Microbial synthesis of health-promoting inositols.

D-chiro-inositol and scyllo-inositol are known for their health-promoting properties and promising as ingredients for functional foods. Strains of Bacillus subtilis and Corynebacterium glutamicum were created by metabolic engineering capable of inexpensive production of these two rare inositols from myo-inositol, which is the most common inositol in nature. In addition, further modifications have enabled the synthesis of the two rare inositols from the much-cheaper carbon sources, glucose or sucrose.

Metabolic adjustments of blood-stage Plasmodium falciparum in response to sublethal pyrazoleamide exposure.

Due to the recurring loss of antimalarial drugs to resistance, there is a need for novel targets, drugs, and combination therapies to ensure the availability of current and future countermeasures. Pyrazoleamides belong to a novel class of antimalarial drugs that disrupt sodium ion homeostasis, although the exact consequences of this disruption in Plasmodium falciparum remain under investigation. In vitro experiments demonstrated that parasites carrying mutations in the metabolic enzyme PfATP4 develop resistance to pyrazoleamide compounds. However, the underlying mechanisms that allow mutant parasites to evade pyrazoleamide treatment are unclear. Here, we first performed experiments to identify the sublethal dose of a pyrazoleamide compound (PA21A092) that caused a significant reduction in growth over one intraerythrocytic developmental cycle (IDC). At this drug concentration, we collected transcriptomic and metabolomic data at multiple time points during the IDC to quantify gene- and metabolite-level alterations in the treated parasites. To probe the effects of pyrazoleamide treatment on parasite metabolism, we coupled the time-resolved omics data with a metabolic network model of P. falciparum. We found that the drug-treated parasites adjusted carbohydrate metabolism to enhance synthesis of myoinositol-a precursor for phosphatidylinositol biosynthesis. This metabolic adaptation caused a decrease in metabolite flux through the pentose phosphate pathway, causing a decreased rate of RNA synthesis and an increase in oxidative stress. Our model analyses suggest that downstream consequences of enhanced myoinositol synthesis may underlie adjustments that could lead to resistance emergence in P. falciparum exposed to a sublethal dose of a pyrazoleamide drug.

SLC5A3-Dependent Myo-inositol Auxotrophy in Acute Myeloid Leukemia.

An enhanced requirement for nutrients is a hallmark property of cancer cells. Here, we optimized an in vivo genetic screening strategy in acute myeloid leukemia (AML), which led to the identification of the myo-inositol transporter SLC5A3 as a dependency in this disease. We demonstrate that SLC5A3 is essential to support a myo-inositol auxotrophy in AML. The commonality among SLC5A3-dependent AML lines is the transcriptional silencing of ISYNA1, which encodes the rate-limiting enzyme for myo-inositol biosynthesis, inositol-3-phosphate synthase 1. We use gain- and loss-of-function experiments to reveal a synthetic lethal genetic interaction between ISYNA1 and SLC5A3 in AML, which function redundantly to sustain intracellular myo-inositol. Transcriptional silencing and DNA hypermethylation of ISYNA1 occur in a recurrent manner in human AML patient samples, in association with IDH1/IDH2 and CEBPA mutations. Our findings reveal myo-inositol as a nutrient dependency in AML caused by the aberrant silencing of a biosynthetic enzyme. SIGNIFICANCE: We show how epigenetic silencing can provoke a nutrient dependency in AML by exploiting a synthetic lethality relationship between biosynthesis and transport of myo-inositol. Blocking the function of this solute carrier may have therapeutic potential in an epigenetically defined subset of AML.This article is highlighted in the In This Issue feature, p. 275.

Genome Features of Asaia sp. W12 Isolated from the Mosquito Anopheles stephensi Reveal Symbiotic Traits.

Asaia bacteria commonly comprise part of the microbiome of many mosquito species in the genera Anopheles and Aedes, including important vectors of infectious agents. Their close association with multiple organs and tissues of their mosquito hosts enhances the potential for paratransgenesis for the delivery of antimalaria or antivirus effectors. The molecular mechanisms involved in the interactions between Asaia and mosquito hosts, as well as Asaia and other bacterial members of the mosquito microbiome, remain underexplored. Here, we determined the genome sequence of Asaia strain W12 isolated from Anopheles stephensi mosquitoes, compared it to other Asaia species associated with plants or insects, and investigated the properties of the bacteria relevant to their symbiosis with mosquitoes. The assembled genome of strain W12 had a size of 3.94 MB, the largest among Asaia spp. studied so far. At least 3585 coding sequences were predicted. Insect-associated Asaia carried more glycoside hydrolase (GH)-encoding genes than those isolated from plants, showing their high plant biomass-degrading capacity in the insect gut. W12 had the most predicted regulatory protein components comparatively among the selected Asaia, indicating its capacity to adapt to frequent environmental changes in the mosquito gut. Two complete operons encoding cytochrome bo3-type ubiquinol terminal oxidases (cyoABCD-1 and cyoABCD-2) were found in most Asaia genomes, possibly offering alternative terminal oxidases and allowing the flexible transition of respiratory pathways. Genes involved in the production of 2,3-butandiol and inositol have been found in Asaia sp. W12, possibly contributing to biofilm formation and stress tolerance.

Subtilisin-Involved Morphology Engineering for Improved Antibiotic Production in Actinomycetes.

In the submerged cultivation of filamentous microbes, including actinomycetes, complex morphology is one of the critical process features for the production of secondary metabolites. Ansamitocin P-3 (AP-3), an antitumor agent, is a secondary metabolite produced by Actinosynnema pretiosum ATCC 31280. An excessive mycelial fragmentation of A. pretiosum ATCC 31280 was observed during the early stage of fermentation. Through comparative transcriptomic analysis, a subtilisin-like serine peptidase encoded gene APASM_4178 was identified to be responsible for the mycelial fragmentation. Mutant WYT-5 with the APASM_4178 deletion showed increased biomass and improved AP-3 yield by 43.65%. We also found that the expression of APASM_4178 is specifically regulated by an AdpA-like protein APASM_1021. Moreover, the mycelial fragmentation was alternatively alleviated by the overexpression of subtilisin inhibitor encoded genes, which also led to a 46.50 ± 0.79% yield increase of AP-3. Furthermore, APASM_4178 was overexpressed in salinomycin-producing Streptomyces albus BK 3-25 and validamycin-producing S. hygroscopicus TL01, which resulted in not only dispersed mycelia in both strains, but also a 33.80% yield improvement of salinomycin to 24.07 g/L and a 14.94% yield improvement of validamycin to 21.46 g/L. In conclusion, our work elucidates the involvement of a novel subtilisin-like serine peptidase in morphological differentiation, and modulation of its expression could be an effective strategy for morphology engineering and antibiotic yield improvement in actinomycetes.

Efficient production of myo-inositol in Escherichia coli through metabolic engineering.

BACKGROUND: The biosynthesis of high value-added compounds using metabolically engineered strains has received wide attention in recent years. Myo-inositol (inositol), an important compound in the pharmaceutics, cosmetics and food industries, is usually produced from phytate via a harsh set of chemical reactions. Recombinant Escherichia coli strains have been constructed by metabolic engineering strategies to produce inositol, but with a low yield. The proper distribution of carbon flux between cell growth and inositol production is a major challenge for constructing an efficient inositol-synthesis pathway in bacteria. Construction of metabolically engineered E. coli strains with high stoichiometric yield of inositol is desirable. RESULTS: In the present study, we designed an inositol-synthesis pathway from glucose with a theoretical stoichiometric yield of 1 mol inositol/mol glucose. Recombinant E. coli strains with high stoichiometric yield (> 0.7 mol inositol/mol glucose) were obtained. Inositol was successfully biosynthesized after introducing two crucial enzymes: inositol-3-phosphate synthase (IPS) from Trypanosoma brucei, and inositol monophosphatase (IMP) from E. coli. Based on starting strains E. coli BW25113 (wild-type) and SG104 (ΔptsG::glk, ΔgalR::zglf, ΔpoxB::acs), a series of engineered strains for inositol production was constructed by deleting the key genes pgi, pfkA and pykF. Plasmid-based expression systems for IPS and IMP were optimized, and expression of the gene zwf was regulated to enhance the stoichiometric yield of inositol. The highest stoichiometric yield (0.96 mol inositol/mol glucose) was achieved from recombinant strain R15 (SG104, Δpgi, Δpgm, and RBSL5-zwf). Strain R04 (SG104 and Δpgi) reached high-density in a 1-L fermenter when using glucose and glycerol as a mixed carbon source. In scaled-up fed-batch bioconversion in situ using strain R04, 0.82 mol inositol/mol glucose was produced within 23 h, corresponding to a titer of 106.3 g/L (590.5 mM) inositol. CONCLUSIONS: The biosynthesis of inositol from glucose in recombinant E. coli was optimized by metabolic engineering strategies. The metabolically engineered E. coli strains represent a promising method for future inositol production. This study provides an essential reference to obtain a suitable distribution of carbon flux between glycolysis and inositol synthesis.

Specialized Metabolism in a Nonmodel Nightshade: Trichome Acylinositol Biosynthesis.

Plants make many biologically active, specialized metabolites, which vary in structure, biosynthesis, and the processes they influence. An increasing number of these compounds are documented to protect plants from insects, pathogens, or herbivores or to mediate interactions with beneficial organisms, including pollinators and nitrogen-fixing microbes. Acylsugars, one class of protective compounds, are made in glandular trichomes of plants across the Solanaceae family. While most described acylsugars are acylsucroses, published examples also include acylsugars with hexose cores. The South American fruit crop naranjilla (lulo; Solanum quitoense) produces acylsugars containing a myoinositol core. We identified an enzyme that acetylates triacylinositols, a function homologous to the last step in the acylsucrose biosynthetic pathway of tomato (Solanum lycopersicum). Our analysis reveals parallels between S. lycopersicum acylsucrose and S. quitoense acylinositol biosynthesis, suggesting a common evolutionary origin.

A bacterial cell factory converting glucose into scyllo-inositol, a therapeutic agent for Alzheimer's disease.

A rare stereoisomer of inositol, scyllo-inositol, is a therapeutic agent that has shown potential efficacy in preventing Alzheimer's disease. Mycobacterium tuberculosis ino1 encoding myo-inositol-1-phosphate (MI1P) synthase (MI1PS) was introduced into Bacillus subtilis to convert glucose-6-phosphate (G6P) into MI1P. We found that inactivation of pbuE elevated intracellular concentrations of NAD+·NADH as an essential cofactor of MI1PS and was required to activate MI1PS. MI1P thus produced was dephosphorylated into myo-inositol by an intrinsic inositol monophosphatase, YktC, which was subsequently isomerized into scyllo-inositol via a previously established artificial pathway involving two inositol dehydrogenases, IolG and IolW. In addition, both glcP and glcK were overexpressed to feed more G6P and accelerate scyllo-inositol production. Consequently, a B. subtilis cell factory was demonstrated to produce 2 g L-1 scyllo-inositol from 20 g L-1 glucose. This cell factory provides an inexpensive way to produce scyllo-inositol, which will help us to challenge the growing problem of Alzheimer's disease in our aging society.

Vitamin requirements and biosynthesis in Saccharomyces cerevisiae.

Chemically defined media for yeast cultivation (CDMY) were developed to support fast growth, experimental reproducibility, and quantitative analysis of growth rates and biomass yields. In addition to mineral salts and a carbon substrate, popular CDMYs contain seven to nine B-group vitamins, which are either enzyme cofactors or precursors for their synthesis. Despite the widespread use of CDMY in fundamental and applied yeast research, the relation of their design and composition to the actual vitamin requirements of yeasts has not been subjected to critical review since their first development in the 1940s. Vitamins are formally defined as essential organic molecules that cannot be synthesized by an organism. In yeast physiology, use of the term "vitamin" is primarily based on essentiality for humans, but the genome of the Saccharomyces cerevisiae reference strain S288C harbours most of the structural genes required for synthesis of the vitamins included in popular CDMY. Here, we review the biochemistry and genetics of the biosynthesis of these compounds by S. cerevisiae and, based on a comparative genomics analysis, assess the diversity within the Saccharomyces genus with respect to vitamin prototrophy.

Synergetic utilization of glucose and glycerol for efficient myo-inositol biosynthesis.

myo-Inositol (MI) as a dietary supplement can provide various health benefits. One major challenge to its efficient biosynthesis is to achieve proper distribution of carbon flux between growth and production. Herein, this challenge was overcome by synergetic utilization of glucose and glycerol. Specifically, glycerol was catabolized to support cell growth while glucose was conserved as the building block for MI production. Growth and production were coupled via the phosphotransferase system, and both modules were optimized to achieve efficient production. First, the optimal enzyme combination was established for the production module. It was observed that enhancing the production module resulted in both increased MI production and better cell growth. In addition, glucose was shown to inhibit glycerol utilization via carbon catabolite repression and the inhibition was released by over-expressing glycerol kinase. Furthermore, the inducible promoter was replaced by strong constitutive promoters to avoid inducer use. With these efforts, the final strain produced MI with both high titer and yield. In fed-batch cultivation, 76 g/L of MI was produced, showing scale-up potential. This study provides a promising strategy to achieve rational distribution of carbon flux.

Regulation of Inositol Biosynthesis: Balancing Health and Pathophysiology.

Inositol is the precursor for all inositol compounds and is essential for viability of eukaryotic cells. Numerous cellular processes and signaling functions are dependent on inositol compounds, and perturbation of their synthesis leads to a wide range of human diseases. Although considerable research has been directed at understanding the function of inositol compounds, especially phosphoinositides and inositol phosphates, a focus on regulatory and homeostatic mechanisms controlling inositol biosynthesis has been largely neglected. Consequently, little is known about how synthesis of inositol is regulated in human cells. Identifying physiological regulators of inositol synthesis and elucidating the molecular mechanisms that regulate inositol synthesis will contribute fundamental insight into cellular processes that are mediated by inositol compounds and will provide a foundation to understand numerous disease processes that result from perturbation of inositol homeostasis. In addition, elucidating the mechanisms of action of inositol-depleting drugs may suggest new strategies for the design of second-generation pharmaceuticals to treat psychiatric disorders and other illnesses.

Osmoregulation by the myo-inositol biosynthesis pathway in turbot Scophthalmus maximus and its regulation by anabolite and c-Myc.

The induction of the myo-inositol biosynthesis (MIB) pathway in euryhaline fishes is an important component of the cellular response to osmotic challenge. The MIPS and IMPA1 genes were sequenced in turbot and found to be highly conserved in phylogenetic evolution, especially within the fish species tested. Under salinity stress in turbot, both MIPS and IMPA1 showed adaptive expression, a turning point in the level of expression occurred at 12 h in all tissues tested. We performed an RNAi assay mediated by long fragment dsRNA prepared by transcription in vitro. The findings demonstrated that knockdown of the MIB pathway weakened the function of gill osmotic regulation, and may induce a genetic compensation response in the kidney and gill to maintain physiological function. Even though the gill and kidney conducted stress reactions or compensatory responses to salinity stress, this inadequately addressed the consequences of MIB knockdown. Therefore, the survival time of turbot under salinity stress after knockdown was obviously less than that under seawater, especially under low salt stress. Pearson's correlation analysis between gene expression and dietary myo-inositol concentration indicated that the MIB pathway had a remarkable negative feedback control, and the dynamic equilibrium mediated by negative feedback on the MIB pathway played a crucial role in osmoregulation in turbot. An RNAi assay with c-Myc in vivo and the use of a c-Myc inhibitor (10058-F4) in vitro demonstrated that c-Myc was likely to positively regulate the MIB pathway in turbot.

Permeabilized Escherichia coli Whole Cells Containing Co-Expressed Two Thermophilic Enzymes Facilitate the Synthesis of scyllo-Inositol from myo-Inositol.

Scyllo-inositol (SI), a stereoisomer of inositol, is regarded as a promising therapeutic agent for Alzheimer's disease. Here, an in vitro cofactor-balance biotransformation for the production of SI from myo-inositol (MI) by thermophilic myo-inositol 2-dehydrogenase (IDH) and scyllo-inositol 2-dehydrogenase (SIDH) is presented. These two enzymes (i.e., IDH and SIDH from Geobacillus kaustophilus) are co-expressed in Escherichia coli BL21(DE3), and E. coli cells containing the two enzymes are permeabilized by heat treatment as whole-cell catalysts to convert MI to SI. After condition optimizations about permeabilized temperature, reaction temperature, and initial MI concentration, about 82 g L-1 of SI is produced from 250 g L-1 of MI within 24 h without any cofactor supplementation. This final titer of SI produced is the highest to the authors' limited knowledge. This study provides a promising method for the large-scale industrial production of SI.

Characterization and engineering control of the effects of reactive oxygen species on the conversion of sterols to steroid synthons in Mycobacterium neoaurum.

The conversion of sterols to steroid synthons by engineered mycobacteria comprises one of the basic ways for the production of steroid medications in the pharmaceutical industry. Here, we revealed that high amounts of reactive oxygen species (ROS) generate during the conversion process of sterols, which impairs the cell viability of mycobacterial cells and thus hinders the conversion of sterols to steroid synthons. Accordingly, the endogenous antioxidants for detoxifying ROS in mycobacteria, ROS scavenging enzymes and low molecular weight thiols, were examined. The results revealed that three antioxidants, catalase (CAT), mycothiol (MSH), and ergothioneine (EGT), demonstrated efficacy toward neutralizing the excessive ROS produced during sterol metabolism. CAT overexpression or MSH or EGT augmentation enhanced the conversion of phytosterols to 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) by 18.9%, 23.8%, and 32.1%, respectively, and also enhanced the cell viability, indicating the benefits of these antioxidants in reducing ROS-induced stress. Further combinatorial augmentation of CAT, MSH, and EGT demonstrated enhanced effects toward intracellular ROS scavenging, resulting in 54.2% greater cell viability and 47.5% enhancement in 4-HBC production. These findings indicated that the excessive ROS induces cell stress, in turn limiting the conversion of sterols, whereas neutralization of the excessive ROS by combined control of CAT, MSH, and EGT serves as an effective strategy to boost the conversion productivity of sterols to steroid synthons.

Engineering transkingdom signalling in plants to control gene expression in rhizosphere bacteria.

The root microbiota is critical for agricultural yield, with growth-promoting bacteria able to solubilise phosphate, produce plant growth hormones, antagonise pathogens and fix N2. Plants control the microorganisms in their immediate environment and this is at least in part through direct selection, the immune system, and interactions with other microorganisms. Considering the importance of the root microbiota for crop yields it is attractive to artificially regulate this environment to optimise agricultural productivity. Towards this aim we express a synthetic pathway for the production of the rhizopine scyllo-inosamine in plants. We demonstrate the production of this bacterial derived signal in both Medicago truncatula and barley and show its perception by rhizosphere bacteria, containing bioluminescent and fluorescent biosensors. This study lays the groundwork for synthetic signalling networks between plants and bacteria, allowing the targeted regulation of bacterial gene expression in the rhizosphere for delivery of useful functions to plants.

Water Deficit Elicits a Transcriptional Response of Genes Governing d-pinitol Biosynthesis in Soybean (Glycine max).

d-pinitol is the most commonly accumulated sugar alcohol in the Leguminosae family and has been observed to increase significantly in response to abiotic stress. While previous studies have identified genes involved in d-pinitol synthesis, no study has investigated transcript expression in planta. The present study quantified the expression of several genes involved in d-pinitol synthesis in different plant tissues and investigated the accumulation of d-pinitol, myo-inositol and other metabolites in response to a progressive soil drought in soybean (Glycine max). Expression of myo-inositol 1-phosphate synthase (INPS), the gene responsible for the conversion of glucose-6-phosphate to myo-inositol-1-phosphate, was significantly up regulated in response to a water deficit for the first two sampling weeks. Expression of myo-inositol O-methyl transferase (IMT1), the gene responsible for the conversion of myo-inositol into d-ononitol was only up regulated in stems at sampling week 3. Assessment of metabolites showed significant changes in their concentration in leaves and stems. d-Pinitol concentration was significantly higher in all organs sampled from water deficit plants for all three sampling weeks. In contrast, myo-inositol, had significantly lower concentrations in leaf samples despite up regulation of INPS suggesting the transcriptionally regulated flux of carbon through the myo-inositol pool is important during water deficit.

Redox regulation by reversible protein S-thiolation in Gram-positive bacteria.

Low molecular weight (LMW) thiols play an important role as thiol-cofactors for many enzymes and are crucial to maintain the reduced state of the cytoplasm. Most Gram-negative bacteria utilize glutathione (GSH) as major LMW thiol. However, in Gram-positive Actinomycetes and Firmicutes alternative LMW thiols, such as mycothiol (MSH) and bacillithiol (BSH) play related roles as GSH surrogates, respectively. Under conditions of hypochlorite stress, MSH and BSH are known to form mixed disulfides with protein thiols, termed as S-mycothiolation or S-bacillithiolation that function in thiol-protection and redox regulation. Protein S-thiolations are widespread redox-modifications discovered in different Gram-positive bacteria, such as Bacillus and Staphylococcus species, Mycobacterium smegmatis, Corynebacterium glutamicum and Corynebacterium diphtheriae. S-thiolated proteins are mainly involved in cellular metabolism, protein translation, redox regulation and antioxidant functions with some conserved targets across bacteria. The reduction of protein S-mycothiolations and S-bacillithiolations requires glutaredoxin-related mycoredoxin and bacilliredoxin pathways to regenerate protein functions. In this review, we present an overview of the functions of mycothiol and bacillithiol and their physiological roles in protein S-bacillithiolations and S-mycothiolations in Gram-positive bacteria. Significant progress has been made to characterize the role of protein S-thiolation in redox-regulation and thiol protection of main metabolic and antioxidant enzymes. However, the physiological roles of the pathways for regeneration are only beginning to emerge as well as their interactions with other cellular redox systems. Future studies should be also directed to explore the roles of protein S-thiolations and their redox pathways in pathogenic bacteria under infection conditions to discover new drug targets and treatment options against multiple antibiotic resistant bacteria.

Increased salt and drought tolerance by D-pinitol production in transgenic Arabidopsis thaliana.

D-ononitol epimerase (OEP) catalyzes the conversion of D-ononitol to D-pinitol, which is the last step in the biosynthetic pathway, where myo-inositol is converted to pinitol in higher plants. In this study, OEP cDNA was isolated from Glycine max (GmOEP) and was functionally characterized, which confirmed that GmOEP expression was induced by high salinity and drought stress treatments. To understand the biological function of GmOEP, transgenic Arabidopsis plants overexpressing this protein were constructed. The transgenic Arabidopsis plants displayed enhanced tolerance to high salinity and drought stress treatments.

The functional interplay of low molecular weight thiols in Mycobacterium tuberculosis.

BACKGROUND: Three low molecular weight thiols are synthesized by Mycobacterium tuberculosis (M.tb), namely ergothioneine (ERG), mycothiol (MSH) and gamma-glutamylcysteine (GGC). They are able to counteract reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). In addition, the production of ERG is elevated in the MSH-deficient M.tb mutant, while the production of MSH is elevated in the ERG-deficient mutants. Furthermore, the production of GGC is elevated in the MSH-deficient mutant and the ERG-deficient mutants. The propensity of one thiol to be elevated in the absence of the other prompted further investigations into their interplay in M.tb. METHODS: To achieve that, we generated two M.tb mutants that are unable to produce ERG nor MSH but are able to produce a moderate (ΔegtD-mshA) or significantly high (ΔegtB-mshA) amount of GGC relative to the wild-type strain. In addition, we generated an M.tb mutant that is unable to produce GGC nor MSH but is able to produce a significantly low level of ERG (ΔegtA-mshA) relative to the wild-type strain. The susceptibilities of these mutants to various in vitro and ex vivo stress conditions were investigated and compared. RESULTS: The ΔegtA-mshA mutant was the most susceptible to cellular stress relative to its parent single mutant strains (ΔegtA and ∆mshA) and the other double mutants. In addition, it displayed a growth-defect in vitro, in mouse and human macrophages suggesting; that the complete inhibition of ERG, MSH and GGC biosynthesis is deleterious for the growth of M.tb. CONCLUSIONS: This study indicates that ERG, MSH and GGC are able to compensate for each other to maximize the protection and ensure the fitness of M.tb. This study therefore suggests that the most effective strategy to target thiol biosynthesis for anti-tuberculosis drug development would be the simultaneous inhibition of the biosynthesis of ERG, MSH and GGC.

Co-suppression of AtMIPS demonstrates cooperation of MIPS1, MIPS2 and MIPS3 in maintaining myo-inositol synthesis.

KEY MESSAGE: Co-suppressed MIPS2 transgenic lines allow bypass of the embryo lethal phenotype of the previously published triple knock-out and demonstrate the effects of MIPS on later stages of development. Regulation of inositol production is of interest broadly for its effects on plant growth and development. The enzyme L-myo-inositol 1-phosphate synthase (MIPS, also known as IPS) isomerizes D-glucose-6-P to D-inositol 3-P, and this is the rate-limiting step in inositol production. In Arabidopsis thaliana, the MIPS enzyme is encoded by three different genes, (AtMIPS1, AtMIPS2 and AtMIPS3), each of which has been shown to produce proteins with biochemically similar properties but differential expression patterns. Here, we report phenotypic and biochemical effects of MIPS co-suppression. We show that some plants engineered to overexpress MIPS2 in fact show reduced expression of AtMIPS1, AtMIPS2 and AtMIPS3, and show altered vegetative phenotype, reduced size and root length, and delayed flowering. Additionally, these plants show reduced inositol, increased glucose levels, and alteration of other metabolites. Our results suggest that the three AtMIPS genes work together to impact the overall synthesis of myo-inositol and overall inositol homeostasis.