Isopropylmalate Synthase Research Articles

Knocking out isopropylmalate synthase simultaneously improves grain appearance and nutritional quality in rice.

Grain appearance and nutritional quality are critical traits for rice marketing. However, how to simultaneously improve grain appearance (slender grain and low chalkiness) and nutritional quality (improved protein and amino acid contents) in rice remains a major challenge. Here, we show that knocking out rice isopropylmalate synthase genes OsIPMS1 and OsIPMS2 can improve both grain appearance and nutritional quality. We find that OsIPMS1 directly interacts with OsIPMS2 to form heterodimers. Meanwhile, we observe that OsIPMS1 and OsIPMS2 influence the expression of genes previously reported to be involved in the determination of grain size and nutritional quality in the developing panicles and grains. Furthermore, we show that Osipms1/2 double mutants exhibit significantly improved grain appearance and nutritional quality in polished rice in both the japonica (Wuyungeng 23) and indica (Huanghuazhan) varieties. Our findings indicate that OsIPMS is a useful target gene for breeding of rice varieties appealing for marketing and with health-benefiting properties.

The effect of epetraborole on the transcriptome and proteome profiles of an Escherichia coli strain overexpressing leuS, Leucyl-tRNA Synthetase

Epetraborole (EP) is a boron-containing antibiotic known for its effectiveness against gram-negative enteric bacteria and Mycobacterium species. It is designed to bind and inhibit the LeuS enzyme (Leucyl-tRNA Synthetase), which is encoded by the essential gene leuS in Escherichia coli. EP inhibits protein translation, impeding bacterial growth. However, when leuS is overexpressed in a recombinant plasmid, the amount of EP required for growth inhibition needs to be increased. This study explored the impact of EP on the transcriptome and proteome of E. coli overexpressing leuS, aiming to reveal additional gene and pathway insights beyond LeuS, shedding light on the biochemical players orchestrating the bacterium’s molecular response. 2D-PAGE Proteomics analysis identified four differentially regulated proteins influenced by EP in the leuS overexpression strain. Notably, LeuA and DeoA emerged as identified proteins. EP may affect LeuA in the cells overexpressing LeuS, which could result in truncated LeuA protein variants. Transcriptomics analyses, based on microarray data, revealed 23 up-regulated and 9 down-regulated genes responding to EP in the overexpression strain (p2). Based on the statistical analyses, the first five up-and down-regulated genes showing the highest fold differences in their mRNA levels are yiaW, mglB, narH, ybiO, flgB and yhdY, deoR, recX, yobB, potF, respectively. Analyses using the Omics Dashboard pathway and String indicate that the EP effect on the leuS overexpressing strain mainly induces alterations in the expression of genes related to the cell exterior, regulation, and response to stimuli. It is suggested that EP and higher levels of LeuS may interfere with the translational and transcriptional regulation of the expression of the leuA gene, which encodes the first enzyme, 2-isopropylmalate synthase, in L-leucine biosynthesis. This study offers new insights into the effects of EP on the bacterium, specifically when the level of the aminoacyl-tRNA synthetase LeuS is increased.

High-level and -yield production of L-leucine in engineered Escherichia coli by multistep metabolic engineering

L-leucine is an essential amino acid widely used in food and pharmaceutical industries. However, the relatively low production efficiency limits its large-scale application. In this study, we rationally developed an efficient L-leucine-producing Escherichia coli strain. Initially, the L-leucine synthesis pathway was enhanced by overexpressing feedback-resistant 2-isopropylmalate synthase and acetohydroxy acid synthase both derived from Corynebacterium glutamicum, along with two other native enzymes. Next, the pyruvate and acetyl-CoA pools were enriched by deleting competitive pathways, employing the nonoxidative glycolysis pathway, and dynamically modulating the citrate synthase activity, which significantly promoted the L-leucine production and yield to 40.69 g/L and 0.30 g/g glucose, respectively. Then, the redox flux was improved by substituting the native NADPH-dependent acetohydroxy acid isomeroreductase, branched chain amino acid transaminase, and glutamate dehydrogenase with their NADH-dependent equivalents. Finally, L-leucine efflux was accelerated by precise overexpression of the exporter and deletion of the transporter. Under fed-batch conditions, the final strain LXH-21 produced 63.29 g/L of L-leucine, with a yield and productivity of 0.37 g/g glucose and 2.64 g/(L h), respectively. To our knowledge, this study achieved the highest production efficiency of L-leucine to date. The strategies presented here will be useful for engineering E. coli strains for producing L-leucine and related products on an industrial scale.

Isopropylmalate synthase <i>NtIPMS</i> as a potential molecular marker for seed vigor in tobacco

Seed vigor is an important trait for tobacco production. However, the evaluation of seed vigor using molecular biomarkers is scarcely reported in tobacco. In this study, the development of molecular marker isopropylmalate synthase NtIPMS was conducted to detect seed ageing degree and seed priming effect in tobacco. Quantitative real-time PCR (qRT-PCR) analysis showed that the expression of NtIPMS was significantly induced at the initial imbibition stage during seed germination. The NtIPMS expression was positively correlated with the degree of seed ageing in non-pelleted and pelleted seeds. The mRNA level of NtIPMS was gradually increased with the increasing degree of seed ageing. The early best effect of gibberellin priming was observed in 30-h primed seeds, and the highest expression of NtIPMS was observed in 12-h primed seeds. The best stop time-point of seed priming is likely at the time 18 h after the relatively higher NtIPMS expression occurred during seed priming process. The NtIPMS mRNA detection has the potential usage as a potential molecular marker for the evaluation of seed vigor in tobacco.

Network and meta-omics reveal the cooperation patterns and mechanisms in an efficient 1,4-dioxane-degrading microbial consortium.

1,4-Dioxane is an emerging wastewater contaminant with probable human carcinogenicity. Our current understanding of microbial interactions during 1,4-dioxane biodegradation process in mixed cultures is limited. Here, we applied metagenomic, metatranscriptomic and co-occurrence network analyses to unraveling the microbial cooperation between degrader and non-degraders in an efficient 1,4-dioxane-degrading microbial consortium CH1. A 1,4-dioxane-degrading bacterium, Ancylobacter polymorphus ZM13, was isolated from CH1 and had a potential of being one of the important degraders due to its high relative abundance, highly expressed monooxygenase genes tmoABCDEF and high betweenness centrality of networks. The strain ZM13 cooperated obviously with 6 bacterial genera in the network, among which Xanthobacter and Mesorhizobium could be involved in the intermediates metabolism with responsible genes encoding alcohol dehydrogenase (adh), aldehyde dehydrogenase (aldh), glycolate oxidase (glcDEF), glyoxylate carboligase (gcl), malate synthase (glcB) and 2-isopropylmalate synthase (leuA) differentially high-expressed. Also, 1,4-dioxane facilitated the shift of biodiversity and function of CH1, and those cooperators cooperated with ZM13 in the way of providing amino acids or fatty acids, as well as relieving environmental stresses to promote biodegradation. These results provide new insights into our understandings of the microbial interactions during 1,4-dioxane degradation, and have important implications for predicting microbial cooperation and constructing efficient and stable synthetic 1,4-dioxane-degrading consortia for practical remediation.

Synthesis of 4-methylvaleric acid, a precursor of pogostone, involves a 2-isobutylmalate synthase related to 2-isopropylmalate synthase of leucine biosynthesis.

We show here that the side chain of pogostone, one of the major components of patchouli oil obtained from Pogostemon cablin and possessing a variety of pharmacological activities, is derived from 4-methylvaleric acid. We also show that 4-methylvaleric acid is produced through the one-carbon α-ketoacid elongation pathway with the involvement of the key enzyme 2-isobutylmalate synthase (IBMS), a newly identified enzyme related to isopropylmalate synthase (IPMS) of leucine (Leu) biosynthesis. Site-directed mutagenesis identified Met132 in the N-terminal catalytic region as affecting the substrate specificity of PcIBMS1. Even though PcIBMS1 possesses the C-terminal domain that in IPMS serves to mediate Leu inhibition, it is insensitive to Leu. The observation of the evolution of IBMS from IPMS, as well as previously reported examples of IPMS-related genes involved in making glucosinolates in Brassicaceae, acylsugars in Solanaceae, and flavour compounds in apple, indicate that IPMS genes represent an important pool for the independent evolution of genes for specialised metabolism.

Two alpha isopropylmalate synthase isozymes with similar kinetic properties are extant in the yeast Lachancea kluyveri.

The first committed step in the leucine biosynthetic pathway is catalyzed by α-isopropylmalate synthase (α-IPMS, EC 2.3.3.13), which in the Saccaromycotina subphylum of Ascomycete yeasts is frequently encoded by duplicated genes. Following a gene duplication event, the two copies may be preserved presumably because the encoded proteins diverge in either functional properties and/or cellular localization. The genome of the petite-negative budding yeast Lachancea kluyveri includes two SAKL0E10472 (LkLEU4) and SAKL0F05170g (LkLEU4BIS) paralogous genes, which are homologous to other yeast α-IPMS sequences. Here, we investigate whether these paralogous genes encode functional α-IPMS isozymes and whether their functions have diverged. Molecular phylogeny suggested that the LkLeu4 isozyme is located in the mitochondria and LkLeu4BIS in the cytosol. Comparison of growth rates, leucine intracellular pools and mRNA levels, indicate that the LkLeu4 isozyme is the predominant α-IPMS enzyme during growth on glucose as carbon source. Determination of the kinetic parameters indicates that the isozymes have similar affinities for the substrates and for the feedback inhibitor leucine. Thus, the diversification of the physiological roles of the genes LkLEU4 and LkLEU4BIS involves preferential transcription of the LkLEU4 gene during growth on glucose and different subcellular localization, although ligand interactions have not diverged.

Glucosinolate biosynthesis: role of MAM synthase and its perspectives.

Glucosinolates, synthesized by the glucosinolate biosynthesis pathway, are the secondary metabolites used as a defence mechanism in the Brassicaceae plants, including Arabidopsis thaliana. The first committed step in the pathway, catalysed by methylthioalkylmalate (MAM) synthase (EC: 2.3.3.17), is to produce different variants of glucosinolates. Phylogenetic analyses suggest that possibly MAM synthases have been evolved from isopropylmalate synthase (IPMS) by the substitutions of five amino acid residues (L143I, H167L, S216G, N250G and P252G) in the active site of IPMS due to point mutations. Considering the importance of MAM synthase in Brassicaceae plants, Petersen et al. (2019) made an effort to characterise the MAM synthase (15 MAM1 variants) in vitro by single substitution or double substitutions. In their study, the authors have expressed the variants in Escherichia coli and analysed the amino acids in the cultures of E. coli in vivo. Since modifying the MAM synthases by transgenic approaches could increase the resistance of Brassicaceae plants for enhancing the defence effect of glucosinolates and their degraded products; hence, MAM synthases should be characterized in detail in vivo in A. thaliana along with the structural analysis of the enzyme for meaningful impact and for its imminent use in vivo.

Free Amino Acids Profile and Expression Analysis of Core Genes Involved in Branched-Chain Amino Acids Metabolism during Fruit Development of Longan (Dimocarpus longan Lour.) Cultivars with Different Aroma Types.

Simple SummaryIn this study, three longan cultivars, including non-aroma types ‘Shixia’ (SX), ‘Lidongben’ (LDB), and strong aroma type ‘Xiangcui’ (XC), were selected to analyze free amino acids (FAAs) variations at six distinct growth stages. The genome-wide identification and expression analysis of genes related to the branched-chain amino acids (BCAA) synthesis pathway were carried out. Results showed that thirty-six FAAs were identified, which increased drastically with fruit development until ripening. During the period of rapid fruit expansion, the aroma of XC changed from light to strong, and the contents of L-alanine and L-leucine were significantly higher than those of SX and LDB. The content of Leu was negatively correlated with the expression of DilBCAT1, -6, and -9 in three varieties, but positively correlated with DilBCAT16, indicating that these four genes may be responsible for the different synthesis and degradation of Leu among cultivars.Amino acids are important component of fruit nutrition and quality. In this study, three longan cultivars, including non-aroma types ‘Shixia’ (SX), ‘Lidongben’ (LDB), and strong aroma type ‘Xiangcui’ (XC), were selected to analyze free amino acids (FAAs) variations at six distinct growth stages (S1–S6). The genome-wide identification and expression analysis of genes related to the branched-chain amino acids (BCAA) synthesis pathway were carried out. Results showed that 36 FAAs were identified, and the total FAAs content ranged from 2601.0 to 9073.5 mg/kg, which increased drastically with fruit development until ripening. L-glutamic acid (Glu), L-alanine (Ala), L-arginine (Arg), γ-Aminobutyric acid (GABA), L-aspartic acid (Asp), L-leucine (Leu), hydroxyl-proline (Hypro), and L-serine (Ser) were the predominant FAAs (1619.9–7213.9 mg/kg) in pulp, accounting for 62.28–92.05% of the total amino acids. During the period of rapid fruit expansion (S2–S4), the aroma of XC changed from light to strong, and the contents of L-alanine (Ala) and L-leucine (Leu) were significantly higher than those of SX and LDB. Furthermore, a total of two 2-isopropyl malate synthase (IPMS), two 3-isopropyl malate dehydrogenase (IPMD), and 16 BCAA transferase (BCAT) genes were identified. The expression levels of DilBCAT1, -6, and -9 genes in XC were significantly higher than those in SX and LDB, while DilBCAT16 in XC was lower. The content of Leu was negatively correlated with the expression of DilBCAT1, -6, and -9 in three varieties, but positively correlated with DilBCAT16, indicating that these four genes may be responsible for the different synthesis and degradation of Leu among cultivars.

Genome-wide identification and characterization of genes involved in the acylsugar pathway in tomato

Acylsugars are allelochemicals secreted by type IV trichomes in leaves and have been associated with resistance to arthropod pests in Solanaceae species. Eighty-seven putative genes were identified in Solanum lycopersicum and 77 in Solanum pennellii involved in the acylsugar pathway, including genes coding key proteins branched-chain keto acid dehydrogenase E2 (BCKD E2), 2-isopropylmalate synthase A (IPMSA), and threonine deaminase/dehydratase (TD). These putative tomato proteins displayed conservation in the conserved domains distribution and amino acids presence of catalytically active site comparing to homologous BCKD E2, IPMSA, and TD proteins from the acylsugar pathway. Phylogenetic analysis of the putative proteins BCKD E2, IPMSA, and TD showed that all putative tomato proteins grouped with their orthologs from the Solanaceae species, corroborating with the distribution of the tree of life. The BCKD E2 and the IPMSA transcripts from S. pennellii showed higher gene expression compared to S. lycopersicum transcripts, suggesting that the acylsugar pathway is more active in wild than in cultivated tomato. In addition, we checked the expression of these genes at three different times (30, 60 and 90 days after planting). The 60-day plants showed higher expression of these genes than the 30- and 90-day plants, which indicates a greater production of metabolites in the flowering stage and suggests that at this development stage the plant demands more defense against pests that may be attracted by flowers. Given the important role of the allelochemicals produced in Solanaceae, the results contribute to a better understanding of acylsugars and their processing pathways and open up opportunities to study their relationship with biotic resistance in these important species and other Solanaceae species.

A common F-box gene regulates the leucine homeostasis of Medicago truncatula and Arabidopsis thaliana.

The F-box domain is a conserved structural protein motif that most frequently interacts with the SKP1 protein, the core of the SCFs (SKP1-CULLIN-F-box protein ligase) E3 ubiquitin protein ligases. As part of the SCF complexes, the various F-box proteins recruit substrates for degradation through ubiquitination. In this study, we functionally characterized an F-box gene (MtF-box) identified earlier in a population of Tnt1 retrotransposon-tagged mutants of Medicago truncatula and its Arabidopsis thaliana homolog (AtF-box) using gain- and loss-of-function plants. We highlighted the importance of MtF-box in leaf development of M. truncatula. Protein-protein interaction analyses revealed the 2-isopropylmalate synthase (IPMS) protein as a common interactor partner of MtF-box and AtF-box, being a key enzyme in the biosynthesis pathway of the branched-chain amino acid leucine. For further detailed analysis, we focused on AtF-box and its role during the cell division cycle. Based on this work, we suggest a mechanism for the role of the studied F-box gene in regulation of leucine homeostasis, which is important for growth.

Citramalate synthase yields a biosynthetic pathway for isoleucine and straight- and branched-chain ester formation in ripening apple fruit

A plant pathway that initiates with the formation of citramalate from pyruvate and acetyl-CoA by citramalate synthase (CMS) is shown to contribute to the synthesis of α-ketoacids and important odor-active esters in apple (Malus × domestica) fruit. Microarray screening led to the discovery of a gene with high amino acid similarity to 2-isopropylmalate synthase (IPMS). However, functional analysis of recombinant protein revealed its substrate preference differed substantially from IPMS and was more typical of CMS. MdCMS also lacked the regulatory region present in MdIPMS and was not sensitive to feedback inhibition. 13C-acetate feeding of apple tissue labeled citramalate and α-ketoacids in a manner consistent with the presence of the citramalate pathway, labeling both straight- and branched-chain esters. Analysis of genomic DNA (gDNA) revealed the presence of two nearly identical alleles in "Jonagold" fruit (MdCMS_1 and MdCMS_2), differing by two nonsynonymous single-nucleotide polymorphisms (SNPs). The mature proteins differed only at amino acid 387, possessing either glutamine387 (MdCMS_1) or glutamate387 (MdCMS_2). Glutamate387 was associated with near complete loss of activity. MdCMS expression was fruit-specific, increasing severalfold during ripening. The translated protein product was detected in ripe fruit. Transient expression of MdCMS_1 in Nicotiana benthamiana induced the accumulation of high levels of citramalate, whereas MdCMS_2 did not. Domesticated apple lines with MdCMS isozymes containing only glutamate387 produced a very low proportion of 2-methylbutanol- and 2-methylbutanoate (2MB) and 1-propanol and propanoate (PROP) esters. The citramalate pathway, previously only described in microorganisms, is shown to function in ripening apple and contribute to isoleucine and 2MB and PROP ester biosynthesis without feedback regulation.

Comparative genomic analysis between newly sequenced Brucella abortus vaccine strain A19 and another Brucella abortus vaccine S19

BackgroundBrucellosis is a bacterial disease caused by Brucella infection. Brucella abortus strain A19 is a spontaneously attenuated vaccine strain that has been used in vaccination of cattle against brucellosis. Until now, the physiological and molecular mechanisms of A19 are still unknown. ResultsIn this paper, the whole-genome sequence of B. abortus A19 was performed using Illumina Hiseq 4000 and PacBio sequencing technology and comparative genomics analysis were carried out with the whole genome sequences of B. abortus strains S19. This analysis indicated that the two vaccine strains have a high degree of similarity in genomic structure. We further analysis of the difference in genomic structure between A19 and S19. And found some differential genes such as eryC, eryD and eryF. Of the other different proteins between A19 and S19, such as outer membrane protein, 2-isopropylmalate synthase, citramalate synthase, GntR family transcriptional regulator and ABC transporters, no clear effects related to bacterial virulence were found, pending further investigation. ConclusionThe data presented here provide a reasonable basis for designing Brucella vaccines that can be used in other strains.

Leucine biosynthesis is required for infection-related morphogenesis and pathogenicity in the rice blast fungus Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae causes one of the most devastating crop diseases world-wide and new control strategies for blast disease are urgently required. We have used insertional mutagenesis in M. oryzae to define biological processes that are critical for blast disease. Here, we report the identification of LEU2A by T-DNA mutagenesis, which putatively encodes 3-isopropylmalate dehydrogenase (3-IPMDH) required for leucine biosynthesis, implicating that synthesis of this amino acid is required for fungal pathogenesis. M. oryzae contains a further predicted 3-IPMDH gene (LEU2B), two 2-isopropylmalate synthase (2-IPMS) genes (LEU4 and LEU9) and an isopropylmalate isomerase (IPMI) gene (LEU1). Targeted gene deletion mutants of LEU1, LEU2A or LEU4 are leucine auxotrophs, and severely defective in pathogenicity. All phenotypes associated with mutants lacking LEU1, LEU2A or LEU4 could be overcome by adding exogenous leucine. The expression levels of LEU1, LEU2A or LEU4 genes were significantly down-regulated by deletion of the transcription factor gene LEU3, an ortholog of Saccharomyces cerevisiae LEU3. We also functionally characterized leucine biosynthesis genes in the wheat pathogen Fusarium graminearum and found that FgLEU1, FgLEU3 and FgLEU4 are essential for wheat head blight disease, suggesting that leucine biosynthesis in filamentous fungal pathogens may be a conserved factor for fungal pathogenicity and, therefore, a potential target for disease control.

Protein acetylation on 2-isopropylmalate synthase from Thermus thermophilus HB27.

Protein lysine Nε-acetylation is one of the important factors regulating cellular metabolism. We performed a proteomic analysis to identify acetylated proteins in the extremely thermophilic bacterium, Thermus thermophilus HB27. A total of 335 unique acetylated lysine residues, including many metabolic enzymes and ribosomal proteins, were identified in 208 proteins. Enzymes involved in amino acid metabolism were the most abundant among acetylated metabolic proteins. 2-Isopropylmalate synthase (IPMS), which catalyzes the first step in leucine biosynthesis, was acetylated at four lysine residues. Acetylation-mimicking mutations at Lys332 markedly decreased IPMS activity in vitro, suggesting that Lys332, which is located in subdomain II, plays a regulatory role in IPMS activity. We also investigated the acetylation-deacetylation mechanism of IPMS and revealed that it was acetylated non-enzymatically by acetyl-CoA and deacetylated enzymatically by TT_C0104. The present results suggest that leucine biosynthesis is regulated by post-translational protein modifications, in addition to feedback inhibition/repression, and that metabolic enzymes are regulated by protein acetylation in T. thermophilus.

Mutations in the Arabidopsis ROL17/isopropylmalate synthase 1 locus alter amino acid content, modify the TOR network, and suppress the root hair cell development mutant lrx1.

The growth and development of organisms must be tightly controlled and adjusted to nutrient availability and metabolic activities. The Target of Rapamycin (TOR) network is a major control mechanism in eukaryotes and influences processes such as translation, mitochondrial activity, production of reactive oxygen species, and the cytoskeleton. In Arabidopsis thaliana, inhibition of the TOR kinase causes changes in cell wall architecture and suppression of phenotypic defects of the cell wall formation mutant lrx1 (leucine-rich repeat extensin 1). The rol17 (repressor of lrx1 17) mutant was identified as a new suppressor of lrx1 that induces also a short root phenotype. The ROL17 locus encodes isopropylmalate synthase 1, a protein involved in leucine biosynthesis. Dependent on growth conditions, mutations in ROL17 do not necessarily alter the level of leucine, but always cause development of the rol17 mutant phenotypes, suggesting that the mutation does not only influence leucine biosynthesis. Changes in the metabolome of rol17 mutants are also found in plants with inhibited TOR kinase activity. Furthermore, rol17 mutants show reduced sensitivity to the TOR kinase inhibitor AZD-8055, indicating a modified TOR network. Together, these data suggest that suppression of lrx1 by rol17 is the result of an alteration of the TOR network.

Distinct mechanisms of substrate selectivity in the DRE-TIM metallolyase superfamily: A role for the LeuA dimer regulatory domain

The use of modular domains in proteins affords nature a simple route to the diversification of protein function, but co-evolution between domains can complicate large-scale functional annotation. The LeuA dimer regulatory domain is primarily responsible for allosteric feedback inhibition of the enzymes isopropylmalate synthase (IPMS) and citramalate synthase (CMS). In addition to this regulatory role, presence of the domain may also affect substrate selectivity in certain members of the enzyme family. To assess the role of the LeuA dimer regulatory domain in substrate selectivity, truncated versions of IPMS and CMS from Methanococcus jannaschii (MjIPMS and MjCMS, respectively) have been created that lack the LeuA dimer regulatory domain. In the case of MjIPMS, loss of the regulatory domain does not affect substrate selectivity, consistent with previous reports identifying conserved active site residues that play this role. Loss of the regulatory domain in MjCMS, however, results in increased functional promiscuity. Both truncated enzymes exhibit a shift in quaternary structure from tetrameric to monomeric forms as judged by size-exclusion chromatography. Kinetic isotope effects reveal that loss of the regulatory domain results in unique effects on catalysis with chemistry becoming more rate-determining in MjIPMS and less rate-determining in MjCMS. Finally, substitution of conserved active site residues in the promiscuous truncated MjCMS affect substrate selectivity while identical substitutions cause no changes in the wild-type enzyme. Overall, the data predicts a more complex role for the LeuA dimer regulatory domain in substrate selectivity through catalytic modulations rather than selectivity through differential binding as a result of extensive co-evolution between the catalytic and regulatory domains.

Metabolic engineering of Bacillus subtilis for l-valine overproduction.

Bacillus subtilis has been commonly applied to industrial enzyme production due to its genetic tractability, "generally recognized as safe (GRAS)" status, and robust growth characteristics. In spite of its ideal attributes as a biomanufacturing platform, B. subtilis has seen limited use in the production of other value-added biochemicals. Here, we report the derivation of engineered strains of B. subtilis for l-valine overproduction using our recently developed CRISPR (clustered regularly interspaced palindromic repeats)-Cas9 (CRISPR-associated [protein] 9) toolkit. We first manipulate the native l-valine biosynthetic pathway by relieving transcriptional and allosteric regulation, resulting in a >14-fold increase in the l-valine titer, compared to the wild-type strain. We subsequently identify and eliminate factors limiting l-valine overproduction, specifically increasing pyruvate availability and blocking the competing l-leucine and l-isoleucine biosynthetic pathways. By inactivating (a) pdhA, encoding the E1α subunit of the pyruvate dehydrogenase complex, to increase the intracellular pyruvate pool, and (b) leuA and ilvA, respectively encoding 2-isopropylmalate synthase and l-threonine dehydratase, to abolish the competing pathways, the l-valine titer reached 4.61 g/L in shake flask cultures. Our engineered l-valine-overproducing strains of B. subtilis are devoid of plasmids and do not sporulate due to the inactivation of sigF, encoding the sporulation-specific transcription factor σ F , making them attractive for large-scale l-valine production. However, acetate dissimilation was identified as limiting l-valine overproduction in ΔpdhA B. subtilis strains, and improving acetate dissimilation or identifying alternate modes of increasing pyruvate pools to enhance l-valine-overproduction should be explored.

Influence of isopropylmalate synthase OsIPMS1 on seed vigour associated with amino acid and energy metabolism in rice.

SummarySeed vigour is an imperative trait for the direct seeding of rice. Isopropylmalate synthase (IPMS) catalyses the committed step of leucine (Leu) biosynthesis, but its effect on seed vigour remains unclear. In this study, rice OsIPMS1 and OsIPMS2 was cloned, and the roles of OsIPMS1 in seed vigour were mainly investigated. OsIPMS1 and OsIPMS2 catalyse Leu biosynthesis, and Leu feedback inhibits their IPMS activities. Disruption of OsIPMS1 resulted in low seed vigour under various conditions, which might be tightly associated with the reduction of amino acids in germinating seeds. Eleven amino acids that associated with stress tolerance, GA biosynthesis and tricarboxylic acid (TCA) cycle were significantly reduced in osipms1 mutants compared with those in wide type (WT) during seed germination. Transcriptome analysis indicated that a total of 1209 differentially expressed genes (DEGs) were altered in osipms1a mutant compared with WT at the early germination stage, wherein most of the genes were involved in glycolysis/gluconeogenesis, protein processing, pyruvate, carbon, fructose and mannose metabolism. Further analysis confirmed that the regulation of OsIPMS1 in seed vigour involved in starch hydrolysis, glycolytic activity and energy levels in germinating seeds. The effects of seed priming were tightly associated with the mRNA levels of OsIPMS1 in priming seeds. The OsIPMS1 might be used as a biomarker to determine the best stop time‐point of seed priming in rice. This study provides novel insights into the function of OsIPMS1 on seed vigour and should have practical applications in seed priming of rice.

Characterization of two 2-isopropylmalate synthase homologs from Thermus thermophilus HB27

2-Isopropylmalate synthase (IPMS) catalyzes the first step of leucine biosynthesis and is regulated via feedback inhibition by leucine. The thermophilic bacterium, Thermus thermophilus HB27, has two IPMS homologous genes: TTC0847 and TTC0849, both of which are in the branched-chain amino acid biosynthetic gene cluster. Since enzymes involved in the leucine biosynthetic pathway are evolutionarily related to those in isoleucine biosynthesis, TTC0847 and TTC0849 are expected to function as IPMS or citramalate synthase, which is the first enzyme in the isoleucine biosynthetic pathway from pyruvate. We characterized these proteins in vitro and in vivo, and revealed that TTC0849 plays a key role in the biosynthesis of leucine and isoleucine, whereas TTC0847 is only involved in that of isoleucine.