Fungal biotechnology

Fungi are used in many industrial processes, such as the production of enzymes, vitamins, polysaccharides, polyhydric alcohols, pigments, lipids, and glycolipids. Some of these products are produced commercially while others are potentially valuable in biotechnology. Fungal secondary metabolites are extremely important to our health and nutrition and have tremendous economic impact. In addition to the multiple reaction sequences of fermentations, fungi are extremely useful in carrying out biotransformation processes. These are becoming essential to the fine-chemical industry in the production of single-isomer intermediates. Recombinant DNA technology, which includes yeasts and other fungi as hosts, has markedly increased markets for microbial enzymes. Molecular manipulations have been added to mutational techniques as a means of increasing titers and yields of microbial processes and in the discovery of new drugs. Today, fungal biology is a major participant in global industry. Moreover, the best is yet to come as genomes of additional species are sequenced at some level (cDNA, complete genomes, expressed sequence tags) and gene and protein arrays become available.     

Biopharmaceuticals in China

The biopharmaceutical industry, whose products are produced mainly by recombinant DNA technology, antibody technologies and cytotechnology, is the most important sector in industrial biotechnology, and is one of the most rapidly growing high-tech industries. The global market for biopharmaceuticals had been growing at annual growth rates of 15-33% over the last 8 years, and sales exceeded 55 billion dollars in 2005. This review presents an overview of the Chinese biopharmaceutical industry, listing the global top-selling biopharmaceuticals in 2005, and briefly describes the major biotech drugs approved by the Chinese State Food and Drug Administration, such as recombinant cytokines, therapeutic antibodies, recombinant vaccines, and gene therapy products.     

植物内生放线菌研究进展

植物内生菌主要包括细菌、真菌和放线菌。放线菌的次生代谢产物,如抗生素、有机酸、生物碱等,在抗癌药物、新型抗生素、植物促生剂、生物农药、纳米新材料等领域取得了一系列研究成果。内生放线菌在生态农业、食品工业、制药工业和环境治理等方面扮演重要角色。本文简要概述了目前植物内生放线菌的研究成果,讨论了存在的问题和发展方向。     

White biotechnology: ready to partner and invest in

It needs three factors to build an industry: market demand, product vision and capital. White biotechnology already produces high volume products such as feed additive amino acids and specialty products like enzymes for enantioselective biocatalysis. It serves large and diverse markets in the nutrition, wellness, pharmaceutical, agricultural and chemical industry. The total volume adds up to $ 50 billion worldwide. In spite of its proven track record, white biotechnology so far did not attract as much capital as red and even green biotechnology. However, the latest finance indicators confirm the continuously growing attractiveness of investment opportunities in white biotechnology. This article discusses white biotechnology's position and potential in the finance market and success factors.     

Formation of Phenolic and Indolic Compounds by Anaerobic Bacteria in the Human Large Intestine

Abstract Batch culture incubations were used to investigate the effects of pH (6.8 or 5.5) and carbohydrate (starch) availability on dissimilatory aromatic amino acid metabolism in human fecal bacteria. During growth on peptide mixtures, tyrosine and phenylalanine fermentations occurred optimally at pH 6.8, while individual metabolic reactions were inhibited by up to 80% in the presence of 10 g l⁻¹ starch. Tryptophan metabolites were not detected in these experiments. When free amino acids replaced peptides, phenol production was increased during carbohydrate fermentation, although formation of p-cresol, another tyrosine metabolite was strongly inhibited. Phenylpropionate, which is produced from phenylalanine, was unaffected by starch. Tryptophan was fermented in these studies, although indole production was reduced in the starch fermentors. The importance of different fermentation substrates (casein, peptide mixtures, free amino acids) on aromatic amino acid metabolism was investigated in incubations of material taken from the proximal bowel. The phenylalanine metabolites, phenylacetate and phenylpropionate, were the principal phenolic compounds formed from all three substrates. Phenol was the major tyrosine metabolite produced in casein and peptide fermentations, while hydroxyphenylpropionate was a more important tyrosine product from free amino acids. Indole was the sole product of tryptophan metabolism, but was formed only from the free amino acid. Bacterial metabolism of individual phenolic and indolic compounds was also investigated. Phenol, p-cresol, phenylacetate, phenylpropionate, 4-ethylphenol, indole, indoleacetate, and indolepropionate were not metabolized by colonic bacteria. However, hydroxyphenylacetate was hydrolyzed to p-cresol, while hydroxyphenylpropionate was transformed into phenylpropionate. Indolepyruvate was either converted to indoleacetate or metabolized into indole. Indolepropionate, and to a lesser degree indoleacetate were produced from indolelactate. These data show that human colonic anaerobes are able to extensively degrade either free or peptide-bound aromatic amino acids, with the concomitant formation of toxic metabolic products. These processes are controlled to a significant degree by environmental factors such as pH and carbohydrate availability, and this ultimately influences the types and amounts of fermentation products that can be formed in different regions of the large bowel. Received: 25 January 1996; Accepted: 8 May 1996     

High-performance liquid chromatography of microbial acid metabolites

The use of high-performance liquid chromatography with a cation-exchange column and effluent monitoring at 210 nm has been evaluated for the profiling of selected microbial metabolites including aliphatic, dicarboxylic, and phenolic acids, as an adjunct to the identification of selected bacteria, detection of bacterial metabolites in foods, and the monitoring of industrial microbial fermentations. Advantages of the technique include the simultaneous require only qualitative or semi-quantitative data. For others, data are given on the day-to-day reproducibility for several acids.     

Introduction of New Tools for Chemical Biology Research on Microbial Metabolites

Recently, high-throughput screening (HTS) has become the mainstream technique for drug discovery. Compounds that are synthesized by combinatorial chemistry might be more suitable than natural products to apply to HTS, because the purification procedure is a drawback of using natural products. Nevertheless, natural products remain an extremely important source of drugs. To overcome the demerits of natural products, we are constructing the RIKEN Natural Products Depository (NPDepo) that is focused primarily on microbial metabolites. In this review, I describe (i) engineering pathways for biosynthetic gene clusters of microbial metabolites, (ii) construction of fraction libraries of microbial metabolites, and (iii) the development of a new screening system using a chemical array and a protein library produced by GLORIA.     

发酵生产7-ACA基因工程菌的构建

头孢菌素类抗牛素是临床用途最广的抗感染药物,其工业生产的重要中间体7－氨基头孢烷酸（7－ACA）采用顶头孢霉发酵产物头孢菌素C为前体,通过化学合成或两步酶法狭得。介绍了在了解头孢菌素C生物合成的前提下,在建赢了顶头孢霉的遗传改造丛础上,运用合成生物学的知识,在头孢菌素C产生菌顶头孢霉中分别构建了三个头孢菌素C酰化酶的表达框架,通过发酵产物的分析并优选表达框架后,再采用传统发酵工艺的优化获得了一株可以直接发酵7-ACA的高产顶头孢霉工程菌。     

Biomass market dynamics supporting the large‐scale deployment of high‐octane fuel production in the United States

US Department of Energy research aimed at co‐optimizing fuels and engine performance identified several bioblendstocks that can improve fuel economy including an aromatic‐rich hydrocarbon derived from woody biomass. This work supports an analysis of its large‐scale deployment implying a production target of approximately 15 billion liters of bioblendstock for the supply of 57 billion liters of high‐octane fuel by 2050. It simulates potential transition pathways to lignocellulosic feedstock market structures capable of supplying a mature biorefining industry at this scale. In the present absence of biorefineries, transitions are modeled via nonbiofuel feedstock markets, so‐called companion markets. The resource distribution across several demand industries is simulated to determine biomass availability and price dynamics over time. Results indicate that the wood supply base is mainly influenced by traditional markets including housing and pulp and paper. The selected companion market of wood pellet combustion for heat and electricity generation is found to positively stimulate biomass mobilization, especially in the initial absence of biorefineries. Eventually, biorefineries are found to be able to out‐compete the companion market. As such, they directly benefit from the processing (i.e., pelleting) capacity established to produce commodity‐type intermediates for the companion market. We conclude that the amount of bioblendstock produced is directly related to the size of the companion market (and its pelleting capacity). An initially larger companion market generates up to 20 million dry tonnes of additional feedstock, equivalent to 27 commercial‐scale biorefineries, or an additional production of 5 billion liters by 2050. Distinguishing between industry‐specific feedstock preferences based on average biomass quality characteristics, this analysis goes beyond past research efforts that assume automatic fungibility across different feedstocks. Improving engine performance is a key driver for the promotion of low‐carbon fuels derived from bioblendstocks. This analysis portrays feedstock market transition pathways for their large‐scale deployment.     

Enhanced submerged Aspergillus ficuum phytase production by implementation of fed-batch fermentation

Phytase is an important feed and food additive, which is both used in animal and human diets. Phytase has been used to increase the absorption of several divalent ions, amino acids, and proteins in the bodies and to decrease the excessive phosphorus release in the manure to prevent negative effects on the environment. To date, microbial phytase has been mostly produced in solid-state fermentations with insignificant production volumes. There are only a few studies in the literature that phytase productions were performed in submerged bench-top reactor scale. In our previous studies, growth parameters (temperature, pH, and aeration) and important fermentation medium ingredients (glucose, Na-phytate, and CaSO₄) were optimized. This study was undertaken for further enhancement of phytase production with Aspergillus ficuum in bench-top bioreactors by conducting fed-batch fermentations. The results showed that addition of 60 g of glucose and 10 g of Na-phytate at 96 h of fermentation increased phytase activity to 3.84 and 4.82 U/ml, respectively. Therefore, the maximum phytase activity was further enhanced with addition of glucose and Na-phytate by 11 and 40 %, respectively, as compared to batch phytase fermentations. It was also reported that phytase activity increased higher in early log stage additions than late log stage additions because of higher microbial activity. In addition, the phytase activity in fed-batch fermentation did not drop significantly as compared to the batch fermentation. Overall, this study shows that fungal phytase can be successfully produced in submerged fed-batch fermentations.     

Effects of long-term antibiotic treatment on mice urinary aromatic amino acid profiles

The gut microbiota–host co-metabolites are good indicators for representing the cross-talk between host and gut microbiota in a bi-direct manner. There is increasing evidence that levels of aromatic amino acids (AAAs) are associated with the alteration of intestinal microbial community though the effects of long-term microbial disturbance remain unclear. Here we monitored the gut microbiota composition and host–microbiota co-metabolites AAA profiles of mice after gentamicin and ceftriaxone treatments for nearly 4 months since their weaning to reveal the relationship between host and microbiome in long- term microbial disturbances. The study was performed employing targeted LC-MS measurement of AAA-related metabolites and 16S RNA sequence of mice cecal contents. The results showed obvious decreased gut microbial diversity and decreased Firmicutes/Bacteroidetes ratio in the cecal contents after long-term antibiotics treatment. The accumulated AAA (tyrosine, phenylalanine and tryptophan) and re-distribution of their downstreaming metabolites that produced under the existence of intestinal flora were found in mice treated with antibiotics for 4 months. Our results suggested that the long-term antibiotic treatment significantly changed the composition of the gut microbiota and destroyed the homeostasis in the intestinal metabolism. And the urinary AAA could be an indicator for exploring interactions between host and gut microbiota.     

Commercialization of plant tissue culture in India

Commercial application of plant tissue culture started in USA with micropropagation of orchids in 1970s. It has seen tremendous expansion globally from 1985 to 1990 in the number of production units as well as the number of plants produced. With an estimated global market of 15 billion US dollars per annum for tissue cultured products, even with exponential expansion in the industry, the demand far exceeds production, leaving enough scope for expansion. This industry appears to be undergoing a pause in growth presently in developed countries as it is finding difficult to remain cost–effective. In US, only half the production capacity is being utilized currently due to high labour costs. In developing countries, with lower wage scales, plants are being produced at much cheaper rates. Indian micropropagation industry, though a late starter by almost a decade, compared to its western counterparts, has expanded exponentially from 5 million annual capacity in 1988 to 190 million in 1996. The facilities now created are at par with the best in leading countries like the Netherlands and USA. To remain in profitable business and to earn the much needed foreign exchange, Indian units need to judiciously mix steady revenue generating items with unique speciality items based on demand in domestic and international markets. This revised version was published online in June 2006 with corrections to the Cover Date.     

Very high gravity ethanol and fatty acid production of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> without amino acid and vitamin

Very high gravity (VHG) fermentation is the mainstream technology in ethanol industry, which requires the strains be resistant to multiple stresses such as high glucose concentration, high ethanol concentration, high temperature and harsh acidic conditions. To our knowledge, it was not reported previously that any ethanol-producing microbe showed a high performance in VHG fermentations without amino acid and vitamin. Here we demonstrate the engineering of a xylose utilizing recombinant Zymomonas mobilis for VHG ethanol fermentations. The recombinant strain can produce ethanol up to 136 g/L without amino acid and vitamin with a theoretical yield of 90 %, which is significantly superior to that produced by all the reported ethanol-producing strains. The intracellular fatty acids of the bacterial were about 16 % of the bacterial dry biomass, with the ratio of ethanol:fatty acids was about 273:1 (g/g). The recombinant strain was achieved by a multivariate-modular strategy tackles with the multiple stresses which are closely linked to the ethanol productivity of Z. mobilis. The over-expression of metB/yfdZ operon enabled the growth of the recombinant Z. mobilis in a chemically defined medium without amino acid and vitamin; and the fatty acids overproduction significantly increased ethanol tolerance and ethanol production. The coupled production of ethanol with fatty acids of the Z. mobilis without amino acid and vitamin under VHG fermentation conditions may permit a significant reduction of the production cost of ethanol and microbial fatty acids.     

单克隆抗体仿制药物的结构分析策略

市场高达数千亿美元的单克隆抗体药物即将结束专利保护期,这对发展中国家产业升级,提高国民医疗水平,都是一次难得的战略机遇。然而,单抗类蛋白仿制药的研发与质控工作难度巨大,必须使用液相色谱-质谱联用技术进行分析。针对单抗仿制药结构必须进行的分析项目(氨基酸序列表达正确性、糖基化修饰形态相似性、以及高级结构统一致性)的液质分析方法进行了系统介绍。此外还就分析工作效率及质控过程的法规依从问题予以讨论。     

Peptidic soil components are a major dietary resource for the humivorous larvae of Pachnoda spp. (Coleoptera: Scarabaeidae)

Humivorous scarab beetle larvae can thrive exclusively on soil organic matter. Feeding experiments have revealed that the larva of Pachnoda ephippiata mineralizes all major humus components except the polyphenolic fraction. High proteolytic activity in the alkaline midgut fluid and an enormous ammonia production during gut passage suggested that peptidic soil components are an important dietary resource for the larva. By comparing acid-hydrolyzable amino acids in food soil and feces, we showed that a significant fraction of the peptides in soil are removed during gut passage. This agrees well with the high concentrations of free amino acids found the midgut section. Incubation experiments revealed the presence of substantial particle-associated proteolytic activity also in the hindgut, most probably due to microbial activity. High rates of ammonia formation in hindgut homogenates and the conversion of radiolabeled amino acids to acetate and propionate indicated that microbial fermentations of soil peptides play an important role in the hindgut. This was corroborated by viable counts of amino-acid-fermenting bacteria, which formed a substantial fraction of the hindgut microbiota. A complete inventory of organic and inorganic nitrogen species before, during, and after gut passage revealed the formation of nitrite and nitrate in midgut and hindgut, and a substantial nitrogen deficit in the feces, suggesting that part of the ammonia formed by mineralization is subjected to oxidation and subsequent denitrification to N2. Together, the results strongly support the hypothesis that peptidic soil components form a major energy and nutrient source for humivorous insects, supplying the animal with microbial fermentation products and essential amino acids.     

Effect of antibiotics on fecal excretion of various products of microbial metabolism of aromatic amino acids

The model experiments on intact albino rats and those exposed to lincomycin, ampicillin and rifampicin showed that intragastric administration of the three antibiotics to the laboratory animals in therapeutic doses for 8 days induced statistically significant changes in the fecal excretion of n-cresol, indole and skatole, microbial metabolites of aromatic amino acids in the gastrointestinal tract of a macroorganism. The results are in favour of using biochemical tests for rapid diagnosis of the intestinal microflora response to antimicrobial agents.     

放线菌中亮氨酸应答调控蛋白的生物学功能及其调控机理

放线菌是一类革兰氏阳性细菌,可产生氨基酸等初级代谢产物和抗生素等次级代谢产物,其广泛用于食品、医药、添加剂及化妆品行业。此外,还有少数放线菌,如分枝杆菌等,是可以引起人和动植物病害的病原菌。亮氨酸应答调控蛋白(Leucine-responsive regulatory protein,Lrp)是一类在氨基酸代谢及其相关代谢过程中的重要转录调控子,能够应答各种氨基酸,参与调控微生物细胞的多个生理过程,例如氨基酸代谢和转运、中心代谢、细菌的持久性和毒力等。本文总结了放线菌Lrp的生物学功能,并综述了放线菌中不同种属Lrp以及天蓝色链霉菌和红色糖多孢菌Lrp调控机理的研究进展。     

Solid-state fermentation: a promising microbial technology for secondary metabolite production

Solid state (substrate) fermentation (SSF) has been used successfully for the production of enzymes and secondary metabolites. These products are associated with the stationary phase of microbial growth and are produced on an industrial scale for use in agriculture and the treatment of disease. Many of these secondary metabolites are still produced by submerged liquid fermentations (SmF) even though production by this method has been shown to be less efficient than SSF. As large-scale production increases further, so do the costs and energy demands. SSF has been shown to produce a more stable product, requiring less energy, in smaller fermenters, with easier downstream processing measures. In this article we review an important area of biotechnology, since the recent evidence indicates that bacteria and fungi, growing under SSF conditions, are more than capable of supplying the growing global demand for secondary metabolites.     

Advances in drug discovery and biochemical studies

Japanese researchers continue to discover new means to combat parasites and make important contributions toward developing tools for global control of parasitic diseases. Streptomyces avermectinius, the source of ivermectin, was discovered in Japan in the early 1970s and renewed and vigorous screening of microbial metabolites in recent years has led to the discovery of new antiprotozoals and anthelminthics, including antimalarial drugs. Intensive studies of parasite energy metabolism, such as NADH-fumarate reductase systems and the synthetic pathways of nucleic acids and amino acids, also contribute to the identification of novel and unique drug targets.