Basic and applied uses of genome-scale metabolic network reconstructions of Escherichia coli.

The genome-scale model (GEM) of metabolism in the bacterium Escherichia coli K-12 has been in development for over a decade and is now in wide use. GEM-enabled studies of E. coli have been primarily focused on six applications: (1) metabolic engineering, (2) model-driven discovery, (3) prediction of cellular phenotypes, (4) analysis of biological network properties, (5) studies of evolutionary processes, and (6) models of interspecies interactions. In this review, we provide an overview of these applications along with a critical assessment of their successes and limitations, and a perspective on likely future developments in the field. Taken together, the studies performed over the past decade have established a genome-scale mechanistic understanding of genotype–phenotype relationships in E. coli metabolism that forms the basis for similar efforts for other microbial species. Future challenges include the expansion of GEMs by integrating additional cellular processes beyond metabolism, the identification of key constraints based on emerging data types, and the development of computational methods able to handle such large-scale network models with sufficient accuracy

Evaluating pathway enumeration algorithms in metabolic engineering case studies

The design of cell factories for the production of compounds involves the search for suitable heterologous pathways. Different strategies have been proposed to infer such pathways, but most are optimization approaches with specific objective functions, not suited to enumerate multiple pathways. In this work, we analyze two pathway enumeration algorithms based on graph representations: the Solution Structure Generation and the Find Path algorithms. Both are capable of enumerating exhaustively multiple pathways using network topology. We study their capabilities and limitations when designing novel heterologous pathways, by applying these methods on two case studies of synthetic metabolic engineering related to the production of butanol and vanillin

novoPathFinder: a webserver of designing novel-pathway with integrating GEM-model

To increase the number of value-added chemicals that can be produced by metabolic engineering and synthetic biology, constructing metabolic space with novel reactions/pathways is crucial. However, with the large number of reactions that existed in the metabolic space and complicated metabolisms within hosts, identifying novel pathways linking two molecules or heterologous pathways when engineering a host to produce a target molecule is an arduous task. Hence, we built a user-friendly web server, novoPathFinder, which has several features: (i) enumerate novel pathways between two specified molecules without considering hosts; (ii) construct heterologous pathways with known or putative reactions for producing target molecule within Escherichia coli or yeast without giving precursor; (iii) estimate novel pathways with considering several categories, including enzyme promiscuity, Synthetic Complex Score (SCScore) and LD50 of intermediates, overall stoichiometric conversions, pathway length, theoretical yields and thermodynamic feasibility. According to the results, novoPathFinder is more capable to recover experimentally validated pathways when comparing other rule-based web server tools. Besides, more efficient pathways with novel reactions could also be retrieved for further experimental exploration. novoPathFinder is available at http://design.rxnfinder.org/novopathfinder/

Systems-level approaches for understanding and engineering of the oleaginous cell factory Yarrowia lipolytica

Concerns about climate change and the search for renewable energy sources together with the goal of attaining sustainable product manufacturing have boosted the use of microbial platforms to produce fuels and high-value chemicals. In this regard, Yarrowia lipolytica has been known as a promising yeast with potentials in diverse array of biotechnological applications such as being a host for different oleochemicals, organic acid, and recombinant protein production. Having a rapidly increasing number of molecular and genetic tools available, Y. lipolytica has been well studied amongst oleaginous yeasts and metabolic engineering has been used to explore its potentials. More recently, with the advancement in systems biotechnology and the implementation of mathematical modeling and high throughput omics data-driven approaches, in-depth understanding of cellular mechanisms of cell factories have been made possible resulting in enhanced rational strain design. In case of Y. lipolytica, these systems-level studies and the related cutting-edge technologies have recently been initiated which is expected to result in enabling the biotechnology sector to rationally engineer Y. lipolytica-based cell factories with favorable production metrics. In this regard, here, we highlight the current status of systems metabolic engineering research and assess the potential of this yeast for future cell factory design development

Metabolic engineering of \u3ci\u3eEscherichia coli\u3c/i\u3e for the \u3ci\u3ede novo\u3c/i\u3e stereospecific biosynthesis of 1,2-propanediol through lactic acid

1,2-propanediol (1,2-PDO) is an industrial chemical with a broad range of applications, such as the production of alkyd and unsaturated polyester resins. It is currently produced as a racemic mixture from nonrenewable petroleum-based feedstocks. We have reported a novel artificial pathway for the biosynthesis of 1,2-PDO via lactic acid isomers as the intermediates. The pathway circumvents the cytotoxicity issue caused by methylglyoxal intermediate in the naturally existing pathway. Successful E. coli bioconversion of lactic acid to 1,2-PDO was shown in previous report. Here, we demonstrated the engineering of E. coli host strains for the de novo biosynthesis of 1,2-PDO through this pathway. Under fermenter-controlled conditions, the R-1,2-PDO was produced at 17.3 g/L with a molar yield of 42.2% from glucose, while the S-isomer was produced at 9.3 g/L with a molar yield of 23.2%. The optical purities of the two isomers were 97.5% ee (R) and 99.3% ee (S), respectively. To the best of our knowledge, these are the highest titers of 1,2-PDO biosynthesized by either natural producer or engineered microbial strains that are published in peer-reviewed journals

Improving flavonoid production in Saccharomyces cerevisiae using synthetic biology tools

Flavonoids are plant secondary metabolites and represent one of the largest classes of natural products. Due to their health-beneficial properties, they have found potential applications in foods, beverages, cosmetics, and pharmaceuticals. Currently, their production is based on extraction from plant material. However, the low abundance of flavonoids in nature hinders efficient extraction and purification, thus inhibiting their market expansion. Chemical synthesis, although possible, relies on the use of harmful chemicals, harsh operating conditions, and high energy consumption.Metabolic engineering of microorganisms to develop so-called “microbial cell factories” has gained increasing attention as a more efficient and sustainable way to produce a variety of chemicals – including flavonoids. Saccharomyces cerevisiae (baker’s yeast) is one of the most well-studied and widely applied eukaryotic organisms for this endeavor. The fact that yeast shares cellular similarities to plants makes it a suitable host for the heterologous expression of flavonoid biosynthetic pathways. In this thesis, I present our efforts to improve the production of flavonoids in S. cerevisiae through the development and application of several synthetic biology tools. First, transcription factor-based biosensors for the isoflavonoid genistein and the flavonoid precursor p-coumaroyl-CoA were established. The latter sensor was used to devise a dynamic regulation strategy for the production of naringenin, a central flavanone and precursor for many flavanone derivatives. Cell growth was improved and naringenin titers were increased significantly. Next, a malonate assimilation pathway was implemented in yeast to enhance the supply of malonyl-CoA, an important precursor for all flavonoid compounds. By expressing a heterologous malonate transporter and malonyl-CoA synthetase, I constructed strains able to grow on externally supplied malonate. The malonate transporter was further evolved through targeted in vivo mutagenesis and beneficial mutations were identified through growth-based enrichment under selective conditions. Lastly, the production of the dihydrochalcone phloretin was explored. Its biosynthesis was accompanied by substantial byproduct formation and product degradation in the yeast cultivation medium. Different strategies, including enzyme scaffolding and antioxidant supplementation, were investigated to improve yeast-based production.Taken together, I addressed some significant challenges within microbial flavonoid production and showcased how synthetic biology tools may overcome these obstacles

Investigation of terminal alkene formation by acyl-CoA dehydrogenases in the biosynthesis of complex natural products

Polyketides are a class of natural products known for their chemical complexity and bioactive properties. They are biosynthesized by an assembly line-like system termed polyketide synthases (PKSs), in which each module consists of various enzymatic domains, each of which has a single catalytic function. The polyketide is extended by two carbons and selectively reduced by each module. These biosynthetic principles make PKSs attractive engineering targets, because a simple swap of one enzymatic domain could change the final polyketide structure and effect an improved or novel bioactivity. However, gaping holes in our understanding of polyketide biosynthesis still exist. In particular, the activities of polyketide-associated enzymes remain relatively under-characterized and are generally not the primary focus of engineering efforts. These enzymes, which include glycosyl transferases, cytochrome P450s, and many others, often are responsible for imparting bioactive functional groups into the polyketide backbone or after the assembly of the “naked” polyketide. Many of these non-canonical functional groups discovered within polyketides are therefore the most desirable engineering targets. In this dissertation, I describe my efforts to characterize a family of terminal alkene-forming enzymes which were originally identified in a polyketide biosynthetic cluster. I show that the enzymes are regioselective and that regioselectivity is controlled by a shift in the protein structure. Finally, I show that these enzymes are widespread in not only polyketide biosynthetic gene clusters, but also in other natural product clusters in the genomes of diverse Actinomycetes

Optimizing pentose utilization in yeast: the need for novel tools and approaches

Hexose and pentose cofermentation is regarded as one of the chief obstacles impeding economical conversion of lignocellulosic biomass to biofuels. Over time, successful application of traditional metabolic engineering strategy has produced yeast strains capable of utilizing the pentose sugars (especially xylose and arabinose) as sole carbon sources, yet major difficulties still remain for engineering simultaneous, exogenous sugar metabolism. Beyond catabolic pathways, the focus must shift towards non-traditional aspects of cellular engineering such as host molecular transport capability, catabolite sensing and stress response mechanisms. This review highlights the need for an approach termed 'panmetabolic engineering', a new paradigm for integrating new carbon sources into host metabolic pathways. This approach will concurrently optimize the interdependent processes of transport and metabolism using novel combinatorial techniques and global cellular engineering. As a result, panmetabolic engineering is a whole pathway approach emphasizing better pathways, reduced glucose-induced repression and increased product tolerance. In this paper, recent publications are reviewed in light of this approach and their potential to expand metabolic engineering tools. Collectively, traditional approaches and panmetabolic engineering enable the reprogramming of extant biological complexity and incorporation of exogenous carbon catabolism

Insights into Triterpene Metabolism in Model Monocotyledonous and Oilseed Plants Genetically Engineered with Genes from \u3cem\u3eBotryococcus Braunii\u3c/em\u3e

Isoprenoids are one of the most diverse classes of natural products and are all derived from universal five carbon, prenyl precursors. Squalene and botryococcene are linear, hydrocarbon triterpenes (thirty carbon compounds with six prenyl units) that have industrial and medicinal values. Squalene is produced by all eukaryotes as it is the first committed precursor to sterols, while botryococcene is uniquely produced by the green algae, Botryococcus braunii (race B). Natural sources for these compounds exist, but there is a desire for more renewable production platforms. The model plant Arabidopsis thaliana was engineered to accumulate botryococcene and squalene in its oil seeds using a combination of biosynthetic genes and subcellular targeting strategies. The model monocotyledonous grass, Brachypodium distachyon, was also engineered to accumulate botryococcene in its leaves, testing a variety of subcellular targeting methods. Both oilseeds and grasses have highly desirable characteristics as production platforms, and both can utilize photosynthesis to power the biosynthesis of these valued hydrocarbons. In each of these efforts, we were able to obtain high levels of triterpene accumulation and uncovered new aspects of isoprenoid metabolism and its regulation. Also investigated was a new gene from the green algae, Botryococcus braunii (race B), encoding for a novel methyltransferase, which in combination with a previously reported methyltransferase, was characterized as converting dimethylated squalene to tetramethylated. Tetramethylated derivatives of squalene (and botryococcene) are highly desirable for industrial applications, and the discovery of the genes encoding for this biosynthetic capacity portends opportunities to engineer other heterologous host organisms and create other amenable production platforms