Biological transformations of steroidal compounds: A review

Microbial transformation is an important tool for structural modification of organic compounds, especially natural products with complex structures like steroids. It can be used to synthesize chemical structures that are difficult to obtain by ordinary methods and as a model of mammalian metabolism due to similarity between mammalian and microbial enzyme systems. During recent years research has been focused on the structural modifications of bioactive steroids by using various microorganisms, in order to obtain biologically potent compounds with diverse structures. Steroidal compounds are responsible for important biological functions in the cells and manifest a variety of activities. This article covers the microbial transformation of sterols, steroidal hormones and some new types of steroids known as bufadienolides. Emphasis has placed on reporting metabolites that may be of general interest and on the practical aspects of work in the field of microbial transformations. The review covers the literature from 1994 to 2011.     

微生物转化制备活性维生素D_3的研究进展

活性维生素D类药物作为一类高效原料药,采用化学合成方法,制备复杂,限制了其广泛应用。采用微生物转化法条件温和,操作简单,对于活性维生素D3的制备具有重要意义。从甾体羟化菌株,生物转化制备不同活性维生素D3、基因工程在生物转化上的应用及转化率的影响因素等方面综述了其研究进展,并对该领域的发展趋势进行了展望。     

生物法合成γ-氨基丁酸的研究策略*

γ-氨基丁酸(γ-aminobutyric acid,GABA)是一种极易溶于水的非蛋白质氨基酸,被广泛应用于食品和制药工业中,市场需求量极大。可通过化学合成法、植物富集法、微生物直接发酵法和生物转化法生产。近年来,因生物法合成GABA具有相对优势,受到研究者们的重视。对GABA的生产方法、生产GABA的微生物、微生物合成GABA的关键代谢途径和GAD酶的定向改造策略进行了论述。     

Microbial biotransformation: a tool for drug designing

For centuries microbial biotransformation has proved to be an imperative tool in alleviating the production of various chemicals used in food, pharmaceutical, agrochemical and other industries. In the field of pharmaceutical research and development, biotransformation studies have been extensively applied to investigate the metabolism of compounds (leads, lead candidates, etc.) using animal models. The microbial biotransformation phenomenon is then commonly employed in comparing metabolic pathways of drugs and scaling up the metabolites of interest discovered in these animal models for further pharmacological and toxicological evaluation. Microorganisms can conveniently afford drugs difficult obtained via synthesis. The plethora of reported microbial biotransformations along with its added benefits has already invoked further research in bioconversion of novel and structurally complex drugs. This review alternatively discusses the prospect of microbial biotransformation studies as a significant element ameliorating drug discovery and design in terms of cost-effectiveness, environment protection and greater structural diversity as compared to animal models used to study metabolism. To explicate the microbial biotransformation paradigm in drug designing 3 main areas in this aspect have been analyzed: 1—lead expansion: obtaining pharmacologically improved metabolites from bioactive molecules; 2—biosynthesis of precursors/intermediates involved in the production of bioactive molecules; 3—resolution of racemic mixture to obtain enantiomers possessing different pharmacological profiles.     

Microbial synthesis of glycosaminoglycans and their oligosaccharides

Compared with chemical synthesis and tissue extraction methods, microbial synthesis of glycosaminoglycans (GAGs) is attractive because of the advantages of eco-friendly processes, production safety, and sustainable development. However, boosting the efficiency of microbial cell factories, precisely regulating GAG molecular weights, and rationally controlling the sulfation degree of GAGs remain challenging. To address these issues, various strategies, including genetic, enzymatic, metabolic, and fermentation engineering, have been developed. In this review, we summarize the recent progress in the construction of efficient GAG-producing microbial cell factories, regulation of the molecular weight of GAGs, and modification of GAG chains. Moreover, future studies, remaining challenges, and potential solutions in this field are discussed.     

Chemical-based fecal source tracking methods: current status and guidelines for evaluation

Fecal source tracking is a rapidly evolving field for which there have been a number of method evaluation studies, workshops, review articles and a book that synthesize information about method efficacy. Chemicals that are specific to human wastewater offer several potential advantages over biologically based methods, but have received less scrutiny. More than 35 chemical analytes have been found to consistently occur in human waste streams and here we review these potential human-origin indicators in context of seven evaluation criteria. Some chemical methods offer advantages over microbial methods: they are generally faster to prepare and analyze, more source-specific because they are not confounded by regrowth in the environment, and some may be more geographically and temporally stable. However, they often require specialized equipment and are usually more expensive regarding sample preparation and analysis. Additionally, most chemicals that are specific to human waste-streams occur at concentrations low enough to be diluted below detection limits once the waste-stream enters the ambient environment. These two factors will likely result in chemical measures being used more often as cross-validation supplements or initial screening approaches, rather than replacements for microbial measures. Cross-validation supplements include several chemicals that are highly specific to human sources and can be important contributors when certainties about human sources are critical, such as in drinking water applications. At least one set of chemicals, fecal sterols and stanols, may have potential for identification of other sources in addition to humans. Of all the chemicals examined to date, optical brighteners (OBs) in detergents have shown considerable promise, especially for screening purposes. Optical brighteners are not as sensitive as most microbial assessments, but can be measured with a hand-held fluorometer, providing near real-time and relatively inexpensive tracking of signals in the field, if the human fecal source contains an OB concentration large enough to produce a measurable signal.     

Microbial steroid transformations: current state and prospects

Studies of steroid modifications catalyzed by microbial whole cells represent a well-established research area in white biotechnology. Still, advances over the last decade in genetic and metabolic engineering, whole-cell biocatalysis in non-conventional media, and process monitoring raised research in this field to a new level. This review summarizes the data on microbial steroid conversion obtained since 2003. The key reactions of structural steroid functionalization by microorganisms are highlighted including sterol side-chain degradation, hydroxylation at various positions of the steroid core, and redox reactions. We also describe methods for enhancement of bioprocess productivity, selectivity of target reactions, and application of microbial transformations for production of valuable pharmaceutical ingredients and precursors. Challenges and prospects of whole-cell biocatalysis applications in steroid industry are discussed.     

Substrate specificity of the biotransformation enzymes in a Nocardia erythropolis culture

Substrate specificity of biotransformation enzymes of culture Nocardia erythropolis was studied. Products of transformation of cholesterol and three sterols of microbial origin: ergosterol, ergosta-5,7-dien-3 beta-ol and ergosta-7,22-dien-3 beta-ol was identified with a help of thin-layer chromatography, UV spectrophotometry and mass-spectrometry. It was established, that delta 22-bond in the side chains of sterols and delta 7-bond slows and delta 5-bond makes impossible cleavage of side chains of sterols.     

Biotechnology: Health care,agriculture, industry,environment

New developments in different branches of biotechnology are discussed. The production of peptide hormones, new interferons and other lymphokines by the microbial and cell cultures, and new enzyme inhibitors of microbial origin are the most important for health care and pharmacy. The main direction in research in the agriculture represents the development of the new, very effective methods of nitrogen fixation and the production of animal growth hormones by genemanipulated microorganisms. One of the most important field of application of biotechnology is the chemical industry, c.f. microbial production of polymers and biotransformation of compounds previously produced by chemical methods (acrylamide, adipic acid, naphtalen conversion, etc.). Several novel methods of degradation of the cellulosic materials are mentioned and exploitation of biotechnology in environmental protection is also discussed.     

MICROALGAL BIOTRANSFORMATION OF STEROIDS1

Microbial biotransformation of steroids is not a new concept, but most studies in this field have focused on fungal and bacterial systems. Microalgae, despite their photosynthetic ability and immense biodiversity, have not received much attention in this aspect until recently. Since the publication of the first article on microalgal biotransformation of steroids about 20 years ago, there have been many reports describing different modifications, including hydroxylation, reduction, side‐chain degradation, and isomerization introduced by these microorganisms on estrane, androstane, and pregnane derivatives. On the other hand, the development of new large‐scale cultivation systems, the adaptation of existing fermentation techniques to microalgae, and the introduction of microalgal genetic manipulation methods have made these organisms promising candidates for a wide range of biotechnological processes, including biotransformations. In this review, we have summarized the steroid transformation patterns of several microalgal strains and present a perspective of the future trends in microalgal biotechnology, including the possibility of adapting relatively new techniques, such as organic media catalysis and cell immobilization, to this specific field.     

Tracing the fate of steroids through a hypersaline microbial mat (Kiritimati,Kiribati/Central Pacific)

Eukaryotic steranes are typically absent or occur in very low concentrations in Precambrian sedimentary rocks. However, it is as yet unclear whether this may reflect low source inputs or a preservational bias. For instance, it has been proposed that eukaryotic lipids were profoundly degraded in benthic microbial mats that were ubiquitous prior to the advent of vertical bioturbation in the Cambrian (“mat‐seal effect”). It is therefore important to test the microbial turnover and degradation of eukaryotic steroids in real‐world microbial mats. Here we assessed steroid inventories in different layers of a microbial mat from a hypersaline lake on Kiritimati (Central Pacific). Various eukaryote‐derived C₂₇‐C₃₀ steroids were detected in all mat layers. These compounds most likely entered the mat system as unsaturated sterols from the water column or the topmost mat, and were progressively altered during burial in the deeper, anoxic mat layers over c. 10³ years. This is reflected by increasing proportions of saturated sterols and sterenes, as well as the presence of thiosteranes in certain horizons. Sterol alteration can partly be assigned to microbial transformation but is also due to chemical reactions promoted by the reducing environment in the deeper mat layers. Notably, however, compounds with a sterane skeleton were similarly abundant in all mat layers and their absolute concentrations did not show any systematic decrease. The observed decrease of steroid/hopanoid ratios with depth may thus rather indicate a progressive “dilution” by lipids derived from heterotrophic bacteria. Further, pyrolysis revealed that steroids, in contrast to hopanoids, were not sequestered into non‐extractable organic matter. This may lead to a preservational bias against steroids during later stages of burial. Taken together, steroid preservation in the microbial mat is not only controlled by heterotrophic degradation, but rather reflects a complex interplay of taphonomic processes.     

Formation of volatile sulfur compounds and metabolism of methionine and other sulfur compounds in fermented food

The formation of volatile sulfur compounds (VSC) in fermented food is a subject of interest. Such compounds are essential for the aroma of many food products like cheeses or fermented beverages, in which they can play an attractive or a repulsive role, depending on their identity and their concentration. VSC essentially arise from common sulfur-bearing precursors, methionine being the most commonly found. In the first section of this paper, the main VSC found in cheese, wine, and beer are reviewed. It is shown that a wide variety of VSC has been evidenced in these food products. Because of their low odor threshold and flavor notes, these compounds impart essential sensorial properties to the final product. In the second section of this review, the main (bio)chemical pathways leading to VSC synthesis are presented. Attention is focused on the microbial/enzymatic phenomena—which initiate sulfur bearing precursors degradation—leading to VSC production. Although chemical reactions could also play an important role in this process, this aspect is not fully developed in our review. The main catabolic pathways leading to VSC from the precursor methionine are presented.     

Biotransformations of steroid compounds by Chaetomium sp. KCH 6651

Biotransformations of steroid compounds: androstenedione, testosterone, progesterone, pregnenolone and DHEA using Chaetomium sp. 1 KCH 6651 strain as a biocatalyst were investigated. The microorganism proved capable of selective hydroxylation of the steroid substrates. Androstenedione was converted to 14α-hydroxyandrost-4-en-3,17-dione (in over 75% yield) and 6β-hydroxyandrost-4-en-3,17-dione (in low yield), while testosterone underwent regioselective hydroxylation at 6β position. Progesterone was transformed to a single product—6β,14α-dihydroxypregnan-4-en-3,20-dione in high yield, whereas biotransformation of DHEA resulted in the formation of 7α-hydroxy derivative, which was subsequently converted to 7α-hydroxyandrost-4-en-3,17-dione.     

酸性矿坑废水及目前研究酸性矿坑废水微生物群落的方法

本文通过对近期文献综述,阐明了酸性矿坑废水（Acid Mine Drainage,AMD）的形成机理以及AMD的微生物多样性。综述了AMD形成过程中的硫化金属矿、黄铁矿以及白铁矿的氧化作用,同时对影响AMD形成的化学、生物学以及物理因素及其过程进行了较为详尽的阐述。对目前使用分子生物学对AMD中微生物群落研究的几种手段进行的总结。最后对研究AMD微生物群落这几种分子生物学方法进行了归纳讨论。阐明了这几种分子生物方法对研究AMD微生物群落的重要意义。     

Microbial production of small peptide: pathway engineering and synthetic biology

Small peptides are a group of natural products with low molecular weights and complex structures. The diverse structures of small peptides endow them with broad bioactivities and suggest their potential therapeutic use in the medical field. The remaining challenge is methods to address the main limitations, namely (i) the low amount of available small peptides from natural sources, and (ii) complex processes required for traditional chemical synthesis. Therefore, harnessing microbial cells as workhorse appears to be a promising approach to synthesize these bioactive peptides. As an emerging engineering technology, synthetic biology aims to create standard, well-characterized and controllable synthetic systems for the biosynthesis of natural products. In this review, we describe the recent developments in the microbial production of small peptides. More importantly, synthetic biology approaches are considered for the production of small peptides, with an emphasis on chassis cells, the evolution of biosynthetic pathways, strain improvements and fermentation.     

Boosting crop yields with plant steroids

Plant sterols and steroid hormones, the brassinosteroids (BRs), are compounds that exert a wide range of biological activities. They are essential for plant growth, reproduction, and responses to various abiotic and biotic stresses. Given the importance of sterols and BRs in these processes, engineering their biosynthetic and signaling pathways offers exciting potentials for enhancing crop yield. In this review, we focus on how alterations in components of sterol and BR metabolism and signaling or application of exogenous steroids and steroid inhibitors affect traits of agronomic importance. We also discuss areas for future research and identify the fine-tuning modulation of endogenous BR content as a promising strategy for crop improvement.     

Microbiological coal desulphurization

Sulphur compounds present in coal impose severe limitations on its utilization since sulphur-containing gases emitted into the atmosphere upon direct combustion of coal cause serious environmental pollution problems. Removal of sulphur compounds from coal by microbial action has many advantages over physical and chemical desulphurization methods. The potential use of various microorganisms for the removal of sulphur compounds from coal is presented. Environmental conditions and major process variables affecting the process performance are identified and their possible effects are discussed. Various process schemes for microbial desulphurization (MDS) of coal are suggested. It is concluded that microbial methods have a high potential in removing sulphur compounds from coal. However, more research and development work is needed in this field to overcome present technological problems.     

酿酒酵母中胆固醇生物合成与优化的研究进展

胆固醇是动物体内积累的主要甾醇化合物,在维持细胞膜功能、合成甾体激素、生产甾体药物中间体等方面具有重要的生物学意义和医学应用价值。传统动物组织提取胆固醇的方法费时费力并存在严重的环境污染问题,而甾醇分子结构的复杂程度也限制了其化学全合成。近些年,人们利用合成生物学方法构建的微生物细胞工厂已成功用于萜类、甾醇类等天然产物的开发与合成。文中综述了胆固醇微生物细胞工厂的研究进展,包括胆固醇生物合成途径的解析、底盘菌株的选择、异源基因元件的挖掘与优化、相关代谢通路的调控等方面,并讨论了当前研究面临的问题,以期为胆固醇的高效生物合成提供参考。     

Microbial production of riboflavin: Biotechnological advances and perspectives

Riboflavin is an essential nutrient for humans and animals, and its derivatives flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are cofactors in the cells. Therefore, riboflavin and its derivatives are widely used in the food, pharmaceutical, nutraceutical and cosmetic industries. Advances in biotechnology have led to a complete shift in the commercial production of riboflavin from chemical synthesis to microbial fermentation. In this review, we provide a comprehensive review of biotechnologies that enhance riboflavin production in microorganisms, as well as representative examples. Firstly, the synthesis pathways and metabolic regulatory processes of riboflavin in microorganisms; and the current strategies and methods of metabolic engineering for riboflavin production are systematically summarized and compared. Secondly, the using of systematic metabolic engineering strategies to enhance riboflavin production is discussed, including laboratory evolution, histological analysis and high-throughput screening. Finally, the challenges for efficient microbial production of riboflavin and the strategies to overcome these challenges are prospected.     

Recent advances in biotechnological production of 2-phenylethanol

2-Phenylethanol (2-PE) is an important aromatic alcohol with a rose-like fragrance. It has been widely applied in the cosmetic, perfume, and food industries and is mainly produced by chemical synthesis. An alternative method for the production of natural flavors and fragrances is the microbial transformation process, which is attracting increasing attention because it is an environmentally friendly process and the products are considered “natural”. The production of 2-PE from L-phenylalanine by biotransformation is possible through the Ehrlich pathway and considerable progress has been made in the development of this process. The present report reviews recent advances in biotechnological production of 2-PE, with emphasis on the strategies used to increase production and the applications of in situ product removal techniques. Future research should focus on product scale-up and product recovery processes for the industrialization of microbial processes.