Reconstruction of methanol and formate metabolic pathway in non-native host for biosynthesis of chemicals and biofuels

One-carbon feedstock such as methanol and formate has attracted much attention as carbon substrate of industrial biotechnology for production of value-added chemicals and biofuels. Productivity improvement of natural one-carbon metabolic pathways in native hosts such as methanotrophs is somewhat difficult due to inefficient genetic tools and low specific growth rate. As an alternative, metabolic engineering can create new and efficient metabolic pathways of one-carbon substrate that can be readily transferred to non-native hosts. In this paper, recent progresses in protein and metabolic engineering for creation of methanol and formate-utilizing synthetic pathways based on RuMP cycle and formolase are reviewed. Perspectives on one-carbon metabolic pathway engineering in non-native host are also discussed.     

Systems biology based metabolic engineering for non-natural chemicals

Production of chemicals in microorganisms is no longer restricted to products arising from native metabolic potential. In this review, we highlight the evolution of metabolic engineering studies, from the production of natural chemicals fermented from biomass hydrolysates, to the engineering of microorganisms for the production of non-natural chemicals. Advances in synthetic biology are accelerating the successful development of microbial cell factories to directly produce value-added chemicals. Here we outline the emergence of novel computational tools for the creation of synthetic pathways, for designing artificial enzymes for non-natural reactions and for re-wiring host metabolism to increase the metabolic flux to products. We also highlight exciting opportunities for applying directed evolution of enzymes, dynamic control of growth and production, growth-coupling strategies as well as decoupled strategies based on orthogonal pathways in the context of non-natural chemicals.     

生物固碳途径研究进展

生物固碳是地球碳循环过程的重要组成部分。自然界已经发现了六条天然生物固碳途径,但自然途径不仅能量利用效率低下,而且人工改造提升固碳效率难度大。随着合成生物学的发展,新的人工固碳途径不断涌现。相对于天然途径,人工固碳途径具有路线短、耗能少、原子经济性高等优点,有望在不久的将来能够替代天然固碳途径,实现固碳效率的大幅提高,是解决人类能源与环境问题的有效途径之一。主要总结了天然固碳途径和人工固碳途径的代谢原理和关键固碳酶的酶学特征,并对未来发展趋势进行展望。     

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.     

Transient expression assays in grapevine: a step towards genetic improvement

In the past few years, the usefulness of transient expression assays has continuously increased for the characterization of unknown gene function and metabolic pathways. In grapevine (Vitis vinifera L.), one of the most economically important fruit crops in the world, recent systematic sequencing projects produced many gene data sets that require detailed analysis. Due to their rapid nature, transient expression assays are well suited for large‐scale genetic studies. Although genes and metabolic pathways of any species can be analysed by transient expression in model plants, a need for homologous systems has emerged to avoid the misinterpretation of results due to a foreign genetic background. Over the last 10 years, various protocols have thus been developed to apply this powerful technology to grapevine. Using cell suspension cultures, somatic embryos, leaves or whole plantlets, transient expression assays enabled the study of the function, regulation and subcellular localization of genes involved in specific metabolic pathways such as the biosynthesis of phenylpropanoids. Disease resistance genes that could be used for marker‐assisted selection in conventional breeding or for stable transformation of elite cultivars have also been characterized. Additionally, transient expression assays have proved useful for shaping new tools for grapevine genetic improvement: synthetic promoters, silencing constructs, minimal linear cassettes or viral vectors. This review provides an update on the different tools (DNA constructs, reporter genes, vectors) and methods (Agrobacterium‐mediated and direct gene transfer methods) available for transient gene expression in grapevine. The most representative results published thus far are then described.     

Biosynthesis and biotechnological application of non-canonical amino acids: Complex and unclear

Compared with the better-studied canonical amino acids, the distribution, metabolism and functions of natural non-canonical amino acids remain relatively obscure. Natural non-canonical amino acids have been mainly discovered in plants as secondary metabolites that perform diversified physiological functions. Due to their specific characteristics, a broader range of natural and artificial non-canonical amino acids have recently been applied in the development of functional materials and pharmaceutical products. With the rapid development of advanced methods in biotechnology, non-canonical amino acids can be incorporated into peptides, proteins and enzymes to improve the function and performance relative to their natural counterparts. Therefore, biotechnological application of non-canonical amino acids in artificial bio-macromolecules follows the central goal of synthetic biology to: create novel life forms and functions. However, many of the non-canonical amino acids are synthesized via chemo- or semi-synthetic methods, and few non-canonical amino acids can be synthesized using natural in vivo pathways. Therefore, further research is needed to clarify the metabolic pathways and key enzymes of the non-canonical amino acids. This will lead to the discovery of more candidate non-canonical amino acids, especially for those that are derived from microorganisms and are naturally bio-compatible with chassis strains for in vivo biosynthesis. In this review, we summarize representative natural and artificial non-canonical amino acids, their known information regarding associated metabolic pathways, their characteristics and their practical applications. Moreover, this review summarizes current barriers in developing in vivo pathways for the synthesis of non-canonical amino acids, as well as other considerations, future trends and potential applications of non-canonical amino acids in advanced biotechnology.     

Metabolomic strategies for the identification of new enzyme functions and metabolic pathways

Recent technological advances in accurate mass spectrometry and data analysis have revolutionized metabolomics experimentation. Activity-based and global metabolomic profiling methods allow simultaneous and rapid screening of hundreds of metabolites from a variety of chemical classes, making them useful tools for the discovery of novel enzymatic activities and metabolic pathways. By using the metabolome of the relevant organism or close species, these methods capitalize on biological relevance, avoiding the assignment of artificial and non-physiological functions. This review discusses state-of-the-art metabolomic approaches and highlights recent examples of their use for enzyme annotation, discovery of new metabolic pathways, and gene assignment of orphan metabolic activities across diverse biological sources.     

The synthetic integron: an in vivo genetic shuffling device

As the field of synthetic biology expands, strategies and tools for the rapid construction of new biochemical pathways will become increasingly valuable. Purely rational design of complex biological pathways is inherently limited by the current state of our knowledge. Selection of optimal arrangements of genetic elements from randomized libraries may well be a useful approach for successful engineering. Here, we propose the construction and optimization of metabolic pathways using the inherent gene shuffling activity of a natural bacterial site-specific recombination system, the integron. As a proof of principle, we constructed and optimized a functional tryptophan biosynthetic operon in Escherichia coli. The trpA-E genes along with ‘regulatory’ elements were delivered as individual recombination cassettes in a synthetic integron platform. Integrase-mediated recombination generated thousands of genetic combinations overnight. We were able to isolate a large number of arrangements displaying varying fitness and tryptophan production capacities. Several assemblages required as many as six recombination events and produced as much as 11-fold more tryptophan than the natural gene order in the same context.     

Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways

Cells are filled with biosensors, molecular systems that measure the state of the cell and respond by regulating host processes. In much the same way that an engineer would monitor a chemical reactor, the cell uses these sensors to monitor changing intracellular environments and produce consistent behavior despite the variable environment. While natural systems derive a clear benefit from pathway regulation, past research efforts in engineering cellular metabolism have focused on introducing new pathways and removing existing pathway regulation. Synthetic biology is a rapidly growing field that focuses on the development of new tools that support the design, construction, and optimization of biological systems. Recent advances have been made in the design of genetically-encoded biosensors and the application of this class of molecular tools for optimizing and regulating heterologous pathways. Biosensors to cellular metabolites can be taken directly from natural systems, engineered from natural sensors, or constructed entirely in vitro. When linked to reporters, such as antibiotic resistance markers, these metabolite sensors can be used to report on pathway productivity, allowing high-throughput screening for pathway optimization. Future directions will focus on the application of biosensors to introduce feedback control into metabolic pathways, providing dynamic control strategies to increase the efficient use of cellular resources and pathway reliability.     

Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for biotechnological production of organic acids and amino acids

Industrial microorganisms have been developed as biocatalysts to provide new or to optimize existing processes for the biotechnological production of chemicals from renewable plant biomass. Rational strain development by metabolic engineering is crucial to successful processes, and is based on efficient genetic tools and detailed knowledge of metabolic pathways and their regulation. This review summarizes recent advances in metabolic engineering of the industrial model bacteria Escherichia coli and Corynebacterium glutamicum that led to efficient recombinant biocatalysts for the production of acetate, pyruvate, ethanol, d- and l-lactate, succinate, l-lysine and l-serine.     

Production of isoprenoid pharmaceuticals by engineered microbes

Throughout human history, natural products have been the foundation for the discovery and development of therapeutics used to treat diseases ranging from cardiovascular disease to cancer. Their chemical diversity and complexity have provided structural scaffolds for small-molecule drugs and have consistently served as inspiration for medicinal design. However, the chemical complexity of natural products also presents one of the main roadblocks for production of these pharmaceuticals on an industrial scale. Chemical synthesis of natural products is often difficult and expensive, and isolation from their natural sources is also typically low yielding. Synthetic biology and metabolic engineering offer an alternative approach that is becoming more accessible as the tools for engineering microbes are further developed. By reconstructing heterologous metabolic pathways in genetically tractable host organisms, complex natural products can be produced from inexpensive sugar starting materials through large-scale fermentation processes. In this Perspective, we discuss ongoing research aimed toward the production of terpenoid natural products in genetically engineered Escherichia coli and Saccharomyces cerevisiae.     

Synthetic regulatory tools for microbial engineering

Microbial engineering requires accurate information about cellular metabolic networks and a set of molecular tools that can be predictably applied to the efficient redesign of such networks. Recent advances in the field of metabolic engineering and synthetic biology, particularly the development of molecular tools for synthetic regulation in the static and dynamic control of gene expression, have increased our ability to efficiently balance the expression of genes in various biological systems. It would accelerate the creation of synthetic pathways and genetic programs capable of adapting to environmental changes in real time to perform the programmed cellular behavior. In this paper, we review current developments in the field of synthetic regulatory tools for static and dynamic control of microbial gene expression.     

Environments that induce synthetic microbial ecosystems

Interactions between microbial species are sometimes mediated by the exchange of small molecules, secreted by one species and metabolized by another. Both one-way (commensal) and two-way (mutualistic) interactions may contribute to complex networks of interdependencies. Understanding these interactions constitutes an open challenge in microbial ecology, with applications ranging from the human microbiome to environmental sustainability. In parallel to natural communities, it is possible to explore interactions in artificial microbial ecosystems, e.g. pairs of genetically engineered mutualistic strains. Here we computationally generate artificial microbial ecosystems without re-engineering the microbes themselves, but rather by predicting their growth on appropriately designed media. We use genome-scale stoichiometric models of metabolism to identify media that can sustain growth for a pair of species, but fail to do so for one or both individual species, thereby inducing putative symbiotic interactions. We first tested our approach on two previously studied mutualistic pairs, and on a pair of highly curated model organisms, showing that our algorithms successfully recapitulate known interactions, robustly predict new ones, and provide novel insight on exchanged molecules. We then applied our method to all possible pairs of seven microbial species, and found that it is always possible to identify putative media that induce commensalism or mutualism. Our analysis also suggests that symbiotic interactions may arise more readily through environmental fluctuations than genetic modifications. We envision that our approach will help generate microbe-microbe interaction maps useful for understanding microbial consortia dynamics and evolution, and for exploring the full potential of natural metabolic pathways for metabolic engineering applications.     

Genetic technologies for extremely thermophilic microorganisms of Sulfolobus,the only genetically tractable genus of crenarchaea

Archaea represents the third domain of life, with the information-processing machineries more closely resembling those of eukaryotes than the machineries of the bacterial counterparts but sharing metabolic pathways with organisms of Bacteria, the sister prokaryotic phylum. Archaeal organisms also possess unique features as revealed by genomics and genome comparisons and by biochemical characterization of prominent enzymes. Nevertheless, diverse genetic tools are required for in vivo experiments to verify these interesting discoveries. Considerable efforts have been devoted to the development of genetic tools for archaea ever since their discovery, and great progress has been made in the creation of archaeal genetic tools in the past decade. Versatile genetic toolboxes are now available for several archaeal models, among which Sulfolobus microorganisms are the only genus representing Crenarchaeota because all the remaining genera are from Euryarchaeota. Nevertheless, genetic tools developed for Sulfolobus are probably the most versatile among all archaeal models, and these include viral and plasmid shuttle vectors, conventional and novel genetic manipulation methods, CRISPR-based gene deletion and mutagenesis, and gene silencing, among which CRISPR tools have been reported only for Sulfolobus thus far. In this review, we summarize recent developments in all these useful genetic tools and discuss their possible application to research into archaeal biology by means of Sulfolobus models.     

Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools

Secondary metabolites are an important source of high-value chemicals, many of which exhibit important pharmacological properties. These valuable natural products are often difficult to synthesize chemically and are commonly isolated through inefficient extractions from natural biological sources. As such, they are increasingly targeted for production by biosynthesis from engineered microorganisms. The budding yeast species Saccharomyces cerevisiae has proven to be a powerful microorganism for heterologous expression of biosynthetic pathways. S. cerevisiae's usefulness as a host organism is owed in large part to the wealth of knowledge accumulated over more than a century of intense scientific study. Yet many challenges are currently faced in engineering yeast strains for the biosynthesis of complex secondary metabolite production. However, synthetic biology is advancing the development of new tools for constructing, controlling, and optimizing complex metabolic pathways in yeast. Here, we review how the coupling between yeast biology and synthetic biology is advancing the use of S. cerevisiae as a microbial host for the construction of secondary metabolic pathways.     

Adaptive evolution of metabolic pathways in Drosophila

The adaptive significance of enzyme variation has been of central interest in population genetics. Yet, how natural selection operates on enzymes in the larger context of biochemical pathways has not been broadly explored. A basic expectation is that natural selection on metabolic phenotypes will target enzymes that control metabolic flux, but how adaptive variation is distributed among enzymes in metabolic networks is poorly understood. Here, we use population genetic methods to identify enzymes responding to adaptive selection in the pathways of central metabolism in Drosophila melanogaster and Drosophila simulans. We report polymorphism and divergence data for 17 genes that encode enzymes of 5 metabolic pathways that converge at glucose-6-phosphate (G6P). Deviations from neutral expectations were observed at five loci. Of the 10 genes that encode the enzymes of glycolysis, only aldolase (Ald) deviated from neutrality. The other 4 genes that were inconsistent with neutral evolution (glucose-6-phosphate dehydrogenase [G6pd]), phosphoglucomutase [Pgm], trehalose-6-phosphate synthetase [Tps1], and glucose-6phosphatase [G6pase] encode G6P branch point enzymes that catalyze reactions at the entry point to the pentose-phosphate, glycogenic, trehalose synthesis, and gluconeogenic pathways. We reconcile these results with population genetics theory and existing arguments on metabolic regulation and propose that the incidence of adaptive selection in this system is related to the distribution of flux control. The data suggest that adaptive evolution of G6P branch point enzymes may have special significance in metabolic adaptation.     

RNA‐Schalter

Riboswitches Synthetic Biology intends to develop novel biological systems such as artificial metabolic pathways. With these, microorganisms are employed to make useful substances such as drugs, biofuels or other chemicals from cheap and renewable resources. Besides genes, regulators are essential parts of genetic circuits. Riboswitches represent a new class of such regulators. They can be developed with customized features using ligand binding RNAs.