Nutraceutical production with food-grade microorganisms

Over the past few years a number of new food ingredients labelled as being nutraceuticals have been launched on the food and pharmaceutical market. These include components that have a proven beneficial effect on human health, such as low-calorie sugars and B vitamins. Lactic acid bacteria, in particular Lactococcus lactis, have been demonstrated to be ideal cell factories for the production of these important nutraceuticals. Developments in the genetic engineering of food-grade microoganisms means that the production of certain nutraceuticals can be enhanced or newly induced through overexpression and/or disruption of relevant metabolic genes.     

基因工程在改善植物油营养价值中的应用

本文介绍了近年来应用基因工程技术对植物油的脂肪酸成分进行改造,进而生产有益于健康的食用油所取得的进展.近10年来,应用基因修饰的方法对植物油进行营养学方面的改良已经取得了很大的进展,随着植物脂肪酸生物合成途径的日趋明确,通过转基因技术可以让植物生产许多含有特殊脂肪酸成分且对人类健康有益的植物油.     

基因工程在改善植物油营养价值中的应用

本文介绍了近年来应用基因工程技术对植物油的脂肪酸成分进行改造, 进而生产有益于健康的食用油所取得的进展。近10年来, 应用基因修饰的方法对植物油进行营养学方面的改良已经取得了很大的进展, 随着植物脂肪酸生物合成途径的日趋明确, 通过转基因技术可以让植物生产许多含有特殊脂肪酸成分且对人类健康有益的植物油。     

Carotenoid metabolism: biosynthesis, regulation, and beyond

Carotenoids are Indispensable to plants and play a critical role in human nutrition and health. Significant progress has been made in our understanding of carotenoid metabolism in plants. The biosynthetic pathway has been extensively studied.Nearly all the genes encoding the biosynthetic enzymes have been isolated and characterized from various organisms. In recent years, there is an increasing body of work on the signaling pathways and plastid development, which might provide global control of carotenoid biosynthesis and accumulation. Herein, we will highlight recent progress on the biosynthesis,regulation, and metabolic engineering of carotenoids in plants, as well as the future research towards elucidating the regulatory mechanisms and metabolic network that control carotenoid metabolism.     

Metabolic pathway engineering in lactic acid bacteria

Lactic acid bacteria (LAB) display a relatively simple carbon and energy metabolism where the sugar source is converted mainly to lactic acid. In Lactococcus lactis metabolic engineering has been very successful in the re-routing of lactococcal pyruvate metabolism to products other than lactic acid. Current metabolic engineering approaches tend to focus on more complex, biosynthetic pathways leading to end-products that generate a health benefit for the consumer (nutraceuticals). Several examples of research on these minor pathways in L. lactis have illustrated the potential of LAB as producers of these metabolites. Whole genome sequencing efforts and corresponding global technologies will have an impact on metabolic engineering in the future.     

Metabolic engineering of isoprenoids

The metabolic engineering of natural products has begun to prosper in the past few years due to genomic research and the discovery of biosynthetic genes. While the biosynthetic pathways and genes for some isoprenoids have been known for many years, new pathways have been found and known pathways have been further investigated. In this article, we review the recent advances in metabolic engineering of isoprenoids, focusing on the molecular genetics that affects pathway engineering the most. Examples in mono- sequi-, and diterpenoid synthesis as well as carotenoid production are discussed.     

Modular co-culture engineering,a new approach for metabolic engineering

With the development of metabolic engineering, employment of a selected microbial host for accommodation of a designed biosynthetic pathway to produce a target compound has achieved tremendous success in the past several decades. Yet, increasing requirements for sophisticated microbial biosynthesis call for establishment and application of more advanced metabolic engineering methodologies. Recently, important progress has been made towards employing more than one engineered microbial strains to constitute synthetic co-cultures and modularizing the biosynthetic labor between the co-culture members in order to improve bioproduction performance. This emerging approach, referred to as modular co-culture engineering in this review, presents a valuable opportunity for expanding the scope of the broad field of metabolic engineering. We highlight representative research accomplishments using this approach, especially those utilizing metabolic engineering tools for microbial co-culture manipulation. Key benefits and major challenges associated with modular co-culture engineering are also presented and discussed.     

The complex network of non-cellulosic carbohydrate metabolism

Partitioning of carbon dominates intracellular fluxes in both photosynthetic and heterotrophic plant tissues, and has vast influence on both plant growth and development. Recently, much progress has been made in elucidating the structures of the biosynthetic and degradative pathways that link the major and minor pools of soluble carbohydrates to cellular polymers such as starch, heteroglycans and fructans. In most cases, the regulatory properties of these pathways have been elucidated and the enzymes involved have been investigated using reverse genetics approaches. Although many of the results from these approaches were merely confirmatory, several of them were highly unexpected. The challenge ahead is to achieve better understanding of metabolic regulation at the network level in order to develop more rational strategies for metabolic engineering.     

Metabolic cartography: experimental quantification of metabolic fluxes from isotopic labelling studies

For the past decade, flux maps have provided researchers with an in-depth perspective on plant metabolism. As a rapidly developing field, significant headway has been made recently in computation, experimentation, and overall understanding of metabolic flux analysis. These advances are particularly applicable to the study of plant metabolism. New dynamic computational methods such as non-stationary metabolic flux analysis are finding their place in the toolbox of metabolic engineering, allowing more organisms to be studied and decreasing the time necessary for experimentation, thereby opening new avenues by which to explore the vast diversity of plant metabolism. Also, improved methods of metabolite detection and measurement have been developed, enabling increasingly greater resolution of flux measurements and the analysis of a greater number of the multitude of plant metabolic pathways. Methods to deconvolute organelle-specific metabolism are employed with increasing effectiveness, elucidating the compartmental specificity inherent in plant metabolism. Advances in metabolite measurements have also enabled new types of experiments, such as the calculation of metabolic fluxes based on (13)CO(2) dynamic labelling data, and will continue to direct plant metabolic engineering. Newly calculated metabolic flux maps reveal surprising and useful information about plant metabolism, guiding future genetic engineering of crops to higher yields. Due to the significant level of complexity in plants, these methods in combination with other systems biology measurements are necessary to guide plant metabolic engineering in the future.     

植物天然产物微生物重组合成研究进展

植物天然产物是小分子药物、营养品、化妆品、香精香料等的主要来源之一,在国民经济中发挥重要的作用。目前植物天然产物主要依赖于植物提取,这种生产方式占用耕地、生长周期长,而且植物活性成分往往含量低、生产成本高。通过解析植物天然产物生物合成途径,在微生物细胞中重构,创建细胞工厂,实现利用可再生原料发酵合成,为植物天然产物的供给提供了新的路线。本文重点介绍了中国科学院天津工业生物技术研究所在萜类、黄酮类、苯丙素类等重要类型植物天然产物微生物重组合成方面的研究进展,简要探讨了当前研究面临的挑战及未来前景。     

Stacking transgenes in forest trees

Huge potential exists for improving plant raw materials and foodstuffs via metabolic engineering. To date, progress has mostly been limited to modulating the expression of single genes of well-studied pathways, such as the lignin biosynthetic pathway, in model species. However, a recent report illustrates a new level of sophistication in metabolic engineering by overexpressing one lignin enzyme while simultaneously suppressing the expression of another lignin gene in a tree, aspen. This novel approach to multi-gene manipulation has succeeded in concurrently improving several wood-quality traits.     

Vitamin production in transgenic plants

Plants are a major source of vitamins in the human diet. Due to their significance for human health and development, research has been initiated to understand the biosynthesis of vitamins in plants. The pathways that are furthest advanced in elucidation are those of provitamin A, vitamin C and vitamin E. There is little knowledge about the regulation, storage, sink and degradation of any vitamin made in plants, or the interaction of vitamin biosynthetic pathways with other metabolic pathways. Researchers as well as life science companies have endeavoured to manipulate levels of vitamins in order to create functional food with enhanced health benefits, and even with the goal of achieving levels worth extracting from plant tissues. Thus far, metabolic engineering has resulted in transgenic plants that contain elevated levels of provitamin A, vitamin C and E, respectively. Additional research is necessary to identify all relevant target genes in order to further improve and tailor plants with elevated vitamin contents at will.     

Metabolic engineering of carotenoid accumulation by creating a metabolic sink

Carotenoids are highly beneficial for human nutrition and health because they provide essential nutrients and important antioxidants in our diets. However, many food crops, especially the major staple crops contain only trace to low amounts of carotenoids. Although significant progress has been made in developing food crops rich in carotenoids by altering the expression of carotenoid biosynthetic genes, in many cases it has proved to be difficult to reach the desired levels of carotenoid enrichment. The recent identification and characterization of a novel gene mutation in cauliflower reveals that creating a metabolic sink to sequester carotenoids is an important mechanism to control carotenoid accumulation in plants. The successful demonstration of increased carotenoid accumulation in association with the formation of sink structures in transgenic crops offers a new and alternative approach to increase carotenoid content. Manipulation of the formation of metabolic sink along with the catalytic activity of the pathway may represent a promising strategy for maximally improving the nutritional quality of food crops.     

Natural products – modifying metabolite pathways in plants

The diversity of plant natural product (PNP) molecular structures is reflected in the variety of biochemical and genetic pathways that lead to their formation and accumulation. Plant secondary metabolites are important commodities, and include fragrances, colorants, and medicines. Increasing the extractable amount of PNP through plant breeding, or more recently by means of metabolic engineering, is a priority. The prerequisite for any attempt at metabolic engineering is a detailed knowledge of the underlying biosynthetic and regulatory pathways in plants. Over the past few decades, an enormous body of information about the biochemistry and genetics of biosynthetic pathways involved in PNPs production has been generated. In this review, we focus on the three large classes of plant secondary metabolites: terpenoids (or isoprenoids), phenylpropanoids, and alkaloids. All three provide excellent examples of the tremendous efforts undertaken to boost our understanding of biosynthetic pathways, resulting in the first successes in plant metabolic engineering. We further consider what essential information is still missing, and how future research directions could help achieve the rational design of plants as chemical factories for high-value products.     

Strain improvement for fermentation and biocatalysis processes by genetic engineering technology

Twenty years ago, the first complete gene cluster encoding the actinorhodin biosynthetic pathway was cloned and characterized. Subsequently, the gene clusters encoding the biosynthetic pathways for many antibiotics were isolated. In the past decade, breakthroughs in technology brought that generation of rationally designed or new hybrid metabolites to fruition. Now, the development of high-throughput DNA sequencing and DNA microarray techniques enables researchers to identify the regulatory mechanisms for the overproduction of secondary metabolites and to monitor gene expression during the fermentation cycle, accelerating the rational application of metabolic pathway engineering. How are the new tools of biotechnology currently being applied to improve the production of secondary metabolites? Where will this progress lead us tomorrow? The use of whole cells or partially purified enzymes as catalysts has been increased significantly for chemical synthesis in pharmaceutical and fine-chemical industries. The development of PCR technologies for protein engineering and DNA shuffling is leading to the generation of new enzymes with increased stability to a wide range of pHs, temperatures and solvents and with increased substrate specificity, reaction rate and enantioselectivity. Where will this emerging technology lead us in the twenty-first century?     

代谢工程在芳香化合物生物合成研究中的应用

生物技术和代谢工程的发展促进了生物合成研究。概述了近年来利用微生物莽草酸途径进行芳香化合物生物合成研究的现况、代谢工程在提高天然芳香化合物产量和扩大合成非天然产生的芳香化合物范围的应用的进展 ,特别是整体代谢工程对提高第二代工程菌产量的作用。指出了生物合成法是生产氨基酸及其它生物小分子如奎尼酸、维生素和抗生素等的未来趋势 ,在工业化生产中有着广阔的应用前景。     

Curating a comprehensive set of enzymatic reaction rules for efficient novel biosynthetic pathway design

Enzyme substrate promiscuity has significant implications for metabolic engineering. The ability to predict the space of possible enzymatic side reactions is crucial for elucidating underground metabolic networks in microorganisms, as well as harnessing novel biosynthetic capabilities of enzymes to produce desired chemicals. Reaction rule-based cheminformatics platforms have been implemented to computationally enumerate possible promiscuous reactions, relying on existing knowledge of enzymatic transformations to inform novel reactions. However, past versions of curated reaction rules have been limited by a lack of comprehensiveness in representing all possible transformations, as well as the need to prune rules to enhance computational efficiency in pathway expansion. To this end, we curated a set of 1224 most generalized reaction rules, automatically abstracted from atom-mapped MetaCyc reactions and verified to uniquely cover all common enzymatic transformations. We developed a framework to systematically identify and correct redundancies and errors in the curation process, resulting in a minimal, yet comprehensive, rule set. These reaction rules were capable of reproducing more than 85% of all reactions in the KEGG and BRENDA databases, for which a large fraction of reactions is not present in MetaCyc. Our rules exceed all previously published rule sets for which reproduction was possible in this coverage analysis, which allows for the exploration of a larger space of known enzymatic transformations. By leveraging the entire knowledge of possible metabolic reactions through generalized enzymatic reaction rules, we are able to better utilize underground metabolic pathways and accelerate novel biosynthetic pathway design to enable bioproduction towards a wider range of new molecules.     

Methods and applications for assembling large DNA constructs

The construction of large DNA molecules that encode pathways, biological machinery, and entire genomes has been limited to the reproduction of natural sequences. However, now that robust methods for assembling hundreds of DNA fragments into constructs > 20 kb are readily available, optimization of large genetic elements for metabolic engineering purposes is becoming more routine. Here, various DNA assembly methodologies are reviewed and some of their potential applications are discussed. We tested the potential of DNA assembly to install rational changes in complex biosynthetic pathways, their potential for generating complex libraries, and consider how various strategies are applicable to metabolic engineering.     

芳香族化合物微生物代谢工程研究进展

代谢工程从20世纪90年代初期发展至今已有近30年历史,对微生物菌种改良和选育工作起到了极大的推动作用.芳香族化合物是一类可以通过微生物发酵生产的化学品,广泛应用于医药、食品、饲料和材料等领域.利用代谢工程手段对莽草酸和芳香族氨基酸合成途径进行理性改造,微生物细胞可以定向地大量积累人们需要的各种芳香族化合物.笔者对近3...