Biocatalytic preparation of natural flavours and fragrances

During the past years biocatalytic production of fine chemicals has been expanding rapidly. Flavours and fragrances belong to many different structural classes and therefore represent a challenging target for academic and industrial research. Here, we present a condensed overview of the potential offered by biocatalysis for the synthesis of natural and natural-identical odorants, highlighting relevant biotransformations using microorganisms and isolated enzymes. The industrial processes based on biocatalytic methods are discussed in terms of their advantages over classical chemical synthesis and extraction from natural sources. Recent applications of the biocatalytic approach to the preparation of the most important fine odorants are comprehensively covered.     

Yeast as an efficient biocatalyst for the production of lipid-derived flavours and fragrances

Responding to consumer' demand for natural products, biotechnology is constantly seeking new biocatalysts. In the field of hydrophobic substrate degradation, some yeast species known some years ago as non-conventional, have acquired their right to be considered as good biocatalysts. These Candida, Yarrowia, Sporobolomyces ... are now used for themselves or for their lipases in processes to produce flavours and fragrances. In this paper we present some examples of use of these biocatalysts to generate high-value compounds and discuss the new trends related to progress in the development of molecular tools or the mastering of the redox characteristics of the medium.     

Biotechnological production of prostaglandins

The metabolism of 20-carbon polyunsaturated fatty acids, particularly arachidonic acid, by prostaglandin H synthase results in a wide range of oxidized products with potent biological activities. Among these metabolites, a group of compounds called prostaglandins has drawn the attention of both scientists and medical practitioners. Prostaglandins can be manufactured from polyunsaturated fatty acids with the help of enzymes from mammals. The yield of the desired prostaglandin can be increased by means of various activators, enzyme recycling, immobilised enzymes or semibatch processes with either continuous or stepwise addition of the substrate. Received: 10 June 1996 / Received revision: 7 November 1996 / Accepted: 10 November 1996     

Biotechnological production of vanillin

Vanillin is one of the most important aromatic flavor compounds used in foods, beverages, perfumes, and pharmaceuticals and is produced on a scale of more than 10 thousand tons per year by the industry through chemical synthesis. Alternative biotechnology-based approaches for the production are based on bioconversion of lignin, phenolic stilbenes, isoeugenol, eugenol, ferulic acid, or aromatic amino acids, and on de novo biosynthesis, applying fungi, bacteria, plant cells, or genetically engineered microorganisms. Here, the different biosynthesis routes involved in biotechnological vanillin production are discussed.     

Biotechnological production of prostaglandin

Prostaglandins (PGs) are the oxidation products of PG endoperoxide (PGH) synthase and other tissue enzymes. They occur in a tissue-specific manner and act as local hormones. Biotechnological production of PGs has been of interest, but not yet fully established. Biological tissues have been used as PG sources, but this disturbs ecological balance, and the cost of production is very high for commercial purposes. On the other hand, various microorganisms have been shown to synthesize them de novo, or biotransform precursors to active molecules, but these processes have not been further evaluated. Using mammalian enzymes in free or immobilized form is a promising new approach to synthesize PG from fatty acid substrates. Rapid enzyme inactivation during the catalysis is the main problem to be solved. Optimization of factors in the reactions and the design of special reactors that will allow removal of products continuously from the reaction medium without affecting enzyme activity need immediate attention from researchers and the pharmaceutical industry.     

Biotechnological production and applications of pullulan

Pullulan is a unique biopolymer with many useful traits and hundreds of patented applications. However, despite the fact that pullulan has been in commercial production for more than 25 years, few of these potential uses have been widely adopted. In large part this may be due to the relatively high price of pullulan. Nevertheless, the last few years have seen a resurgence in interest in pullulan, particularly for higher-value health and pharmaceutical applications.Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable     

Biotechnological production and applications of phytases

Phytases decompose phytate, which is the primary storage form of phosphate in plants. More than 10 years ago, the first commercial phytase product became available on the market. It offered to help farmers reduce phosphorus excretion of monogastric animals by replacing inorganic phosphates by microbial phytase in the animal diet. Phytase application can reduce phosphorus excretion by up to 50%, a feat that would contribute significantly toward environmental protection. Furthermore, phytase supplementation leads to improved availability of minerals and trace elements. In addition to its major application in animal nutrition, phytase is also used for processing of human food. Research in this field focuses on better mineral absorption and technical improvement of food processing. All commercial phytase preparations contain microbial enzymes produced by fermentation. A wide variety of phytases were discovered and characterized in the last 10 years. Initial steps to produce phytase in transgenic plants were also undertaken. A crucial role for its commercial success relates to the formulation of the enzyme solution delivered from fermentation. For liquid enzyme products, a long shelf life is achieved by the addition of stabilizing agents. More comfortable for many customers is the use of dry enzyme preparations. Different formulation technologies are used to produce enzyme powders that retain enzyme activity, are stable in application, resistant against high temperatures, dust-free, and easy to handle.     

Biotechnological production and applications of statins

Statins are a group of extremely successful drugs that lower cholesterol levels in blood; decreasing the risk of heath attack or stroke. In recent years, statins have also been reported to have other biological activities and numerous potential therapeutic uses. Natural statins are lovastatin and compactin, while pravastatin is derived from the latter by biotransformation. Simvastatin, the second leading statin in the market, is a lovastatin semisynthetic derivative. Lovastatin is mainly produced by Aspergillus terreus strains, and compactin by Penicillium citrinum. Lovastatin and compactin are produced industrially by liquid submerged fermentation, but can also be produced by the emerging technology of solid-state fermentation, that displays some advantages. Advances in the biochemistry and genetics of lovastatin have allowed the development of new methods for the production of simvastatin. This lovastatin derivative can be efficiently synthesized from monacolin J (lovastatin without the side chain) by a process that uses the Aspergillus terreus enzyme acyltransferase LovD. In a different approach, A. terreus was engineered, using combinational biosynthesis on gene lovF, so that the resulting hybrid polyketide synthase is able to in vivo synthesize 2,2-dimethylbutyrate (the side chain of simvastatin). The resulting transformant strains can produce simvastatin (instead of lovastatin) by direct fermentation.     

Biotechnological production and application of fructooligosaccharides

Currently, prebiotics are all carbohydrates of relatively short chain length. One important group is the fructooligosaccharides (FOS), a special kind of prebiotic associated to the selective stimulation of the activity of certain groups of colonic bacteria. They have a positive and beneficial effect on intestinal microbiota, reducing the incidence of gastrointestinal infections and also possessing a recognized bifidogenic effect. Traditionally, these prebiotic compounds have been obtained through extraction processes from some plants, as well as through enzymatic hydrolysis of sucrose. However, different fermentative methods have also been proposed for the production of FOS, such as solid-state fermentations utilizing various agro-industrial by-products. By optimizing the culture parameters, FOS yields and productivity can be improved. The use of immobilized enzymes and cells has also been proposed as being an effective and economic method for large-scale production of FOS. This article is an overview of the results considering recent studies on FOS biosynthesis, physicochemical properties, sources, biotechnological production and applications.     

Biotechnological production of plant-based insecticides

The demand for natural and nonpersistent insecticides is increasing day by day. Plant cell cultures could be an alternative to conventional methods of production of insecticides from field-grown plants. In vitro cultured plant cells produce a wide array of insecticides as a part of their secondary metabolism. Their ability to synthesize key enzymes and the manipulation of these could lead to the enhanced production of many insecticides of industrial importance. The development of a high-yielding hairy root culture system for thiophenes, nicotine, and phytoecdysones is of considerable interest. In this article, the current literature on various factors that influence the growth, production, and secretion of six insecticidal compounds, namely, pyrethrins, azadirachtin, thiophenes, nicotine, rotenoids, and phytoecdysones which have been prospects for the scale-up of cell cultures, genetic engineering to obtain transgenic plants, and metabolically engineered plants for increased production of bio-molecules, has been discussed. Environmental safety clearance and the future prospects of application of biomolecules for plant-derived insecticides are presented.     

Biotechnological production of itaconic acid

Itaconic acid (IA) is an unsaturated dicarbonic organic acid. It can easily be incorporated into polymers and may serve as a substitute for petrochemical-based acrylic or methacrylic acid. It is used at 1-5% as a co-monomer in resins and also in the manufacture of synthetic fibres, in coatings, adhesives, thickeners and binders. The favoured production process is fermentation of carbohydrates by fungi, with a current market volume of about 15,000 t/a. Due to the high price of about US$ 4/kg, the use of IA is restricted. At present, the production rates do not exceed 1 g l(-1) h(-1), accompanied by product concentrations of about 80 g l(-1). New biotechnology approaches, such as immobilisation techniques, screening programmes and genetic engineering, could lead to higher productivity. Also, the use of alternative substrates may reduce costs and thus open the market for new and increased applications.     

Biotechnological production of pyruvic acid

Pyruvic acid is an important organic acid widely used in the chemical and drug, as well as agrochemical, industries. Compared with the chemical method, biotechnological production of pyruvic acid is an alternative approach because of the low cost. An overview of biotechnological production of pyruvate, including direct fermentative production employing eukaryotic and prokaryotic microorganisms, production by a resting cell method and an enzymatic method as well as the recovery of pyruvate, is discussed. A multi-vitamin auxotrophic yeast strain, Torulopsis glabrata. has been used in the commercial production of pyruvate; emphasis is therefore placed on the mechanism and characteristics of pyruvate production by this strain.     

Biotechnological production of 2-phenylethanol

2-Phenylethanol (2-PE) is an important flavour and fragrance compound with a rose-like odour. Most of the world's annual production of several thousand tons is synthesised by chemical means but, due to increasing demand for natural flavours, alternative production methods are being sought. Harnessing the Ehrlich pathway of yeasts by bioconversion of L-phenylalanine to 2-PE could be an option, but in situ product removal is necessary due to product inhibition. This review describes the microbial production of 2-PE, and also summarizes the chemical syntheses and the market situation.     

Biotechnological intensification of biogas production

The importance of syntrophic relationships among microorganisms participating in biogas formation has been emphasized, and the regulatory role of in situ hydrogen production has been recognized. It was assumed that the availability of hydrogen may be a limiting factor for hydrogenotrophic methanogens. This hypothesis was tested under laboratory and field conditions by adding a mesophilic (Enterobacter cloacae) or thermophilic hydrogen-producing (Caldicellulosyruptor saccharolyticus) strain to natural biogas-producing consortia. The substrates were waste water sludge, dried plant biomass from Jerusalem artichoke, and pig manure. In all cases, a significant intensification of biogas production was observed. The composition of the generated biogas did not noticeably change. In addition to being a good hydrogen producer, C. saccharolyticus has cellulolytic activity; hence, it is particularly suitable when cellulose-containing biomass is fermented. The process was tested in a 5-m³ thermophilic biogas digester using pig manure slurry as a substrate. Biogas formation increased at least 160–170% upon addition of the hydrogen-producing bacteria as compared to the biogas production of the spontaneously formed microbial consortium. Using the hydrogenase-minus control strain provided evidence that the observed enhancement was due to interspecies hydrogen transfer. The on-going presence of C. saccharolyticus was demonstrated after several months of semicontinuous operation.     

Natural flavours production: a biotechnological approach

In a growing world market for flavours, and one in which there is a distinct trend towards ‘natural’ compounds, the production of flavours via biotechnological processes offers a number of advantages. This review discusses how consumer choice, regulatory definitions and technical advances are combining to present opportunities for the commercial exploitation of biological technology for flavour production.     

Biotechnological production of l-tyrosine and derived compounds

The aromatic amino acid l-tyrosine is a compound with multiple applications in the food, pharmaceutical, cosmetic and chemical industries. This review summarizes the current knowledge on the metabolic pathways involved in the synthesis of this amino acid and the strategies employed to develop and improve microbial production strains. Common strategies for l-tyrosine overproduction include the elimination of negative feedback control in key pathway enzymes and increasing the pool of the aromatic precursors phosphoenolpyruvate and erythrose-4-phosphate. Following these approaches, production strains have been generated that allow the synthesis of l-tyrosine with a yield from glucose corresponding to 80% of the theoretical maximum. Recent developments in the utilization of l-tyrosine as a substrate for microbial and enzymatic conversion into valuable products are also presented and discussed. For example, the production of the aromatic polymer melanin has been reported by the bioconversion of l-tyrosine using an Escherichia coli strain expressing a gene encoding the enzyme tyrosinase from Rhizobium etli. Metabolic engineering by expressing genes encoding the enzyme p-hydroxyphenylacetate 3-hydroxylase in an E. coli strain modified for l-tyrosine production from glucose results in the capacity to synthesize l-3,4-dihydroxyphenylalanine, a compound employed for treating Parkinson's disease.     

Biotechnological production of lutein and its applications

Lutein is an antioxidant that has gathered increasing attention due to its potential role in preventing or ameliorating age-related macular degeneration. Currently, it is produced from marigold oleoresin, but continuous reports of lutein-producing microalgae pose the question if those microorganisms can become an alternative source. Several microalgae have higher lutein contents than most marigold cultivars and have been shown to yield productivities hundreds of times higher than marigold crops on a per square meter basis. Microalgae and marigold are opposite alternatives in the use of resources such as land and labor and the prevalence of one or the other could change in the future as the lutein demand rises and if labor or land becomes more restricted or expensive in the producing countries. The potential of microalgae as a lutein source is analyzed and compared to marigold. It is suggested that, in the current state of the art, microalgae could compete with marigold even without counting on any of the improvements in microalgal technology that can be expected in the near future.     

Biotechnological production of erythritol and its applications

Erythritol, a four-carbon polyol, is a biological sweetener with applications in food and pharmaceutical industries. It is also used as a functional sugar substitute in special foods for people with diabetes and obesity because of its unique nutritional properties. Erythritol is produced by microbial methods using mostly osmophilic yeasts and has been produced commercially using mutant strains of Aureobasidium sp. and Pseudozyma tsukubaensis. Due to the high yield and productivity in the industrial scale of production, erythritol serves as an inexpensive starting material for the production of other sugars. This review focuses on the approaches for the efficient erythritol production, strategies used to enhance erythritol productivity in microbes, and the potential biotechnological applications of erythritol.     

Biotechnological production of mannitol and its applications

Mannitol, a naturally occurring polyol (sugar alcohol), is widely used in the food, pharmaceutical, medical, and chemical industries. The production of mannitol by fermentation has become attractive because of the problems associated with its production chemically. A number of homo- and heterofermentative lactic acid bacteria (LAB), yeasts, and filamentous fungi are known to produce mannitol. In particular, several heterofermentative LAB are excellent producers of mannitol from fructose. These bacteria convert fructose to mannitol with 100% yields from a mixture of glucose and fructose (1:2). Glucose is converted to lactic acid and acetic acid, and fructose is converted to mannitol. The enzyme responsible for conversion of fructose to mannitol is NADPH- or NADH-dependent mannitol dehydrogenase (MDH). Fructose can also be converted to mannitol by using MDH in the presence of the cofactor NADPH or NADH. A two enzyme system can be used for cofactor regeneration with simultaneous conversion of two substrates into two products. Mannitol at 180 g l⁻¹ can be crystallized out from the fermentation broth by cooling crystallization. This paper reviews progress to date in the production of mannitol by fermentation and using enzyme technology, downstream processing, and applications of mannitol.