Single-mutations at the galactose-binding site of enzymes GalK, GalU, and LgtC enable the efficient synthesis of UDP-6-azido-6-deoxy-d-galactose and azido-functionalized Gb3 analogs.

Lysosomal accumulation of the glycosphingolipid globotriaosylceramide Gb3 is linked to the deficient activity of the α-galactosidase A in the Anderson-Fabry disease and an elevated level of deacylated Gb3 is a hallmark of this condition. Localization of Gb3 in the plasma membrane is critical for studying how the membrane organization and its dynamics are affected in this genetic disorder. Gb3 analogs containing a terminal 6-azido-functionalized galactose in its head group globotriose (αGal1, 4ßGal1, and 4Glc) are attractive chemical reporters for bioimaging, as the azido-group may act as a chemical tag for bio-orthogonal click chemistry. We report here the production of azido-Gb3 analogs employing mutants of galactokinase, UTP-glucose-1-phosphate uridylyltransferase, and α-1,4-galactosyltransferase LgtC, which participate in the synthesis of the sugar motif globotriose. Variants of enzymes galactokinase/UTP-glucose-1-phosphate uridylyltransferase generate UDP-6-azido-6-deoxy-d-galactose, which is the galactosyl-donor used by LgtC for transferring the terminal galactose moiety to lactosyl-acceptors. Residues at the galactose-binding site of the 3 enzymes were modified to facilitate the accommodation of azido-functionalized substrates and variants outperforming the wild-type enzymes were characterized. Synthesis of 6-azido-6-deoxy-d-galactose-1-phosphate, UDP-6-azido-6-deoxy-d-galactose, and azido-Gb3 analogs by variants GalK-E37S, GalU-D133V, and LgtC-Q187S, respectively, is 3-6-fold that of their wild-type counterparts. Coupled reactions with these variants permit the production of the pricy, unnatural galactosyl-donor UDP-6-azido-6-deoxy-d-galactose with ~90% conversion yields, and products azido-globotriose and lyso-AzGb3 with substrate conversion of up to 70%. AzGb3 analogs could serve as precursors for the synthesis of other tagged glycosphingolipids of the globo-series.

Galactose induces formation of cell wall stubs and cell death in Arabidopsis roots.

MAIN CONCLUSION: Arabidopsis seedlings growing on low concentration of galactose stop regular root growth. Incomplete cell division with cell wall stubs, binuclear and giant cells and lignified root tips are observed. Galactose is a sugar abundant in root cell walls of Arabidopsis. Nevertheless, we found that the germination of Arabidopsis seedlings on galactose containing media causes a strong modification of the root development, as shown by analysing the root with microscopy methods ranging from the bright field over confocal to transmission electron microscopy. At concentrations of about 1 mM, the growth of the primary root stops after a few days though stem cell markers like WOX5 are still expressed. The root tip swells and forms a slightly opaque, partially lignified structure in parts of the cortex and the central cylinder. The formation of the cell plate after mitosis is impaired, often leading to cell wall stubs and binuclear cells. Some cells in the cortex and the central cylinder degenerate, while some rhizodermal and cortical cells increase massively in size. The galactose toxicity phenotype in Arabidopsis depends on the activity of galactokinase and is completely diminished in galactokinase knock-out lines. From the comparison of the galactose toxicity phenotype with those of cytokinesis mutants and plants treated with appropriate inhibitors we speculate that the toxicity syndrome of galactose is caused by interference with intracellular vesicle transport or cell wall biogenesis.

Hexose transporter SWEET5 confers galactose sensitivity to Arabidopsis pollen germination via a galactokinase.

Galactose is an abundant and essential sugar used for the biosynthesis of many macromolecules in different organisms, including plants. Galactose metabolism is tightly and finely controlled, since excess galactose and its derivatives are inhibitory to plant growth. In Arabidopsis (Arabidopsis thaliana), root growth and pollen germination are strongly inhibited by excess galactose. However, the mechanism of galactose-induced inhibition during pollen germination remains obscure. In this study, we characterized a plasma membrane-localized transporter, Arabidopsis Sugars Will Eventually be Exported Transporter 5, that transports glucose and galactose. SWEET5 protein levels started to accumulate at the tricellular stage of pollen development and peaked in mature pollen, before rapidly declining after pollen germinated. SWEET5 levels are responsible for the dosage-dependent sensitivity to galactose, and galactokinase is essential for these inhibitory effects during pollen germination. However, sugar measurement results indicate that galactose flux dynamics and sugar metabolism, rather than the steady-state galactose level, may explain phenotypic differences between sweet5 and Col-0 in galactose inhibition of pollen germination.

4-Deoxy-4-fluoro-GalNAz (4FGalNAz) Is a Metabolic Chemical Reporter of O-GlcNAc Modifications, Highlighting the Notable Substrate Flexibility of O-GlcNAc Transferase.

Bio-orthogonal chemistries have revolutionized many fields. For example, metabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain a bio-orthogonal functionality, such as azides or alkynes. MCRs are metabolically incorporated into glycoproteins by living systems, and bio-orthogonal reactions can be subsequently employed to install visualization and enrichment tags. Unfortunately, most MCRs are not selective for one class of glycosylation (e.g., N-linked vs O-linked), complicating the types of information that can be gleaned. We and others have successfully created MCRs that are selective for intracellular O-GlcNAc modification by altering the structure of the MCR and thus biasing it to certain metabolic pathways and/or O-GlcNAc transferase (OGT). Here, we attempt to do the same for the core GalNAc residue of mucin O-linked glycosylation. The most widely applied MCR for mucin O-linked glycosylation, GalNAz, can be enzymatically epimerized at the 4-hydroxyl to give GlcNAz. This results in a mixture of cell-surface and O-GlcNAc labeling. We reasoned that replacing the 4-hydroxyl of GalNAz with a fluorine would lock the stereochemistry of this position in place, causing the MCR to be more selective. After synthesis, we found that 4FGalNAz labels a variety of proteins in mammalian cells and does not perturb endogenous glycosylation pathways unlike 4FGalNAc. However, through subsequent proteomic and biochemical characterization, we found that 4FGalNAz does not widely label cell-surface glycoproteins but instead is primarily a substrate for OGT. Although these results are somewhat unexpected, they once again highlight the large substrate flexibility of OGT, with interesting and important implications for intracellular protein modification by a potential range of abiotic and native monosaccharides.

What makes a histone variant a variant: Changing H2A to become H2A.Z.

Chromatin structure and underlying DNA accessibility is modulated by the incorporation of histone variants. H2A.Z, a variant of the H2A core histone family, plays a distinct and essential role in a diverse set of biological functions including gene regulation and maintenance of heterochromatin-euchromatin boundaries. Although it is currently unclear how the replacement of H2A with H2A.Z can regulate gene expression, the variance in their amino acid sequence likely contributes to their functional differences. To tease apart regions of H2A.Z that confer its unique identity, a set of plasmids expressing H2A-H2A.Z hybrids from the native H2A.Z promoter were examined for their ability to recapitulate H2A.Z function. First, we found that the H2A.Z M6 region was necessary and sufficient for interaction with the SWR1-C chromatin remodeler. Remarkably, the combination of only 9 amino acid changes, the H2A.Z M6 region, K79 and L81 (two amino acids in the α2-helix), were sufficient to fully rescue growth phenotypes of the htz1Δ mutant. Furthermore, combining three unique H2A.Z regions (K79 and L81, M6, C-terminal tail) was sufficient for expression of H2A.Z-dependent heterochromatin-proximal genes and GAL1 derepression. Surprisingly, hybrid constructs that restored the transcription of H2A.Z-dependent genes, did not fully recapitulate patterns of H2A.Z-specific enrichment at the tested loci. This suggested that H2A.Z function in transcription regulation may be at least partially independent of its specific localization in chromatin. Together, this work has identified three regions that can confer specific H2A.Z-identity to replicative H2A, furthering our understanding of what makes a histone variant a variant.

A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress.

Upon replication stress, budding yeast checkpoint kinase Mec1ATR triggers the downregulation of transcription, thereby reducing the level of RNA polymerase (RNAP) on chromatin to facilitate replication fork progression. Here, we identify a hydroxyurea-induced phosphorylation site on Mec1, Mec1-S1991, that contributes to the eviction of RNAPII and RNAPIII during replication stress. The expression of the non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII through a TAP fusion to its Rpb3 subunit, suggesting that lethality in mec1-S1991A mutants arises from replication-transcription conflicts. Coincident with a failure to repress gene expression on hydroxyurea in mec1-S1991A cells, highly transcribed genes such as GAL1 remain bound at nuclear pores. Consistently, we find that nuclear pore proteins and factors controlling RNAPII and RNAPIII are phosphorylated in a Mec1-dependent manner on hydroxyurea. Moreover, we show that Mec1 kinase also contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase by promoting degradation of the Rpb1 subunit.

Structure-Based Optimization of Small Molecule Human Galactokinase Inhibitors.

Classic galactosemia is a rare disease caused by inherited deficiency of galactose-1 phosphate uridylyltransferase (GALT). Accumulation of galactose-1 phosphate (gal-1P) is thought to be the major cause of the chronic complications associated with this disease, which currently has no treatment. Inhibiting galactokinase (GALK1), the enzyme that generates galactose-1 phosphate, has been proposed as a novel strategy for treating classic galactosemia. Our previous work identified a highly selective unique dihydropyrimidine inhibitor against GALK1. With the determination of a co-crystal structure of this inhibitor with human GALK1, we initiated a structure-based structure-activity relationship (SAR) optimization campaign that yielded novel analogs with potent biochemical inhibition (IC50 < 100 nM). Lead compounds were also able to prevent gal-1P accumulation in patient-derived cells at low micromolar concentrations and have pharmacokinetic properties suitable for evaluation in rodent models of galactosemia.

Huntingtin Polyglutamine Fragments Are a Substrate for Hsp104 in Saccharomyces cerevisiae.

The aggregation of huntingtin fragments with expanded polyglutamine repeat regions (HttpolyQ) that cause Huntington's disease depends on the presence of a prion with an amyloid conformation in yeast. As a result of this relationship, HttpolyQ aggregation indirectly depends on Hsp104 due to its essential role in prion propagation. We find that HttQ103 aggregation is directly affected by Hsp104 with and without the presence of [RNQ+] and [PSI+] prions. When we inactivate Hsp104 in the presence of prion, yeast cells have only one or a few large HttQ103 aggregates rather than numerous smaller aggregates. When we inactivate Hsp104 in the absence of prion, there is no significant aggregation of HttQ103, whereas with active Hsp104, HttQ103 aggregates accumulate slowly due to the severing of spontaneously nucleated aggregates by Hsp104. We do not observe either effect with HttQ103P, which has a polyproline-rich region downstream of the polyglutamine region, because HttQ103P does not spontaneously nucleate and Hsp104 does not efficiently sever the prion-nucleated HttQ103P aggregates. Therefore, the only role of Hsp104 in HttQ103P aggregation is to propagate yeast prion. In conclusion, because Hsp104 efficiently severs the HttQ103 aggregates but not HttQ103P aggregates, it has a marked effect on the aggregation of HttQ103 but not HttQ103P.

Heterologous Expression and Auto-Activation of Human Pro-Inflammatory Caspase-1 in Saccharomyces cerevisiae and Comparison to Caspase-8.

Caspases are a family of cysteine proteases that play an essential role in inflammation, apoptosis, cell death, and development. Here we delve into the effects caused by heterologous expression of human caspase-1 in the yeast Saccharomyces cerevisiae and compare them to those of caspase-8. Overexpression of both caspases in the heterologous model led to their activation and caused mitochondrial hyperpolarization, damage to different organelles, and cell death. All these effects were dependent on their protease activity, and caspase-8 was more aggressive than caspase-1. Growth arrest could be at least partially explained by dysfunction of the actin cytoskeleton as a consequence of the processing of the yeast Bni1 formin, which we identify here as a likely direct substrate of both caspases. Through the modulation of the GAL1 promoter by using different galactose:glucose ratios in the culture medium, we have established a scenario in which caspase-1 is sufficiently expressed to become activated while yeast growth is not impaired. Finally, we used the yeast model to explore the role of death-fold domains (DD) of both caspases in their activity. Peculiarly, the DDs of either caspase showed an opposite involvement in its intrinsic activity, as the deletion of the caspase activation and recruitment domain (CARD) of caspase-1 enhanced its activity, whereas the deletion of the death effector domain (DED) of caspase-8 diminished it. We show that caspase-1 is able to efficiently process its target gasdermin D (GSDMD) when co-expressed in yeast. In sum, we propose that S. cerevisiae provides a manageable tool to explore caspase-1 activity and structure-function relationships.

Primary angle closure glaucoma is characterized by altered extracellular matrix homeostasis in the iris.

PURPOSE: To characterize the proteome of the iris in primary angle closure glaucoma (PACG). EXPERIMENTAL DESIGN: In this cross-sectional study, iris samples were obtained from surgical iridectomy of 48 adults with PACG and five normal controls. Peptides from iris were analysed using liquid chromatography-tandem mass spectrometry on an Orbitrap Q Exactive Plus mass spectrometer. Verification of proteins of interest was conducted using selected reaction monitoring on a triple quadrupole mass spectrometer. The main outcome was proteins with a log2 two-fold difference in expression in iris between PACG and controls. RESULTS: There were 3,446 non-redundant proteins identified in human iris, of which 416 proteins were upregulated and 251 proteins were downregulated in PACG compared with controls. Thirty-two upregulated proteins were either components of the extracellular matrix (ECM) (fibrillar collagens, EMILIN-2, fibrinogen, fibronectin, matrilin-2), matricellular proteins (thrombospondin-1), proteins involved in cell-matrix interactions (integrins, laminin, histidine-rich glycoprotein, paxillin), or protease inhibitors known to modulate ECM turnover (α-2 macroglobulin, tissue factor pathway inhibitor 2, papilin). Two giant proteins, titin and obscurin, were up- and down-regulated, respectively, in the iris in PACG compared with controls. CONCLUSIONS AND CLINICAL RELEVANCE: This proteomic study shows that ECM composition and homeostasis are altered in the iris in PACG.

Galactokinase deficiency: a treatable cause of bilateral cataracts.

Congenital cataract can be caused by several systemic diseases and differential diagnosis should be done between infections, genetic or metabolic diseases. We present a case of a 12-month-old girl with bilateral nuclear cataracts that was referred for investigation. Since she did not present a family history of congenital cataracts or metabolic diseases, and her physical examination was normal, a systemic evaluation was performed. Biochemical studies disclosed abnormal galactose metabolism signs. The diagnosis of galactokinase (GALK1) deficiency was considered and the study of the GALK1 gene allowed identifying a pathogenic genetic variant and a predictably pathogenic missense mutation, previously not described. Dietary measures were imposed with a good evolution.

FACT interacts with Set3 HDAC and fine-tunes GAL1 transcription in response to environmental stimulation.

The histone chaperone facilitates chromatin transactions (FACT) functions in various DNA transactions. How FACT performs these multiple functions remains largely unknown. Here, we found, for the first time, that the N-terminal domain of its Spt16 subunit interacts with the Set3 histone deacetylase complex (Set3C) and that FACT and Set3C function in the same pathway to regulate gene expression in some settings. We observed that Spt16-G132D mutant proteins show defects in binding to Set3C but not other reported FACT interactors. At the permissive temperature, induction of the GAL1 and GAL10 genes is reduced in both spt16-G132D and set3Δ cells, whereas transient upregulation of GAL10 noncoding RNA (ncRNA), which is transcribed from the 3' end of the GAL10 gene, is elevated. Mutations that inhibit GAL10 ncRNA transcription reverse the GAL1 and GAL10 induction defects in spt16-G132D and set3Δ mutant cells. Mechanistically, set3Δ and FACT (spt16-G132D) mutants show reduced histone acetylation and increased nucleosome occupancy at the GAL1 promoter under inducing conditions and inhibition of GAL10 ncRNA transcription also partially reverses these chromatin changes. These results indicate that FACT interacts with Set3C, which in turn prevents uncontrolled GAL10 ncRNA expression and fine-tunes the expression of GAL genes upon a change in carbon source.

Decoupling transcription factor expression and activity enables dimmer switch gene regulation.

Gene-regulatory networks achieve complex mappings of inputs to outputs through mechanisms that are poorly understood. We found that in the galactose-responsive pathway in Saccharomyces cerevisiae, the decision to activate the transcription of genes encoding pathway components is controlled independently from the expression level, resulting in behavior resembling that of a mechanical dimmer switch. This was not a direct result of chromatin regulation or combinatorial control at galactose-responsive promoters; rather, this behavior was achieved by hierarchical regulation of the expression and activity of a single transcription factor. Hierarchical regulation is ubiquitous, and thus dimmer switch regulation is likely a key feature of many biological systems. Dimmer switch gene regulation may allow cells to fine-tune their responses to multi-input environments on both physiological and evolutionary time scales.

Deuterium-depletion has no significant impact on the mutation rate of Escherichia coli, deuterium abundance therefore has a probabilistic, not deterministic effect on spontaneous mutagenesis.

Deuterium (D), the second most abundant isotope of hydrogen is present in natural waters at an approximate concentration of 145-155 ppm (ca. 1.5E-4 atom/atom). D is known to influence various biological processes due to its physical and chemical properties, which significantly differ from those of hydrogen. For example, increasing D-concentration to >1000-fold above its natural abundance has been shown to increase the frequency of genetic mutations in several species. An interesting deterministic hypothesis, formulated with the intent of explaining the mechanism of D-mutagenicity is based on the calculation that the theoretical probability of base pairs to comprise two adjacent D-bridges instead of H-bridges is 2.3E-8, which is equal to the mutation rate of certain species. To experimentally challenge this hypothesis, and to infer the mutagenicity of D present at natural concentrations, we investigated the effect of a nearly 100-fold reduction of D concentration on the bacterial mutation rate. Using fluctuation tests, we measured the mutation rate of three Escherichia coli genes (cycA, ackA and galK) in media containing D at either <2 ppm or 150 ppm concentrations. Out of 15 pair-wise fluctuation analyses, nine indicated a significant decrease, while three marked the significant increase of the mutation/culture value upon D-depletion. Overall, growth in D-depleted minimal medium led to a geometric mean of 0.663-fold (95% confidence interval: 0.483-0.911) change in the mutation rate. This falls nowhere near the expected 10,000-fold reduction, indicating that in our bacterial systems, the effect of D abundance on the formation of point mutations is not deterministic. In addition, the combined results did not display a statistically significant change in the mutation/culture value, the mutation rate or the mutant frequency upon D-depletion. The potential mutagenic effect of D present at natural concentrations on E. coli is therefore below the limit of detection using the indicated methods.

Fragment Screening Reveals Starting Points for Rational Design of Galactokinase 1 Inhibitors to Treat Classic Galactosemia.

Classic galactosemia is caused by loss-of-function mutations in galactose-1-phosphate uridylyltransferase (GALT) that lead to toxic accumulation of its substrate, galactose-1-phosphate. One proposed therapy is to inhibit the biosynthesis of galactose-1-phosphate, catalyzed by galactokinase 1 (GALK1). Existing inhibitors of human GALK1 (hGALK1) are primarily ATP-competitive with limited clinical utility to date. Here, we determined crystal structures of hGALK1 bound with reported ATP-competitive inhibitors of the spiro-benzoxazole series, to reveal their binding mode in the active site. Spurred by the need for additional chemotypes of hGALK1 inhibitors, desirably targeting a nonorthosteric site, we also performed crystallography-based screening by soaking hundreds of hGALK1 crystals, already containing active site ligands, with fragments from a custom library. Two fragments were found to bind close to the ATP binding site, and a further eight were found in a hotspot distal from the active site, highlighting the strength of this method in identifying previously uncharacterized allosteric sites. To generate inhibitors of improved potency and selectivity targeting the newly identified binding hotspot, new compounds were designed by merging overlapping fragments. This yielded two micromolar inhibitors of hGALK1 that were not competitive with respect to either substrate (ATP or galactose) and demonstrated good selectivity over hGALK1 homologues, galactokinase 2 and mevalonate kinase. Our findings are therefore the first to demonstrate inhibition of hGALK1 from an allosteric site, with potential for further development of potent and selective inhibitors to provide novel therapeutics for classic galactosemia.

Ancient balancing selection maintains incompatible versions of the galactose pathway in yeast.

Metabolic pathways differ across species but are expected to be similar within a species. We discovered two functional, incompatible versions of the galactose pathway in Saccharomyces cerevisiae We identified a three-locus genetic interaction for growth in galactose, and used precisely engineered alleles to show that it arises from variation in the galactose utilization genes GAL2, GAL1/10/7, and phosphoglucomutase (PGM1), and that the reference allele of PGM1 is incompatible with the alternative alleles of the other genes. Multiloci balancing selection has maintained the two incompatible versions of the pathway for millions of years. Strains with alternative alleles are found primarily in galactose-rich dairy environments, and they grow faster in galactose but slower in glucose, revealing a trade-off on which balancing selection may have acted.

Galactokinase deficiency: lessons from the GalNet registry.

PURPOSE: Galactokinase (GALK1) deficiency is a rare hereditary galactose metabolism disorder. Beyond cataract, the phenotypic spectrum is questionable. Data from affected patients included in the Galactosemias Network registry were collected to better characterize the phenotype. METHODS: Observational study collecting medical data of 53 not previously reported GALK1 deficient patients from 17 centers in 11 countries from December 2014 to April 2020. RESULTS: Neonatal or childhood cataract was reported in 15 and 4 patients respectively. The occurrence of neonatal hypoglycemia and infection were comparable with the general population, whereas bleeding diathesis (8.1% versus 2.17-5.9%) and encephalopathy (3.9% versus 0.3%) were reported more often. Elevated transaminases were seen in 25.5%. Cognitive delay was reported in 5 patients. Urinary galactitol was elevated in all patients at diagnosis; five showed unexpected Gal-1-P increase. Most patients showed enzyme activities ≤1%. Eleven different genotypes were described, including six unpublished variants. The majority was homozygous for NM_000154.1:c.82C>A (p.Pro28Thr). Thirty-five patients were diagnosed following newborn screening, which was clearly beneficial. CONCLUSION: The phenotype of GALK1 deficiency may include neonatal elevation of transaminases, bleeding diathesis, and encephalopathy in addition to cataract. Potential complications beyond the neonatal period are not systematically surveyed and a better delineation is needed.

BARMR1/FAM92A1, a novel gene encoding BAR domain protein with multi-functions.

BARMR1/FAM92A1 encodes a novel BAR domain protein, and is widely expressed during embryonic development and highly expressed in tumor cells. Mutation or deletion of BARMR1/FAM92A1 caused developmental disorder and the BARMR1/FAM92A1 overexpression in tumor cells is associated with poor prognosis. The subcellular location of BARMR1/FAM92A1 determined its biological functions by interacting with different proteins. When colocalized and interacted with CBY at the centrioles/basal bodies of primary cilia, BARMR1/FAM92A1 facilitate ciliogenesis, whilst binded to GAL1 in the nuclei, it promotes cell proliferation, migration, and malignancy of tumor cells.

Chromatin regulatory genes differentially interact in networks to facilitate distinct GAL1 activity and noise profiles.

Controlling chromatin state constitutes a major regulatory step in gene expression regulation across eukaryotes. While global cellular features or processes are naturally impacted by chromatin state alterations, little is known about how chromatin regulatory genes interact in networks to dictate downstream phenotypes. Using the activity of the canonical galactose network in yeast as a model, here, we measured the impact of the disruption of key chromatin regulatory genes on downstream gene expression, genetic noise and fitness. Using Trichostatin A and nicotinamide, we characterized how drug-based modulation of global histone deacetylase activity affected these phenotypes. Performing epistasis analysis, we discovered phenotype-specific genetic interaction networks of chromatin regulators. Our work provides comprehensive insights into how the galactose network activity is affected by protein interaction networks formed by chromatin regulators.

Design and Characterization of Rapid Optogenetic Circuits for Dynamic Control in Yeast Metabolic Engineering.

The use of optogenetics in metabolic engineering for light-controlled microbial chemical production raises the prospect of utilizing control and optimization techniques routinely deployed in traditional chemical manufacturing. However, such mechanisms require well-characterized, customizable tools that respond fast enough to be used as real-time inputs during fermentations. Here, we present OptoINVRT7, a new rapid optogenetic inverter circuit to control gene expression in Saccharomyces cerevisiae. The circuit induces gene expression in only 0.6 h after switching cells from light to darkness, which is at least 6 times faster than previous OptoINVRT optogenetic circuits used for chemical production. In addition, we introduce an engineered inducible GAL1 promoter (PGAL1-S), which is stronger than any constitutive or inducible promoter commonly used in yeast. Combining OptoINVRT7 with PGAL1-S achieves strong and light-tunable levels of gene expression with as much as 132.9 ± 22.6-fold induction in darkness. The high performance of this new optogenetic circuit in controlling metabolic enzymes boosts production of lactic acid and isobutanol by more than 50% and 15%, respectively. The strength and controllability of OptoINVRT7 and PGAL1-S open the door to applying process control tools to engineered metabolisms to improve robustness and yields in microbial fermentations for chemical production.