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

AGRICULTURAL AND Article FOOD CHEMISTRY Cie This:Agrie Food Chem.19,7,63-27 pubs acs.ora/JAFC Investigation of Glutamate Dependence Mechanism for Poly-y- glutamic Acid Production in Bacillus subtilis on the Basis of Transcriptome Analysis Xiaohai Feng, State Key Laboratory of Materials-Oriented Chemical Engineering Nanjing 1 People's Republic of Chin 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 Supporting Information ent of c ction by glutan the glutamate d e first sy ges I led th ctncntoofghtanmai elutamate-dependent strai erin PGA titer n 1ed1021±0.4 and reveals potential molecular targets for increasing economical y-PGA production. KEYWORDS:poly-y-glutamic acid,glutamate dependence,Bacillus subtilis NX-2,transcriptome 1.INTRODUCTION glutamat a cell a enzyme) of D- mic acid units that in B.an ight' 291-fold in B.amylo rmore,the With excellen NK-I nainly synthesized by microbial fe eering method is pro on of has e,and efforts have been ma related metabolic pathw ays,the y-PGA production in the much l than that of th Th andrenewgbleabstatespndcoptimiationofthefementatie egy to hoose glutamate-dependent strains forth ction of cations.the cost of bioproduc tior is the mai ctor dete ng the economi GA- oducing strain euaye into two s,which can meg the cost of s is cru ial for ind pplications. Over the past fe ng t cost o 56 ndent well as on imp nent oft y-PGA yield ovo from carbor urces without the Mushroom untreated can mo and nice March 19,2019 plihed fermenta the glutamate-independen May 12,2 to in -PGA roduction in the PaMy12019 ACS Publications 010A 636 1AkoamCR28a6to2

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 em￾ployed.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 compo￾nents 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

Journal of Agricultural and Food Chemistry Table 1.Strains and Plasmids Used in This Study d-typ e strain,CGMCC No.083: E celi GM2163 FlaeY-galkE4 rpl.136(S)dm1:Tn NX- X-2 derivat n NX-22 of putM and NX-2-AputM n of putM gen X- utM with putM at E col and B he c twf an pa 6

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

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 626

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 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

Journal of Agricultural and Food Chemistry Article Amplite Col nyvale,CA, NAD/NADH quanti g to the the stral e total rna o a by AUSA)T d with D bion. )to DN RN the 30 otinythte random DNA (FPKM) and DE ficant dif at p (GOY Ency RNA apparent morpnology ned as PCR RN h the in intra ular ATP DPH, and g of the 2.5.Anab ATP.NADPH,and nat ached (0.105 D)of the n4.12±1.93 ver 3.89-fold y-PGA B).T aphy (HPLC)( 491+03 L)was than that of NX-2 (403+037 VL) ar pher ith appearing in the other y as perfo ng pr of gut ate may 3.RESULTS AND DISCUSSION adies aled that the en nt of ATP.NADPH and P it hat nit fy-PGAPo by B. glutamate addition on B. btilis,we compar ate red the in ted from the and NX-2(Gl of y-P de ate (NX- after ubation for v train NX. (Glut obs differ ent in batio the othe hand. tamate is ess id nd hat gluta ddition plays mp yed to mateaddition were reflected not only in the ed that the

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 tran￾scriptase (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 glutamate￾dependent 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 intra￾cellular 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

Journal of Agricultural and Food Chemistry 1,5-lactone-6 Glucose 122 comA-P Fructose-6- 2 17 Fructose-1,5-P D-nbulose- L◆→ Acetyl-CoA 22 re 2 PPP.TC DH) nent respe ht be the key mo ular dnv of y n role in th ransc lyses o n NADPE DD to Thus,th a p K-2(Glu in Tab the analy The activat s pro the nt ro the asid (T et have the -PGA svnthesis tion of y-PGA T thes RT-PCR gh dependen y in e results showed that 13 genes related to glucose 626

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 tran￾scriptome 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 ATP￾dependent 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

Journal of Agricultural and Food Chemistry Article Figure 3.Effects of dif re and glu : withoutgta nants in (a away:(E) of gitA PGA dule.The as ate synth five ay e the ause of the gl deper ent differenc n ,ther diti of ger eason may be that the on mption and the gro rate,thus further on 7-PGA p ge subtilis.It sh uld be note d that genes rela ed to glutamate genes pdhC were grea the nated as NX-2-pgi.NX-2-PfkA.NX-2-pdhA,NX-2-pdhB. sis and the glutamat increased glu tion and the h rate in the w contr the glu Modules PGA Production without Glutamate Addition. furthe in strains NX-2-pdhA. NX. -pdhB, NX-2 T overexpre of r-PG depende A repre the exp on of (encodin itat sythase)and 26

transport, ammonium transport, and glutamate synthesis were mutated. Thus, they predicted that these changes may be the cause of the glutamate-dependent difference. In contrast, there were no significant differences in the transcript level of genes involved in the glucose transport and ammonium transport pathways in this study. The reason may be that the glucose transport and ammonium transport pathways might have relatively little influence on the glutamate dependence in B. subtilis. It should be noted that genes related to glutamate synthesis undergo great changes at both the genomic and transcript levels, suggesting a potential link between the intracellular glutamate synthesis and the glutamate-dependent difference in B. subtilis. Further analysis was needed to clarify the molecular interactions between the expression changes and glutamate-dependent performance. 3.3. Identification of Key Modules for γ-PGA Production without Glutamate Addition. To further elucidate the mechanism of glutamate dependence in B. subtilis during γ-PGA-producing fermentation, it was decided to overexpress the significantly upregulated genes involved in five metabolic pathways (glycolysis, PPP, TCA cycle, glutamate synthesis, and γ-PGA synthesis) under no-glutamate con￾ditions. 3.3.1. Enhancement of the Glycolytic Pathway. Previous studies have revealed that the glycolytic pathway is critical for carbon consumption and the growth rate, thus further influencing the γ-PGA productivity.31 To investigate the effects of enhanced glycolytic flux on γ-PGA production, genes such as pgi, pfkA, pdhA, pdhB, and pdhC were individually overexpressed, and the resultant strains were designated as NX-2-pgi, NX-2-pfkA, NX-2-pdhA, NX-2-pdhB, and NX-2-pdhC, respectively. As depicted in Figure 3A, overexpression of pgi and pfkA significantly (p < 0.05) increased glucose consumption and the growth rate in comparison to the wild-type strain. In contrast, the glucose utilization efficiency and the growth rate were obviously lower in strains NX-2-pdhA, NX-2-pdhB, and NX-2-pdhC. The overexpression of pdhA, pdhB, and pdhC led to acetyl￾coenzyme A (acetyl-CoA) accumulation, which may further repress the expression of citZ (encoding citrate synthase) and Figure 3. Effects of different recombinants on γ-PGA production, biomass, and glucose consumption without glutamate addition: (A) recombinants involved in glycolytic pathway; (B) recombinants involved in pentose phosphate pathway; (C) recombinants involved in TCA cycle; (D) recombinants involved in glutamate synthesis pathway; (E) effects of the overexpression of gltA, gltB, putM, and rocA on the intracellular glutamate, arginine, and proline accumulation; (F) recombinants involved in γ-PGA synthesis module. The 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 6268

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 626

thus 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

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

Journal of Agricultural and Food Chemistry 7-PGA Apparent conversion rate of glu 90 ration (g/L d the e add 021)/x y-PGA of th 200 at p Table 3.Comparison of-PGA Production by Some Glutamate-Dependent and Glutamate-Independent Bacills spp .PGA (L) ate-Dep dent Stra 37℃，96b 45 a48 mate (30),matose ()y 30L40℃，90h 350 02 R subtils NX.220 e (80).glutamate (40).NHCl (8) 7℃ 42.0 035 0.35 RN24(仙 glucose (40),glutamate (40),(NH).SO.(5) flask,32 C,48 h 35.5 074 04 B.subtils GXG-55 10L,50℃，34h 195 20037℃ 4.4 0) 18 k32C,66h 8 thesis n y had almost no effec -PGA produe NX-23.The hig ony the amate synthesi obt ous ov tA nalysis.we can co hat c fNX-7 while. as well (T 2. este sis for Highe PGA titer and productivity.In rec ars. al stra the emote the for eato GA 11 was enhance by 18 in B. their t the fou ease studies have this distinct result ompa NX-Z-pi (th only on path of the sing stra with thes 124 6271

synthesis pathway had almost no effect on γ-PGA production, whereas only the enhancement of glutamate synthesis increased γ-PGA production from 0 g/L to a maximum of 4.21 ± 0.35 g/L. Given the above analysis, we can conclude that the efficiency of intracellular glutamate synthesis is the key regulator for glutamate dependence in B. subtilis for γ-PGA production. 3.4. Combinatorial Engineering of Glutamate Syn￾thesis for Higher γ-PGA Production. The aforementioned results confirmed that the enhanced expression of glutamate synthesis genes could promote the production of γ-PGA. These data pointed to the increased necessity of intracellular glutamate for a higher γ-PGA yield. Therefore, it was decided to coexpress gltA, gltB, rocA, and putM to test their influence on the intracellular glutamate synthesis and γ-PGA production. As shown in Table 2, different combinations of the four genes produced distinct results. In comparison to NX-2-putM (the best γ-PGA producer with single-gene overexpression), the γ- PGA titer of the putM + rocA overexpressing strain, NX-22, was higher by 81.24%. Then, a slight increase in the γ-PGA titer was detected in the putM + rocA + gltB overexpressing strain, NX-23. The highest γ-PGA titer of 10.21 ± 0.42 g/L was obtained via simultaneous overexpression of gltA, gltB, rocA, and putM, which manifested a 1.43-fold enhancement over that of NX-2-putM. Meanwhile, the intracellular glutamate concentration increased to 25.32 ± 1.92 mg/gDCW, and the glucose consumption rate and cell growth were significantly (p < 0.05) higher as well (Table 2). These results suggested that systematic enhancement of glutamate synthesis led to a higher γ-PGA titer and productivity. In recent years, several strategies have been proposed to improve the intracellular glutamate supply for improvement of γ-PGA production. For instance, a γ-PGA yield was enhanced by 18.5% in B. amyloliquefaciens LL3 via downregulation of rocG (encoding glutamate dehydrogenase for glutamate degradation), leading to an increase in the concentration of intracellular glutamate.12 Although several studies have been conducted on this topic, most of them focused only on the major biosynthetic pathway of glutamate synthesis, ignoring other efficient supply pathways (such as proline metabolism). In comparison with these reports, the advantage of this study is the focus on a systematic combination of different pathways for glutamate accumulation. Figure 5. Effects of glutamate concentration on biomass, γ-PGA production, residual glutamate, and apparent conversion rate of glutamate. The amount of γ-PGA and the number of the added glutamate are defined as Y and X, respectively. The apparent conversion rate is defined as R = (Y − 10.21)/X × 100%. Asterisks indicate the statistical significance of differences at p < 0.05. Table 3. Comparison of γ-PGA Production by Some Glutamate-Dependent and Glutamate-Independent Bacillus spp γ-PGA strain nutrients (g/L) culture conditions production (g/L) productivity (g/(L h)) yield on total sugar (g/g) Glutamate-Dependent Strains B. subtilis F-2−0144 glucose (20), glutamic acid (150), veal infusion broth (1) flask, 37 °C, 96 h 45.5 0.48 0.27 B. subtilis (natto) MR- 14145 sodium glutamate (30), maltose (60), soy sauce (70) 30 L, 40 °C, 90 h 35.0 0.39 0.22 B. subtilis NX-226 glucose (80), glutamate (40), NH4Cl (8) 7.5 L, 32 °C, 120 h 42.0 0.35 0.35 B. subtilis NX-24 (this study) glucose (40), glutamate (40), (NH4)2SO4 (5) flask, 32 °C, 48 h 35.52 0.74 0.44 Glutamate-Independent Strains B. subtilis GXG-515 glucose (25), NH4NO3 (25) 10 L, 50 °C, 34 h 19.5 0.57 0.78 B. amyloliquefaciens LL38 sucrose (50), (NH4)2SO4 (2) 200 L, 37 °C, 48 h 4.4 0.091 0.088 B. licheniformis A3510 glucose (75), NH4C1 (18) flask, 30 °C, 120 h 8.16 0.068 0.108 B. subtilis NX-24 (this study) glucose (40), (NH4)2SO4 (5) flask, 32 °C, 66 h 10.21 0.16 0.26 Journal of Agricultural and Food Chemistry Article DOI: 10.1021/acs.jafc.9b01755 J. Agric. Food Chem. 2019, 67, 6263−6274 6271

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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