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甘肃农业大学：食品科学与工程学院（文献讲义）Investigation of Glutamate Dependence Mechanism for Poly-γglutamic Acid Production in Bacillus subtilis on the Basis of Transcriptome Analysis

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Investigation of Glutamate Dependence Mechanism for Poly-γ- glutamic Acid Production in Bacillus subtilis on the Basis of Transcriptome Analysis Yuanyuan Sha,†,‡ Tao Sun,†,‡ Yibin Qiu,†,‡ Yifan Zhu,†,‡ Yijing Zhan,†,‡,§ Yatao Zhang,†,‡ Zongqi Xu,†,‡ Sha Li,†,‡ Xiaohai Feng,†,‡ and Hong Xu*,†,‡ † State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing 211816, People’s Republic of China ‡ College of Food Science and Light Industry, Nanjing Tech University, Nanjing 211816, People’s Republic of China § Nanjing Shineking Biotech Co., Ltd., Nanjing 210061, People’s Republic of China *S Supporting Information ABSTRACT: The development of commercial poly-γ-glutamic acid (γ-PGA) production by glutamate-dependent strains requires understanding the glutamate dependence mechanism in the strains. Here, we first systematically analyzed the response pattern of Bacillus subtilis to glutamate addition by comparative transcriptomics. Glutamate addition induced great changes in intracellular metabolite concentrations and significantly upregulated genes involved in the central metabolic pathways. Subsequent gene overexpression experiments revealed that only the enhancement of glutamate synthesis pathway successfully led to γ-PGA accumulation without glutamate addition, indicating the key role of intracellular glutamate for γ-PGA synthesis in glutamate-dependent strains. Finally, by a combination of metabolic engineering targets, the γ-PGA titer reached 10.21 ± 0.42 g/L without glutamate addition. Exogenous glutamate further enhanced the γ-PGA yield (35.52 ± 0.26 g/L) and productivity (0.74 g/(L h)) in shake-flask fermentation. This work provides insights into the glutamate dependence mechanism in B. subtilis and reveals potential molecular targets for increasing economical γ-PGA production. KEYWORDS: poly-γ-glutamic acid, glutamate dependence, Bacillus subtilis NX-2, transcriptome 1. INTRODUCTION Poly-γ-glutamic acid (γ-PGA) is a natural multifunctional biopolymer composed of D- and/or L-glutamic acid units that are connected by γ-amide linkages. The biopolymer has a molecular weight ranging from 100 to over 1000 kDa and is mainly synthesized by microbial fermentation.1 With excellent characteristics such as water solubility, biocompatibility, and edibility, γ-PGA has been widely applied in food, medicine, agriculture, and cosmetics.2 Substantial efforts have been made in the large-scale production of γ-PGA: e.g., screening of producers with good productive performance, use of cheaper and renewable substrates, and optimization of the fermentation process.3−5 Selection of γ-PGA-overproducing strains is one of the most effective ways to improve γ-PGA productivity. The species of Bacillus are the main γ-PGA-producing strains.2 On the basis of their glutamic acid requirement, the γ- PGA-producing strains are usually classified into two groups: (1) the glutamate-dependent strains, which can produce γ- PGA only with exogenous L-glutamate supplementation, including Bacillus subtilis NX-2,4 Bacillus licheniformis ATCC 9945a,6 and Bacillus licheniformis WX-02,7 and (2) the glutamate-independent strains that can synthesize γ-PGA de novo from carbon sources without the addition of exogenous glutamate, e.g., Bacillus amyloliquefaciens LL3,8 B. subtilis C10,9 and Bacillus licheniformis A35.10 Due to their economics and simplified fermentation process, the glutamate-independent strains have attracted considerable attention. Several strategies have been developed to improve γ-PGA production in the glutamate-independent strains. For instance, double deletion of genes cwlO (encodes a cell wall−lytic enzyme) and epsA-O cluster (responsible for extracellular polysaccharide synthesis) in B. amyloliquefaciens LL3 results in a 63.2% increase in the production of γ-PGA.11 Furthermore, the γ-PGA titer is increased 2.91-fold in B. amyloliquefaciens NK-1 when a systematic modular pathway engineering method is employed.12 Although the production of γ-PGA has been significantly improved via modification of γ-PGA synthesis related metabolic pathways, the γ-PGA production in the glutamate-independent strains was much lower than that of the glutamate-dependent strains. Therefore, it is a promising strategy to choose glutamate-dependent strains for the industrial production of γ-PGA. For a broad range of applications, the cost of bioproduct production is the main factor determining the economic viability of a fermentation process.13 Reducing the cost of biopolymer production by optimizing the medium components is crucial for industrial applications. Over the past few years, several studies have focused on reducing the cost of fermentation via development of cheaper and greener carbon sources as well as on improvement of the γ-PGA yield. Mushroom residues,4 untreated cane molasses,5 and rice Received: March 19, 2019 Revised: May 12, 2019 Accepted: May 15, 2019 Published: May 15, 2019 Article Cite This: J. Agric. Food Chem. 2019, 67, 6263−6274 pubs.acs.org/JAFC © 2019 American Chemical Society 6263 DOI: 10.1021/acs.jafc.9b01755 J. Agric. Food Chem. 2019, 67, 6263−6274 Downloaded via JIANGNAN UNIV on October 19, 2019 at 05:39:06 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles

Table 1. Strains and Plasmids Used in This Study strain or plasmid relevant properties source Strains B. subtilis NX-2 wild-type strain, CGMCC No.0833 CGMCC B. subtilis 168 Tthe strain with P43 promoter this lab E. coli DH5α F−,φ80dlacZΔM1, Δ(lacZYA-argF) U169, deoR, recA1, endA1, hsdR17(rk−, mk+ ), phoA, supE44, λ−thi-1, gyrA96, relA1 this lab E. coli GM2163 F−, ara-14 leuB6 thi-1 fhuA31 lacY1 tsx-78 galK2 galT22 supE44 hisG4 rpsL 136 (StrR ) xyl-5 mtl-1 dam13::Tn9 (CamR ) dcm-6 mcrB1 hsdR2 mcrA this lab NX-2-zwf NX-2 derivative, overexpression of zwf this study NX-2-pgl NX-2 derivative, overexpression of pgl this study NX-2-gnd NX-2 derivative, overexpression of gnd this study NX-2-pgi NX-2 derivative, overexpression of pgi this study NX-2-pfkA NX-2 derivative, overexpression of pfkA this study NX-2-pdhA NX-2 derivative, overexpression of pdhA this study NX-2-pdhB NX-2 derivative, overexpression of pdhB this study NX-2-pdhC NX-2 derivative, overexpression of pdhC this study NX-2-citA NX-2 derivative, overexpression of citA this study NX-2-citB NX-2 derivative, overexpression of citB this study NX-2-icd NX-2 derivative, overexpression of icd this study NX-2-sdhA NX-2 derivative, overexpression of sdhA this study NX-2-fumC NX-2 derivative, overexpression of fumC this study NX-2-gltA NX-2 derivative, overexpression of gltA this study NX-2-gltB NX-2 derivative, overexpression of gltB this study NX-2-putM NX-2 derivative, overexpression of putM this study NX-2-rocA NX-2 derivative, overexpression of rocA this study NX-2-racE NX-2 derivative, overexpression of racE this study NX-2-DegQ NX-2 derivative, overexpression of DegQ this study NX-2-DegU NX-2 derivative, overexpression of DegU this study NX-2-DegS NX-2 derivative, overexpression of DegS this study NX-2-pgsBCA NX-2 derivative, overexpression of pgsBCA this study NX-22 NX-2 derivative, combinatorial overexpression of putM and rocA this study NX-23 NX-2 derivative, combinatorial overexpression of putM, rocA and gltB this study NX-24 NX-2 derivative, combinatorial overexpression of putM, rocA, gltB and gltA this study NX-2-ΔputM NX-2 derivative, deletion of putM gene this study NX-2-ΔrocA NX-2 derivative, deletion of rocA gene this study NX-2-ΔputM-M NX-2-ΔputM derivative, complemented with putM at original locus this study NX-2-ΔrocA-A NX-2-ΔrocA derivative, complemented with rocA at original locus this study Plasmids pHY300PLK E. coli and B. subtilis shuttle vector; AmpR , TetR TaKaRa, Dalian, China pDR-pheS* pDR with P43-pheS* cassette inserted in the multicloning sites EcoRI and SpeI this laboratory pHY-zwf pHY300PLK containing P43 promoter, the gene zwf and amyL terminator this study pHY-pgl pHY300PLK containing P43 promoter, the gene pgl and amyL terminator this study pHY-gnd pHY300PLK containing P43 promoter, the gene gnd and amyL terminator this study pHY-pgi pHY300PLK containing P43 promoter, the gene pgi and amyL terminator this study pHY-pfkA pHY300PLK containing P43 promoter, the gene pfkA and amyL terminator this study pHY-pdhA pHY300PLK containing P43 promoter, the gene pdhA and amyL terminator this study pHY-pdhB pHY300PLK containing P43 promoter, the gene pdhB and amyL terminator this study pHY-pdhC pHY300PLK containing P43 promoter, the gene pdhC and amyL terminator this study pHY-citA pHY300PLK containing P43 promoter, the gene citA and amyL terminator this study pHY-citB pHY300PLK containing P43 promoter, the gene citB and amyL terminator this study pHY-icd pHY300PLK containing P43 promoter, the gene icd and amyL terminator this study pHY-sdhA pHY300PLK containing P43 promoter, the gene sdhA and amyL terminator this study pHY-fumC pHY300PLK containing P43 promoter, the gene fumC and amyL terminator this study pHY-gltA pHY300PLK containing P43 promoter, the gene gltA and amyL terminator this study pHY-gltB pHY300PLK containing P43 promoter, the gene gltB and amyL terminator this study pHY-putM pHY300PLK containing P43 promoter, the gene putM and amyL terminator this study pHY-rocA pHY300PLK containing P43 promoter, the gene rocA and amyL terminator this study pHY-racE pHY300PLK containing P43 promoter, the gene racE and amyL terminator this study pHY-DegQ pHY300PLK containing P43 promoter, the gene DegQ and amyL terminator this study pHY-DegU pHY300PLK containing P43 promoter, the gene DegU and amyL terminator this study pHY-DegS pHY300PLK containing P43 promoter, the gene DegS and amyL terminator this study Journal of Agricultural and Food Chemistry Article DOI: 10.1021/acs.jafc.9b01755 J. Agric. Food Chem. 2019, 67, 6263−6274 6264

straw14 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 Lglutamate remains the major obstacle limiting the large-scale production of γ-PGA. Thus, it is necessary to reveal the mechanisms underlying glutamate dependence in glutamatedependent 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 dependence. 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 fermentative 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-PGAproducing 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 highosmolarity 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-putMDNF/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-OUTF/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

Amplite Colorimetric NAD/NADH Ratio Assay Kit, AAT Bioquest, Sunnyvale, CA, USA). Intracellular glutamate was quantified by a previously reported method.21 The intracellular concentration of proline was determined according to the method described by Moses et al.,22 and quantitative determination of ornithine was carried out by reversed phase HPLC as described by Georgi et al.23 2.4. Transcriptomic Analysis. B. subtilis NX-2 was cultured in the fermentation medium in 500 mL flasks with and without glutamate addition for 36 h. All of the strains were cultured in triplicate. The total RNA of these strains was extracted by means of the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The isolated RNA was digested with DNaseI (Ambion, Carlsbad, CA, USA) to remove any possible extra DNA contamination. The RNA was then precipitated by ethanol and resuspended in 300 μL of RNase-free water. The concentration of the total RNA was determined on a spectrophotometer (752 N, Shanghai, China), and RNA quality was checked on a 1% RNA agarose gel. The first cDNA strand was synthesized by reverse transcription with incubation of pure mRNA, biotinylated random hexamers, and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA), and then the second cDNA strand was synthesized by primer extension by ExTaq polymerase. DNA sequencing was performed on a HiSeq 2500 sequencing system (Illumina) by the Beijing Genomics Institute (BGI), Shenzhen, China. The fragments per kilobase of exon per million fragments mapped (FPKM) values for gene expression were calculated, and statistically significant differences in gene expression were detected in the DESeq software with the following criteria: |log2 Fold Change| > 1.0 and p value <0.05. Downstream functional classification was achieved by gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Real-time reverse transcription (qRT-PCR) was applied to confirm the RNA-Seq results. The procedure of qRT-PCR was performed as described before.40 The specific primers used for qRT-PCR are presented in Table S2. The 16 s rRNA gene was used as the internal standard to normalize the gene expression. The expression level of genes in the experimental group was compared with that of the control group after normalization to the reference gene. 2.5. Analytical Methods. Fermented samples were withdrawn from shaking flasks for analysis at regular intervals. The optical density (OD) of the fermentation solution at a wavelength of 660 nm (OD600) was determined on a spectrophotometer (752 N, Shanghai, China). The γ-PGA concentrations were determined by gel permeation chromatography (GPC) on a Superpose 6 column (Shimadzu Corp.) with an RI-10 refractive-index detector. The glucose concentrations were analyzed by high-performance liquid chromatography (HPLC) (Agilent 1200, USA) on a BP-100 Pb2+ column (Benson Polymeric Inc., USA) with a refractive index detector, and HPLC analysis was performed by following previously described procedures.5 3. RESULTS AND DISCUSSION 3.1. Changes in Morphological Characteristics and Intracellular Metabolites in B. subtilis upon Glutamate Addition. Microbial synthesis of γ-PGA by a glutamatedependent B. subtilis strain was determined with and without exogenous glutamate addition. To investigate the effects of glutamate addition on B. subtilis, we compared the morphological characteristics of the strain on agar containing glutamate (NX-2(Glutamate)) with those of the strain on agar not containing glutamate (NX-2) after incubation for varied periods. As shown in Figure 1A, large differences in colony morphology were observed between different incubation conditions; colonies of NX-2(Glutamate) appeared to be notably more mucoid and larger than those of NX-2. This result confirmed that glutamate addition plays an important role in γ-PGA production in glutamate-dependent strains. The effects of glutamate addition were reflected not only in the differences in apparent morphology but also in the levels of intracellular metabolites. Therefore, the concentrations of intracellular ATP, NADPH, and glutamate were measured in this study. As illustrated in Figure 1B, there were obvious increases in intracellular ATP, NADPH, and glutamate levels after glutamate addition. The final concentrations of intracellular ATP, NADPH, and glutamate reached (0.105 ± 0.004) × 10−4 mmol/gDCW (grams of dry cell weight), 14.28 ± 0.83 μmol/gDCW, and 45.12 ± 1.93 mg/gDCW, respectively; these numbers were 1.28-fold, 1.03-fold, and 3.89-fold higher than the corresponding levels in the control group, strain NX-2. In addition, the cell growth of B. subtilis NX-2 was also investigated (Figure 1B). The DCW of NX-2(Glutamate) (4.91 ± 0.33 g/L) was higher than that of NX-2 (4.03 ± 0.32 g/L), a similar phenomenon appearing in the other γ-PGA fermentation process with glutamate addition.26 These results indicated that the addition of glutamate may increase the intracellular ATP, NADPH, and glutamate supply. Several studies have revealed that the enhancement of ATP, NADPH, and glutamate supply can be effective for product synthesis, especially for γ-PGA.20,24,25 Glutamate, the direct precursor of γ-PGA, is essential for the effective production of γ-PGA by glutamate-dependent strains. In our previous studies, it has been reported that the unit of γ-PGA produced by B. subtilis NX-2 came from two parts, extracellular glutamate and intracellular glutamate converted from the glucose,26 and only approximately 9% of the units of γ-PGA were derived from glucose. This result suggested that most of the increased intracellular glutamate in strain NX-2(Glutamate) were converted from the extracellular glutamate added in the media. On the other hand, glutamate is essential for maintaining the intracellular carbon−nitrogen cycle. Thus, when glutamate was supplied in the medium, several key genes involved in the central metabolic pathways were upregulated to adapt to changes in intracellular metabolism. Thus, we hypothesized that the efficient supply of intracellular ATP, Figure 1. Effects of glutamate addition on colony morphology (A) and intracellular metabolites (B) in B. subtilis NX-2. Asterisks indicate the statistical significance of differences at p < 0.05. Journal of Agricultural and Food Chemistry Article DOI: 10.1021/acs.jafc.9b01755 J. Agric. Food Chem. 2019, 67, 6263−6274 6266

NADPH, or glutamate might be the key molecular driver of γ- PGA production in the glutamate-dependent strains. 3.2. Transcriptome Analyses of B. subtilis in Response to Glutamate Addition. To systematically analyze the global gene expression changes in response to glutamate addition during fermentative γ-PGA production, a comparative transcriptome analysis was performed between groups NX-2 and NX-2(Glutamate). The results of the differential gene expression analysis obtained by the differentially expressed gene filtering analysis and by GO functional and KEGG pathway analyses are shown in Figure S1. An overview of the most strongly responding transcripts is compiled in Table S3. The differentially expressed genes involved in central metabolism are shown in Figure 2. When glutamate was supplied as a substrate, most genes related to glycolysis, the pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, glutamate synthesis, and the γ-PGA synthesis pathway were found to be upregulated, whereas genes related to glutamate degradation were downregulated. To verify the upregulation of these genes, qRT-PCR was applied (Figure S3). In general, the enhanced glycolysis would upregulate the total carbon flux and the glucose consumption rate with a concomitant increase in the specific growth rate, thereby elevating volumetric productivity in B. subtilis. 27 Cofactors ATP and NADPH play an important role in the synthesis of γ- PGA. One of the most well-studied strategies to increase the levels of ATP and NADPH has been the metabolic manipulation of stimulating carbon flow into the PPP and TCA cycle in B. subtilis. 28,29 Thus, the activation of PPP and TCA is a promising way to improve glucose assimilation for γ- PGA production in B. subtilis. For γ-PGA-producing bacteria, the process of endogenous glutamate synthesis is critical. As expected, expression of the genes involved in the glutamate synthesis pathway was found to be upregulated in the NX- 2(Glutamate) group; this result is consistent with the findings of the analysis described above. The activation of glutamate synthesis provided abundant precursors for γ-PGA production. In addition, the genes related to the γ-PGA synthesis pathway were also upregulated. Osera et al. have revealed that the regulators DegQ, DegS, and DegU play an important role in the activation of γ-PGA polymerase expression.30 These changes in the expression of genes involved in glycolysis, the PPP, TCA cycle, glutamate synthesis, and the γ-PGA synthesis pathway might be crucial factors for glutamate dependence in B. subtilis. Previously, Zeng et al. analyzed the difference between a glutamate-dependent strain and a glutamate-independent strain in terms of glutamate dependence at the genomic level.15 The results showed that 13 genes related to glucose Figure 2. Schematic of genes involved in metabolisms (glycolysis, PPP, TCA cycle, glutamate synthesis, and γ-PGA synthesis) and their expression patterns in response to glutamate addition. The numbers are the expression ratios (log2) in B. subtilis NX-2 strain at 50 g/L vs 0 g/L glutamate. Red indicates up-regulation and green down-regulation. Definitions: zwf, encodes glucose-6-phosphate dehydrogenase; pgl, encodes 6- phosphogluconolactonase; gnd, encodes 6-phosphogluconate dehydrogenase; pgi, encodes glucose-6-phosphate isomerase; pfkA, encodes ATPdependent phosphofructokinase; pdhA, encodes E1α subunit of pyruvate dehydrogenase; pdhB, encodes E1β subunit of pyruvate dehydrogenase; pdhC, encodes E2 subunit of pyruvate dehydrogenase; citA, encodes citrate synthase isoenzymes; citB, encodes aconitate hydratase; icd, encodes isocitrate dehydrogenase; odhA, encodes a subunit of 2-ketoglutarate dehydrogenase; sdhA, encodes succinate dehydrogenase; fumC, encodes fumarate hydratase; gltA, encodes the large subunit of glutamate synthase (GOGAT); gltB, encodes the small subunit of glutamate synthase (GOGAT); rocG, encodes glutamate dehydrogenase (GDH); gudB, encodes cryptic glutamate dehydrogenase (GDH); argJ, encodes ornithine acetyltransferase; putM, encodes proline dehydrogenase; rocA, encodes Δ1 -pyrroline-5-carboxylate dehydrogenase; proB, encodes γ-glutamate kinase; racE, encodes glutamate racemase; DegQ, encodes pleiotropic regulator; DegU, encodes two-component response regulator; DegS, encodes two-component sensor histidine kinase; pgsB, pgsC, and pgsA, encode γ-PGA synthetase operon. Journal of Agricultural and Food Chemistry Article DOI: 10.1021/acs.jafc.9b01755 J. Agric. Food Chem. 2019, 67, 6263−6274 6267

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