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.     

Comparison of culture methods on exopolysaccharide production in the submerged culture of Cordyceps militaris and process optimization

Aims: To improve exopolysaccharides (EPS) production of Cordyceps militaris (C. militaris), effects of different culture method on mycelial biomass and EPS production in the submerged culture of C. militaris were investigated. Methods and Results: A new two‐stage fermentation process for EPS production of C. militaris was designed in this work. Central composite design (CCD) was utilized to optimize the two‐stage fermentation process. The results showed that the two‐stage fermentation process for EPS production was superior to other culture method (conventional static culture and shake culture). CCD revealed that the optimum values of the test variables for EPS production were shaken for 140 h followed by 130‐h static culture. The maximum EPS production reached 3·2 g l^?1 under optimized two‐stage culture and was about 2·3‐fold and 1·6‐fold in comparison with those of original static culture and shake culture. Conclusions: It was indicated that a new two‐stage culture method obtained in this work possessed a high potential for the industrial production for EPS of C. militaris. Significance and Impact of the Study: The fundamental information obtained in this work is complementary to those of previous investigations on the submerged culture of C. militaris for the production of bioactive metabolites.     

Production of bioactive metabolites by submerged fermentation of the medicinal mushroom Antrodia cinnamomea: recent advances and future development

Edible and medicinal mushrooms have usually been considered as a sustainable source of unique bioactive metabolites, which are valued as promising provisions for human health. Antrodia cinnamomea is a unique edible and medicinal fungus widespread in Taiwan, which has attracted much attention in recent years for its high value in both scientific research and commercial applications owing to its potent therapeutic effects, especially for its hepatic protection and anticancer activity. Due to the scarcity of the fruiting bodies, the cultivation of A. cinnamomea by submerged fermentation appears to be a promising substitute which possesses some unique advantages, such as short culture time period and its high feasibility for scale-up production. However, the amount of fungal bioactive metabolites derived from the cultured mycelia of A. cinnamomea grown by submerged fermentation is much less than those obtained from the wild fruiting bodies. Hence, there is an urgent need to bridge such a discrepancy on bioactive metabolites between the wild fruiting bodies and the cultured mycelia. The objective of this article is to review recent advances and the future development of the mycelial submerged fermentation of A. cinnamomea in terms of enhancement for the production of fungal bioactive components by the optimization of culture conditions and the regulation of fungal metabolism. This review provides valuable information for further biotechnological applications of A. cinnamomea as well as other mushrooms being the source of bioactive ingredients by submerged fermentation.     

Bipolar heterothallism,a principal mating system of<Emphasis Type="Italic">Cordyceps militaris in vitro</Emphasis>

Interest inin vitro study of entomopathogenic fungi, includingCordyceps species, has been increasing due to their valuable bioactive compounds and biocontrol effects. AmongCordyceps species,in vitro stromata ofC. militaris has been successfully produced and cultivated for industrial purposes. However, genetic study onin vitro stromata formation ofC. militaris has not been carried out yet. Here, relationship between mating system and perithecial stromata formation ofC. militaris is reported. Mating system was determined by observing perithecial stromata formation from mono-ascospore cultures and their pair-wise combinations. Certain combinations of mono-ascospore strains produced perithecial club-shaped stromata, whereas other combinations produced either no stromata or only abnormal non-perithecial stromata. Similarly, monoascospore cultures without combination produced either no stromata or only abnormal nonperithecial stromata. Despite obvious heterothallism, self-fertility was occasionally observed in few strains ofC. militaris. These observations indicated thatC. militaris behaves as a bipolar heterothallic fungus and requires two mating compatible strains in order to produce regular clubshaped perithecial stromata, a fundamental requirement for its industrial cultivation.     

蛹虫草饲料添加剂的研究现状及展望

蛹虫草饲料添加剂包括蛹虫草子实体、蛹虫草培养残基、蛹虫草及其培养残基提取物、蛹虫草菌固液发酵产物、 微生物发酵蛹虫草残基等产品。蛹虫草饲料添加剂含有粗蛋白、粗脂肪、氨基酸等营养成分,以及虫草素、腺苷、多糖等活性成分,在畜禽、反刍动物、水产品等动物养殖中的应用均获得较好的 效果。对蛹虫草子实体、蛹虫草培养残基、蛹虫草及其培养残基提取物、利用蛹虫草菌及培养残基制作发酵饲料等蛹虫草饲料添加剂在动物养殖中的研究应用进行了总结,对存在的问题及发展前景进行了探讨及展望。     

The medicinal fungus Cordyceps militaris: research and development

Ophiocordyceps sinensis (syn. Cordyceps sinensis) is a highly valued medicinal fungus. This entomopathogen has a limited distribution, has been overharvested in the wild, and its stromata have not been artificially cultivated. Another entomopathogenic fungus, Cordyceps militaris (commonly known as orange caterpillar fungus), has chemical capacities similar to those of O. sinensis, but unlike O. sinensis, its stromata can be easily cultivated. Consequently, C. militaris is being studied as an alternative to O. sinensis, and the large-scale production of stromata is receiving substantial attention. Significant research has been conducted on the genetic resources, nutritional and environmental requirements, mating behavior, and biochemical and pharmacological properties of C. militaris. The complete genome of C. militaris has recently been sequenced. This fungus has been the subject of many reviews, but few have focused on its biology. The current paper reviews the biological aspects of the fungus including host range, mating system, cytology and genetics, insect- and non-insect nutritional requirements, environmental influence on stroma development, and commercial development.     

Na_2SeO_4对蛹虫草子实体生长的影响

蛹虫草是一种药食两用真菌,具有与冬虫夏草相似的功能,且富硒能力较强。本研究通过大量的人工栽培试验,旨在探究不同浓度Na_2SeO_4对新疆本地蛹虫草子实体生长的影响。试验表明,质量浓度为20 mg/L的Na_2SeO_4对蛹虫草的生长不产生显著影响,但蛹虫草各项生物学指标均随着培养基中外源Na_2SeO_4浓度的增加而呈下降趋势,说明随着外源Na_2SeO_4浓度的增加会对蛹虫草的生长产生抑制效应,当外源Na_2SeO_4质量浓度达到200 mg/L时,生产的蛹虫草已不具备商品价值。由此可见,20 mg/L的质量浓度是以Na_2SeO_4为硒源进行蛹虫草富硒研究的安全浓度。该研究为富硒产品开发寻找新的硒源开辟了新思路,为新疆地区进一步大规模栽培富硒蛹虫草提供一定的参考,但是对以Na_2SeO_4为硒源的最佳富硒浓度还有待于进一步研究。     

Targeted Gene Deletion in <Emphasis Type="Italic">Cordyceps militaris</Emphasis> Using the Split-Marker Approach

The macrofungus Cordyceps militaris contains many kinds of bioactive ingredients that are regulated by functional genes, but the functions of many genes in C. militaris are still unknown. In this study, to improve the frequency of homologous integration, a genetic transformation system based on a split-marker approach was developed for the first time in C. militaris to knock out a gene encoding a terpenoid synthase (Tns). The linear and split-marker deletion cassettes were constructed and introduced into C. militaris protoplasts by PEG-mediated transformation. The transformation of split-marker fragments resulted in a higher efficiency of targeted gene disruption than the transformation of linear deletion cassettes did. The color phenotype of the Tns gene deletion mutants was different from that of wild-type C. militaris. Moreover, a PEG-mediated protoplast transformation system was established, and stable genetic transformants were obtained. This method of targeted gene deletion represents an important tool for investigating the role of C. militaris genes.     

Biologically active components and nutraceuticals in the <Emphasis Type="Italic">Monascus</Emphasis>-fermented rice: a review

Monascus-fermented rice has traditionally been used as a natural food colorant and food preservative of meat and fish for centuries. It has recently become a popular dietary supplement because of many of its bioactive constituents being discovered, including a series of active drug compounds, monacolins, indicated as the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors for reducing serum cholesterol level. The controversy of its safety has been provoked because a mycotoxin, citrinin, is also produced along with the Monascus secondary metabolites by certain strains or under certain cultivation conditions. This review introduces the basic production process and addresses on the compounds with bioactive functions. Current advances in avoiding the harmful ingredient citrinin are also discussed.     

基于农业废弃物培育优质蛹虫草的研究

利用农业废弃物甘薯藤及蛹虫草培养基废弃物作为培养基的主要原料进行蛹虫草菌种驯化。蛹虫草子实体培养基中添加不同比例蛹虫草培养基废弃物及甘薯藤粒,通过适宜的培养条件,废弃物中淀粉、蛋白质、糖类、氨基酸等营养物质及蛹虫草胞外酶酶解产生的小分子物质被充分利用,以培育优质蛹虫草子实体。当一级种子中加入蛹虫草培养基废弃物20 g/L和甘薯藤粉10 g/L,二级种中加入蛹虫草培养基废弃物20 g/L、甘薯藤粉10 g/L,使蛹虫草子实体栽培料中蛹虫草培养基废弃物占32%～45%,甘薯藤粒占10%～15%,二者比例为(3～4)：1时,栽培效果最好。本研究蛹虫草培养基替代原料资源丰富易得,并可节约生产用粮,降低原料成本,从而实现农用废弃物再利用,减少环境污染,也符合绿色环保可持续发展的理念。     

Optimization of culture conditions and medium components for the production of mycelial biomass and exo-polysaccharides with Cordyceps militaris in liquid culture

Both crude exo-biopolymers and mycelial biomass, produced by liquid culture of Cordyceps species, are believed to possess several potential health benefits. As a result of its known biological activities, Cordyceps militaris has been extensively characterized in regards to potential medicinal applications. However, optimized liquid culture conditions for enhanced polysaccharide productivity have yet to be developed, which is a necessary step for industrial applications. Therefore, in this study, the liquid culture conditions were optimized for maximal production of mycelial biomass and exo-polysaccharide (EPS) by C. militaris. The effects of medium composition, environmental factors, and C/N ratio were investigated. Among these variables 80 g, glucose; 10 g, yeast extract; 0.5 g, MgSO₄·7H₂O; and 0.5 g, KH₂PO₄ in 1 L distilled water were found to be the most suitable carbon, nitrogen, and mineral sources, respectively. The optimal temperature, initial pH, agitation, and aeration were determined to be 24°C, uncontrolled pH, 200 rpm, and 1.5 vvm, respectively. Under these optimal conditions, mycelial growth in shake flask cultures and 5 L jar bioreactors was 29.43 and 40.60 g/L, respectively, and polysaccharide production in shake flask cultures and 5 L jar bioreactors was 2.53 and 6.74 g/L, respectively.     

Exploring the bioprospecting and biotechnological potential of white-rot and anaerobic Neocallimastigomycota fungi: peptidases,esterases, and lignocellulolytic enzymes

Fungi constitute an invaluable natural resource for scientific research, owing to their diversity; they offer a promising alternative for bioprospecting, thus contributing to biotechnological advances. For a long time, extensive information has been exploited and fungal products have been tested as a source of natural compounds. In this context, enzyme production remains a field of interest, since it offers an efficient alternative to the hazardous processes of chemical transformations. Owing to their vast biodiversity and peculiar biochemical characteristics, two fungal categories, white-rot and anaerobic Neocallimastigomycota, have gathered considerable attention for biotechnological applications. These fungi are known for their ability to depolymerize complex molecular structures and are used in degradation of lignocellulosic biomass, improvement of animal feed digestibility, biogas and bioethanol production, and various other applications. However, there are only limited reports that describe proteolytic enzymes and esterases in these fungi and their synergistic action with lignocellulolytic enzymes on degradation of complex polymers. Thus, in this minireview, we focus on the importance of these organisms in enzyme technology, their bioprospecting, possibility of integration of their enzyme repertoire, and their prospects for future biotechnological innovation.

     

Tanshinone biosynthesis in Salvia miltiorrhiza and production in plant tissue cultures

Salvia miltiorrhiza Bunge (Lamiaceae) root, generally called Danshen, is an important herb in Chinese medicine widely used for treatment of cardiovascular diseases. Diterpenoid tanshinons are major bioactive constituents of Danshen with notable pharmacological activities and the potential as new drug candidates against some important human diseases. The importance of Danshen for traditional and modern medicines has motivated the research interest over two decades in the biosynthesis and biotechnological production of tanshinones. Although diterpenes in plants are presumably derived from the non-mevalonate (MVA) pathway, tanshinone biosynthesis in S. miltiorrhiza may also depend on the MVA pathway based on some key enzymes and genes detected in the early steps of these pathways. Plant tissue cultures are the major biotechnological processes for rapid production of tanshinones and other bioactive compounds in the herb. Various in vitro cultures of S. miltiorrhiza have been established, including cell suspension, adventitious root, and hairy root cultures, which can accumulate the major tanshinones as in the plant roots. Tanshinone production in cell and hairy root cultures has been dramatically enhanced with various strategies, including medium optimization, elicitor stimulation, and nutrient feeding operations. This review will summarize the above developments and also provide our views on future trends.     

蛹虫草线粒体DNA与细胞核DNA进化关系的比较

【目的】分析蛹虫草是否存在核内线粒体DNA片段,比较蛹虫草线粒体DNA与细胞核DNA的碱基变异程度及所反映的菌株间的系统发育关系。【方法】通过本地BLAST或LAST对蛹虫草线粒体基因组和核基因组进行序列相似性搜索;从10个已知线粒体基因组的蛹虫草菌株中分别扩增7个细胞核蛋白编码基因片段,并与其在14个线粒体蛋白编码基因上的碱基变异情况进行比较。【结果】蛹虫草核基因组中存在5处较短的核内线粒体DNA片段,总长只有278bp。蛹虫草核DNA的变异频率整体上高于线粒体DNA。核DNA和线粒体DNA所反映的蛹虫草菌株间的系统发育关系存在显著差异。【结论】蛹虫草线粒体DNA与核DNA间不存在长片段的基因交流,二者变异频率不同,所反映的蛹虫草菌株间的系统发育关系也有差异。本研究增加了对蛹虫草线粒体与细胞核DNA进化关系的认识。     

Expression and export: recombinant protein production systems for Aspergillus

Several Aspergillus species, in particular Aspergillus niger and Aspergillus oryzae, are widely used as protein production hosts in various biotechnological applications. In order to improve the expression and secretion of recombinant proteins in these filamentous fungi, several novel genetic engineering strategies have been developed in recent years. This review describes state-of-the-art genetic manipulation technologies used for strain improvement, as well as recent advances in designing the most appropriate engineering strategy for a particular protein production process. Furthermore, current developments in identifying bottlenecks in the protein production and secretion pathways are described and novel approaches to overcome these limitations are introduced. An appropriate combination of expression vectors and optimized host strains will provide cell factories customized for each production process and expand the great potential of Aspergilli as biotechnology workhorses to more complex multi-step industrial applications.     

<Emphasis Type="Italic">Acinetobacter</Emphasis> lipases: molecular biology,biochemical properties and biotechnological potential

Lipases (EC 3.1.1.3) have received increased attention recently, evidenced by the increasing amount of information about lipases in the current literature. The renewed interest in this enzyme class is due primarily to investigations of their role in pathogenesis and their increasing use in biotechnological applications [38]. Also, many microbial lipases are available as commercial products, the majority of which are used in detergents, cosmetic production, food flavoring, and organic synthesis. Lipases are valued biocatalysts because they act under mild conditions, are highly stable in organic solvents, show broad substrate specificity, and usually show high regio- and/or stereo-selectivity in catalysis. A number of lipolytic strains of Acinetobacter have been isolated from a variety of sources and their lipases possess many biochemical properties similar to those that have been developed for biotechnological applications. This review discusses the biology of lipase expression in Acinetobacter, with emphasis on those aspects relevant to potential biotechnology applications.     

Molecular cloning and sequence analysis of the β-1,3-glucan synthase catalytic subunit gene from a medicinal fungus, Cordyceps militaris

The entomopathogenic fungus Cordyceps militaris belongs to vegetable wasps and plant worms and is used as herbal medicine, but β-1,3-glucan biosynthesis has been poorly studied in C. militaris. The fungal FKS1 gene encodes an integral membrane protein that is the catalytic subunit of β-1,3-glucan synthase. Here, we isolated cDNA clones encoding a full-length open reading frame of C. militaris FKS1. Cordyceps militaris Fks1 protein is a 1981 amino acid protein that shows significant similarity with other fungal Fks proteins. This study is the first report of molecular cloning of the β-1,3-glucan synthase catalytic subunit gene from vegetable wasps and plant worms.     

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.     

The Genus Gluconobacter Oxydans: Comprehensive Overview of Biochemistry and Biotechnological Applications

ABSTRACT

The genus Gluconobacter comprises some of the most frequently used microorganisms when it comes to biotechnological applications. Not only has it been involved in “historical” production processes, such as vinegar production, but in the last decades many bioconversion routes for special and rare sugars involving Gluconobacter have been developed. Among the most recent are the biotransformations involved in the production of L-ribose and miglitol, both very promising pharmaceutical lead molecules. Most of these processes make use of Gluconobacter's membrane-bound polyol dehydrogenases. However, recently other enzymes have also caught the eye of industrial biotechnology. Among them are dextran dextrinase, capable of transglucosylating substrate molecules, and intracellular NAD-dependent polyol dehydrogenases, of interest for co-enzyme regeneration. As such, Gluconobacter is an important industrial microbial strain, but it also finds use in other fields of biotechnology, such as biosensor-technology. This review aims to give an overview of the myriad of applications for Gluconobacter, with a special focus on some recent developments.     

Pyruvate production in Candida glabrata: manipulation and optimization of physiological function

Candida glabrata, a multi-vitamin auxotrophic yeast, can accumulate a large amount of pyruvate extracellularly using glucose as the carbon source, a characteristic that has facilitated the cost-effective biotechnological production of pyruvate on an industrial scale. In this review, we describe the current advances in further improving the performance of C. glabrata for efficient pyruvate production, which includes: optimization of the vitamin and dissolved oxygen concentrations, regulation of intracellular cofactor levels and improvement of the environmental robustness of C. glabrata. We also discuss the current efforts using systems biology to understand the metabolism of C. glabrata. Finally, perspectives on engineering and exploiting C. glabrata as a cell factory for efficiently producing various chemicals and materials are discussed.