glutamate dependence mechanism better and will provide clues regarding molecular targets for rational strain improvement. 2. MATERIALS AND METHODS 2.1. Microorganisms, Media, and Cultivation Conditions. All of the bacterial strains and plasmids used in this work are given in Table 1. B. subtilis NX-2 (CGMCC No.0833) and Escherichia coli DH5α were grown at 37 °C in Luria−Bertani (LB) medium for routine strain construction and maintenance. For γ-PGA production in B. subtilis, fermentation was carried out in a fermentation medium consisting of the following: 40 g/L glucose, 50 g/L glutamate, 5 g/L (NH4)2SO4, 2 g/L K2HPO4·3H2O, 0.1 g/L MgSO4, and 0.03 g/L MnSO4. 14 The seed culture (2%, v/v) was transferred into 80 mL of the fermentation medium in 500 mL shaking flasks. The fermentation was carried out at 32 °C with an agitation rate of 220 rpm for 66 h. When glutamate was added to the fermentation medium, the fermentation flask was incubated at 32 °C for 48 h. In addition, the relevant antibiotic (100 μg/mL ampicillin or 20 μg/mL tetracycline) was added to the medium when necessary. 2.2. Construction of Plasmid. The primers used in this study are given in Table S1. The expression vectors were constructed on the basis of pHY-300PLK. First, the P43 promoter (K02174.1) and α- amylase terminator TamyE (938356) from B. subtilis 168 and the pgi gene (937165) from B. subtilis NX-2 were amplified with the corresponding primers. The amplified fragments were ligated by splicing overlap extension PCR (SOE-PCR) with primers P43-F and TamyE-R and then cloned into pHY300PLK at the restriction sites EcoRI and HindIII, thus generating pHY-pgi. The recombinant plasmid pHY-pgi was then transferred into B. subtilis NX-2 by high￾osmolarity electroporation.17 The recombinant strain NX-2-pgi was confirmed by PCR and plasmid extraction.18 The other strains were constructed by the same method as that for NX-2-pgi and were denoted NX-2-n (n represents the name of the gene). The gene deletion and complementation vectors were constructed according to our previously reported method.18 Briefly, the homology arms of gene putM were amplified from B. subtilis NX-2 genome with primer pairs pDR-putMUP-F/pDR-putMUP-R and pDR-putMDN￾F/pDR-putMDN-R. The fragments were then fused using SOE-PCR. The resulting fragment was cloned into pDR-pheS* using the EcoRI and XhoI sites, generating pDR-pheS*-ΔputM. After sequence validation, the putM deletion plasmid was transferred into B. subtilis NX-2. After the selection of single- and double-exchange transformers, the clones obtained were verified by PCR using primers putM-OUT￾F/putM-OUT-R. Similarly, the deletion of rocA was constructed with the same method. The putM and rocA complementation strains NX-2- ΔputM-M and NX-2-ΔrocA-A were constructed by introducing the genes putM and rocA into the original locus, respectively. 2.3. Analysis of Intracellular Metabolites. The concentration of intracellular metabolites (ATP, NADPH, and glutamate) was determined using a method reported previously.19−21 The cells in the fermentation broth were harvested by centrifugation (4 °C, 8000g for 20 min) and washed three times with PBS (pH 7.0). Then, the cell pellets were resuspended in PBS to attain the desired optical density, followed by their disruption in a sonicator (600 W for 30 min with cycles of 3 s sonication followed by a 5 s pause). The broken cells were centrifuged at 8000g for 3 min, and then the concentrations of ATP and NADPH in the supernatants were measured with commercial assay kits (ATP Assay Kit, Beyotime, Jiangsu, China; Table 1. continued strain or plasmid relevant properties source Plasmids pHY-pgsBCA pHY300PLK containing P43 promoter, the gene pgsBCA and amyL terminator this study pHY-putMA pHY300PLK containing P43, the genes putM, rocA and amyL terminator this study pHY-putMA-gltB pHY300PLK containing P43, the genes putM, rocA, gltB and amyL terminator this study pHY- putMA-gltAB pHY300PLK containing P43, the genes putM, rocA, gltB, gltA and amyL terminator this study pDR-pheS*-ΔputM pDR-pheS* derivate, with homology arms for the deletion of putM gene this study pDR-pheS*-ΔrocA pDR-pheS* derivate, with homology arms for the deletion of rocA gene this study pDR-pheS*-ΔputM-M pDR-pheS* derivate, with homology arms for the complementation of putM gene this study pDR-pheS*-ΔrocA-A pDR-pheS* derivate, with homology arms for the complementation of rocA gene this study Journal of Agricultural and Food Chemistry Article DOI: 10.1021/acs.jafc.9b01755 J. Agric. Food Chem. 2019, 67, 6263−6274 6265