Constructing de novo biosynthetic pathways for chemical synthesis inside living cells

Living organisms have evolved a vast array of catalytic functions that make them ideally suited for the production of medicinally and industrially relevant small-molecule targets. Indeed, native metabolic pathways in microbial hosts have long been exploited and optimized for the scalable production of both fine and commodity chemicals. Our increasing capacity for DNA sequencing and synthesis has revealed the molecular basis for the biosynthesis of a variety of complex and useful metabolites and allows the de novo construction of novel metabolic pathways for the production of new and exotic molecular targets in genetically tractable microbes. However, the development of commercially viable processes for these engineered pathways is currently limited by our ability to quickly identify or engineer enzymes with the correct reaction and substrate selectivity as well as the speed by which metabolic bottlenecks can be determined and corrected. Efforts to understand the relationship among sequence, structure, and function in the basic biochemical sciences can advance these goals for synthetic biology applications while also serving as an experimental platform for elucidating the in vivo specificity and function of enzymes and reconstituting complex biochemical traits for study in a living model organism. Furthermore, the continuing discovery of natural mechanisms for the regulation of metabolic pathways has revealed new principles for the design of high-flux pathways with minimized metabolic burden and has inspired the development of new tools and approaches to engineering synthetic pathways in microbial hosts for chemical production.     

Metabolic pathway analysis of S. pneumoniae: an in silico approach towards drug-design

The emergence of multidrug resistant varieties of Streptococcus pneumoniae (S. pneumoniae) has led to a search for novel drug targets. An in silico comparative analysis of metabolic pathways of the host Homo sapiens (H. sapiens) and the pathogen S. pneumoniae have been performed. Enzymes from the biochemical pathways of S. pneumoniae from the KEGG metabolic pathway database were compared with proteins from the host H. sapiens, by performing a BLASTp search against the non-redundant database restricted to the H. sapiens subset. The e-value threshold cutoff was set to 0.005. Enzymes, which do not show similarity to any of the host proteins, below this threshold, were filtered out as potential drug targets. Five pathways unique to the pathogen S. pneumoniae when compared to the host H. sapiens have been identified. Potential drug targets from these pathways could be useful for the discovery of broad-spectrum drugs. Potential drug targets were also identified from pathways related to lipid metabolism, carbohydrate metabolism, amino acid metabolism, energy metabolism, vitamin and cofactor biosynthetic pathways and nucleotide metabolism. Of the 161 distinct targets identified from these pathways, many are in various stages of progress at the Microbial Genome Database. However, 44 of the targets are new and can be considered for rational drug design. The study was successful in listing out potential drug targets from the S. pneumoniae proteome involved in vital aspects of the pathogen's metabolism, persistence, virulence and cell wall biosynthesis. This systematic evaluation of metabolic pathways of host and pathogen through reliable and conventional bioinformatics approach can be extended to other pathogens of clinical interest.     

代谢工程——生物工程学科的新兴研究领域

代谢工程包括推理性代谢工程及逆代谢工程。针对限制生物活性的因素 ,从不同的途径设计代谢改变策略 ,采用重组DNA技术解除或削弱该影响 ,提高目的产物的产率 ,是生化工程学科提高生物活性的新兴研究领域。讨论了推理性代谢工程及逆代谢工程的设计及应用。     

Leveraging algal omics to reveal potential targets for augmenting TAG accumulation

Ongoing global efforts to commercialize microalgal biofuels have expedited the use of multi-omics techniques to gain insights into lipid biosynthetic pathways. Functional genomics analyses have recently been employed to complement existing sequence-level omics studies, shedding light on the dynamics of lipid synthesis and its interplay with other cellular metabolic pathways, thus revealing possible targets for metabolic engineering. Here, we review the current status of algal omics studies to reveal potential targets to augment TAG accumulation in various microalgae. This review specifically aims to examine and catalog systems level data related to stress-induced TAG accumulation in oleaginous microalgae and inform future metabolic engineering strategies to develop strains with enhanced bioproductivity, which could pave a path for sustainable green energy.     

植物次生代谢物途径及其研究进展

植物次生代谢是植物在长期进化过程中与环境相互作用的结果,由初生代谢派生。萜类、生物碱类、苯丙烷类为植物次生代谢物的主要类型,其代谢途径多以代谢频道形式存在,具有种属、生长发育期等特异性。从植物次生代谢物的分类、代谢途径及代谢调控基因工程等方面展开论述,重点介绍了次生代谢物的生物合成途径,以及利用基因工程等技术对植物次生代谢途径进行遗传改良等方面的研究进展,为全面认识植物代谢网络、合理定位次生代谢及其关键酶、促进野生植物资源可持续利用等提供理论依据。     

Metabolic Flux Analysis using <Superscript>13</Superscript>C Isotopes: III. Significance for Systems Biology and Metabolic Engineering

At present, ¹³C-MFA is a primary method for quantitatively characterizing intracellular carbon fluxes in cells in vivo under steady-state conditions. The method has been successfully used to investigate both the fundamental characteristics of prokaryotic and eukaryotic cell metabolism and to improve producer strains for more than twenty years. This publication is the last in a set of reviews that describe various aspects of the method. Here, the authors highlight recent achievements that involved using ¹³C-MFA to elucidate bacterial metabolism. Analyses of well-characterized bacterial model strains revealed that central metabolism robustness is provided by a set of alternative metabolic pathways; these analyses also helped develop a better understanding of the physiological significance of these pathways and identified previously unknown functions of well-studied metabolic pathways. Several examples of ¹³C-MFA-based fundamental investigations of poorly characterized bacteria are also analyzed. In applied investigations, flux analysis of strains that produce amino acids, vitamins and antibiotics indicated targets for modifications, suggested unconventional metabolic engineering approaches, and, most importantly, confirmed their utility. In the last section of this article, ¹³C-MFA prospects, including the monitoring of the dynamics of metabolic flux distribution during culture growth, are discussed.     

燃料乙醇的代谢工程研究进展

乙醇是来自可再生资源的最有发展前景的液态燃料,目前采用生物发酵法生产乙醇仍然是最重要的途径。利用代谢工程技术改造乙醇代谢网络、提高乙醇产量是生物工程科学家的研究重点。从扩展代谢途径和构建新的代谢途径等方面全面阐述了代谢工程技术在燃料乙醇生产中的应用。     

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.     

Rethinking glycolysis: on the biochemical logic of metabolic pathways

Metabolic pathways may seem arbitrary and unnecessarily complex. In many cases, a chemist might devise a simpler route for the biochemical transformation, so why has nature chosen such complex solutions? In this review, we distill lessons from a century of metabolic research and introduce new observations suggesting that the intricate structure of metabolic pathways can be explained by a small set of biochemical principles. Using glycolysis as an example, we demonstrate how three key biochemical constraints--thermodynamic favorability, availability of enzymatic mechanisms and the physicochemical properties of pathway intermediates--eliminate otherwise plausible metabolic strategies. Considering these constraints, glycolysis contains no unnecessary steps and represents one of the very few pathway structures that meet cellular demands. The analysis presented here can be applied to metabolic engineering efforts for the rational design of pathways that produce a desired product while satisfying biochemical constraints.     

Pathway Engineering in Secondary Metabolite-Producing Actinomycetes

Abstract

Actinomycetes represent the microbial group richest in production of variable secondary metabolites. These mostly bioactive molecules are the end products of complex multistep biosynthetic pathways. Recent progress in the molecular genetics and biochemistry of the biosynthetic capacities of actinomycetes enables first attempts to redesign these pathways in a directed fashion. However, in contrast to several examples of designed biochemical improvement of primary metabolic processes in microorganisms, none of the products or strains derived from pathway engineering in actinomycetes discussed herein have reached pilot or production scale. The main reasons for this slow progress are the complicated pathways themselves, their complex regulation during the actinomycete cell cycle, and their uniqueness, as most pathways and products are specific for a strain rather than for a given species or larger taxonomic group. However, the modular use of a minimum of very similar enzymes and their conversion of similar intermediates to form the building blocks for the production of a maximum of divergent end products gives hope for the future application of these genetic models for the redesign of complex pathways for modified or new natural products. Several strategies that can be followed to reach this aim are discussed, mainly for the variable 6-deoxyhexose metabolism as an ubiquitously applicable example.     

Engineering the spatial organization of metabolic enzymes: mimicking nature's synergy

A growing body of evidence indicates that many cellular reactions within metabolic pathways are catalyzed not by free-floating 'soluble' enzymes, but via one or more membrane-associated multienzyme complexes. This type of macromolecular organization has important implications for the overall efficiency, specificity, and regulation of metabolic pathways. An ever-increasing number of biochemical and genetic studies on primary and secondary metabolism have laid a solid foundation for this model, providing compelling evidence in favor of the so-called channeling of intermediates between enzyme active sites and colocalization of enzymes inside a cell. In this review, we discuss several of nature's most notable multifunctional enzyme systems including the AROM complex and tryptophan synthase, each of which provides new fundamental insights into the structural organization of metabolic machinery within living cells. We then focus on the growing body of literature related to engineering strategies using protein chimeras and post-translational assembly mechanisms. Common among these techniques is the desire to mimic natural enzyme organization for optimizing the production of valuable metabolites with industrial and medical importance.     

Metabolic engineering

Metabolic engineering has developed as a very powerful approach to optimising industrial fermentation processes through the introduction of directed genetic changes using recombinant DNA technology. Successful metabolic engineering starts with a careful analysis of cellular function; based on the results of this analysis, an improved strain is designed and subsequently constructed by genetic engineering. In recent years some very powerful tools have been developed, both for analysing cellular function and for introducing directed genetic changes. In this paper, some of these tools are reviewed and many examples of metabolic engineering are presented to illustrate the power of the technology. The examples are categorised according to the approach taken or the aim: (1) heterologous protein production, (2) extension of substrate range, (3) pathways leading to new products, (4) pathways for degradation of xenobiotics, (5) improvement of overall cellular physiology, (6) elimination or reduction of by-product formation, and (7) improvement of yield or productivity.     

Nutrimetabolomics: integrating metabolomics in nutrition to disentangle intake of animal-based foods

Food intake and metabolization of foods is a complex and multi-facetted process that encompasses the introduction of new metabolite compounds in our body, initiation or alterations in endogenous metabolic processes and biochemical pathways, and likely also involving the activity of the gut microbial community that we host. The explorative nature of metabolomics makes it a superior tool for examining the whole response to food intake in a more thorough way and has led to the introduction of the term nutrimetabolomics. Protein derived from animal sources constitutes an important part of our diet, and there is therefore an interest in understanding how these animal-derived dietary sources influence us metabolically. This review aims to illuminate how the introduction of nutrimetabolomics has contributed to gain novel insight into metabolic and nutritional aspects related to intake of animal-based foods.     

Genetic engineering of polyamine and carbohydrate metabolism for osmotic stress tolerance in higher plants

Plant growth and productivity are greatly affected by various stress factors. The molecular mechanisms of stress tolerance in plant species have been well established. Metabolic pathways involving the synthesis of metabolites such as polyamines, carbohydrates, proline and glycine betaine have been shown to be associated with stress tolerance. Introduction of the stress-induced genes involved in these pathways from tolerant species to sensitive plants seems to be a promising approach to confer stress tolerance in plants. In cases where single gene is not enough to confer tolerance, metabolic engineering necessitates the introduction of multiple transgenes in plants.     

Evolution of the C(4) photosynthetic mechanism: are there really three C(4) acid decarboxylation types?

Some of the most productive plants on the planet use a variant of photosynthesis known as the C(4) pathway. This photosynthetic mechanism uses a biochemical pump to concentrate CO(2) to levels up to 10-fold atmospheric in specialized cells of the leaf where Rubisco, the primary enzyme of C(3) photosynthesis, is located. The basic biochemical pathways underlying this process, discovered more than 40 years ago, have been extensively studied and, based on these pathways, C(4) plants have been subdivided into two broad groups according to the species of C(4) acid produced in the mesophyll cells and into three groups according to the enzyme used to decarboxylate C(4) acids in the bundle sheath to release CO(2). Recent molecular, biochemical, and physiological data indicate that these three decarboxylation types may not be rigidly genetically determined, that the possibility of flexibility between the pathways exists and that this may potentially be both developmentally and environmentally controlled. This evidence is synthesized here and the implications for C(4) engineering discussed.     

PTEN and SHIP2 phosphoinositide phosphatases as negative regulators of insulin signalling

Insulin resistance in peripheral tissues is the primary cause responsible for onset of type II diabetes mellitus. Recently, the genetic and biochemical dissection of intracellular signalling pathways transducing the metabolic and mitogenic effects of insulin has contributed to the understanding of the molecular causes of this insulin resistance. In particular, important efforts have been developed to comprehend the role of negative regulators of insulin signalling, since they might represent future therapeutical targets to reduce insulin resistance in peripheral tissues. Herein, we will briefly review major intracellular signalling pathways activated by insulin and how they are negatively regulated by distinct mechanisms. In particular, the role of PTEN and SHIP2, two phosphoinositide phosphatases recently implicated as negative modulators of insulin signalling, is in focus. Current knowledge on the role of PTEN and SHIP2 in insulin resistance, type II diabetes and related disorders will also be discussed.     

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.