Microbial production of poly-γ-glutamic acid

Poly-γ-glutamic acid (γ-PGA) is a natural, biodegradable and water-soluble biopolymer of glutamic acid. This review is focused on nonrecombinant microbial production of γ-PGA via fermentation processes. In view of its commercial importance, the emphasis is on l-glutamic acid independent producers (i.e. microorganisms that do not require feeding with the relatively expensive amino acid l-glutamic acid to produce γ-PGA), but glutamic acid dependent production is discussed for comparison. Strategies for improving production, reducing costs and using renewable feedstocks are discussed.     

Biochemistry and molecular genetics of poly-γ-glutamate synthesis

Current research into poly-gamma-glutamate (PGA) and its biosynthesis is reviewed. In PGA-producing Bacillus subtilis, glutamate racemase supplies abundant DL-glutamate, the substrate for PGA synthesis. The pgsBCA genes of PGA-producing B. subtilis, which encode the membrane-associated PGA synthetase complex PgsBCA, were characterized and the enzyme complex was suggested to be an atypical amide ligase based on its structure and function. A novel reaction mechanism of PGA synthesis is proposed.     

Cooperative adsorption of critical metal ions using archaeal poly-γ-glutamate

Antimony, beryllium, chromium, cobalt (Co), gallium (Ga), germanium, indium (In), lithium, niobium, tantalum, the platinoids, the rare-earth elements (including dysprosium, Dy), and tungsten are generally regarded to be critical (rare) metals, and the ions of some of these metals are stabilized in acidic solutions. We examined the adsorption capacities of three water-soluble functional polymers, namely archaeal poly-γ-glutamate (L-PGA), polyacrylate (PAC), and polyvinyl alcohol (PVA), for six valuable metal ions (Co²⁺, Ni²⁺, Mn²⁺, Ga³⁺, In³⁺, and Dy³⁺). All three polymers showed apparently little or no capacity for divalent cations, whereas L-PGA and PAC showed the potential to adsorb trivalent cations, implying the beneficial valence-dependent selectivity of anionic polyelectrolytes with multiple carboxylates for metal ions. PVA did not adsorb metal ions, indicating that the crucial role played by carboxyl groups in the adsorption of crucial metal ions cannot be replaced by hydroxyl groups under the conditions. In addition, equilibrium studies using the non-ideal competitive adsorption model indicated that the potential for L-PGA to be used for the removal (or collection) of water-soluble critical metal ions (e.g., Ga³⁺, In³⁺, and Dy³⁺) was far superior to that of any other industrially-versatile PAC materials.     

Genetic and metabolic engineering for microbial production of poly-γ-glutamic acid

Poly-γ-glutamic acid (γ-PGA) is a natural biopolymer of glutamic acid. The repeating units of γ-PGA may be derived exclusively from d-glutamic acid, or l-glutamic acid, or both. The monomer units are linked by amide bonds between the α-amino group and the γ-carboxylic acid group. γ-PGA is biodegradable, edible and water-soluble. It has numerous existing and emerging applications in processing of foods, medicines and cosmetics. This review focuses on microbial production of γ-PGA via genetically and metabolically engineered recombinant bacteria. Strategies for improving production of γ-PGA include modification of its biosynthesis pathway, enhancing the production of its precursor (glutamic acid), and preventing loss of the precursor to competing byproducts. These and other strategies are discussed. Heterologous synthesis of γ-PGA in industrial bacterial hosts that do not naturally produce γ-PGA is discussed. Emerging trends and the challenges affecting the production of γ-PGA are reviewed.     

Microbial transformation of α-pinene

Summary A bacterium capable of utilizing -pinene as a sole carbon and energy source was isolated from soil. This strain, named strain S201-1, which was identified as Pseudomonas maltophilia on the basis of its taxonomical properties, accumulated limonene, borneol, camphor, perillic acid, and 2-(4-methyl-3-cyclohexenylidene) propionic acid from -pinene in the culture broth. It was demonstrated that -pinene, -pinene, borneol, camphor, and a number of p-menthane derivatives were oxidized by this strain. Relations between the protonation of -pinene and the formation of the products by the microbe are discussed.     

Microbial production of poly-β-hydroxybutyrate by marine microbes isolated from various marine environments

Considering the industrial interest of Poly-β-hydroxybutyrate (PHB), bacteria isolated from the various marine arenas were screened for their ability to accumulate PHB and were compared with Wausteria eutropha (MTCC-1285). Among the 42 isolates, four strains showed the accumulation of PHB. The maximum PHB producer Vibrio sp. (MK4) was further studied in detail. To increase the productivity, steps were taken to evaluate the effect of carbon sources, nitrogen sources, pH and sodium chloride concentration on PHB productivity by MK4. The optimized conditions were further used for the batch fermentation over a period of 72 h. Significantly higher maximum biomass of 9.1 g/L with a PHB content of 4.223 g/L was obtained in a laboratory-scale bioreactor at 64 h, thus giving a productivity of 0.065 g/L/h. The extracted polymer was compared with the authentic PHB and was confirmed to be PHB using FTIR analysis and ¹H NMR analysis. Thus, the study highlights the potential of the use of Vibrio sp (MK4) in the commercial production of PHB.     

Analytical approaches to poly-γ-glutamate: quantification, molecular size determination, and stereochemistry investigation

Poly-γ-glutamate, a nylon-like polyamide material typically consisting of both enantiomers of glutamate, exhibits reasonable biodegradability and its multi-functionality is attracting particular attention. Thus, its industrial application as a versatile and chiral polymer is in increasing demands. Poly-γ-glutamate is presently synthesized using several biocatalysts (e.g., bacterial cells), but the uncontrollable structural diversity of such biosynthesized materials is an area of concern. This review highlights analytical approaches of interest to assure the functional and structural reproducibility of poly-γ-glutamate.     

Isolation of Bacillus subtilis (chungkookjang), a poly-γ-glutamate producer with high genetic competence

A bacterium with high poly-gamma-glutamate (PGA) productivity was isolated from the traditional Korean seasoning, Chung-Kook-Jang. This bacterium could be classified as a Bacillus subtilis, but sporulation in culture was infrequent in the absence of Mn2+. It was judged to be a variety of B. subtilis and designated B. subtilis (chungkookjang). L-Glutamate significantly induced PGA production, and highly elongated PGAs were synthesized. The volumetric yield reached 13.5 mg ml(-1) in the presence of 2% L-glutamate. The D-glutamate content was over 50% in every PGA produced under the conditions used. During PGA production, glutamate racemase activity was found in the cells, suggesting that the enzyme is involved in the D-glutamate supply. Molecular sizes of PGAs were changed by the salt concentration in the medium; PGAs with comparatively low molecular masses were produced in culture media containing high concentrations of NaCl. B. subtilis (chungkookjang) harbors no plasmid and is the first B. subtilis strain reported with both naturally high PGA productivity and high genetic competence.     

Microbial transformation of acetyl-11-keto-β-boswellic acid and their inhibitory activity on LPS-induced NO production

The capabilities of 20 strains of fungi to transform acetyl-11-keto-β-boswellic (AKBA) were screened. And biotransformation of AKBA by Cunninghamella blakesleana AS 3.970 afforded five metabolites (1–5), while two metabolites (6, 7) were isolated from biotransformation of Cunninghamella elegans AS 3.1207. The chemical structures of these metabolites were identified by spectral methods including 2D NMR and their structures were elucidated as 7β-hydroxy-3-acety-11-keto-β-boswellic acid (1), 21β-dihydroxy-3-acety-11-keto-β-boswellic acid (2), 7β,22α-dihydroxy-3-acety-11-keto-β-boswellic acid (3), 7β,16α-dihydroxy-3-acety-11-keto-β-boswellic acid (4), 7β,15α-dihydroxy-3-acety-11-keto-β-boswellic acid (5); 7β,15α,21β-trihydroxy-3-acety-11-keto-β-boswellic acid (6) and 15α,21β-dihydroxy-3-acety-11-keto-β-boswellic acid (7). All these products are previously unknown. Their primary structure–activity relationships (SAR) of inhibition activity on LPS-induced NO production in RAW 264.7 macrophage cells were evaluated.     

Mutations suppressing the loss of DegQ function in Bacillus subtilis (natto) poly-γ-glutamate synthesis

The degQ gene of Bacillus subtilis (natto), encoding a small peptide of 46 amino acids, is essential for the synthesis of extracellular poly-gamma-glutamate (γPGA). To elucidate the role of DegQ in γPGA synthesis, we knocked out the degQ gene in Bacillus subtilis (natto) and screened for suppressor mutations that restored γPGA synthesis in the absence of DegQ. Suppressor mutations were found in degS, the receptor kinase gene of the DegS-DegU two-component system. Recombinant DegS-His(6) mutant proteins were expressed in Escherichia coli cells and subjected to an in vitro phosphorylation assay. Compared with the wild type, mutant DegS-His(6) proteins showed higher levels of autophosphorylation (R208Q, M195I, L248F, and D250N), reduced autodephosphorylation (D250N), reduced phosphatase activity toward DegU, or a reduced ability to stimulate the autodephosphorylation activity of DegU (R208Q, D249G, M195I, L248F, and D250N) and stabilized DegU in the phosphorylated form. These mutant DegS proteins mimic the effect of DegQ on wild-type DegSU in vitro. Interestingly, DegQ stabilizes phosphorylated DegS only in the presence of DegU, indicating a complex interaction of these three proteins.     

Molecular cloning of murine folylpoly-γ-glutamate synthetase

Folylpoly-γ-glutamate synthetase (FPGS) is essential for mammalian cell survival and is a major determinant of cytotoxicity and selectivity for folate antimetabolites. Here we describe the cloning of a cDNA encoding murine FPGS isolated from L1210 leukemia cells. The amino acid sequence of murine FPGS is 82% identical to human FPGS [1] with identical discrete regions of up to 41 residues. Murine FPGS contains two AUG initiation codons, shown to be responsible for mitochondrial and cytosolic forms of the enzyme in human cells [2]. Previous studies indicated species, tissue, and tumor specific differences in mammalian FPGS. The availability of murine FPGS expands the knowledge and understanding of the spectrum of these variations.     

Glutamic acid independent production of poly-γ-glutamic acid by Bacillus amyloliquefaciens LL3 and cloning of pgsBCA genes

A new glutamic acid independent poly-γ-glutamic acid (γ-PGA) producing strain, which was identified as Bacillusamyloliquefaciens LL3 by analysis of 16S rDNA and gyrase subunit A gene (gyrA), was isolated from fermented food. The product had a molecular weight of 470, 801 and l-glutamate monomer content of 98.47%. The pre-optimal medium, based on single-factor tests and orthogonal design, contained 50 g/L sucrose, 2 g/L (NH₄)₂SO₄, 0.6 g/L MgSO₄, and provided well-balanced changes in processing parameters and a γ-PGA yield of 4.36 g/L in 200 L system. The γ-PGA synthetase genes pgsBCA were cloned from LL3, and successfully expressed by pTrcLpgs vector in Escherichia coli JM109, resulting the synthesis of γ-PGA without glutamate. This study demonstrates the designedly improved yield of γ-PGA in 200 L system and the first report of pgsBCA from glutamic acid independent strain, which will benefit the metabolized mechanism investigation and the wide-ranging application of γ-PGA.     

Crystal structure of bacteriophage ϕNIT1 zinc peptidase PghP that hydrolyzes γ-glutamyl linkage of bacterial poly-γ-glutamate

Poly‐γ‐glutamate hydrolase P (PghP) of Bacillus subtilis bacteriophage ΦNIT1 hydrolyzes the γ‐glutamyl peptide linkage of extracellular poly‐γ‐glutamate produced by bacilli, which facilitates infection and propagation of phage progenies. Crystal structure of PghP was determined at a resolution of 1.9 Å. Structure of PghP was elucidated as a globular protein with an open α/β mixed core structure and a seven‐stranded parallel/anti‐parallel β‐sheet. The β‐sheet contained a core four‐stranded parallel β‐sheet. A zinc‐binding motif, His‐Glu‐His, was identified at the C‐terminal end of the β‐sheet. Structure analysis demonstrated that PghP, which had not been previously classified into any peptidase/protease family due to lack of amino acid sequence similarity with known enzymes, had a catalytic center containing a zinc ion and an overall topology resembling mammalian carboxypeptidase A and related enzymes. Structural comparisons indicated important amino acid residues of PghP for catalysis and recognition of the γ‐peptide bond of poly‐γ‐glutamate, which was confirmed by site‐directed mutagenesis of PghP. Proteins 2011. © 2012 Wiley Periodicals, Inc.     

Microbial growth and production kinetics of Streptomyces antibioticus Tü 6040

Streptomyces antibioticus Tü 6040 is the producer of simocyclinones, which belong to a novel family of angucyclinone antibiotics some of which show antitumor activities. Growth and antibiotic production is dependent on the medium composition, especially on the carbon and nitrogen source, and on the fermentation conditions. The best results with respect to antibiotic productivity were achieved using a chemically defined medium with glycerol and L-lysine as carbon and nitrogen source, respectively, in an airlift fermenter with minimised shear stress at low gas flow rates withour oxygen limitation. These conditions led to a homogeneous formation of pellets of 1–2 mm in diameter and guaranteed reproducible product yields of the main compound, simocyclinone D8, in the range of 300 mg/l.     

Sequential production of two biopolymers-levan and poly-ε-lysine by microbial fermentation

Sequential fermentation for the production of two invaluable biopolymers, levan and poly-ε-lysine (ε-PL), has been successfully developed. It involves fermentation of Bacillus subtilis (natto) Takahashi in sucrose medium to produce levan, separation of levan product from small remaining sugar molecules by ultrafiltration and fermentation of the remnant from levan production by Streptomyces albulus to produce ε-PL. In the process, 50-60 g/L of levan was produced (100% recovery after precipitation by ethanol). The remnant from levan production with glucose adjusted to 30 g/L and with combined use of yeast extract (10 g/L), (NH₄)₂SO₄ (2 g/L) and basal salts was proven to be suitable for ε-PL production. 4.37 g/L of ε-PL accumulation (85% recovery after purification) was reached in 72 h using two-stage fermentation with control of pH. The process of using remnant (waste) from levan fermentation for the second biopolymer (ε-PL) production is unprecedented and the products obtained are environmental-friendly.     

Folylpoly-γ-glutamate synthesis by bacteria and mammalian cells

Summary The purification and properties of folylpolyglutamate synthetase fromCorynebacterium sp, and some properties of partially purified enzyme fromLactobacillus casei, Streptococcus faecalis, Neurospora crassa, pig liver, and Chinese hamster ovary cells, are described.TheCorynebacterium enzyme catalyzes a MgATP-dependent addition of glutamate to a variety of reduced pteroate and pteroylmono-, di-, and triglutamate substrates, with the concomitant production of MgADP and phosphate. Although glutamate moieties are added in a sequential fashion, the kinetic mechanism, which is Ordered Ter Ter, precludes the sequential addition of glutamate moieties to enzyme-bound folate. It is suggested that catalysis precedes via the formation of a pteroyl--glutamyl phosphate intermediate.Thein vivo distribution of folylpolyglutamates in bacteria and mammalian cells, which differ from source to source, appear to be a reflection of the ability of folylpolyglutamates to act as substrates for folylpolyglutamate synthetases from different sources.Only one enzyme appears to be involved in the conversion of pteroylmonoglutamates to polyglutamate forms in both bacteria and mammalian cells. Bacterial folylpolyglutamate synthetases use a variety of pteroylmonoglutamates as their preferred monoglutamate substrate, but use 5,10-methylenetetrahydropteroylpolyglutamates as their preferred, and sometimes only, polyglutamate substrate. Mono- and polyglutamyl forms of tetrahydrofolate are the preferred substrates of mammalian folylpolyglutamate synthetases.     

A newly isolated Bacillus siamensis SB1001 for mass production of poly-γ-glutamic acid

Poly-γ-glutamic acid (γ-PGA) is a retaining agent; it has applications in the food, medicine, agriculture, cosmetics and wastewater treatment industries. Most of the γ-PGA producing strains belong to the genus Bacillus. This study reports on a novel γ-PGA producing species. Bacillus siamensis SB1001 was screened and isolated from organically cultivated soybeans exhibiting a high γ-PGA producing ability. The fermentation medium and culture parameters for γ-PGA production by Bacillus siamensis SB1001 were optimized by statistical methods. The sucrose, l-glutamic acid and dipotassium phosphate in the medium were shown to be the significant factors of the γ-PGA production, and the optimum medium obtained consisted of the following: 106.86 g/L sucrose, 69.84 g/L l-glutamic acid and 2.39 g/L dipotassium phosphate. Using the optimized medium, 25.22 g/L γ-PGA were produced with a productivity of 1.05 g/L/h. The γ-PGA obtained had a molecular weight of 7.9 × 10⁵ Da and a polydispersity index of 2.34, and the ratio of d-/L-glutamic acid was 89.71%:10.29%. To the best of our knowledge, this is the first report of γ-PGA production by B. siamensis strain. B. siamensis SB1001 has great potential as an industrial γ-PGA producer.     

Effect of phosphate supply and aeration on poly-β-hydroxybutyrate production in Azotobacter chroococcum

The effects of phosphate supply and aeration on cell growth and PHB accumulation were investigated in Azotobacter chroococcum 23 with the aim of increasing PHB production. Phosphate limitation favoured PHB formation in Azotobacter chroococcum 23, but inhibited growth. Azotobacter chroococcum 23 cells demonstrated intensive uptake of orthophosphate during exponential growth. At the highest phosphate concentration (1·5 g/litre) and low aeration the amount of intracellular orthophosphate/g residual biomass was highest. Under conditions of fed-batch fermentation the possibility of controlling the PHB production process by the phosphate level in the cultivation medium was demonstrated. A 36 h fed-batch fermentation resulted in a biomass yield of 110 g/litre with a PHB cellular concentration of 75% dry weight, PHB content 82·5 g/litre, PHB yield Y_P/S = 0·24 g/g and process productivity 2·29 g/litre·h.     

Microbial transglutaminase—a review of its production and application in food processing

Transglutaminase (EC 2.3.2.13) catalyses an acyl-transfer reaction in which the -carboxamide groups of peptide-bound glutaminyl residues are the acyl donors. The enzyme catalyses in vitro cross-linking in whey proteins, soya proteins, wheat proteins, beef myosin, casein and crude actomysin refined from mechanically deboned poultry meat. In recent years, on the basis of the enzyme's reaction to gelatinize various food proteins through the formation of cross-links, this enzyme has been used in attempts to improve the functional properties of foods. Up to now, commercial transglutaminase has been merely obtained from animal tissues. The complicated separation and purification procedure results in an extremely high price for the enzyme, which hampers a wide application in food processing. Recently studies on the production of transglutaminase by microorganisms have been started. The enzyme obtained from microbial fermentation has been applied in the treatment of food of different origins. Food treated with microbial transglutaminase appeared to have an improved flavour, appearance and texture. In addition, this enzyme can increase shelf-life and reduce allergenicity of certain foods. This paper gives an overview of the development of microbial transglutaminase production, including fermentation and down-stream processing, as well as examples of how to use this valuable enzyme in processing foods of meat, fish and plant origin.