Microbial production of cyanophycin: From enzymes to biopolymers

Cyanophycin is an attractive biopolymer with chemical and material properties that are suitable for industrial applications in the fields of food, medicine, cosmetics, nutrition, and agriculture. For efficient production of cyanophycin, considerable efforts have been exerted to characterize cyanophycin synthetases (CphAs) and optimize fermentations and downstream processes. In this paper, we review the characteristics of diverse CphAs from cyanobacteria and non-cyanobacteria. Furthermore, strategies for cyanophycin production in microbial strains, including Escherichia coli, Pseudomonas putida, Ralstonia eutropha, Rhizopus oryzae, and Saccharomyces cerevisiae, heterologously expressing different cphA genes are reviewed. Additionally, chemical and material properties of cyanophycin and its derivatives produced through biological or chemical modifications are reviewed in the context of their industrial applications. Finally, future perspectives on microbial production of cyanophycin are provided to improve its cost-effectiveness.     

Microbial and bioconversion production of D-xylitol and its detection and application

D-Xylitol is found in low content as a natural constituent of many fruits and vegetables. It is a five-carbon sugar polyol and has been used as a food additive and sweetening agent to replace sucrose, especially for non-insulin dependent diabetics. It has multiple beneficial health effects, such as the prevention of dental caries, and acute otitis media. In industry, it has been produced by chemical reduction of D-xylose mainly from photosynthetic biomass hydrolysates. As an alternative method of chemical reduction, biosynthesis of D-xylitol has been focused on the metabolically engineered Saccharomyces cerevisiae and Candida strains. In order to detect D-xylitol in the production processes, several detection methods have been established, such as gas chromatography (GC)-based methods, high performance liquid chromatography (HPLC)-based methods, LC-MS methods, and capillary electrophoresis methods (CE). The advantages and disadvantages of these methods are compared in this review.     

Microbial Transformation of Antibiotics

Microbial transformation of the nucleoside analogue antibiotic showdomycin was performed using some Streptomyces species. Both the growing culture and the resting cells of Streptomyces sp. No. 383 arrested the antibacterial activity of showdomycin. The inactivated showdomycin was isolated from the reaction mixture by carbon chromatography and was identified with an isoshowdomycin sample which has been chemically derived from showdomycin. It is conjectured that the conversion of showdomycin to isoshowdomycin results from isomerization by an enzyme of Streptomyces sp. No. 383.     

Microbial production of dihydroxyacetone

Dihydroxyacetone is extensively used in cosmetic industry as an artificial suntan besides having clinical and biological applications. Thus, it is important to meet the commercial demand of dihydroxyacetone at an economical and qualitative level. Microbial route of production is found to be more favorable for dihydroxyacetone as compared to chemical methods. This review gives detailed information about the microbial route of dihydroxyacetone production. Till date the microorganism which is most utilized for dihydroxyacetone production is Gluconobacter oxydans. Some limitations associated with dihydroxyacetone production by G. oxydans like substrate inhibition, product inhibition and oxygen limitation are discussed here. Various fermentation modes and culture conditions have been tried for their ability to overcome these limitations. It has been found that fed-batch mode of fermentation provides a better yield as compared to batch mode for dihydroxyacetone production. Two-stage repeated fed-batch mode of fermentation has been found to be the most optimized mode. Immobilization has also been recognized as a much better alternative for fermentation since it avoids the problem of substrate and product inhibition to a greater extent. Although these methods have increased the dihydroxyacetone production to a prominent level yet the production has not reached the level required to meet the commercial demand. One looks for future prospects of developing recombinant microbial method for dihydoxyacetone production.     

Microbial production of glutathione

World Journal of Microbiology and Biotechnology - The present work aims to identify the microbial diversity associated with six Indian Drosophila species using next generation sequencing (NGS)...     

Microbial production of fatty alcohols

Fatty alcohols have numerous commercial applications, including their use as lubricants, surfactants, solvents, emulsifiers, plasticizers, emollients, thickeners, and even fuels. Fatty alcohols are currently produced by catalytic hydrogenation of fatty acids from plant oils or animal fats. Microbial production of fatty alcohols may be a more direct and environmentally-friendly strategy since production is carried out by heterologous enzymes, called fatty acyl-CoA reductases, able to reduce different acyl-CoA molecules to their corresponding primary alcohols. Successful examples of metabolic engineering have been reported in Saccharomyces cerevisiae and Escherichia coli in which the production of fatty alcohols ranged from 1.2 to 1.9 g/L, respectively. Due to their metabolic advantages, oleaginous yeasts are considered the best hosts for production of fatty acid-derived chemicals. Some of these species can naturally produce, under specific growth conditions, lipids at high titers (>50 g/L) and therefore provide large amounts of fatty acyl-CoAs or fatty acids as precursors. Very recently, taking advantage of such features, over 8 g/L of C₁₆–C₁₈ fatty alcohols have been produced in Rhodosporidium toruloides. In this review we summarize the different metabolic engineering strategies, hosts and cultivation conditions used to date. We also point out some future trends and challenges for the microbial production of fatty alcohols.     

Microbial production of sialic acid and sialylated human milk oligosaccharides: Advances and perspectives

Sialic acids (SAs) are important functional sugars, and monomers of sialylated human milk oligosaccharides (sialylated HMOs or sialyllactoses), which are crucial for improving infant development and can facilitate infant brain development, maintain brain health, and enhance immunity. The most common form of SA is N-acetylneuraminic acid (NeuAc), and the main forms of sialyllactoses are 6′-sialyllactose (6′-SL) and 3′-sialyllactose (3′-SL). As functional food additive, the demand for NeuAc and sialyllactoses will continuously increase due to their wide and important fields of application. However, NeuAc and sialyllactoses produced by traditional extraction methods are inefficient and may cause allergen contamination, and cannot keep up with the rapidly increasing market demand. Therefore, the production of NeuAc and sialyllactoses by sustainable biotechnological methods have attracted increasing attention. In particular, the development of metabolic engineering and synthetic biology techniques and strategies have promoted efficient biosynthesis of NeuAc and sialyllactoses. In this review, we first discussed the application of NeuAc and sialyllactoses. Secondly, metabolic engineering and protein engineering-fueled progress of whole-cell catalysis and de novo synthesis of NeuAc and sialyllactoses were systematically summarized and compared. Furthermore, challenges of efficient microbial production of NeuAc and sialyllactoses as well as strategies for overcoming the challenges were discussed, such as clustered regularly interspaced short palindromic repeats interference (CRISPRi)-aided identification of key precursor transport pathways, synergistically debottleneck of kinetic and thermodynamic limits in synthetic pathways, and dynamic regulation of metabolic pathways for balancing cell growth and production. We hope this review can further facilitate the understanding of limiting factors that hampered efficient production of sialic acid and sialyllactoses, as well as contribute to the development of strategies for the construction of efficient production hosts for high-level production of sialic acid and sialyllactose based on synthetic biology tools and strategies.     

From miracle fruit to transgenic tomato: mass production of the taste-modifying protein miraculin in transgenic plants

The utility of plants as biofactories has progressed in recent years. Some recombinant plant-derived pharmaceutical products have already reached the marketplace. However, with the exception of drugs and vaccines, a strong effort has not yet been made to bring recombinant products to market, as cost-effectiveness is critically important for commercialization. Sweet-tasting proteins and taste-modifying proteins have a great deal of potential in industry as substitutes for sugars and as artificial sweeteners. The taste-modifying protein, miraculin, functions to change the perception of a sour taste to a sweet one. This taste-modifying function can potentially be used not only as a low-calorie sweetener but also as a new seasoning that could be the basis of a new dietary lifestyle. However, miraculin is far from inexpensive, and its potential as a marketable product has not yet been fully developed. For the last several years, biotechnological production of this taste-modifying protein has progressed extensively. In this review, the characteristics of miraculin and recent advances in its production using transgenic plants are summarized, focusing on such topics as the suitability of plant species as expression hosts, the cultivation method for transgenic plants, the method of purifying miraculin and future advances required to achieve industrial use.     

Ethanol sensitivity of NMDA receptors

NMDA receptors are ionotropic glutamate receptors assembled of subunits of the NR1 and of the NR2 family (NR2A–NR2D). The subunit diversity largely affects the pharmacological properties of NMDA receptors and, hence, gives rise to receptor heterogeneity. As an overall result of studies on recombinant and native NMDA receptors, ethanol inhibits the function of receptors containing the subunits NR2A and/or NR2B to a greater extent than those containing NR2C or NR2D. For example, in rat cultured mesencephalic neurons, NR2C expression was developmentally increased, whereas expression of NR2A and NR2B was decreased. These changes coincided with a developmental loss of sensitivity of NMDA responses to ethanol and ifenprodil, a non-competitive NMDA receptor antagonist that shows selectivity for NR2B-containing receptors. Also in rat locus coeruleus neurons, the low ethanol sensitivity of somatic NMDA receptors could be explained by a prominent expression of NR2C. The inhibitory site of action for ethanol on the NMDA receptor is not yet known. Patch–clamp studies suggest a target site exposed to or only accessible from the extracellular environment. Apparently, amino acid residue Phe⁶³⁹, located in the TM3 domain of NR1, plays a crucial role in the inhibition of NMDA receptor function by ethanol. Since this phenylalanine site is common to all NMDA and non-NMDA receptor (AMPA/kainate receptor) subunits, this observation is consistent with accumulating evidence for a similar ethanol sensitivity of a variety of NMDA and non-NMDA receptors, but it cannot explain the differences in ethanol sensitivity observed with different NR2 subunits.     

Microbial production and application of sophorolipids

Sophorolipids are surface-active compounds synthesized by a selected number of yeast species. They have been known for over 40 years, but because of growing environmental awareness, they recently regained attention as biosurfactants due to their biodegradability, low ecotoxicity, and production based on renewable resources. In this paper, an overview is given of the producing yeast strains and various aspects of fermentative sophorolipid production. Also, the biochemical pathways and regulatory mechanisms involved in sophorolipid biosynthesis are outlined. To conclude, a summary is given on possible applications of sophorolipids, either as native or modified molecules.     

Microbial challenges of poultry meat production

Food safety and shelf-life are both important microbial concerns in relation to broiler meat production. Focus is mainly placed on the absence or control of potentially pathogenic microbes such as Salmonella spp. and Campylobacter spp. but, from the commercial point of view, other spoilage bacteria also play a role as potential threats. Regarding food safety, the primary target should be the production of pathogen-free live animals, thus allowing slaughter plants to keep the processing line free of those microorganisms.Consumers believe that quality of foods from organic production is superior to foods from conventional production. The aim of the present study was to evaluate and compare the bacterial quality of chicken meat from organic and conventional production on the basis of traditional meat quality criteria. Fresh free grazing broiler carcasses were purchased directly from rural households (n = 80) and fresh retail chicken parts from conventional broiler carcasses from the local supermarkets in the region of Epirus (Poultry Producers Association. Arta) (n = 200).The samples were microbiologically tested for the presence of bacteria such as: Salmonella spp., Listeria monocytogenes, Staphylococcus aureus, Enterobacteriaceae, Escherichia coli, Campylobacter spp., and C. perfringens. Total count of aerobic mesophilic bacteria was also determined. Bacteriological tests were performed by means of standard methods of isolation and identification of individual species of bacteria according to ISO requirements. API-tests (bioMerieux) and Vitek 2 Identification System (bioMerieux) were used for biochemical determination. High levels of microbial contamination and occurrence of pathogenic bacteria at then fresh free grazing broiler carcasses reflect the poor hygienic quality of the slaughter conditions in the rural households.     

Microbial production of (+)-trans-isochorismic acid

Two methods are described for the preparation of enantiomerically pure (+)-trans-isochorismic acid, an important metabolite of the postchorismate pathway. Both methods can be employed to prepare isotopically labeled isochorismic acid. One of the two methods is suitable to prepare bulk quantities of isochorismic acid using a recombinant strain of Klebsiella pneumoniae 62-1. (c) 1995 John Wiley & Sons, Inc.     

Microbial production of vitamin B12

One of the most alluring and fascinating molecules in the world of science and medicine is vitamin B12 (cobalamin), which was originally discovered as the anti pernicious anemia factor and whose enigmatic complex structure is matched only by the beguiling chemistry that it mediates. The biosynthesis of this essential nutrient is intricate, involved and, remarkably, confined to certain members of the prokaryotic world, seemingly never have to have made the eukaryotic transition. In humans, the vitamin is required in trace amounts (approximately 1 microg/day) to assist the actions of only two enzymes, methionine synthase and (R)-methylmalonyl-CoA mutase; yet commercially more than 10 t of B12 are produced each year from a number of bacterial species. The rich scientific history of vitamin B12 research, its biological functions and the pathways employed by bacteria for its de novo synthesis are described. Current strategies for the improvement of vitamin B12 production using modern biotechnological techniques are outlined.     

Microbial production of isoquinoline from indene

A purified microbial isolate, identified as a strain of Rhodococcus sp., metabolized indene primarily to iso quinoline and lesser amounts of indandiol and indanone. Isoquinoline production was dependent on the presence of microbial culture, indene, and ammonium ions as the source of nitrogen in the molecule. The ability to produce isoquinoline was induced by growth on benzene or naphthalene and by the presence of indene itself. The culture produced compounds tentatively identified as 3-methylisoquinoline and 3-ethylisoquinoline from 2-methylindene and from 2-ethylindene, respectively. Deuterated indene was converted to deuterated isoquinoline, deuterated indanone, and deuterated indandiol. Experiments with [15N]ammonium nitrate and ammonium [15N]nitrate confirmed ammonium as the source of nitrogen in the isoquinoline products.     

Microbial and enzymatic production of l-carnitine

l-Carnitine (vitamin B_T) plays a vital role in the transportation of long-chain fatty acids into mitochondrial matrix in mammals. l-Carnitine has a wide range of applications in pharmaceuticals, food products, and feed additives. Due to the increasing demand for l-carnitine in food and pharmaceutical applications, production of this compound has become prominent. However, very little has been reported on the production of l-carnitine. This review discusses the microbial and the enzymatic production of l-carnitine from different starting materials (substrates).