Functional Caracterization of CapBCA in Controlling Poly-γ-Glutamic Acid Synthesis in Corynebacterium Glutamicum

Poly-γ-glutamic acid (γ-PGA) is a natural anionic biopolymer widely used in various elds, including medicine, food, cosmetics, and environmental protection. The γ-PGA synthase complex, CapBCA, is the only polyprotein complex responsible for γ-PGA synthesis. However, systematic and in-depth research on the function of each component involved in γ-PGA synthesis is scarce, which limits enhanced production of γ-PGA. functions associated with maintaining

At present, microbial fermentation is the predominant method for the production of commercial γ-PGA owing to its advantages such as cheap raw materials, less environmental pollution, and higher purity of natural products. The main γ-PGA producers are Bacilli spp. In general, γ-PGA-producing strains can be divided into two types based on the requirement of glutamic acid as a precursor in the production process, namely, glutamate-dependent strains [8,9] and glutamate-independent strains [10][11][12]. Different strategies have been developed to increase γ-PGA yield of glutamate-dependent strains, including screening of mutant strains, optimizing the culture medium and fermentation process, and increasing the expression level of γ-PGA synthase. However, external supply of glutamic acid signi cantly increases the cost of γ-PGA production [13][14][15]. To solve this problem, glutamate-independent strains have attracted increasing attention, and researchers have screened and identi ed naturally occurring glutamateindependent strains. Subsequently, most studies have focused on increasing the ATP supply via metabolic engineering and knock out of degrading enzymes genes, achieving further improvement in γ-PGA production [16,17]. As only γ-PGA synthase complex can polymerize glutamic acid to γ-PGA [2,18], the expression of γ-PGA synthase in heterologous strains has been investigated, and γ-PGA synthesis using glucose as substrate has been successfully achieved in Escherichia coli and Corynebacterium glutamicum [19][20][21]. In our recent study, we accomplished de novo synthesis of γ-PGA to achieve a nal titer of 21.3 g/L using an industrial C. glutamicum strain as the chassis cell [22]. However, the unclear relationship between the function of γ-PGA synthase and rate of γ-PGA synthesis limits further improvement in γ-PGA production.
γ-PGA synthase is encoded by capB, capC, and capA genes, and their homologs in Bacillus spp. are pgsBCA. In our earlier research, CapB, CapC, and CapA of Bacillus licheniformis ATCC9945a were found to share 90.08%, 89.93%, and 65.30% identity with PgsB, PgsC, and PgsA of Bacillus subtilis Ia1a, respectively [22]. The functional analysis of γ-PGA synthase is the key to increase γ-PGA synthesis. Based on the amino acid sequence characteristics, CapBCA has been speculated to be a membrane crosslinking enzyme [23]. It has been presumed that CapB and CapC jointly form the catalytic site, whereas CapA transports γ-PGA to the outside of the membrane to achieve γ-PGA chain extension [1,18]. Although γ-PGA synthase has always been considered as a membrane cross-linking enzyme, there is still a lack of visual evidence. While membrane localization of γ-PGA synthase had been observed in E. coli expressing pgsBCA heterologously, localization of γ-PGA synthase in Gram-positive bacteria is still vague [26]. In addition, it remains unclear whether capB, capC, and capA are necessary for the synthesis of γ-PGA. Ashiuchi et al. [24] reported that γ-PGA production was detected only in strains with complete pgsBCA, suggesting that all the enzyme components are essential for γ-PGA synthesis. By contrast, Sawada et al. [25] demonstrated that B. subtilis (lacking genomic pgsBCA genes) introduced with pgsBC genes could produce 26.0 g/L γ-PGA. Although the functional study of enzymes is the key to improve γ-PGA production, the unclear role of the three components of γ-PGA synthase in γ-PGA synthesis is a major obstacle and still remains unclear.
To address these issues, in the present study, we performed a systematic investigation of the γ-PGA synthase CapBCA. In particular, we explored the localization of CapBCA in C. glutamicum, functional requirement of CapBCA, and in uence of the expression level of each component of CapBCA on γ-PGA production. Through bioinformatics analysis, the transmembrane region of each component was identi ed, and visual evidence of protein membrane localization was obtained by using reporter genes translationally fused to CapB, CapC, and CapA. Subsequently, CapBCA was con rmed to be necessary for γ-PGA synthesis through the expression of different combinations of each component. Finally, the expression intensity of each component was individually regulated, and the effect of each component's expression on the synthesis of γ-PGA was determined. The results obtained in this study help to better understand γ-PGA synthase localization and its in uence on γ-PGA synthesis, thus further facilitating strain design to enhance the production of γ-PGA.

Results
Analysis of CapBCA localization on the membrane In this study, a series of bioinformatics methods were used to analyze the structure of CapBCA protein and predict their localization. The transmembrane helices and possible signal peptides of CapB, CapC, and CapA were analyzed by TOPCONS, and the results are shown in Additional le 2: Fig. S1. CapC exhibited ve transmembrane-spanning helices, while CapB and CapA presented one transmembranespanning helix at the amino terminus, respectively. The subcellular localization of CapB, CapC, and CapA was analyzed by PSORTb, and the results are shown in Table 1. With a total probability score of 10, the probability that CapB, CapC, and CapA were localized on the plasma membrane was 8.78, 10.00, and 9.51, respectively. To con rm the localization of CapB, CapC, and CapA proteins, each component fused with a uorescent protein (superfolder green uorescent protein (sfGFP)) was expressed in C. glutamicum. sfGFP is an e cient and stable folding variant, which uoresces in the bacterial periplasmic space [27]. The sfgfp gene was cloned into the pZM1 vector resulting in plasmid pZM1-S. The C-terminus of CapB, CapC, and CapA protein was fused with sfGFP to construct plasmid pZM1-BS, -CS, and -AS, respectively (Fig. 1a). The recombinant plasmids were transferred into C. glutamicum F343 to obtain the strains F343-S, -BS, -CS, and -AS, respectively. The cell morphology was observed under laser confocal microscope, and the successful expression of sfGFP, CapB-sfGFP, CapC-sfGFP, and CapA-sfGFP proteins was con rmed (Fig. 1b). The strain F343-S exhibited uorescence in the entire cell, while strains F343-BS, -CS, and -AS only presented uorescence at the cell edge, which proved that CapB, CapC, and CapA proteins were localized on the cell membrane in C. glutamicum. Subsequently, the membrane and cytoplasmic proteins of F343-B, -C, -A, -S, -BS, -CS, and -AS were extracted and their uorescence intensities were measured. The uorescence of F343-S was mainly detected in the cytoplasmic protein, while that of F343-BS, -CS, and -AS was predominantly identi ed in the membrane protein, further con rming that CapB, CapC, and CapA were localized on the cell membrane of C. glutamicum (Fig. 1c).
Investigation of functional necessity of CapBCA for γ-PGA synthesis To determine whether each component of CapBCA is necessary for γ-PGA synthesis, we expressed CapB, CapC, and CapA proteins separately and in combination in C. glutamicum F343. The recombinant strains F343-B, -C, -A, -BC, -CA, -BA, and -BCA were constructed and their fermentation products were veri ed. Gel permeation chromatography (GPC) revealed that strain F343-BCA produced 5.82 g/L γ-PGA after 24 h of fermentation. In contrast, strains F343-B, -C, -A, -BC, -CA, and -BA did not produce γ-PGA during the entire fermentation period (Table. 2). These ndings suggested that all the components of γ-PGA (CapB, CapC, and CapA) have crucial roles in γ-PGA synthesis.
To con rm whether all the components (CapB, CapC, and CapA) of γ-PGA synthase are necessary for γ-PGA synthesis, the fermentation broth of each recombinant strain was puri ed according to the γ-PGA puri cation method to obtain freeze-dried products. Samples were prepared at a concentration of 5 mg/mL to determine their molecular weight. The results showed that the molecular weight of the fermentation product of F343-BCA was 1189.41 kDa, while that of the fermentation product of F343-B, -C, -A, -BC, -CA, and -BA was about 1.00 kDa for each (Additional le 2: Figure S2).
Subsequently, the puri ed fermentation products of the engineered strains were analyzed. The results of proton nuclear magnetic resonance ( 1 H NMR) con rmed the presence of γ-PGA, and the protons at "a,", "b," and "c" positions in Fig. 2a correspond to the characteristic peaks in Fig. 3b, respectively [22]. The 1 H NMR analysis of the fermentation products of the recombinant strains is shown in Fig. 2b. The results revealed that the fermentation product of F343-BCA was consistent with the characteristic peak of γ-PGA, indicating that the product was γ-PGA. However, there was no characteristic peak at the corresponding position for products produced by the recombinant strains F343-B, -C, -A, -BC, -CA, and -BA, indicating that γ-PGA was not produced by these strains. Therefore, it can be concluded that all the components of γ-PGA synthase are essential for γ-PGA production.
In uence of different expression levels of CapBCA on γ-PGA synthesis The expression intensity of each of CapB, CapC, and CapA was regulated to explore the in uence of γ-PGA synthetase components on γ-PGA production. The intensity of each gene was divided into three increase in γ-PGA yield, while a further increase in the expression level of CapA (from medium to high) led to a 10.36% decrease in γ-PGA production (Fig. 3). To explain the contribution of CapB, CapC, and CapA to γ-PGA yield, we analyzed the three factors by analysis of variance (ANOVA). The ndings showed that the contribution of CapB, CapC, and CapA was 20.03%, 68.24%, and 11.73%, respectively, indicating that CapC expression had the greatest impact on γ-PGA yield, followed by CapB and CapA expressions (Fig. 4).

Discussion
γ-PGA, a natural biopolymer in which D-and/or L-glutamic acids are coupled to each other via γ-amide bonds, has a wide range of applications, such as in foods, medicine, cosmetics, and environmental protection. The enzyme γ-PGA synthase has been identi ed as the sole machinery responsible for the synthesis of γ-PGA. However, the unclear relationship between the function of γ-PGA synthase and γ-PGA synthesis rate limits further optimization and improvement of γ-PGA production. Based on the fusion of reporter genes, combined expression, and regulation, the present study performed a detailed analysis of the localization and function of γ-PGA synthase, and elucidated the in uence of CapB, CapC, and CapA on γ-PGA biosynthesis. The results of this study are highly valuable for explaining the interaction among the γ-PGA synthase components and mechanism of γ-PGA synthesis.
Ashiuchi et al. [24] detected glutamate-dependent ATPase activity in E. coli cell membrane complexes heterologously expressing pgsBCA, and concluded that PgsBCA complex is localized on the cell membrane. In 2020, membrane localization of PgsBCA was observed in E. coli that heterologously expressed pgsBCA [26]. Considering the difference in the cell membrane composition, the location of CapBCA in C. glutamicum was explored in the present study. We rst predicted the subcellular localization and transmembrane regions of CapBCA through bioinformatics. Then, C. glutamicum was used as a host to express the C-terminal fusion uorescent protein of CapBCA. The uorescence intensity results showed that CapB, CapC, and CapA were localized on the cell membrane, and clear membrane localization was observed under laser confocal microscope. These ndings clearly demonstrated the localization of γ-PGA synthase and provided a basis for further research on protein function.
Previous studies have con rmed that pgsB, pgsC, and pgsA genes are all necessary for γ-PGA production [28][29][30]. However, Sawada et al. [25] found that B. subtilis (lacking the genomic pgsBCA genes) introduced with pgsBC genes could produce 26.0 g/L γ-PGA. Therefore, to clarify whether capB, capC, and capA are essential for the synthesis of γ-PGA, in the present study, we expressed different combinations of the γ-PGA synthase components in C. glutamicum which did not produce γ-PGA. Our results were noted to be in agreement with those reported in previous studies, which indicated that all pgsBCA genes are essential for γ-PGA synthesis. Moreover, by using 1 H NMR, we further proved that γ-PGA could be synthesized only in the presence of all the capBCA genes [21].
Consequently, it is crucial to elucidate the relationship between the expression intensity of each component of γ-PGA synthase and γ-PGA synthesis. In E. coli, PgsBCA under the regulation of constitutive HCE promoter presented higher catalytic activity and higher γ-PGA concentration [31].
Besides, γ-PGA yield has been reported to increase with increasing the expression of γ-PGA synthase [32]. Moreover, the strength of pgsB and pgsC expression has been noted to have a greater impact on γ-PGA synthesis [14,20,[33][34][35]. In the present study, we systematically analyzed the in uence of changes in the expression intensity of each component of γ-PGA synthase on γ-PGA synthesis, and observed that enhancement of the transcription levels of CapB and CapC (from low to high) alone led to a 35.44% and 76.53% increase in γ-PGA yield, respectively. However, moderate increase in the transcription levels of CapA (from low to medium) led to 35.01% increase in γ-PGA yield, whereas a further increase in the expression of CapA (from medium to high) led to a 10.36% decrease in γ-PGA production. In particular, CapC had the greatest in uence on γ-PGA synthesis, accounting for 68.24% (based on ANOVA).
In summary, we systematically studied the localization and function of γ-PGA synthase complex, and determined membrane localization of γ-PGA synthase and the effect of each enzyme component on γ-

Strains, media, and culture conditions
All the strains and plasmids used in this study are listed in Additional le 1: Table S1. E. coli JM109 was used for plasmid construction and C. glutamicum F343 was employed for gene expression.
Luria-Bertani (LB) solid medium (0.5% yeast extract, 1% tryptone, 1% NaCl, and 2% agar) was used as the solid growth medium for E. coli JM109 and C. glutamicum F343. The seeding medium for C. glutamicum All E. coli JM109 strains were cultured in LB medium at 37 ℃ for plasmid propagation. All C. glutamicum strains were cultured in seed medium for 11 h at 32°C. Then, the preculture was inoculated into the fermentation medium at an initial density (OD 600 ) of 1 and cultured at 32°C and 120 rpm. After 2 h, IPTG was added to a nal concentration of 1 mM and the temperature was increased to 37°C.

Construction of recombinant plasmid
All the primer sequences used for plasmid construction are shown in Additional le 1: Table S2. The sfgfp gene was obtained by PCR using the primer pair sfgfp-Nde-F and sfgfp-BamH-R. The fragment was digested with NdeI and BamHI and ligated to pZM1 digested with NdeI and BamHI to obtain the plasmid pZM1-S. The recombinant plasmids pZM1-BS, pZM1-CS, and pZM1-AS were constructed with Hieff Clone® Plus One Step Cloning Kit (YEASEN, Shanghai, China). The plasmid pZM1-BCA was used as a template to amplify capB, capC, and capA genes. For example, to achieve the fusion of CapB and sfGFP, the fragment of capB with the terminator removed was ampli ed by PCR using the primer pair capB-gfp-F/R, and ligated to the plasmid pZM1-sfgfp digested with NdeI using Hieff Clone® Plus One Step Cloning Kit (YEASEN, Shanghai, China).

Bioinformatics analysis
The subcellular localization was predicted through PSORTb subcellular localization prediction tool Membrane and cytoplasmic proteins extraction and uorescence measurements The cells were washed twice, resuspended in PBS (pH 7.4), and disrupted by sonication (Scientz-IID, Scientz, Ningbo, China). Membrane and cytoplasmic proteins were respectively extracted using membrane and cytoplasmic protein extraction kits (Sangon, Shanghai, China), and the protein concentration and uorescence intensity were determined. Modi ed BCA protein assay kits (Beyotime, Nanjing, China) were used to determine the protein concentration, and the uorescence intensity was measured using uorescence spectrophotometer (Synergy H4; BioTek, Winooski, VT, USA).
Puri cation of γ-PGA Ethanol precipitation was employed for γ-PGA puri cation. After centrifuging the fermentation broth at 8760 g for 35 min, four volumes of ethanol were added to the supernatant and incubated overnight at 4°C. Then, the precipitate was centrifuged, dissolved in water, and dialyzed to remove impurities. Finally, γ-PGA was obtained after freeze-drying the solution.

Analytical methods
The samples were collected at indicated time points and diluted, and their cell density (OD 600 ) was measured by using a Spectrophotometer (AOE Instruments Co. Inc., Shanghai, China). γ-PGA concentration was determined on a Waters 1515 HPLC system with a refractive index detector. The TSKgel series columns (TSKgel SuperAW-H, TSKgel super Aw 4 000, TSKgel super Aw 5 000) were used for separation at a column temperature of 35°C.
The weight-average molecular weight of γ-PGA was evaluated by GPC. In brief, the sample was ltered through a 0.45-µm lter membrane and analyzed using Agilent 1260 In nity Availability of data and materials The datasets used and analyzed in this study are available from the corresponding author on request.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests. (C. glutamicum F343 expressing CapB-sfGFP fusion protein with pZM1), F343-CS (C. glutamicum F343 expressing CapC-sfGFP fusion protein with pZM1). Scale bar = 20 μm. c Fluorescence intensity for the detection of cytoplasmic and membrane proteins.  CapA on γ-PGA titer. c Effect of individual regulation of CapB, CapC, and CapA on γ-PGA yield. Cyan represents the relative transcription level and effect of capB on γ-PGA production; blue denotes the relative transcription level and effect of capC on γ-PGA production; and purple the indicates relative transcription level and effect of capA on γ-PGA production. The color from light to dark represents the corresponding gene expression level from low to high and its in uence on γ-PGA production. Asterisks indicate statistically signi cant differences. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 4
Contribution of CapB, CapC, and CapA to γ-PGA synthesis. The table on the left represents the effect of the expression level of γ-PGA synthase components on γ-PGA yield. Data are the means ± standard