Scheffersomyces stipitis ability to valorize different residual biomasses for vitamin B9 production

Sugar beet pulp (SBP), sugar beet molasses (SBM) and unfermented grape marcs (UGM) represent important waste in the agro-food sector. If suitably pre-treated, hexose and pentose sugars can be released in high quantities and can subsequently be used by appropriate cell factories as growth media and for the production of (complex) biomolecules, accomplishing the growing demand for products obtained from sustainable resources. One example is vitamin B₉ or folate, a B-complex vitamin currently produced by chemical synthesis, almost exclusively in the oxidized form of folic acid (FA). It is therefore desirable to develop novel competitive strategies for replacing its current fossil-based production with a sustainable bio-based process. In this study, we assessed the production of natural folate by the yeast Scheffersomyces stipitis, investigating SBM, SBP and UGM as potential growth media. Pre-treatment of SBM and SBP had previously been optimized in our laboratory; thus, here we focused only on UGM pre-treatment and hydrolysis strategies for the release of fermentable sugars. Then, we optimized the growth of S. stipitis on the three media formulated from those biomasses, working on inoculum pre-adaptation, oxygen availability and supplementation of necessary nutrients to support the microorganism. Folate production, measured with a microbiological assay, reached 188.2 ± 24.86 μg/L on SBM, 130.6 ± 1.34 μg/L on SBP and 101.9 ± 6.62 μg/L on UGM. Here, we demonstrate the flexibility of S. stipitis in utilizing different residual biomasses as growth media. Moreover, we assessed the production of folate from waste, and to the best of our knowledge, we obtained the highest production of folate from residual biomasses ever reported, providing the first indications for the future development of this microbial production process.     

<Emphasis Type="SmallCaps">d</Emphasis>-Galacturonic acid catabolism in microorganisms and its biotechnological relevance

d-Galacturonic acid is the main constituent of pectin, a naturally abundant compound. Pectin-rich residues accumulate when sugar is extracted from sugar beet or juices are produced from citrus fruits. It is a cheap raw material but currently mainly used as animal feed. Pectin has the potential to be an important raw material for biotechnological conversions to fuels or chemicals. In this paper, we review the microbial pathways for the catabolism of d-galacturonic acid that would be relevant for the microbial conversion to useful products.     

Crypthecodinium cohnii with emphasis on DHA production: a review

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid (PUFA) that belongs to the ω-3 group. In recent years, DHA has attracted much attention because of its recognized beneficial effect on human health. At present, fish oil is the major source of DHA, but it may be produced by microorganisms with additional benefits. Marine microorganisms may contain large amounts of DHA and are considered a potential source of this important fatty acid. Some of these organisms can be grown heterotrophically on organic substrates without light, offering the possibility of greatly increasing microalgal cell concentration under controlled and monitored conditions, resulting in a very high quality product. Among the heterotrophic marine dinoflagellates, Crypthecodinium cohnii has been identified as a prolific producer of DHA. The organism is extraordinary in that it produces no other PUFAs than DHA in its cell lipid in any significant amount, which makes the DHA purification process very attractive, particularly for pharmaceutical and nutraceutical applications. This paper reviews recent advances in the biotechnological production of DHA by C. cohnii.     

Gel-forming algal polysaccharides: structural characterisation and technological applications

Abstract

Some aspects of both the chemical and physico-chemical properties of gelling algal polysaccharides are reviewed in connection with their technological and biotechnological uses. In fact, besides the traditional applications, which are mainly in the field of food industry, recent sophisticated biotechnological manipulations opened the way for the exploitation of these molecules for very advanced applications like the formulation of micro spheres for cell incubation. Some aspects of the structure of alginate gel beads used for cell immobilisation are illustrated.     

Cell death and mitochondrial dysfunction induced by the dietary non-proteinogenic amino acid l-azetidine-2-carboxylic acid (Aze)

In addition to the 20 protein amino acids that are vital to human health, hundreds of naturally occurring amino acids, known as non-proteinogenic amino acids (NPAAs), exist and can enter the human food chain. Some NPAAs are toxic through their ability to mimic protein amino acids and this property is utilised by NPAA-containing plants to inhibit the growth of other plants or kill herbivores. The NPAA l-azetidine-2-carboxylic acid (Aze) enters the food chain through the use of sugar beet (Beta vulgaris) by-products as feed in the livestock industry and may also be found in sugar beet by-product fibre supplements. Aze mimics the protein amino acid l-proline and readily misincorporates into proteins. In light of this, we examined the toxicity of Aze to mammalian cells in vitro. We showed decreased viability in Aze-exposed cells with both apoptotic and necrotic cell death. This was accompanied by alterations in endosomal–lysosomal activity, changes to mitochondrial morphology and a significant decline in mitochondrial function. In summary, the results show that Aze exposure can lead to deleterious effects on human neuron-like cells and highlight the importance of monitoring human Aze consumption via the food chain.

     

Genetic approaches to sustainable pest management in sugar beet (Beta vulgaris)

Sugar beet (Beta vulgaris) is an important arable crop, traditionally used for sugar extraction, but more recently, for biofuel production. A wide range of pests, including beet cyst nematode (Heterodera schachtii), root‐knot nematodes (Meloidogyne spp.), green peach aphids (Myzus persicae) and beet root maggot (Tetanops myopaeformis), infest the roots or leaves of sugar beet, which leads to yield loss directly or through transmission of beet pathogens such as viruses. Conventional pest control approaches based on chemical application have led to high economic costs. Development of pest‐resistant sugar beet varieties could play an important role towards sustainable crop production while minimising environmental impact. Intensive Beta germplasm screening has been fruitful, and genetic lines resistant to nematodes, aphids and root maggot have been identified and integrated into sugar beet breeding programmes. A small number of genes responding to pest attack have been cloned from sugar beet and wild Beta species. This trend will continue towards a detailed understanding of the molecular mechanism of insect–host plant interactions and host resistance. Molecular biotechnological techniques have shown promise in developing transgenic pest resistance varieties at an accelerated speed with high accuracy. The use of transgenic technology is discussed with regard to biodiversity and food safety.     

Biosynthesis of 2-hydroxyisobutyric acid (2-HIBA) from renewable carbon

Nowadays a growing demand for green chemicals and cleantech solutions is motivating the industry to strive for biobased building blocks. We have identified the tertiary carbon atom-containing 2-hydroxyisobutyric acid (2-HIBA) as an interesting building block for polymer synthesis. Starting from this carboxylic acid, practically all compounds possessing the isobutane structure are accessible by simple chemical conversions, e. g. the commodity methacrylic acid as well as isobutylene glycol and oxide. During recent years, biotechnological routes to 2-HIBA acid have been proposed and significant progress in elucidating the underlying biochemistry has been made. Besides biohydrolysis and biooxidation, now a bioisomerization reaction can be employed, converting the common metabolite 3-hydroxybutyric acid to 2-HIBA by a novel cobalamin-dependent CoA-carbonyl mutase. The latter reaction has recently been discovered in the course of elucidating the degradation pathway of the groundwater pollutant methyl tert -butyl ether (MTBE) in the new bacterial species Aquincola tertiaricarbonis. This discovery opens the ground for developing a completely biotechnological process for producing 2-HIBA. The mutase enzyme has to be active in a suitable biological system producing 3-hydroxybutyryl-CoA, which is the precursor of the well-known bacterial bioplastic polyhydroxybutyrate (PHB). This connection to the PHB metabolism is a great advantage as its underlying biochemistry and physiology is well understood and can easily be adopted towards producing 2-HIBA. This review highlights the potential of these discoveries for a large-scale 2-HIBA biosynthesis from renewable carbon, replacing conventional chemistry as synthesis route and petrochemicals as carbon source.     

Biotechnological production of <Emphasis Type="SmallCaps">d</Emphasis>-glyceric acid and its application

Glycerol is currently produced in large amounts as a by-product during fat splitting and biodiesel fuel production. Over the past decade, both chemical and biotechnological processes to convert glycerol to value-added chemicals have been increasingly explored. This mini-review provides recent information about the biotechnological production of a glycerol derivative, d-glyceric acid (d-GA), and its possible applications. Little is known about GA as a bioproduct, but it is naturally found in different kinds of plants as a phytochemical constituent and is reported to have some biological activity. A racemic mixture of dl-GA can be obtained from glycerol via chemical oxidation; however, d-GA is mainly biotechnologically produced with the aid of bacteria. Under aerobic conditions, some acetic acid bacteria transform glycerol into d-GA, and optimization of initial glycerol concentration and aeration rate provided a yield of more than 80 g/l d-GA, using a strain of Gluconobacter frateurii.     

Immobilized Nucleases

Abstract

Nucleases are important analytical enzymes and are used widely for the determination of nucleic acid structure. Their application depends on the specificity and mode of action of the particular enzyme. Nucleases have also found application in the production of flavor enhancers like 5′ IMP and 5′ GMP, removal of nucleic acids in single cell protein preparations, and as therapeutic agents. Immobilization of nucleases and the use of immobilized nucleases for various biotechnological applications have been reviewed.     

果胶酶亲和吸附剂的制备及其性能比较

以甜菜渣、果胶、海藻酸钠为原料 ,与表氯醇在碱性条件下交联 ,制备了果胶酶亲和吸附剂并应用这几种亲和吸附剂对草酸青霉果胶酶和黑曲霉果胶酶进行亲和层析实验。结果表明 :用小于 60目的甜菜渣粉末和海藻酸钠制备的果胶酶亲和吸附剂 (分别简写为SBP吸附剂和SA吸附剂 )具有较好的纯化果胶酶性能 ,其中SBP吸附剂性能稳定 ,而SA吸附剂膨胀系数较大 ,在流动相改变时引起流速不稳。     

Ferulic Acid: An Antioxidant Found Naturally in Plant Cell Walls and Feruloyl Esterases Involved in its Release and Their Applications

ABSTRACT

Ferulic acid is the most abundant hydroxycinnamic acid in the plant world and maize bran with 3.1% (w/w) ferulic acid is one of the most promising sources of this antioxidant. The dehydrodimers of ferulic acid are important structural components in the plant cell wall and serve to enhance its rigidity and strength. Feruloyl esterases are a subclass of the carboxylic acid esterases that hydrolyze the ester bond between hydroxycinnamic acids and sugars present in plant cell walls and they have been isolated from a wide range of microorganisms, when grown on complex substrates such as cereal brans, sugar beet pulp, pectin and xylan. These enzymes perform a function similar to alkali in the deesterification of plant cell wall and differ in their specificities towards the methyl esters of cinnamic acids and ferulolylated oligosaccharides. They act synergistically with xylanases and pectinases and facilitate the access of hydrolases to the backbone of cell wall polymers. The applications of ferulic acid and feruloyl esterase enzymes are many and varied. Ferulic acid obtained from agricultural byproducts is a potential precursor for the production of natural vanillin, due to the lower production cost.     

Lipid and fatty acid metabolism in Ralstonia eutropha: relevance for the biotechnological production of value-added products

Lipid and fatty acid metabolism has been well studied in model microbial organisms like Escherichia coli and Bacillus subtilis. The major precursor of fatty acid biosynthesis is also the major product of fatty acid degradation (β-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from β-oxidation allows for the biosynthesis of natural products of biotechnological importance. Ralstonia eutropha can utilize acetyl-CoA from fatty acid metabolism to produce intracellular polyhydroxyalkanoate (PHA). R. eutropha can also be engineered to utilize fatty acid metabolism intermediates to produce different PHA precursors. Metabolism of lipids and fatty acids can be rerouted to convert carbon into other value-added compounds like biofuels. This review discusses the lipid and fatty acid metabolic pathways in R. eutropha and how they can be used to construct reagents for the biosynthesis of products of industrial importance. Specifically, how the use of lipids or fatty acids as the sole carbon source in R. eutropha cultures adds value to these biotechnological products will be discussed here.     

Biotechnological production and applications of microbial phenylalanine ammonia lyase: a recent review

Abstract

Phenylalanine ammonia lyase (PAL) catalyzes the nonoxidative deamination of l-phenylalanine to form trans-cinnamic acid and a free ammonium ion. It plays a major role in the catabolism of l-phenylalanine. The presence of PAL has been reported in diverse plants, some fungi, Streptomyces and few Cyanobacteria. In the past two decades, PAL has gained considerable significance in several clinical, industrial and biotechnological applications. Since its discovery, much knowledge has been gathered with reference to the enzyme’s importance in phenyl propanoid pathway of plants. In contrast, there is little knowledge about microbial PAL. Furthermore, the commercial source of the enzyme has been mainly obtained from the fungi. This study focuses on the recent advances on the physiological role of microbial PAL and the improvements of PAL biotechnological production both from our laboratory and many others as well as the latest advances on the new applications of microbial PAL.     

Two glucuronoyl esterases of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

The white-rot fungus Phanerochaete chrysosporium produces glucuronoyl esterase, a recently discovered carbohydrate esterase, during growth on sugar beet pulp. Two putative genes encoding this enzyme, ge1 and ge2, were isolated and cloned. Heterologous expression in Aspergillus vadensis, Pycnoporus cinnabarinus and Schizophyllum commune resulted in extracellular glucuronoyl esterase activity, demonstrating that these genes encode this enzymatic function. The amino acid sequence of GE1 was used to identify homologous genes in the genomes of twenty-four fungi. Approximately half of the genomes, both from ascomycetes and basidiomycetes, contained putative orthologues, but their presence could not be assigned to any of fungal class or subclass. Comparison of the amino acid sequences of identified and putative glucuronoyl esterases to other types of carbohydrate esterases (CE) confirmed that they form a separate family of CEs. These enzymes are interesting candidates for biotechnological applications such as the separation of lignin and hemicellulose.     

Biotechnological routes based on lactic acid production from biomass

Lactic acid, the most important hydroxycarboxylic acid, is now commercially produced by the fermentation of sugars present in biomass. In addition to its use in the synthesis of biodegradable polymers, lactic acid can be regarded as a feedstock for the green chemistry of the future. Different potentially useful chemicals such as pyruvic acid, acrylic acid, 1,2-propanediol, and lactate ester can be produced from lactic acid via chemical and biotechnological routes. Here, we reviewed the current status of the production of potentially valuable chemicals from lactic acid via biotechnological routes. Although some of the reactions described in this review article are still not applicable at current stage, due to their “greener” properties, biotechnological processes for the production of lactic acid derivatives might replace the chemical routes in the future.     

Release of ferulic acid and feruloylated oligosaccharides from sugar beet pulp by Streptomyces tendae

Given several promising industrial applications of ferulic acid, this study was designed to identify actinomycete strains able to release high levels of this acid from sugar beet pulp (SBP). Out of 47 strains tested, 37% were found to release free ferulic acid from the growth substrate. One strain, identified as Streptomyces tendae by 16S RNA gene sequencing, was capable of releasing 80% of the ferulic acid ester-linked to the pectin in SBP after 5 days of growth. These data suggest that some actinomycetes are able to release ferulic acid and feruloylated oligosaccharides from SBP. During growth on SBP, it seems that Streptomyces species solubilize and release feruloylated oligosaccharides by specific carbohydrase activities before de-esterification and release of free ferulic acid.     

Metabolic Engineering of Plant L-Ascorbic Acid Biosynthesis: Recent Trends and Applications

ABSTRACT

Vitamin C (L-ascorbic acid; AsA) is the major soluble antioxidant found in plants and is also an essential component of human nutrition. Although numerous biotechnological methods have been exploited to increase its yield, pressures such as commercial competition and environmental concerns make it urgent to find a new way for industrial production of plant-derived AsA. Engineering plant AsA has now become feasible because of our increased understanding of its biosynthetic pathway. Several possible strategies could be followed to increase AsA production, such as overcoming the rate limiting steps in the biosynthetic pathway, promoting recycling, and reducing catabolism. For these purposes, genes of plant, microbial and animal origins have been successfully used. Several examples will be given to illustrate these various approaches. The existing and potential achievements in increasing AsA production would provide the opportunity for enhancing nutritional quality and stress tolerance of crop plants.     

Biotechnological production and applications of the ω-3 polyunsaturated fatty acid docosahexaenoic acid

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid composed of 22 carbon atoms and six double bonds. Because the first double bond, as counted from the methyl terminus, is at position three, DHA belongs to the so-called -3 group. In recent years, DHA has attracted much attention because of its beneficial effect on human health. At present, fish oil is the major source of DHA, but alternatively it may be produced by use of microorganisms. Marine microorganisms may contain large quantities of DHA and are considered a potential source of this important fatty acid. Some of these organisms can be grown heterotrophically on organic substrates without light. These processes can be well controlled and DHA with constant quality can be produced all year round. This paper reviews recent advances in the biotechnological production of DHA by marine microorganisms.     

Bioprocesses for 2-phenylethanol and 2-phenylethyl acetate production: current state and perspectives

2-Phenylethanol (2-PE) and 2-phenethyl acetate (2-PEA) are valuable generally recognized as safe flavoring agents widely used in industry. Perfumes, pharmaceuticals, polishes, and personal care products, are some of the final products using these compounds as additives due to their rose-like odor. Also, 2-PE is used in disinfectants, pest control, and cleaning products due to its biocide capability. Although most of these additives production are derived from chemical synthesis, the current trend of consumers to prefer natural products has contributed to the development of biotechnological approaches as an alternative way to obtain natural 2-PE and 2-PEA. The most efficient route to bioproduce these compounds is through the bioconversion of L-phenylalanine via the Ehrlich pathway, and most of the advances have been focused on the development of this process. This review compiles the most recent developments in the biotechnological production of 2-PE and 2-PEA, indicating the most studied strains producing 2-PE and 2-PEA, the current advances in the in situ product recovery in liquid systems, an overview of the strain developments, and the progress in the use of residue-based systems. Future research should address the need for more sustainable and economic systems such as those using wastes as raw materials, as well as the scale-up of the proposed technologies.

     

Biotechnological production of muconic acid: current status and future prospects

Muconic acid (MA), a high value-added bio-product with reactive dicarboxylic groups and conjugated double bonds, has garnered increasing interest owing to its potential applications in the manufacture of new functional resins, bio-plastics, food additives, agrochemicals, and pharmaceuticals. At the very least, MA can be used to produce commercially important bulk chemicals such as adipic acid, terephthalic acid and trimellitic acid. Recently, great progress has been made in the development of biotechnological routes for MA production. This present review provides a comprehensive and systematic overview of recent advances and challenges in biotechnological production of MA. Various biological methods are summarized and compared, and their constraints and possible solutions are also described. Finally, the future prospects are discussed with respect to the current state, challenges, and trends in this field, and the guidelines to develop high-performance microbial cell factories are also proposed for the MA production by systems metabolic engineering.