Poly-γ-glutamic acid induces system tolerance to drought stress by promoting abscisic acid accumulation in Brassica napus L.

As a new plant biostimulant, poly-γ-glutamic acid (γ-PGA) may be an effective anti-drought agent that can efficiently alleviate the damage to plants under drought stress. In this study, the effects of γ-PGA on the physiological responses of oilseed rape (Brassica napus L.) seedlings under drought stress were investigated using hydroponics. Growth and development of the rape seedlings were significantly inhibited in a polyethylene glycol-simulated drought environment. However, 12 d after application of γ-PGA under drought stress, the fresh weight, chlorophyll content, and relative water content of rape seedlings all markedly increased. Moreover, proline content and antioxidant enzyme activity were all markedly enhanced, and the malondialdehyde content was significantly reduced in rape seedlings treated with γ-PGA. Furthermore, the content of the important anti-drought response hormone, abscisic acid (ABA), as well as the expression levels of the ABA metabolism regulation genes BnNCED3, BnZEP, and BnAAO4, significantly increased. These results indicate that γ-PGA may induce elements of a tolerance system to drought stress by promoting ABA accumulation in B. Napus.

A Dual Network Hydrogel Sunscreen Based on Poly-γ-glutamic Acid/Tannic Acid Demonstrates Excellent Anti-UV, Self-Recovery, and Skin-Integration Capacities.

Novel sunscreen products based on bioadhesive/gel systems that can prevent the skin penetration behaviors of UV filters have attracted increasing attention in recent years. However, integration is very difficult to achieve and control on the wet surface of the skin under sweaty/dynamic physiological conditions, resulting in functional failure. Herein, we demonstrated the fabrication of a novel dual-network hydrogel sunscreen (DNHS) based on poly-γ-glutamic acid (γ-PGA) and tannic acid (TA), which demonstrated prominent UV protection properties across broad UVA and UVB regions (360-275 nm). Due to a three-dimensional network microstructure and a highly hydrated nature that mimics the extracellular matrix of natural skin, DNHS can perfectly match the skin surface without irritation and sensitization. In addition, the intermolecular hydrogen bond interactions of γ-PGA and TA provide an important driving force for coacervation, which endows the DNHS with remarkable self-recovery properties (within 60 s). Moreover, due to the multiple interfacial interactions between γ-PGA/TA and the protein-rich skin tissue surfaces, DNHS simultaneously possesses excellent skin-integration and water-resistance capacities, and it can be readily removed on demand. Our results highlight the potential of the DNHS to be used in next-generation sunscreens by providing long-term and stable UV protection functions even under sweaty/dynamic physiological conditions.

Improving poly-(γ-glutamic acid) production from a glutamic acid-independent strain from inulin substrate by consolidated bioprocessing.

To excavate the application of Jerusalem artichoke on poly(γ-glutamic acid) (γ-PGA) production, a γ-PGA producing strain Bacillus amyloliquefaciens NX-2S154 was obtained through atmospheric and room temperature plasma mutagenesis, which produced 14.83 ± 0.31 g/L of γ-PGA in batch fermentation with raw inulin extract. Simultaneous saccharification and fermentation (SSF) by adding commercial inulinase were further investigated for γ-PGA fermentation. Results showed SSF could eliminate the ineffective utilization of inulin while avoiding inhibition effect of high concentration substrate, which made γ-PGA concentration reach 18.54 ± 0.39 g/L with the process being shortened by 17%. Finally, an immobilized column for reducing inulinase cost was introduced to γ-PGA production. Repeated batch cultures showed the novel bioreactor exhibited higher stability and simplicity and gave average γ-PGA concentration and productivity of 19.40 ± 0.37 g/L and 0.27 ± 0.008 g/L/h, respectively. This work proposes a productive method for efficient γ-PGA production using Jerusalem artichoke feedstock.

The bio-processing of soybean dregs by solid state fermentation using a poly γ-glutamic acid producing strain and its effect as feed additive.

Soybean dregs are restricted as feed additives because they contain anti-nutrient factors. Herein, soybean dreg was bio-transformed by solid-state fermentation (SSF) using a poly γ-glutamic acid (γ-PGA) producing stain Bacillus amyloliquefaciens NX-2S. The maximum γ-PGA production of 65.79 g/kg was reached in a 5 L fermentation system while the conditions are 70% initial moisture of soybean dregs with addition of molasses meal, 12% inoculum size, 30 °C fermentation temperature, initial pH of 8, and 60 h fermentation time. Meanwhile, continuous batch fermentation was proved feasible. After SSF, the anti-nutritional factors such as trypsin inhibitor, phytic acid and tannin were reduced by 98.7%, 97.8%, and 63.2%, respectively. Compared with unfermented soybean dregs, adding fermented soybean dregs to feed increased the average weight gain of rats by 15.6% and reduced the ratio of feed to meat by 11.3%. Therefore, this study provided a feasible strategy for processing soybean dregs as feed additive.

Systematic unravelling of the inulin hydrolase from Bacillus amyloliquefaciens for efficient conversion of inulin to poly-(γ-glutamic acid).

BACKGROUND: Bacillus amyloliquefaciens NB is a newly discovered strain, which produces poly-(γ-glutamic acid) (γ-PGA) from raw extracted inulin of Jerusalem artichoke tubers; however, the underlying mechanisms remain unknown. To address this problem, we identified the inulin hydrolase in wild-type strain B. amyloliquefaciens NB. RESULTS: The novel inulin hydrolase (CscA) was discovered from strain NB, with high inulinase activity (987.0 U/mg at 55 °C) and strong resistance at pH values between 8.0 and 11.0, suggesting the potential application of CscA in Jerusalem artichoke biorefinery. CscA exhibited a k cat/K m of (6.93 ± 0.27) × 103 for inulin; its enzymatic activity was stimulated by metal ions, like K+, Mn2+, or Ca2+. Similar to their role in glycoside hydrolase 32 family enzymes, the conserved Asp37, Asp161, and Glu215 residues of CscA contribute to its catalytic activity. Targeted disruption of CscA gene suppressed inulin utilization by strain NB. Overexpression of CscA significantly enhanced the γ-PGA generation by 19.2% through enhancement in inulin consumption. CONCLUSIONS: The inulin hydrolase CscA is critical for inulin metabolism in B. amyloliquefaciens and indicates potential application in Jerusalem artichoke biorefinery.

Investigation of Glutamate Dependence Mechanism for Poly-γ-glutamic Acid Production in Bacillus subtilis on the Basis of Transcriptome Analysis.

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.

Characterization of a Regulator pgsR on Endogenous Plasmid p2Sip and Its Complementation for Poly(γ-glutamic acid) Accumulation in Bacillus amyloliquefaciens.

Bacillus amyloliquefaciens NX-2S154 is a promising poly(γ-glutamic acid) (γ-PGA) producing strain discovered in previous studies. However, the wild-type strain contains an unknown endogenous plasmid, p2Sip, which causes low transformation efficiency and instability of exogenous plasmids. In our study, p2Sip is 5622 bp with 41% G+C content and contains four putative open reading frames (ORFs), including genes repB, hsp, and mobB and γ-PGA-synthesis regulator, pgsR. Elimination of p2Sip from strain NX-2S154 delayed γ-PGA secretion and decreased production of γ-PGA by 18.1%. Integration of a pgsR expression element into the genomic BamHI locus using marker-free manipulation based on pheS* increased the γ-PGA titer by 8%. pgsR overexpression upregulated the expression of γ-PGA synthase pgsB, regulator degQ, and glutamic acid synthase gltA, thus increasing the γ-PGA production in B. amyloliquefaciens NB. Our results indicated that pgsR from p2Sip plays an important regulatory role in γ-PGA synthesis in B. amyloliquefaciens.

The Endophyte Pantoea alhagi NX-11 Alleviates Salt Stress Damage to Rice Seedlings by Secreting Exopolysaccharides.

Endophytes have the potential to enhance the ability of plants to resist salt stress, improving crop development and yield. Therefore, in this study, we isolated an endophyte that produced large amounts of exopolysaccharides (EPSs) from the roots of sea rice and examined its effects on the physiological responses of rice (Oryza sativa L. ssp. japonica "Nipponbare") seedlings to salt stress using hydroponic experiments. The endophyte was named Pantoea alhagi NX-11 based on its morphological characteristics and 16S ribosomal DNA (rDNA) sequence alignment. Rice seedlings that had been inoculated with P. alhagi NX-11 exhibited a 30.3% increase in fresh weight, a 28.6% increase in root length, a 51.6% increase in shoot length, and a 26.3% increase in chlorophyll content compared with control seedlings under normal conditions. In addition, inoculated rice seedlings had a 37.5% lower malondialdehyde content, a 133% higher K+/Na+ ratio, and a 52.8% higher proline content after 7 days under salt stress, as well as up-regulated expression of proline synthase, down-regulated expression of proline dehydrogenase, and enhanced antioxidant enzyme activities. Interestingly, rice seedlings that were inoculated with an EPS-deficient strain named NX-11eps- that was obtained by atmospheric and room temperature plasma (ARTP) mutagenesis were damaged by salt stress and had similar physiological and biochemical indicators to the control group. Therefore, we speculate that the ability of P. alhagi NX-11 to enhance the salt tolerance of rice seedlings is related to the EPSs it produces.

Efficient Biosynthesis of Low-Molecular-Weight Poly-γ-glutamic Acid by Stable Overexpression of PgdS Hydrolase in Bacillus amyloliquefaciens NB.

Low-molecular-weight poly-γ-glutamic acid (LMW-γ-PGA) has attracted much attention owing to its great potential in food, agriculture, medicine, and cosmetics. Current methods of LMW-γ-PGA production, including enzymatic hydrolysis, are associated with low operational stability. Here, an efficient method for stable biosynthesis of LMW-γ-PGA was conceived by overexpression of γ-PGA hydrolase in Bacillus amyloliquefaciens NB. To establish stable expression of γ-PGA hydrolase (PgdS) during fermentation, a novel plasmid pNX01 was constructed with a native replicon from endogenous plasmid p2Sip, showing a loss rate of 4% after 100 consecutive passages. Subsequently, this plasmid was applied in a screen of high activity PgdS hydrolase, leading to substantial improvements to γ-PGA titer with concomitant decrease in the molecular weight. Finally, a satisfactory yield of 17.62 ± 0.38 g/L LMW-γ-PGA with a weight-average molecular weight of 20-30 kDa was achieved by direct fermentation of Jerusalem artichoke tuber extract. Our study presents a potential method for commercial production of LMW-γ-PGA.

Recent advances in bio-based multi-products of agricultural Jerusalem artichoke resources.

The Jerusalem artichoke is a perennial plant that belongs to the sunflower family. As a non-grain crop, Jerusalem artichoke possesses a number of desirable characteristics that make it a valuable feedstock for biorefinery, such as inulin content, rapid growth, strong adaptability, and high yields. This review provides a comprehensive introduction to renewable Jerusalem artichoke-based biomass resources and recent advances in bio-based product conversion. Furthermore, we discuss the latest in the development of inulinase-producing microorganisms and enhanced inulin hydrolysis capacity of microbes by genetic engineering, which lead to a more cost-effective Jerusalem artichoke biorefinery. The review is aimed at promoting Jerusalem artichoke industry and new prospects for higher value-added production.

Poly-γ-glutamic acid, a bio-chelator, alleviates the toxicity of Cd and Pb in the soil and promotes the establishment of healthy Cucumis sativus L. seedling.

Poly-γ-glutamic acid (γ-PGA) can be used as a chemical stabilizer to chelate heavy metals in polluted soils. We investigated the effects of γ-PGA on cucumber seedlings under Cd and Pb stresses. γ-PGA effectively reduced the growth inhibitory effects of Cd and Pb on cucumber seedlings. Cd and Pb absorption in cucumber seedlings was also decreased. Further, γ-PGA decreased the malondialdehyde content, and increased the proline content and the total antioxidant capacity of cucumber seedlings in a dose-dependent manner. Infrared spectral characterization of γ-PGA-Cd and γ-PGA-Pb showed that Cd2+ and Pb2+ bind to free carboxyl groups on γ-PGA. Furthermore, γ-PGA-Cd and γ-PGA-Pb were degraded by 22.02 and 24.68%, respectively, within 28 weeks. The chelating rate of γ-PGA-Pb and γ-PGA-Cd reached 27.26 and 14.28%, respectively. Further, γ-PGA alleviated the negative effects of Cd and Pb on soil microorganisms. Thus, γ-PGA can effectively reduce the accumulation of heavy metals in crops caused by heavy metal pollution of farmland, and has significant application value.

Exogenous application of poly-γ-glutamic acid enhances stress defense in Brassica napus L. seedlings by inducing cross-talks between Ca2+, H2O2, brassinolide, and jasmonic acid in leaves.

Poly-γ-glutamic acid (γ-PGA) is a microbe-secreted isopeptide shown to promote growth and enhance crop stress tolerance. However, its downstream signaling pathways are unknown. Here, we studied γ-PGA-induced tolerance to salt and cold stresses. Pretreatment with γ-PGA contributed to enhance stress tolerance of canola seedlings by promoting proline accumulation and total antioxidant capacity (T-AOC) improvement. Further, Ca2+, H2O2, brassinolide, and jasmonic acid were found to be involved in the γ-PGA-induced process. First, using signal blockers, we concluded that γ-PGA activated Ca2+ fluctuations in canola seedling leaves. Second, the activated Ca2+ further elicited H2O2 production by Ca2+-binding proteins CBL9, CPK4, and CPK5. Third, the H2O2 signal promoted brassinolide and jasmonic acid biosynthesis by upregulating key genes (DWF4 and LOX2, respectively) for synthesizing these compounds. Lastly, brassinolide and jasmonic acid increased H2O2 which promoted proline accumulation and T-AOC improvement and further enhanced Ca2+-binding proteins including CaM, CBL10, and CPK9.

Development of Jerusalem artichoke resource for efficient one-step fermentation of poly-(γ-glutamic acid) using a novel strain Bacillus amyloliquefaciens NX-2S.

This study aimed to develop non-food fermentation for the cost-effective production of poly-(γ-glutamic acid) (γ-PGA) using a novel strain of Bacillus amyloliquefaciens NX-2S. The new isolate assimilated inulin more efficiently than other carbohydrates from Jerusalem artichoke, without hydrolytic treatment. To investigate the effect of inulin on γ-PGA production, the transcript levels of γ-PGA synthetase genes (pgsB, pgsC, pgsA), regulatory genes (comA, degQ, degS), and the glutamic acid biosynthesis gene (glnA) were analyzed; inulin addition upregulated these key genes. Without exogenous glutamate, strain NX-2S could produce 6.85±0.22g/L of γ-PGA during fermentation. Exogenous glutamate greatly enhances the γ-PGA yield (39.4±0.38g/L) and productivity (0.43±0.05g/L/h) in batch fermentation. Our study revealed a potential method of non-food fermentation to produce high-value products.

Enzyme-induced dual-network ε-poly-l-lysine-based hydrogels with robust self-healing and antibacterial performance.

An enzyme-induced strategy is reported to construct novel self-mending hydrogels based on ε-poly-l-lysine with both excellent self-healing properties (95%) and antibacterial capacity. Most importantly, the hydrogels are able to accelerate wound healing efficiently, which shows great potential in myriad biomedical fields, such as wound repair, artificial skin, and tissue engineering.

The microbe-secreted isopeptide poly-γ-glutamic acid induces stress tolerance in Brassica napus L. seedlings by activating crosstalk between H2O2 and Ca2.

Poly-γ-glutamic acid (γ-PGA) is a microbe-secreted isopeptide that has been shown to promote growth and enhance stress tolerance in crops. However, its site of action and downstream signaling pathways are still unknown. In this study, we investigated γ-PGA-induced tolerance to salt and cold stresses in Brassica napus L. seedlings. Fluorescent labeling of γ-PGA was used to locate the site of its activity in root protoplasts. The relationship between γ-PGA-induced stress tolerance and two signal molecules, H2O2 and Ca2+, as well as the γ-PGA-elicited signaling pathway at the whole plant level, were explored. Fluorescent labeling showed that γ-PGA did not enter the cytoplasm but instead attached to the surface of root protoplasm. Here, it triggered a burst of H2O2 in roots by enhancing the transcription of RbohD and RbohF, and the elicited H2O2 further activated an influx of Ca2+ into root cells. Ca2+ signaling was transmitted via the stem from roots to leaves, where it elicited a fresh burst of H2O2, thus promoting plant growth and enhancing stress tolerance. On the basis of these observation, we propose that γ-PGA mediates stress tolerance in Brassica napus seedlings by activating an H2O2 burst and subsequent crosstalk between H2O2 and Ca2+ signaling.

Analysis of the Metabolic Pathways Affected by Poly(γ-glutamic Acid) in Arabidopsis thaliana Based on GeneChip Microarray.

Plant growth is promoted by poly(γ-glutamic acid) (γ-PGA). However, the molecular mechanism underlying such promotion is not yet well understood. Therefore, we used GeneChip microarrays to explore the effects of γ-PGA on gene transcription in Arabidopsis thaliana. Our results revealed 299 genes significantly regulated by γ-PGA. These differently expressed genes participate mainly in metabolic and cellular processes and in stimuli responses. The metabolic pathways linked to these differently expressed genes were also investigated. A total of 64 of the 299 differently expressed genes were shown to be directly involved in 24 pathways such as brassinosteroid biosynthesis, α-linolenic acid metabolism, phenylpropanoid biosynthesis, and nitrogen metabolism, all of which were influenced by γ-PGA. The analysis demonstrated that γ-PGA promoted nitrogen assimilation and biosynthesis of brassinosteroids, jasmonic acid, and lignins, providing a better explanation for why γ-PGA promotes growth and enhances stress tolerance in plants.

Improvement of poly-γ-glutamic acid biosynthesis in a moving bed biofilm reactor by Bacillus subtilis NX-2.

The production of poly-γ-glutamic acid (γ-PGA) by Bacillus subtilis NX-2 using a moving bed biofilm reactor (MBBR) system was tested for the first time in this study. Polypropylene TL-2 was chosen as a suitable carrier, and γ-PGA concentration of 42.7±0.86g/L and productivity of 0.59±0.06g/(Lh) were obtained in batch fermentation. After application of the strategy of dissolved oxygen (DO)-stat feeding, higher γ-PGA concentration and productivity were achieved than with glucose feedback feeding. Finally, the repeated fed-batch cultures implemented in the MBBR system showed high stability, and the maximal γ-PGA concentration and productivity of 74.2g/L and 1.24g/(Lh) were achieved, respectively. In addition, the promotion of oxygen transfer by an MBBR carrier was well explained by a computational fluid dynamics (CFD) simulation. These results suggest that an MBBR system could be applied to large-scale γ-PGA production.

Enhanced poly(γ-glutamic acid) production by H2 O2 -induced reactive oxygen species in the fermentation of Bacillus subtilis NX-2.

Effects of reactive oxygen species (ROS) on cell growth and poly(γ-glutamic acid) (γ-PGA) synthesis were studied by adding hydrogen peroxide to a medium of Bacillus subtilis NX-2. After optimizing the addition concentration and time of H2 O2 , a maximum concentration of 33.9 g/L γ-PGA was obtained by adding 100 µM H2 O2 to the medium after 24 H. This concentration was 20.6% higher than that of the control. The addition of diphenyleneiodonium chloride (ROS inhibitor) can interdict the effect of H2 O2 -induced ROS. Transcriptional levels of the cofactors and relevant genes were also determined under ROS stress to illustrate the possible metabolic mechanism contributing to the improve γ-PGA production. The transcriptional levels of genes belonging to the tricarboxylic acid cycle and electron transfer chain system were significantly increased by ROS, which decreased the NADH/NAD+ ratio and increased the ATP levels, thereby providing more reducing power and energy for γ-PGA biosynthesis. The enhanced γ-PGA synthetic genes also directly promoted the formation of γ-PGA. This study was the first to use the ROS control strategy for γ-PGA fermentation and provided valuable information on the possible mechanism by which ROS regulated γ-PGA biosynthesis in B. subtilis NX-2.

Systematic unravelling of the biosynthesis of poly (L-diaminopropionic acid) in Streptomyces albulus PD-1.

Poly(L-diaminopropionic acid) (PDAP) is one of the four homopoly(amino acid)s that have been discovered in nature. However, the molecular mechanism of PDAP biosynthesis has yet to be described. In this work, the general layout of the PDAP biosynthetic pathway is characterised in Streptomyces albulus PD-1 by genome mining, gene disruption, heterologous expression and in vitro feeding experiments. As a result, L-diaminopropionic acid (L-DAP), which is the monomer of PDAP, is shown to be jointly synthesised by two protein homologues of cysteine synthetase and ornithine cyclodeaminase. Then, L-DAP is assembled into PDAP by a novel nonribosomal peptide synthetase (NRPS) with classical adenylation and peptidyl carrier protein domains. However, instead of the traditional condensation or thioesterase domain of NRPSs, this NRPS has seven transmembrane domains surrounding three tandem soluble domains at the C-terminus. As far as we know, this novel single-module NRPS structure has only been reported in poly(ε-L-lysine) synthetase. The similar NRPS structure of PDAP synthetase and poly(ε-L-lysine) synthetase may be a common characteristic of homopoly(amino acid)s synthetases. In this case, we may discover and/or design more homopoly(amino acid)s by mining this kind of novel NRPS structure in the future.

Effect of Poly(γ-glutamic acid) on the Physiological Responses and Calcium Signaling of Rape Seedlings (Brassica napus L.) under Cold Stress.

Cold stress adversely affects plant growth and development. Poly(γ-glutamic acid) (γ-PGA) is a potential plant growth regulator that may be an effective cryoprotectant that prevents crops from damage during cold weather. In this study, the effects of γ-PGA on the physiological responses of rape seedlings subject to cold stress were investigated using hydroponic experiments. We determined that the malondialdehyde content was decreased by 33.4% and the proline content was increased by 62.5% by γ-PGA after 144 h under cold stress. Antioxidant enzymes activities were also evidently enhanced after treatment with γ-PGA. These responses counteracted increases in the fresh weight and chlorophyll content of rape seedlings, which increased by 24.5 and 50.9%, respectively, after 144 h, which meant that growth inhibition caused by cold was mitigated by γ-PGA. Our results also showed that γ-PGA also regulated Ca(2+) concentrations in the cytoplasm and calcium-dependent protein kinases, which are associated with cold resistance. In conclusion, we suggest that the Ca(2+)/CPKs signal pathway is involved in the γ-PGA-mediated enhancement of cold resistance in rape seedlings.