Yeast Metabolic Pathways Research Articles

Modulation of Aromatic Amino Acid Metabolism by Indigenous Non-Saccharomyces Yeasts in Croatian Maraština Wines.

This study aimed to provide novel information on the impact of indigenous non-Saccharomyces yeasts, including Metschnikowia chrysoperlae, Metschnikowia sinensis/shanxiensis, Metschnikowia pulcherrima, Lachancea thermotolerans, Hanseniaspora uvarum, Hanseniaspora guilliermondii, and Pichia kluyveri, on metabolites related to the metabolism of tryptophan, phenylalanine, and tyrosine. The experiment included two fermentation practices: monoculture and sequential fermentation with commercial Saccharomyces cerevisiae, using sterile Maraština grape juice. A targeted approach through ultrahigh-resolution liquid chromatography associated with mass spectrometry was used to quantify 38 metabolites. All the indigenous yeasts demonstrated better consumption of tryptophan in monoculture than in interaction with S. cerevisiae. M. sinensis/shanxiensis was the only producer of indole-3-carboxylic acid, while its ethyl ester was detected in monoculture fermentation with H. guilliermondii. H. guilliermondii consumed the most phenylalanine among the other isolates. 5-hydroxy-L-tryptophan was detected in fermentations with M. pulcherrima and M. sinensis/shanxiensis. M. pulcherrima significantly increased tryptophol content and utilised tyrosine in monoculture fermentations. Sequential fermentation with M. sinensis/shanxiensis and S. cerevisiae produced higher amounts of N-acetyl derivatives of tryptophan and phenylalanine, while H. guilliermondii-S. cerevisiae fermentation resulted in wines with the highest concentrations of L-kynurenine and 3-hydroxyanthranilic acid. P. kluyveri produced the highest concentration of N-acetyl-L-tyrosine in monoculture fermentations. These findings highlight the different yeast metabolic pathways.

New regulatory role of Znf1 in transcriptional control of pentose phosphate pathway and ATP synthesis for enhanced isobutanol and acid tolerance.

To develop a cost-effective microbial cell factory for the production of biofuels and biochemicals, an understanding of tolerant mechanisms is vital for the construction of robust host strains. Here, we characterized a new function of a key metabolic transcription factor named Znf1 and its involvement in stress response in Saccharomyces cerevisiae to enhance tolerance to advanced biofuel, isobutanol. RNA-sequencing analysis of the wild-type versus the znf1Δ deletion strains in glucose revealed a new role for transcription factor Znf1 in the pentose phosphate pathway (PPP) and energy generation. The gene expression analysis confirmed that isobutanol induces an adaptive cell response, resulting in activation of ATP1-3 and COX6 expression. These genes were Znf1 targets that belong to the electron transport chain, important to produce ATPs. Znf1 also activated PPP genes, required for the generation of key amino acids, cellular metabolites, and maintenance of NADP/NADPH redox balance. In glucose, Znf1 also mediated the upregulation of valine biosynthetic genes of the Ehrlich pathway, namely ILV3, ILV5, and ARO10, associated with the generation of key intermediates for isobutanol production.Using S. cerevisiae knockout collection strains, cells with deleted transcriptional regulatory gene ZNF1 or its targets displayed hypersensitivity to isobutanol and acid inhibitors; in contrast, overexpression of ZNF1 enhanced cell survival. Thus, the transcription factor Znf1 functions in the maintenance of energy homeostasis and redox balance at various checkpoints of yeast metabolic pathways. It ensures the rapid unwiring of gene transcription in response to toxic products/by-products generated during biofuel production. Importantly, we provide a new approach to enhance strain tolerance during the conversion of glucose to biofuels.

Electrostatic Fermentation: Molecular Response Insights for Tailored Beer Production.

Electrostatic fermentation avoids the cellular redox imbalance of traditional fermentation, but knowledge gaps exist. This study explores the impact of electrostatic fermentation on the growth, volatile profile, and genetic response of Saccharomyces pastorianus Saflager S-23. The applied voltage (15 and 30 V) in the electrostatic fermentation system increased the growth and substrate utilization of S. pastorianus while decreasing ethanol production. The aromas typically associated with traditional fermentation, such as alcoholic, grape, apple, and sweet notes, were diminished, while aromas like roses, fruits, flowers, and bananas were augmented in electrostatic fermentation. RNA-seq analysis revealed upregulation of genes involved in cell wall structure, oxidoreductase activity, and iron ion binding, while genes associated with protein synthesis, growth control, homeostasis, and membrane function were downregulated under the influence of applied voltage. The electrostatic fermentation system modulates genetic responses and metabolic pathways in yeast, rendering it a promising method for tailored beer production. Demonstrating feasibility under industrial-scale and realistic conditions is crucial for advancing towards commercialization.

Artificial Self-assembling Nanocompartment for Organizing Metabolic Pathways in Yeast.

Metabolic pathways are commonly organized by sequestration into discrete cellular compartments. Compartments prevent unfavorable interactions with other pathways and provide local environments conducive to the activity of encapsulated enzymes. Such compartments are also useful synthetic biology tools for examining enzyme/pathway behavior and for metabolic engineering. Here, we expand the intracellular compartmentalization toolbox for budding yeast (Saccharomyces cerevisiae) with Murine polyomavirus virus-like particles (MPyV VLPs). The MPyV system has two components: VP1 which self-assembles into the compartment shell and a short anchor, VP2C, which mediates cargo protein encapsulation via binding to the inner surface of the VP1 shell. Destabilized green fluorescent protein (GFP) fused to VP2C was specifically sorted into VLPs and thereby protected from host-mediated degradation. An engineered VP1 variant displayed improved cargo capture properties and differential subcellular localization compared to wild-type VP1. To demonstrate their ability to function as a metabolic compartment, MPyV VLPs were used to encapsulate myo-inositol oxygenase (MIOX), an unstable and rate-limiting enzyme in d-glucaric acid biosynthesis. Strains with encapsulated MIOX produced ∼20% more d-glucaric acid compared to controls expressing “free” MIOX—despite accumulating dramatically less expressed protein—and also grew to higher cell densities. This is the first demonstration in yeast of an artificial biocatalytic compartment that can participate in a metabolic pathway and establishes the MPyV platform as a promising synthetic biology tool for yeast engineering.

Vitamins in wine: Which, what for, and how much?

Vitamins are essential compounds to yeasts, and notably in winemaking contexts. Vitamins are involved in numerous yeast metabolic pathways, including those of amino acids, fatty acids, and alcohols, which suggests their notable implication in fermentation courses, as well as in the development of aromatic compounds in wines. Although they are major components in the course of those microbial processes, their significance and impact have not been extensively studied in the context of winemaking and wine products, as most of the studies focusing on the subject in the past decades have relied on relatively insensitive and imprecise analytical methods. Therefore, this review provides an extensive overview of the current knowledge regarding the impacts of vitamins on grape must fermentations, wine-related yeast metabolisms, and requirements, as well as on the profile of wine sensory characteristics. We also highlight the methodologies and techniques developed over time to perform vitamin analysis in wines, and assess the importance of precisely defining the role played by vitamins in winemaking processes, to ensure finer control of the fermentation courses and product characteristics in a highly complex matrix.

The yeast platform engineered for synthetic gRNA-landing pads enables multiple gene integrations by a single gRNA/Cas9 system

Saccharomyces cerevisiae is a versatile microbial platform to build synthetic metabolic pathways for production of diverse chemicals. To expedite the construction of complex metabolic pathways by multiplex CRISPR-Cas9 genome-edit, eight desirable intergenic loci, located adjacent to highly expressed genes selected from top 100 expressers, were identified and fully characterized for three criteria after integrating green fluorescent protein (GFP) gene - CRISPR-mediated GFP integration efficiency, expression competency assessed by levels of GFP fluorescence, and assessing growth rates of GFP integrated strains. Five best performing intergenic loci were selected to build a multiplex CRISPR platform, and a synthetic 23-bp DNA comprised of 20-bp synthetic DNA with a protospacer adjacent motif (PAM) was integrated into the five loci using CRISPR-Cas9 in a sequential manner. This process resulted in five different yeast strains harbouring 1–5 synthetic gRNA-binding sites in their genomes. Using these pre-engineered yeast strains, simultaneous integrations of 2-, 3-, 4-, or 5-genes to the targeted loci were demonstrated with efficiencies from 85% to 98% using beet pigment betalain (3-gene pathway), hygromycin and geneticin resistance markers. Integrations of the multiple, foreign genes in the targeted loci with 100% precision were validated by genotyping. Finally, we further developed the strain to have 6th synthetic gRNA-binding site, and the resulting yeast strain was used to generate a yeast strain producing a sesquiterpene lactone, kauniolide by simultaneous 6-gene integrations. This study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in yeast.

Chinese Hamster Cells in Biotechnological and Gerontological Research

One of the most frequently used model objects in gerontology is yeast, primarily Saccharomyces cerevisiae. Ample data indicating that the lesions in yeast undergoing chronological or stationary phase aging are similar to the age-related lesions in metazoan cells have been accumulated. However, yeast, similarly to any other study objects, also has drawbacks; in particular, although yeast cells are eukaryotes, they are evolutionarily far from mammals. This imposes limitations on the studies of non-conserved metabolic pathways in yeast. In some cases, mammalian cells (for example, Chinese hamster cells) are more suitable for chronological model experiments. They are widely used in industry for manufacturing monoclonal antibodies and recombinant proteins. A significant proportion of these products are produced after cessation of proliferation which initiates chronological aging of the culture. The accumulated data on the features of cell metabolism, as well as growth and duration of the functional activity of the cell culture, are extremely valuable for gerontologists. The exchange of information between these two branches—biotechnological and gerontological—will be beneficial to both of them.

Effects of nitrogen supplementation on Saccharomyces cerevisiae JP14 fermentation for mead production

Abstract Honey must supplementation is necessary for mead production due to the deficiency in nitrogen materials in this feedstock, despite its high fermentative sugar content. The nitrogen limitation can halt or slow fermentation and lead to the production of unpleasant sensorial compounds, such as sulfur derivatives. The yeast JP14, a Saccharomyces cerevisiae isolated from Jatai bee’s pollen, was inoculated in 25 °Brix honey must with – 0 (control); 0,3; 0,7 and 1,0 g∙L-1 – of di-ammonium phosphate (DAP) and ammonium sulfate (AS). The addition of both supplements resulted in increased cell viability in the first 5 days of fermentation at 20 °C, but did not affect the final acidity of the produced meads. Supplementation also leads to increased sugar consumption, and sugar conversion into ethanol increased as nitrogen supplementation increased, especially with DAP. This indicates that these compounds also regulate yeast metabolic pathways. Supplementary nitrogen acts both in protein anabolism and the gene expression of glycolytic and fermentative pathway components, favoring, in this case, sugar conversion into ethanol. This is the first work describing how different DAP and AS concentrations influences mead production and showing the comparison between these two supplements.

Assembly and Multiplex Genome Integration of Metabolic Pathways in Yeast Using CasEMBLR.

Genome integration is a vital step for implementing large biochemical pathways to build a stable microbial cell factory. Although traditional strain construction strategies are well established for the model organism Saccharomyces cerevisiae, recent advances in CRISPR/Cas9-mediated genome engineering allow much higher throughput and robustness in terms of strain construction. In this chapter, we describe CasEMBLR, a highly efficient and marker-free genome engineering method for one-step integration of in vivo assembled expression cassettes in multiple genomic sites simultaneously. CasEMBLR capitalizes on the CRISPR/Cas9 technology to generate double-strand breaks in genomic loci, thus prompting native homologous recombination (HR) machinery to integrate exogenously derived homology templates. As proof-of-principle for microbial cell factory development, CasEMBLR was used for one-step assembly and marker-free integration of the carotenoid pathway from 15 exogenously supplied DNA parts into three targeted genomic loci. As a second proof-of-principle, a total of ten DNA parts were assembled and integrated in two genomic loci to construct a tyrosine production strain, and at the same time knocking out two genes. This new method complements and improves the field of genome engineering in S. cerevisiae by providing a more flexible platform for rapid and precise strain building.

Promotion of artemisinin content in Artemisia annua by overexpression of multiple artemisinin biosynthetic pathway genes

Artemisinin, isolated from an annual herbaceous plant Artemisia annua L., is an effective antimalarial compound. However, artemisinin is accumulated in small amounts (0.01–0.1% leaf dry weight) in A. annua, resulting in constant high artemisinin price. Although metabolic engineering of partial artemisinin metabolic pathway in yeast achieved great success, artemisinin from A. annua is still the important business resource. Here, we report on the generation of transgenic plants with simultaneously overexpressing four artemisinin biosynthetic pathway genes, amorpha-4,11-diene synthase gene (ADS), amorpha-4,11-diene 12-monooxygenase gene (CYP71AV1), cytochrome P450 reductase gene (CPR), and aldehyde dehydrogenase 1 gene (ALDH1) via Agrobacterium-mediated transformation. The qRT-PCR analysis demonstrated that the introduced four genes of the transgenic lines were all highly expressed. Through high-performance liquid chromatography analysis, the artemisinin contents were increased markedly in transformants, with the highest being 3.4-fold higher compared with non-converter. These results indicate that overexpression of multiple artemisinin biosynthetic pathway genes is a promising approach to improve artemisinin yield in A. annua.

CRISPR-Cas RNA Scaffolds for Transcriptional Programming in Yeast.

CRISPR scaffold RNAs (scRNAs) provide a modular system for locus-specific transcriptional programming. scRNAs are generated by extending CRISPR guide RNA sequences with domains that recruit RNA-binding proteins, thus physically linking DNA binding and protein recruitment activities. A single scRNA molecule encodes information about the target locus and instructions about what regulatory function to execute at that locus. Sets of scRNA constructs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. Such programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point, and this approach has been recently applied to flexibly redirect flux through a complex branched metabolic pathway in yeast. This protocol describes how to construct multi-scRNA transcriptional programs in yeast, including target site selection, cloning strategies, and yeast engineering.

Flavour-active volatile compounds in beer: production, regulation and control

Many classes of compounds have been shown to play an important role in the development of flavour characteristics of beer. This significantly influences its taste and sensory properties, thus affecting its market performance. Despite the intensive research aimed at unravelling the precise mechanism and regulation of flavour formation in beer, current knowledge remains far from complete. Several reviews are available on the general composition of beer and yeast metabolic pathways involved in the production of volatile compounds in beer. However, a limited amount of work has been reported concerning the influence of some of the beer constituents and other important parameters on beer flavour. This paper reviews the current knowledge of the biochemistry behind flavour formation in beer and discusses the different factors that influence its formation and how it can be controlled during brewery fermentation. Copyright © 2017 The Institute of Brewing & Distilling

Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae.

Efficient microbial utilization of cellulosic sugars is essential for the economic production of biofuels and chemicals. Although the yeast Saccharomyces cerevisiae is a robust microbial platform widely used in ethanol plants using sugar cane and corn starch in large-scale operations, glucose repression is one of the significant barriers to the efficient fermentation of cellulosic sugar mixtures. A recent study demonstrated that intracellular utilization of cellobiose by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular β-glucosidase (encoded by gh1-1) can alleviate glucose repression, resulting in the simultaneous cofermentation of cellobiose and nonglucose sugars. Here we report enhanced cellobiose fermentation by engineered yeast expressing cdt-1 and gh1-1 through laboratory evolution. When cdt-1 and gh1-1 were integrated into the genome of yeast, the single copy integrant showed a low cellobiose consumption rate. However, cellobiose fermentation rates by engineered yeast increased gradually during serial subcultures on cellobiose. Finally, an evolved strain exhibited a 15-fold-higher cellobiose fermentation rate. To identify the responsible mutations in the evolved strain, genome sequencing was performed. Interestingly, no mutations affecting cellobiose fermentation were identified, but the evolved strain contained 9 copies of cdt-1 and 23 copies of gh1-1 We also traced the copy numbers of cdt-1 and gh1-1 of mixed populations during the serial subcultures. The copy numbers of cdt-1 and gh1-1 in the cultures increased gradually with similar ratios as cellobiose fermentation rates of the cultures increased. These results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step in engineered yeast and copies of genes coding for metabolic enzymes might be amplified in yeast if there is a growth advantage. This study indicates that on-demand gene amplification might be an efficient strategy for yeast metabolic engineering. In order to enable rapid and efficient fermentation of cellulosic hydrolysates by engineered yeast, we delve into the limiting factors of cellobiose fermentation by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular β-glucosidase (encoded by gh1-1). Through laboratory evolution, we isolated mutant strains capable of fermenting cellobiose much faster than a parental strain. Genome sequencing of the fast cellobiose-fermenting mutant reveals that there are massive amplifications of cdt-1 and gh1-1 in the yeast genome. We also found positive and quantitative relationships between the rates of cellobiose consumption and the copy numbers of cdt-1 and gh1-1 in the evolved strains. Our results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step for efficient cellobiose fermentation. We demonstrate the feasibility of optimizing not only heterologous metabolic pathways in yeast through laboratory evolution but also on-demand gene amplification in yeast, which can be broadly applicable for metabolic engineering.

Proteome-wide quantitative multiplexed profiling of protein expression: carbon-source dependency in Saccharomyces cerevisiae.

The global proteomic alterations in the budding yeast Saccharomyces cerevisiae due to differences in carbon sources can be comprehensively examined using mass spectrometry-based multiplexing strategies. In this study, we investigate changes in the S. cerevisiae proteome resulting from cultures grown in minimal media using galactose, glucose, or raffinose as the carbon source. We used a tandem mass tag 9-plex strategy to determine alterations in relative protein abundance due to a particular carbon source, in triplicate, thereby permitting subsequent statistical analyses. We quantified more than 4700 proteins across all nine samples; 1003 proteins demonstrated statistically significant differences in abundance in at least one condition. The majority of altered proteins were classified as functioning in metabolic processes and as having cellular origins of plasma membrane and mitochondria. In contrast, proteins remaining relatively unchanged in abundance included those having nucleic acid-related processes, such as transcription and RNA processing. In addition, the comprehensiveness of the data set enabled the analysis of subsets of functionally related proteins, such as phosphatases, kinases, and transcription factors. As a resource, these data can be mined further in efforts to understand better the roles of carbon source fermentation in yeast metabolic pathways and the alterations observed therein, potentially for industrial applications, such as biofuel feedstock production.

Metabolomic Measurements at Three Time Points of a Chardonnay Wine Fermentation withSaccharomyces cerevisiae

The transformation of grape juice to wine is a complex metabolic relationship between two species, the grape plant Vitis vinifera and yeast, primarily Saccharomyces cerevisiae . The final molecular composition resulting from the grape–yeast relationship contributes to the flavor, aroma, and mouthfeel of the wine. In this study, we examined this complex relationship by determining the exo- and endo-metabolome (the collection of metabolites present extra- and intracellularly, respectively) of yeast at three time points (days 4, 9, and 15) of a Chardonnay wine fermentation. We identified and tracked 227 metabolites in the exo-metabolome and 404 metabolites in the endo-metabolome, and each metabolite was grouped into metabolic pathways or into metabolite families. Considerable metabolic variation was present at each stage of the fermentation, illuminating metabolic patterns suggesting that regulation of the yeast metabolic pathways is coupled to the fermentation progress. Analysis of the differential utilization and production of primary and secondary metabolites during a wine fermentation in this work provides a key understanding of cell-communication mechanisms relevant to metabolic engineering and industrial biotechnological processes.

Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds

Eukaryotic cells execute complex transcriptional programs in which specific loci throughout the genome are regulated in distinct ways by targeted regulatory assemblies. We have applied this principle to generate synthetic CRISPR-based transcriptional programs in yeast and human cells. By extending guide RNAs to include effector protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. We apply this approach to flexibly redirect flux through a complex branched metabolic pathway in yeast. Moreover, these programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point. CRISPR-associated RNA scaffolds provide a powerful way to construct synthetic gene expression programs for a wide range of applications, including rewiring cell fates or engineering metabolic pathways.

Metabolic engineering of Saccharomyces cerevisiae for the production of triacetic acid lactone

Biobased chemicals have become attractive replacements for their fossil-fuel counterparts. Recent studies have shown triacetic acid lactone (TAL) to be a promising candidate, capable of undergoing chemical conversion to sorbic acid and other valuable intermediates. In this study, Saccharomyces cerevisiae was engineered for the high-level production of TAL by overexpression of the Gerbera hybrida 2-pyrone synthase (2-PS) and systematic engineering of the yeast metabolic pathways. Pathway analysis and a computational approach were employed to target increases in cofactor and precursor pools to improve TAL synthesis. The pathways engineered include those for energy storage and generation, pentose biosynthesis, gluconeogenesis, lipid biosynthesis and regulation, cofactor transport, and fermentative capacity. Seventeen genes were selected for disruption and independently screened for their effect on TAL production; combinations of knockouts were then evaluated. A combination of the pathway engineering and optimal culture parameters led to a 37-fold increase in titer to 2.2g/L and a 50-fold increase in yield to 0.13 (g/g glucose). These values are the highest reported in the literature, and provide a 3-fold improvement in yield over previous reports using S. cerevisiae. Identification of these metabolic bottlenecks provides a strategy for overproduction of other acetyl-CoA-dependent products in yeast.

SIRT1 – An Anti-Inflammatory Pathway at the Crossroads Between Metabolic Disease and Atherosclerosis

Atherosclerosis is a chronic inflammatory disease that is based on the interaction between inflammatory cell subsets and specific cells in the arterial wall. SIRT1 deacetylates histone and non-histone proteins and has been implicated in protective effects of caloric restriction on lifespan and metabolic pathways in yeast, nematodes, and mice. In the vasculature of rodents, SIRT1 mediates vasodilatation through the release of endothelial nitric oxide synthase-derived nitric oxide and scavenges reactive oxygen species. Using a genetic loss-of-function approach, SIRT1 has been shown to interfere with crucial steps of endothelial activation and atherogenesis by suppressing NFκB signaling: Partial SIRT1 deletion in ApoE-/- mice prevented expression of endothelial adhesion molecules thereby hampering the extravasation of circulating monocytes. In monocyte-derived macrophages SIRT1 deletion reduced the expression of the scavenger receptor lectin-like oxidized low-density lipoprotein receptor 1 (Lox-1) resulting in reduced foam cell formation and atherosclerosis. Moreover, it was reported that SIRT1 regulates the activity of liver X-receptor, thereby promoting ABCA1-driven reverse cholesterol transport in plaque-resident macrophages slowing foam cell formation. Finally, SIRT1 suppressed the expression of endothelial tissue factor, and thus exerted anti-thrombotic properties during induced carotid thrombosis in mice. These findings indicate protective effects of SIRT1 in atherogenesis and thrombosis at an experimental level and highlight the opportunity to translate this concept from bench to bedside. Indeed, SIRT1 activators are available and have been shown to exert beneficial effects at the preclinical level in obesity and type 2 diabetes mellitus (T2DM). SIRT1 activators are currently being evaluated in phase II clinical trials in patients with T2DM. The concept of SIRT1 activation appears a promising strategy for novel therapeutic approaches in patients with atherothrombosis.

Metabolic engineering of Saccharomyces cerevisiae for alkaloid production

Natural products or natural‐product inspired molecules comprise most successful drugs. However, these high‐value molecules are difficult to synthesize chemically and are often isolated through inefficient extractions from natural 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. Yet many challenges are currently faced in engineering yeast strains for the biosynthesis of complex secondary metabolite production. Synthetic biology is advancing the development of new tools for constructing, controlling, and optimizing complex metabolic pathways in yeast.I will describe recent work in engineering yeast as a production host for diverse alkaloid compounds, and in particular the benzylisoquinoline alkaloids (BIAs). Yeast strains engineered to express enzymes from diverse organisms, including plants, bacteria, and humans, have been demonstrated to synthesize numerous intermediates and end‐products in this biosynthetic pathway. In addition, I will describe the development of synthetic biology platforms that are providing new approaches to optimizing complex biosynthetic pathways.

Regulatory architecture determines optimal regulation of gene expression in metabolic pathways

In response to environmental changes, the connections ("arrows") in gene regulatory networks determine which genes modulate their expression, but the quantitative parameters of the network ("the numbers on the arrows") are equally important in determining the resulting phenotype. What are the objectives and constraints by which evolution determines these parameters? We explore these issues by analyzing gene expression changes in a number of yeast metabolic pathways in response to nutrient depletion. We find that a striking pattern emerges that couples the regulatory architecture of the pathway to the gene expression response. In particular, we find that pathways controlled by the intermediate metabolite activation (IMA) architecture, in which an intermediate metabolite activates transcription of pathway genes, exhibit the following response: the enzyme immediately downstream of the regulatory metabolite is under the strongest transcriptional control, whereas the induction of the enzymes upstream of the regulatory intermediate is relatively weak. This pattern of responses is absent in pathways not controlled by an IMA architecture. The observation can be explained by the constraint imposed by the fundamental feedback structure of the network, which places downstream enzymes under a negative feedback loop and upstream ones under a positive feedback loop. This general design principle for transcriptional control of a metabolic pathway can be derived from a simple cost/benefit model of gene expression, in which the observed pattern is an optimal solution. Our results suggest that the parameters regulating metabolic enzyme expression are optimized by evolution, under the strong constraint of the underlying regulatory architecture.