José L. Avalos

Crystal structure of the eukaryotic strong inward-rectifier K+ channel Kir2.2 at 3.1 A resolution.

Bio-Diodes Inward rectifier potassium channels conduct K+ ions into the cell at internal negative membrane voltages, but at internal positive membrane voltages they are blocked by intracellular multivalent ions. These channels control the resting membrane voltage and are required for the healthy function of many electrically excitable cells. Mutations can result in transient paralysis causing, for example, heart problems. Tao et al. (p. 1668) now report a 3.1 angstrom resolution structure of the inward rectifier, Kir2.2 from chicken, which has a similar structure to the human equivalent. The combination of observations of conductive and inhibitory ion binding sites with electrophysiological data finally explains the mechanism of action of these long-studied channels and reveals how they maintain their low sensitivity to toxins, as well as provides a basis for the design of therapeutic drugs. A structure reveals the basis of diode-like conduction properties and toxin insensitivity of these channels. Inward-rectifier potassium (K+) channels conduct K+ ions most efficiently in one direction, into the cell. Kir2 channels control the resting membrane voltage in many electrically excitable cells, and heritable mutations cause periodic paralysis and cardiac arrhythmia. We present the crystal structure of Kir2.2 from chicken, which, excluding the unstructured amino and carboxyl termini, is 90% identical to human Kir2.2. Crystals containing rubidium (Rb+), strontium (Sr2+), and europium (Eu3+) reveal binding sites along the ion conduction pathway that are both conductive and inhibitory. The sites correlate with extensive electrophysiological data and provide a structural basis for understanding rectification. The channel’s extracellular surface, with large structured turrets and an unusual selectivity filter entryway, might explain the relative insensitivity of eukaryotic inward rectifiers to toxins. These same surface features also suggest a possible approach to the development of inhibitory agents specific to each member of the inward-rectifier K+ channel family.

Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols.

Efforts to improve the production of a compound of interest in Saccharomyces cerevisiae have mainly involved engineering or overexpression of cytoplasmic enzymes. We show that targeting metabolic pathways to mitochondria can increase production compared with overexpression of the enzymes involved in the same pathways in the cytoplasm. Compartmentalization of the Ehrlich pathway into mitochondria increased isobutanol production by 260%, whereas overexpression of the same pathway in the cytoplasm only improved yields by 10%, compared with a strain overproducing enzymes involved in only the first three steps of the biosynthetic pathway. Subcellular fractionation of engineered strains revealed that targeting the enzymes of the Ehrlich pathway to the mitochondria achieves greater local enzyme concentrations. Other benefits of compartmentalization may include increased availability of intermediates, removing the need to transport intermediates out of the mitochondrion and reducing the loss of intermediates to competing pathways.

Structure of a Sir2 enzyme bound to an acetylated p53 peptide

Sir2 proteins are NAD(+)-dependent protein deacetylases that play key roles in transcriptional regulation, DNA repair, and life span regulation. The structure of an archaeal Sir2 enzyme, Sir2-Af2, bound to an acetylated p53 peptide reveals that the substrate binds in a cleft in the enzyme, forming an enzyme-substrate beta sheet with two flanking strands in Sir2-Af2. The acetyl-lysine inserts into a conserved hydrophobic tunnel that contains the active site histidine. Comparison with other structures of Sir2 enzymes suggests that the apoenzyme undergoes a conformational change upon substrate binding. Based on the Sir2-Af2 substrate complex structure, mutations were made in the other A. fulgidus sirtuin, Sir2-Af1, that increased its affinity for the p53 peptide.

Harnessing yeast organelles for metabolic engineering

Each subcellular compartment in yeast offers a unique physiochemical environment and metabolite, enzyme, and cofactor composition. While yeast metabolic engineering has focused on assembling pathways in the cell cytosol, there is growing interest in embracing subcellular compartmentalization. Beyond harnessing distinct organelle properties, physical separation of organelles from the cytosol has the potential to eliminate metabolic crosstalk and enhance compartmentalized pathway efficiency. In this Perspective we review the state of the art in yeast subcellular engineering, highlighting the benefits of targeting biosynthetic pathways to subcellular compartments, including mitochondria, peroxisomes, the ER and/or Golgi, vacuoles, and the cell wall, in different yeast species. We compare the performances of strains developed with subcellular engineering to those of native producers or yeast strains previously engineered with cytosolic pathways. We also identify important challenges that lie ahead, which need to be addressed for organelle engineering to become as mainstream as cytosolic engineering in academia and industry.

Optogenetic regulation of engineered cellular metabolism for microbial chemical production

The optimization of engineered metabolic pathways requires careful control over the levels and timing of metabolic enzyme expression. Optogenetic tools are ideal for achieving such precise control, as light can be applied and removed instantly without complex media changes. Here we show that light-controlled transcription can be used to enhance the biosynthesis of valuable products in engineered Saccharomyces cerevisiae. We introduce new optogenetic circuits to shift cells from a light-induced growth phase to a darkness-induced production phase, which allows us to control fermentation with only light. Furthermore, optogenetic control of engineered pathways enables a new mode of bioreactor operation using periodic light pulses to tune enzyme expression during the production phase of fermentation to increase yields. Using these advances, we control the mitochondrial isobutanol pathway to produce up to 8.49 ± 0.31 g l−1 of isobutanol and 2.38 ± 0.06 g l−1 of 2-methyl-1-butanol micro-aerobically from glucose. These results make a compelling case for the application of optogenetics to metabolic engineering for the production of valuable products.

Uncovering the role of branched-chain amino acid transaminases in Saccharomyces cerevisiae isobutanol biosynthesis

Isobutanol and other branched-chain higher alcohols (BCHAs) are promising advanced biofuels derived from the degradation of branched-chain amino acids (BCAAs). The yeast Saccharomyces cerevisiae is a particularly attractive host for the production of BCHAs due to its high tolerance to alcohols and prevalent use in the bioethanol industry. Degradation of BCAAs begins with transamination reactions, catalyzed by branched-chain amino acid transaminases (BCATs) located in the mitochondria (Bat1p) and cytosol (Bat2p). However, the roles that these transaminases play in isobutanol production remain poorly understood and obscured by conflicting reports in the literature. In this work, we elucidate the influence of BCATs on isobutanol production in two genetic backgrounds (CEN.PK2-1C and BY4741). In the process, we uncover and characterize two competing isobutanol pathways, which can be manipulated by overexpressing or deleting BAT1 or BAT2, and adding or removing valine from the fermentation media. We show that deletion of BAT1 alone increases isobutanol production by 14.2-fold over wild type strains in media lacking valine, and examine how interactions between valine and the regulatory protein Ilv6p affect isobutanol production. Compartmentalizing the five-gene isobutanol biosynthetic pathway in mitochondria of BAT1 deletion strains results in an additional 2.1-fold increase in isobutanol production in the absence of valine. While valine inhibits isobutanol production, it boosts 2-methyl-1-butanol production. This work clarifies the role of transamination activity in BCHA biosynthesis, and develops valuable strategies and strains for future optimization of isobutanol production.

Traditional and novel tools to probe the mitochondrial metabolism in health and disease

Mitochondrial metabolism links energy production to other essential cellular processes such as signaling, cellular differentiation, and apoptosis. In addition to producing adenosine triphosphate (ATP) as an energy source, mitochondria are responsible for the synthesis of a myriad of important metabolites and cofactors such as tetrahydrofolate, α‐ketoacids, steroids, aminolevulinic acid, biotin, lipoic acid, acetyl‐CoA, iron‐sulfur clusters, heme, and ubiquinone. Furthermore, mitochondria and their metabolism have been implicated in aging and several human diseases, including inherited mitochondrial disorders, cardiac dysfunction, heart failure, neurodegenerative diseases, diabetes, and cancer. Therefore, there is great interest in understanding mitochondrial metabolism and the complex relationship it has with other cellular processes. A large number of studies on mitochondrial metabolism have been conducted in the last 50 years, taking a broad range of approaches. In this review, we summarize and discuss the most commonly used tools that have been used to study different aspects of the metabolism of mitochondria: ranging from dyes that monitor changes in the mitochondrial membrane potential and pharmacological tools to study respiration or ATP synthesis, to more modern tools such as genetically encoded biosensors and trans‐omic approaches enabled by recent advances in mass spectrometry, computation, and other technologies. These tools have allowed the large number of studies that have shaped our current understanding of mitochondrial metabolism. WIREs Syst Biol Med 2017, 9:e1373. doi: 10.1002/wsbm.1373

Current and future modalities of dynamic control in metabolic engineering

Metabolic engineering aims to maximize production of valuable compounds using cells as biological catalysts. When incorporating engineered pathways into host organisms, an inherent conflict is presented between maintenance of cellular health and generation of products. This challenge has been addressed through two main modalities of dynamic control: decoupling growth from production via two-phase fermentations and autoregulation of pathways to optimize product formation. However, dynamic control can offer even greater potential for metabolic engineering through open-loop and closed-loop control modalities of the production phase. Here we review recent applications of dynamic control strategies in metabolic engineering. We then explore the potential of integrating biosensors and computer-assisted feedback control as a promising future modality of dynamic control.

Metabolic engineering: Biosensors get the green light

A novel approach recruits the largest prokaryotic family of ligand-induced transcriptional regulators to develop a new class of biosensors in yeast based on transcriptional activation, vastly expanding the repertoire of biosensors that could function in eukaryotic hosts.

Metabolic pathway engineering

Organisms can be engineered to produce a wide variety of compounds by either enhancing endogenous metabolic pathways, or by introducing exogenous pathways that are either borrowed from other organism, or de novo-designed pathways unknown to nature. While overexpression of bottleneck enzymes and deletion of competing pathways remain at the core of metabolic pathway engineering, there are many other key elements that need to be considered to successfully develop strains for the production of valuable products. Some of these key elements include the choice of host organism, whichmust take into account the natural advantages and disadvantages of the host for a specific application; computational methods to discover, design, and optimize metabolic pathways; transcriptional control engineering, to finetune the levels and timing of enzyme expression; enzyme engineering to design novel enzymatic activities required in specific metabolic pathways; development of new synthetic biology tools to increase the capabilities and expedience of genetic interventions; and innovative strategies to reduce metabolic burden. This special issue of Synthetic and Systems Biotechnology, focusing on “Metabolic Pathway Engineering” comprises eight review articles and five original research studies that provide excellent examples of how these key elements support and augment metabolic engineering.