Poly (glutamic acid)--an emerging biopolymer of commercial interest

Poly (γ-glutamic acid) (PGA) is water-soluble, anionic, biodegradable, and edible biopolymer produced by Bacillus subtilis. It has multifarious potential applications in foods, pharmaceuticals, healthcare, water treatment and other fields. The production of PGA has already been established on the industrial scale. Various studies regarding the fermentative production, downstream processing and characterization of PGA have been reported in the literature. This review provides updated information on fermentative production of PGA by various bacterial strains and effect of fermentation conditions and media component on production of PGA in submerged as well as solid state fermentation. Information on the application of genetic engineering for enhancement of yield of PGA, kinetic studies for production of PGA in submerged fermentation and recovery and purification of PGA is included. An attempt has also been made to review the current and potential applications of PGA. This review may contribute to further development of this commercially and academically interesting biopolymer.     

Effect of phenylacetic acid on the growth and production of penicillin G acylase of recombinant and host strains derived from Escherichia coli W

The industrial production strain Escherichia coli RE3(pKA18) for penicillin G acylase (PGA) bears simultaneously the pga gene on the chromosome as an inducible gene pgaⁱ, (the inductor is phenylacetic acid, PAA) and on the recombinant plasmid pKA18 as a constitutively expressed gene pga^c. Under non-selective conditions, plasmid-less strains (P⁻) appeared in 17th successive batch culture. However, the population was over taken by P⁻ cells already in fourth culture if the medium was supplemented with PAA. The rate of plasmid loss from the culture depends on the PAA concentration and on the expression of pgaⁱ, not on PGA overproduction from pga^c. PAA at inducing concentration has a negative effect on PGA expression and plasmid stability in the high-expression self-cloning system RE3(pKA18) which results in the reduction of: (1) the specific growth rate of a culture and biomass concentration, (2) the synthesis of PGA (e.g. the specific activity of the strain) and (3) the copy number of the recombinant plasmid and promotion of the plasmid loss from the culture. Segregational stability of pKA18 increases in P⁺ persisting clones and in re-transformed P⁻ clones segregated during the selection in the presence of PAA.     

Plant aging and excess light enhance flavan-3-ol content in Cistus clusii

Physiological studies on aging in perennials are mainly focused either on the primary metabolism or the hormonal regulation of the process. However, to our knowledge, the involvement of the secondary metabolism in this process has not yet been explored. Cistus clusii, a Mediterranean sclerophyllous evergreen bush, shows considerable amounts of flavan-3-ols in leaves. In the present study, we aimed at determining the impact of environmental conditions and plant aging in the flavan-3-ol content in C. clusii plants grown in field conditions, which included summer drought and recovery periods. Six-year-old plants suffered more from photo-oxidative stress, especially during excess light periods, and showed lower maximum photosynthetic rates than 1-year-old plants. C. clusii leaves accumulated (−)-epigallocatechin gallate in early summer, in a strong positive correlation with both the photon flux density and the photoperiod, but not with the plant water status. Moreover, C. clusii plants accumulated proanthocyanidins (polymeric flavan-3-ols) in leaves during summer. Older plants showed higher levels of proanthocyanidins and (−)-epicatechin, but only during late spring and summer. From the result of the present study, we conclude that excess light enhances flavan-3-ol content in C. clusii, a process enhanced as plants age due to increased excess light stress.     

粪产碱杆菌青霉素G酰化酶在大肠杆菌中组成型表达及分离纯化

将粪产碱杆菌青霉素G酰化酶基因构建重组表达质粒pKKFPGA ,pKKFPGA再转化宿主菌DH5α,所得重组菌不需诱导便能高效表达青霉素G酰化酶 ,表达量达 2590u L ,比野生型粪产碱杆菌表达量高432倍 ,其菌体比活力达300 (u L) A₆₀₀。菌体破碎后的上清液经DEAE-SepharoseCL 6B离子交换层析和Butyl-SepharoseCL 4B疏水层析 ,即可得纯度提高 20倍、比活为 686u mg的青霉素G酰化酶 ,两步纯化的总收率达 91%。Western印迹分析表明5%的原前体青霉素酰化酶在胞内形成了包涵体 ,说明其成熟的限速步骤在胞内的运输阶段.     

High-level expression of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas diminuta NK703 in Escherichia coli by combined optimization strategies

In this work, a glutaryl-7-aminocephalosporanic acid acylase (GLA) coding gene was cloned from Pseudomonas diminuta NK703 which was screened from oilfield. The concerted effects of the expression system, inducing condition and culture medium on the expression of NK703 GLA in E. coli were firstly investigated. The best combination was the recombinant E. coli strain of pET-28a + GLA/BL21(DE3) with 2.0% (w/v) lactose inducing in YT medium at 25 °C. Then, by optimizing the components of culture medium, a synthetic medium with dextrin and a feeding medium with glycerol as the carbon sources were developed to further enhance the GLA yield and improve the GLA solubility. In the end, the NK703 GLA activity increased about 50-fold, reached 14,470 ± 465 U/L, and the GLA productivity and the proportion of soluble GLA to the total soluble protein attained 206.0 ± 9.033 U L⁻¹ h⁻¹ and 60.13%, respectively. Scaling up the GLA production in 3.7 L fermenter under the optimized conditions identified in shake flask, the GLA activity also reached 12,406 ± 521 U/L, which was the highest report at fermenter level.     

The penicillin G acylase production by B. megaterium is amino acid consumption dependent

Aiming at to enhance the production of penicillin G acylase (PGA) by Bacillus megaterium, we have performed flasks experiments using different medium composition. Using 51 g/L of casein hydrolyzed with Alcalase and 2.7 g/L of phenylacetic acid (PhAc), the following carbon substrates were tested, individually and combined: glucose, glycerol, and lactose (present in cheese whey). Glycerol and glucose showed to be effective nutrients for the microorganism growth but delayed the PGA production. Cheese whey always increased enzyme production and cell mass. However, lactose (present in cheese whey) was not a significant carbon source for B. megaterium. PhAc, amino acids, and small peptides present in the hydrolyzed casein were the actual carbon sources for enzyme production. Replacement of hydrolyzed casein by free amino acids, 10.0 g/L, led to a significant increase in enzyme production (app. 150%), with a preferential consumption of alanine, aspartic acid, glycine, serine, arginine, threonine, lysine, and glutamic acid. A decrease of the enzyme production was observed when 20.0 g/L of amino acids were used. Using the single omission technique, it was shown that none of the 18 tested amino acids was essential for enzyme production. The use of a medium containing eight of the preferentially consumed amino acids lead to similar enzyme production level obtained when using 18 amino acids. PhAc, up to 2.7 g/L, did not inhibit enzyme production, even if added at the beginning of the cultivation.     

Two methionine aminopeptidases from Acinetobacter baumannii are functional enzymes

Drug resistance in Gram-negative bacteria, such as Acinetobacter baumannii, is emerging as a significant healthcare problem. New antibiotics with a novel mechanism of action are urgently needed to overcome the drug resistance. Methionine aminopeptidase (MetAP) carries out an essential cotranslational methionine excision in many bacteria and is a potential target to develop such novel antibiotics. Two putative MetAP genes were identified in A. baumannii genome, but whether they actually function as MetAP enzymes was not known. Therefore, we established an efficient E. coli expression system for their production as soluble and metal-free proteins for biochemical characterization. We demonstrated that both could carry out the metal-dependent catalysis and could be activated by divalent metal ions with the order Fe(II) ≈ Ni(II) > Co(II) > Mn(II) for both. By using a set of metalloform-selective inhibitors discovered on other MetAP enzymes, potency and metalloform selectivity on the A. baumannii MetAP proteins were observed. The similarity of their catalysis and inhibition to other MetAP enzymes confirmed that both may function as competent MetAP enzymes in A. baumannii and either or both may serve as the potential drug target.     

cis-Jasmone induces accumulation of defence compounds in wheat, Triticum aestivum

Liquid phase extraction (LPE) and vapor phase extraction (VPE) methodologies were used to evaluate the impact of the plant activator, cis-jasmone, on the secondary metabolism of wheat, Triticum aestivum, var. Solstice. LPE allowed the measurement of benzoxazinoids, i.e. 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one (HMBOA) and 6-methoxy-benzoxazolin-2-one (MBOA), and phenolic acids such as trans-p-coumaric acid, syringic acid, p-hydroxybenzoic acid, vanillic acid and cis- and trans-ferulic acid. Using LPE, a significantly higher level of DIMBOA was found in aerial parts and roots of T. aestivum following treatment with cis-jasmone, when compared with untreated plants. Similar results were obtained for phenolic acids, such as trans-ferulic acid and vanillic acid in roots. Using VPE, it was possible to measure levels of 2-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (HBOA), benzoxazolin-2(3H)-one (BOA), ferulic acid, syringic acid and coumaric acid. The levels of HBOA in aerial parts and roots were significantly greater in cis-jasmone treated plants compared to untreated plants. cis-Jasmone is known to be a plant activator in terms of production of defence-related volatile semiochemicals that repel aphids and increase the foraging activity of aphid parasitoids. These results show, for the first time, that cis-jasmone also induces selective production of secondary metabolites that are capable of directly reducing development of pests, diseases and weeds.     

Acyl-homoserine lactone-dependent eavesdropping promotes competition in a laboratory co-culture model

Many Proteobacteria use acyl-homoserine lactone (AHL)-mediated quorum sensing to activate the production of antibiotics at high cell density. Extracellular factors like antibiotics can be considered public goods shared by individuals within a group. Quorum-sensing control of antibiotic production may be important for protecting a niche or competing for limited resources in mixed bacterial communities. To begin to investigate the role of quorum sensing in interspecies competition, we developed a dual-species co-culture model using the soil saprophytes Burkholderia thailandensis (Bt) and Chromobacterium violaceum (Cv). These bacteria require quorum sensing to activate the production of antimicrobial factors that inhibit growth of the other species. We demonstrate that quorum-sensing-dependent antimicrobials can provide a competitive advantage to either Bt or Cv by inhibiting growth of the other species in co-culture. Although the quorum-sensing signals differ for each species, we show that the promiscuous signal receptor encoded by Cv can sense signals produced by Bt, and that this ability to eavesdrop on Bt can provide Cv an advantage in certain situations. We use an in silico approach to investigate the effect of eavesdropping in competition, and show conditions where early activation of antibiotic production resulting from eavesdropping can promote competitiveness. Our work supports the idea that quorum sensing is important for interspecies competition and that promiscuous signal receptors allow eavesdropping on competitors in mixed microbial habitats.     

Structural Insights into the Substrate Specificity of (S)-Ureidoglycolate Amidohydrolase and Its Comparison with Allantoate Amidohydrolase

In plants, the ureide pathway is a metabolic route that converts the ring nitrogen atoms of purine into ammonia via sequential enzymatic reactions, playing an important role in nitrogen recovery. In the final step of the pathway, (S)-ureidoglycolate amidohydrolase (UAH) catalyzes the conversion of (S)-ureidoglycolate into glyoxylate and releases two molecules of ammonia as by-products. UAH is homologous in structure and sequence with allantoate amidohydrolase (AAH), an upstream enzyme in the pathway with a similar function as that of an amidase but with a different substrate. Both enzymes exhibit strict substrate specificity and catalyze reactions in a concerted manner, resulting in purine degradation. Here, we report three crystal structures of Arabidopsis thaliana UAH (bound with substrate, reaction intermediate, and product) and a structure of Escherichia coli AAH complexed with allantoate. Structural analyses of UAH revealed a distinct binding mode for each ligand in a bimetal reaction center with the active site in a closed conformation. The ligand directly participates in the coordination shell of two metal ions and is stabilized by the surrounding residues. In contrast, AAH, which exhibits a substrate-binding site similar to that of UAH, requires a larger active site due to the additional ureido group in allantoate. Structural analyses and mutagenesis revealed that both enzymes undergo an open-to-closed conformational transition in response to ligand binding and that the active-site size and the interaction environment in UAH and AAH are determinants of the substrate specificities of these two structurally homologous enzymes.     

Purification and partial characterization of novel penicillin V acylase from Acinetobacter sp. AP24 isolated from Loktak Lake,an Indo-Burma biodiversity hotspot

Members of the bacterial genus Acinetobacter have attracted great attention over the past few decades, on account of their various biotechnological applications and clinical implications. In this study, we are reporting the first experimental penicillin V acylase (PVA) activity from this genus. Penicillin acylases are pharmaceutically important enzymes widely used in the synthesis of semisynthetic beta-lactam antibiotics. The bacterium, identified as Acinetobacter sp. AP24, was isolated from the water of Loktak Lake (Manipur, India), an Indo-Burma biodiversity hotspot. PVA production was increased threefold in an optimized medium with 0.2% sodium glutamate and 1% glucose as nitrogen and carbon sources respectively, after 24 hr of fermentation at 28°C and pH 7.0 with shaking at 180 rpm. The enzyme was purified to homogeneity by cation-exchange chromatography using SP-sepharose resin. The PVA is a homotetramer with subunit molecular mass of 34 kD. The enzyme was highly specific toward penicillin V with optimal hydrolytic activity at 40°C and pH 7.5. The enzyme was stable from pH 5.0 to 9.0 at 25 °C for 2 hr. The enzyme retained 75% activity after 1 hr of incubation at 40°C at pH 7.5.     

重组青霉素G酰化酶在枯草芽孢杆菌中的表达条件优化

 为获得巨大芽孢杆菌青霉素 G酰化酶 (PGA)的高产菌株和条件 ,构建了分泌表达 PGA的基因工程枯草杆菌菌株 ,对表达条件进行了优化 .以 LB作为初始培养基 ,考察了温度、苯乙酸、装液量、碳源对于工程菌 PGA产量的影响 .实验发现重组细胞产酶不再需要变温和苯乙酸诱导 .充足的通气量和适当浓度的淀粉可使细胞密度及 PGA表达量大为提高 .表达条件优化后 ,菌体 A60 0由 3提高到 2 0 ,PGA的表达量由 3～ 6U/ml提高到 35～ 40 U/ml,为目前生产用巨大芽孢杆菌表达量的 6倍 .     

Crystal structure of malonyl CoA-Acyl carrier protein transacylase from Xanthomanous oryzae pv. oryzae and its proposed binding with ACP

Xanthomonas oryzae pv. oryzae (Xoo) is a plant bacterial pathogen that causes bacterial blight (BB) disease, resulting in serious production losses of rice. The crystal structure of malonyl CoA-acyl carrier protein transacylase (XoMCAT), encoded by the gene fabD (Xoo0880) from Xoo, was determined at 2.3 Å resolution in complex with N-cyclohexyl-2-aminoethansulfonic acid. Malonyl CoA-acyl carrier protein transacylase transfers malonyl group from malonyl CoA to acyl carrier protein (ACP). The transacylation step is essential in fatty acid synthesis. Based on the rationale, XoMCAT has been considered as a target for antibacterial agents against BB. Protein-protein interaction between XoMCAT and ACP was also extensively investigated using computational docking, and the proposed model revealed that ACP bound to the cleft between two XoMCAT subdomains.     

Divergence of cofactor recognition across evolution: coenzyme A binding in a prokaryotic arylamine N-acetyltransferase

Arylamine N-acetyltransferase (NAT) enzymes are widespread in nature. They serve to acetylate xenobiotics and/or endogenous substrates using acetyl coenzyme A (CoA) as a cofactor. Conservation of the architecture of the NAT enzyme family from mammals to bacteria has been demonstrated by a series of prokaryotic NAT structures, together with the recently reported structure of human NAT1. We report here the cloning, purification, kinetic characterisation and crystallographic structure determination of NAT from Mycobacterium marinum, a close relative of the pathogenic Mycobacterium tuberculosis. We have also determined the structure of M. marinum NAT in complex with CoA, shedding the first light on cofactor recognition in prokaryotic NATs. Surprisingly, the principal CoA recognition site in M. marinum NAT is located some 30 Å from the site of CoA recognition in the recently deposited structure of human NAT2 bound to CoA. The structure explains the Ping-Pong Bi-Bi reaction mechanism of NAT enzymes and suggests mechanisms by which the acetylated enzyme intermediate may be protected. Recognition of CoA in a much wider groove in prokaryotic NATs suggests that this subfamily may accommodate larger substrates than is the case for human NATs and may assist in the identification of potential endogenous substrates. It also suggests the cofactor-binding site as a unique subsite to target in drug design directed against NAT in mycobacteria.     

Probing electron transfer reactions between two azurins from Alcaligenes xylosoxidans GIFU 1051 with optically active Ru complexes as molecular recognition probes: Importance of the 43rd residue

Electron transfer reactions between optically-active Ru^II/III complexes incorporating (S)-/(R)-amino acids, and the two azurins, azurin-1 (az-1Cu) and azurin-2 (az-2Cu) isolated from Alcaligenes xylosoxidans GIFU 1051, have been studied to probe molecular recognition sites on the two azurins. The Ru^II/III complexes are K[Ru^II(L)(bpy)] and [Ru^III(L)(bpy)], and have a tripodal ligand (L) derived from the (S)-/(R)-amino acids, which are in turn exchanged for other functional substituent groups, such as (S)-/(R)-phenylalanine, -leucine, -valine, -alanine, and -glutamic acid (L = (S)-/(R)-BCMPA, -BCMLE, -BCMVA, -BCMAL, and -BCMGA). In the oxidation reaction of az-1Cu^I promoted by the Ru^III complexes, the kinetic parameters exhibited enantio- and stereo-selectivities, while the same reaction of az-2Cu^I was less enantio- and stereo-selective. These differences suggest that the processes of formation of the activated states are different for the two azurins. On the other hand, such a difference has not been observed for az-1 and az-2 with respect to the reduction reactions promoted by both azurins Cu^II by the Ru^II complexes within the experimental error. This suggests that the neutrality of the Ru complexes is important for precise molecular recognition of azurins. His117 has been proposed as the electron transfer site. The local structures in the vicinity of the His117 side chain in the two azurins, are essentially identical with the exception of the 43rd residue, Val43 and Ala43 for az-1 and az-2, respectively. Electron transfer reactions between Ru^III complexes and a mutant azurin, V43A-az-1, were also carried out. Interestingly, the activation parameters estimated were very similar to those of az-2, indicating that the 43rd residue acts as the electron transfer site in azurins and provides rationalization for the different mechanisms of az-1 and az-2 in redox reactions.     

Metabolic engineering of Escherichia coli: A sustainable industrial platform for bio-based chemical production

In order to decrease carbon emissions and negative environmental impacts of various pollutants, more bulk and/or fine chemicals are produced by bioprocesses, replacing the traditional energy and fossil based intensive route. The Gram-negative rod-shaped bacterium, Escherichia coli has been studied extensively on a fundamental and applied level and has become a predominant host microorganism for industrial applications. Furthermore, metabolic engineering of E. coli for the enhanced biochemical production has been significantly promoted by the integrated use of recent developments in systems biology, synthetic biology and evolutionary engineering. In this review, we focus on recent efforts devoted to the use of genetically engineered E. coli as a sustainable platform for the production of industrially important biochemicals such as biofuels, organic acids, amino acids, sugar alcohols and biopolymers. In addition, representative secondary metabolites produced by E. coli will be systematically discussed and the successful strategies for strain improvements will be highlighted. Moreover, this review presents guidelines for future developments in the bio-based chemical production using E. coli as an industrial platform.     

生物法合成达泊西汀

通过固定化青霉素G酰化酶(PGA)对(±)-N-苯乙酰基-3-氨基-3-苯基丙酸进行酶法拆分,得到合成达泊西汀的中间体(S)-3-氨基-3-苯基丙酸,(S)-3-氨基-3-苯基丙酸经过还原、甲基化、缩合等多步化学合成得到最终产物达泊西汀。(±)-N-苯乙酰基-3-氨基-3-苯基丙酸的最佳拆分条件:底物(±)-N-苯乙酰基-3-氨基-3-苯基丙酸2.83 g,固定化的青霉素酰化酶2.66 g,pH 7.5,25℃反应4 h,(S)-3-氨基-3-苯基丙酸收率为89.4%,e.e.值99.3%。达泊西汀的总收率25.5%,e.e.值96.7%。     

Monoclonal antibody against the poly-γ-d-glutamic acid capsule of Bacillus anthracis protects mice from enhanced lethal toxin activity due to capsule and anthrax spore challenge

Background

The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT.

Methods

To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with K_d values of 0.8 μM and 2.6 μM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed.

Results

Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen.

General significance

Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.     

Production of (R)-3-hydroxybutyric acid by fermentation and bioconversion processes with Azohydromonas lata

Feasibility of producing (R)-3-hydroxybutyric acid ((R)-3-HB) using wild type Azohydromonas lata and its mutants (derived by UV mutation) was investigated. A. lata mutant (M5) produced 780 mg/l in the culture broth when sucrose was used as the carbon source. M5 was further studied in terms of its specificity with various bioconversion substrates for production of (R)-3-HB. (R)-3-HB concentration produced in the culture broth by M5 mutant was 2.7-fold higher than that of the wild type strain when sucrose (3% w/v) and (R,S)-1,3-butanediol (3% v/v) were used as carbon source and bioconversion substrate, respectively. Bioconversion of resting cells (M5) with glucose (1% v/w), ethylacetoacetate (2% v/v), and (R,S)-1,3-butanediol (3% v/v), resulted in (R)-3-HB concentrations of 6.5 g/l, 7.3 g/l and 8.7 g/l, respectively.