Branched-chain amino acid synthesis is coupled to TOR activation early in the cell cycle in yeast.

How cells coordinate their metabolism with division determines the rate of cell proliferation. Dynamic patterns of metabolite synthesis during the cell cycle are unexplored. We report the first isotope tracing analysis in synchronous, growing budding yeast cells. Synthesis of leucine, a branched-chain amino acid (BCAA), increases through the G1 phase of the cell cycle, peaking later during DNA replication. Cells lacking Bat1, a mitochondrial aminotransferase that synthesizes BCAAs, grow slower, are smaller, and are delayed in the G1 phase, phenocopying cells in which the growth-promoting kinase complex TORC1 is moderately inhibited. Loss of Bat1 lowers the levels of BCAAs and reduces TORC1 activity. Exogenous provision of valine and, to a lesser extent, leucine to cells lacking Bat1 promotes cell division. Valine addition also increases TORC1 activity. In wild-type cells, TORC1 activity is dynamic in the cell cycle, starting low in early G1 but increasing later in the cell cycle. These results suggest a link between BCAA synthesis from glucose to TORC1 activation in the G1 phase of the cell cycle.

Conformational interdomain flexibility in a bacterial α-isopropylmalate synthase is necessary for leucine biosynthesis.

α-Isopropylmalate synthase (IPMS) catalyzes the first step in leucine (Leu) biosynthesis and is allosterically regulated by the pathway end product, Leu. IPMS is a dimeric enzyme with each chain consisting of catalytic, accessory, and regulatory domains, with the accessory and regulatory domains of each chain sitting adjacent to the catalytic domain of the other chain. The IPMS crystal structure shows significant asymmetry because of different relative domain conformations in each chain. Owing to the challenges posed by the dynamic and asymmetric structures of IPMS enzymes, the molecular details of their catalytic and allosteric mechanisms are not fully understood. In this study, we have investigated the allosteric feedback mechanism of the IPMS enzyme from the bacterium that causes meningitis, Neisseria meningitidis (NmeIPMS). By combining molecular dynamics simulations with small-angle X-ray scattering, mutagenesis, and heterodimer generation, we demonstrate that Leu-bound NmeIPMS is in a rigid conformational state stabilized by asymmetric interdomain polar interactions. Furthermore, we found removing these polar interactions by mutagenesis impaired the allosteric response without compromising Leu binding. Our results suggest that the allosteric inhibition of NmeIPMS is achieved by restricting the flexibility of the accessory and regulatory domains, demonstrating that significant conformational flexibility is required for catalysis.

Improvement of acetyl-CoA supply and glucose utilization increases l-leucine production in Corynebacterium glutamicum.

BACKGROUND: l-Leucine is an important essential amino acid with multiple industrial applications, whose market requirements cannot be met because of the low productivity. MAIN METHODS AND MAJOR RESULTS: In this study, a strain of Corynebacterium glutamicum with high l-leucine yield was constructed to enhance its acetyl-CoA supply and glucose utilization. One copy of leuA under the control of a strong promoter was incorporated into the C. glutamicum genome. Then, acetyl-CoA supply was increased by the integration of a terminator in front of gltA and by the heterogeneous overexpression of acetyl-CoA synthetase (ACS) and deacetylase (CobB) derived from Escherichia coli. Next, the transcriptional regulator sugR was deleted to enhance glucose uptake via a phosphotransferase-mediated route. In fed-batch fermentation performed in a 5-L reactor, l-leucine production of 40.11 ± 0.73 g L-1  was achieved under the optimized conditions, with an l-leucine yield and productivity of 0.25 g g-1 glucose and 0.59 g L-1 h-1 , respectively. CONCLUSIONS AND IMPLICATIONS: These results represent a significant improvement in the l-leucine production of C. glutamicum, indicating that the process possesses high potential for industrial application. These strategies can be also expanded to enable the production of other value-added biochemicals derived from the intermediates of central carbon metabolism.

Duplication and Functional Divergence of Branched-Chain Amino Acid Biosynthesis Genes in Aspergillus nidulans.

Fungi, bacteria, and plants, but not animals, synthesize the branched-chain amino acids: leucine, isoleucine, and valine. While branched-chain amino acid (BCAA) biosynthesis has been well characterized in the yeast Saccharomyces cerevisiae, it is incompletely understood in filamentous fungi. The three BCAAs share several early biosynthesis steps before divergence into specific pathways. In Aspergillus nidulans, the genes for the first two dedicated steps in leucine biosynthesis have been characterized, but the final two have not. We used sequence searches of the A. nidulans genome to identify two genes encoding ß-isopropylmalate dehydrogenase, which catalyzes the penultimate step of leucine biosynthesis, and six genes encoding BCAA aminotransferase, which catalyzes the final step in biosynthesis of all three BCAA. We have used combinations of gene knockouts to determine the relative contribution of each of these genes to BCAA biosynthesis. While both ß-isopropylmalate dehydrogenase genes act in leucine biosynthesis, the two most highly expressed BCAA aminotransferases are responsible for BCAA biosynthesis. We have also characterized the expression of leucine biosynthesis genes using reverse transcriptase-quantitative PCR and found regulation in response to leucine availability is mediated through the Zn(II)2Cys6 transcription factor LeuB. IMPORTANCE Branched-chain amino acid (BCAA) biosynthesis is important for pathogenic fungi to successfully cause disease in human and plant hosts. The enzymes for their production are absent from humans and, therefore, provide potential antifungal targets. While BCAA biosynthesis is well characterized in yeasts, it is poorly understood in filamentous fungal pathogens. Developing a thorough understanding of both the genes encoding the metabolic enzymes for BCAA biosynthesis and how their expression is regulated will inform target selection for antifungal drug development.

The anti-obesity effects exerted by different fractions of Artemisia sphaerocephala Krasch polysaccharide in diet-induced obese mice.

Artemisia sphaerocephala Krasch polysaccharide (ASKP) consists of two main fractions, 60P (molecular weight at 551 kDa) and 60S (molecular weight at 39 kDa). The anti-obesity effects of ASKP and its two fractions were investigated in high-fat-diet-fed mice and showed similar capability in efficiently preventing the development of obesity. The final body weight and body weight gain of obesity mice model were reduced by 12.44% and 35.33% by ASKP, 10.63% and 34.35% by 60P, and 7.82% and 20.04% by 60S. They also showed similar efficiency to ameliorate dyslipidemia, systematic inflammation, and gut dysbiosis. The colonic genes of barrier integrity were significantly upregulated and the genes of hepatic lipid metabolism and that of colonic inflammatory response were suppressed. They attenuated the gut dysbiosis in obese mice, such as the significant enrichment of beneficial genera (Bifidobacterium and Olsenella) and suppression of harmful ones (Mucispirillum and Helicobacter). Significant enrichment of carbohydrate metabolism associated with the promotion of short-chain fatty acid production and decrease of the metabolisms related to obesity and gut dysbiosis (valine, leucine, and isoleucine biosynthesis, and nitrogen metabolism) were also observed by the administration of ASKP, 60P, and 60S. Overall, these polysaccharides showed potential in acting as prebiotics in preventing high-fat-diet-induced obesity.

Effect of low-level ultrasound treatment on the production of L-leucine by Corynebacterium glutamicum in fed-batch culture.

Various process intensification methods were proposed to improve the yield, quality, and safety of fermented products. Here, we report the enhancement of L-leucine production by Corynebacterium glutamicum CP using ultrasound-assisted fed-batch fermentation. Response surface methodology was employed to optimize the sonication conditions. At an ultrasonic power density of 94 W/L, frequency of 25 kHz, interval of 31 min, and duration of 37 s, C. glutamicum CP produced 52.89 g/L of L-leucine in 44 h, representing a 21.6% increase compared with the control. The production performance of L-leucine was also improved under ultrasonic treatment. Moreover, the effects of ultrasound treatment on the fermentation performance of L-leucine were studied in terms of cell morphology, cell membrane permeability, and enzyme activity. The results indicate that ultrasonication is an efficient method for the intensification of L-leucine production by C. glutamicum CP.

Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine.

BACKGROUND: Biosynthesis of L-tert-leucine (L-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of L-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. RESULTS: In this work, a novel fusion enzyme (GDH-R3-LeuDH) for the efficient biosynthesis of L-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH-R3-LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of L-tle by GDH-R3-LeuDH was all enhanced by twofold. Finally, the space-time yield of L-tle catalyzing by GDH-R3-LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). CONCLUSIONS: It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize L-tle and reach the highest space-time yield up to now. These results demonstrated the great potential of the GDH-R3-LeuDH fusion enzyme for the efficient biosynthesis of L-tle.

Biosynthetic L-tert-leucine using Escherichia coli co-expressing a novel NADH-dependent leucine dehydrogenase and a formate dehydrogenase

BACKGROUND: L-tert-Leucine has been widely used in pharmaceutical, chemical, and other industries as a vital chiral intermediate. Compared with chemical methods, enzymatic methods to produce L-tert-leucine have unparalleled advantages. Previously, we found a novel leucine dehydrogenase from the halophilic thermophile Laceyella sacchari (LsLeuDH) that showed good thermostability and great potential for the synthesis of L-tertleucine in the preliminary study. Hence, we manage to use the LsLeuDH coupling with a formate dehydrogenase from Candida boidinii (CbFDH) in the biosynthesis of L-tert-leucine through reductive amination in the present study. RESULT: The double-plasmid recombinant strain exhibited higher conversion than the single-plasmid recombinant strain when resting cells cultivated in shake flask for 22 h were used. Under the optimized conditions, the double-plasmid recombinant E. coli BL21 (pETDute-FDH-LDH, pACYCDute-FDH) transformed 1 mol·L-1 trimethylpyruvate (TMP) completely into L-tert-leucine with greater than 99.9% ee within 8 h. CONCLUSIONS: The LsLeuDH showed great ability to biosynthesize L-tert-leucine. In addition, it provided a new option for the biosynthesis of L-tert-leucine.

Rational modification of the carbon metabolism of Corynebacterium glutamicum to enhance L-leucine production.

L-Leucine is an essential amino acid that has wide and expanding applications in the industry. It is currently fast-growing market demand that provides a powerful impetus to further increase its bioconversion productivity and production stability. In this study, we rationally engineered the metabolic flux from pyruvate to L-leucine synthesis in Corynebacterium glutamicum to enhance both pyruvate availability and L-leucine synthesis. First, the pyc (encoding pyruvate carboxylase) and avtA (encoding alanine-valine aminotransferase) genes were deleted to weaken the metabolic flux of the tricarboxylic acid cycle and reduce the competitive consumption of pyruvate. Next, the transcriptional level of the alaT gene (encoding alanine aminotransferase) was down regulated by inserting a terminator to balance L-leucine production and cell growth. Subsequently, the genes involved in L-leucine biosynthesis were overexpressed by replacing the native promoters PleuA and PilvBNC of the leuA gene and ilvBNC operon, respectively, with the promoter Ptuf of eftu (encoding elongation factor Tu) and using a shuttle expression vector. The resulting strain WL-14 produced 28.47 ± 0.36 g/L L-leucine in shake flask fermentation.

2,3-Dihydroxyisovalerate production by Klebsiella pneumoniae.

2,3-Dihydroxyisovalerate is an intermediate of valine and leucine biosynthesis pathway; however, no natural microorganism has been found yet that can accumulate this compound. Klebsiella pneumoniae is a useful bacterium that can be used as a workhorse for the production of a range of industrially desirable chemicals. Dihydroxy acid dehydratase, encoded by the ilvD gene, catalyzes the reaction of 2-ketoisovalerate formation from 2,3-dihydroxyisovalerate. In this study, an ilvD disrupted strain was constructed which resulted in the inability to synthesize 2-ketoisovalerate, yet accumulate 2,3-dihydroxyisovalerate in its culture broth. 2,3-Butanediol is the main metabolite of K. pneumoniae and its synthesis pathway and the branched-chain amino acid synthesis pathway share the same step of the α-acetolactate synthesis. By knocking out the budA gene, carbon flow into the branched-chain amino acid synthesis pathway was upregulated, which resulted in a distinct increase in 2,3-dihydroxyisovalerate levels. Lactic acid was identified as a by-product of the process and by blocking the lactic acid synthesis pathway, a further increase in 2,3-dihydroxyisovalerate levels was obtained. The culture parameters of 2,3-dihydroxyisovalerate fermentation were optimized, which include acidic pH and medium level oxygen supplementation to favor 2,3-dihydroxyisovalerate synthesis. At optimal conditions (pH 6.5, 400 rpm), 36.5 g/L of 2,3-dihydroxyisovalerate was produced in fed-batch fermentation over 45 h, with a conversion ratio of 0.49 mol/mol glucose. Thus, a biological route of 2,3-dihydroxyisovalerate production with high conversion ratio and final titer was developed, providing a basis for an industrial process. Key Points • A biological route of 2,3-dihydroxyisovalerate production was setup. • Disruption of budA causes 2,3-dihydroxuisovalerate accumulation in K. pneumoniae. • Disruption of ilvD prevents 2,3-dihydroxyisovalerate reuse by the cell. • 36.5 g/L of 2,3-dihydroxyisovalerate was obtained in fed-batch fermentation.

Deciphering the crucial roles of AraC-type transcriptional regulator Cgl2680 on NADPH metabolism and L-lysine production in Corynebacterium glutamicum.

Lysine is widely used in food, medical and feed industries. The biosynthesis of L-lysine is closely related to NADPH level, but the regulation mechanism between the biosynthesis of L-lysine in C. glutamicum and the cofactor NADPH is still not clear. Here, a high intracellular NADPH level strain C. glutamicum XQ-5Δpgi::(zwf-gnd) was constructed by blocking the glycolytic pathway and overexpressing the pentose phosphate pathway in the lysine-producing strain C. glutamicum XQ-5, and the intracellular NADPH level in strain XQ-5Δpgi::(zwf-gnd) was increased from 3.57 × 10-5 nmol/(104 cells) to 1.8 × 10-4 nmol/(104 cell). Transcriptome analyses pointed to Cgl2680 as an important regulator of NADPH levels and L-lysine biosynthesis in C. glutamicum. By knocking out the gene Cgl2680, the intracellular NADPH level of the recombinant C. glutamicum lysCfbr ΔCgl2680 was raised from 7.95 × 10-5 nmol/(104 cells) to 2.04 × 10-4 nmol/(104 cells), consequently leading to a 2.3-fold increase in the NADPH/NADP+ ratio. These results indicated that the regulator Cgl2680 showed the negative regulation for NADPH regeneration. In addition, Cgl2680-deficient strain C. glutamicum lysCfbr ΔCgl2680 showed the increase of yield of both L-lysine and L-leucine as well as the increase of H2O2 tolerance. Collectively, our data demonstrated that Cgl2680 plays an important role in negatively regulating NADPH regeneration, and these results provides new insights for breeding L-lysine or L-leucine high-yielding strain.

Mitochondrial Compartmentalization Confers Specificity to the 2-Ketoacid Recursive Pathway: Increasing Isopentanol Production in Saccharomyces cerevisiae.

Recursive elongation pathways produce compounds of increasing carbon-chain length with each iterative cycle. Of particular interest are 2-ketoacids derived from recursive elongation, which serve as precursors to a valuable class of advanced biofuels known as branched-chain higher alcohols (BCHAs). Protein engineering has been used to increase the number of iterative elongation cycles completed, yet specific production of longer-chain 2-ketoacids remains difficult to achieve. Here, we show that mitochondrial compartmentalization is an effective strategy to increase specificity of recursive pathways to favor longer-chain products. Using 2-ketoacid elongation as a proof of concept, we show that overexpression of the three elongation enzymes-LEU4, LEU1, and LEU2-in mitochondria of an isobutanol production strain results in a 2.3-fold increase in the isopentanol to isobutanol product ratio relative to overexpressing the same elongation enzymes in the cytosol, and a 31-fold increase relative to wild-type enzyme expression. Reducing the loss of intermediates allows us to further boost isopentanol production to 1.24 ± 0.06 g/L of isopentanol. In this strain, isopentanol accounts for 86% of the total BCHAs produced, while achieving the highest isopentanol titer reported for Saccharomyces cerevisiae. Localizing the elongation enzymes in mitochondria  enables the development of strains in which isopentanol constitutes as much as 93% of BCHA production. This work establishes mitochondrial compartmentalization as a new approach to favor high titers and product specificities of larger products from recursive pathways.

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.

The leucine biosynthetic pathway is crucial for adaptation to iron starvation and virulence in Aspergillus fumigatus.

In contrast to mammalia, fungi are able to synthesize the branched-chain amino acid leucine de novo. Recently, the transcription factor LeuB has been shown to cross-regulate leucine biosynthesis, nitrogen metabolism and iron homeostasis in Aspergillus fumigatus, the most common human mold pathogen. Moreover, the leucine biosynthetic pathway intermediate α-isopropylmalate (α-IPM) has previously been shown to posttranslationally activate LeuB homologs in S. cerevisiae and A. nidulans. Here, we demonstrate that in A. fumigatus inactivation of both leucine biosynthetic enzymes α-IPM synthase (LeuC), which disrupts α-IPM synthesis, and α-IPM isomerase (LeuA), which causes cellular α-IPM accumulation, results in leucine auxotrophy. However, compared to lack of LeuA, lack of LeuC resulted in increased leucine dependence, a growth defect during iron starvation and decreased expression of LeuB-regulated genes including genes involved in iron acquisition. Lack of either LeuA or LeuC decreased virulence in an insect infection model, and inactivation of LeuC rendered A. fumigatus avirulent in a pulmonary aspergillosis mouse model. Taken together, we demonstrate that the lack of two leucine biosynthetic enzymes, LeuA and LeuC, results in significant phenotypic consequences indicating that the regulator LeuB is activated by α-IPM in A. fumigatus and that the leucine biosynthetic pathway is an attractive target for the development of antifungal drugs.

MoLEU1, MoLEU2, and MoLEU4 regulated by MoLEU3 are involved in leucine biosynthesis, fungal development, and pathogenicity in Magnaporthe oryzae.

Amino acids are vital components in cell metabolism. Leucine is a regulatory factor that generates significant impact on protein synthesis/turnover, modulates diverse cellular signalling pathways and participates in oxidative processes and immune responses. Here, we identified and characterized the functions of a leucine-associated Zn2 Cys6 -type transcription factor, MoLeu3. Disruption of MoLEU3 resulted in significantly reduced pathogenicity in barley and rice. Quantitative RT-PCR showed that the expression levels of the putative leucine biosynthesis-related genes, MoLEU1, MoLEU2 and MoLEU4 were downregulated in the ΔMoleu3 mutant. We used high-throughput gene knockout method to generate the null mutants of MoLEU1, MoLEU2 and MoLEU4 respectively. The ΔMoleu1, ΔMoleu2 and ΔMoleu4 mutants are leucine auxotroph and showed similar phenotypic characterizations, including reduced conidiation, delayed mobilization and degradation of glycogen and lipid droplets, limited appressorium-mediated penetration, and restricted invasive hyphae growth within host cells. Collectively, MoLEU1, MoLEU2, and MoLEU4 regulated by MoLEU3 play crucial roles in fungal development and infectious processes through modulation of leucine biosynthesis in Magnaporthe oryzae.

The ilvGMEDA Operon Is Regulated by Transcription Attenuation in Vibrio alginolyticus ZJ-T.

Bacteria synthesize amino acids according to their availability in the environment or, in the case of pathogens, within the host. We explored the regulation of the biosynthesis of branched-chain amino acids (BCAAs) (l-leucine, l-valine, and l-isoleucine) in Vibrio alginolyticus, a marine fish and shellfish pathogen and an emerging opportunistic human pathogen. In this species, the ilvGMEDA operon encodes the main pathway for biosynthesis of BCAAs. Its upstream regulatory region shows no sequence similarity to the corresponding region in Escherichia coli or other Enterobacteriaceae, and yet we show that this operon is regulated by transcription attenuation. The translation of a BCAA-rich peptide encoded upstream of the structural genes provides an adaptive response similar to the E. coli canonical model. This study of a nonmodel Gram-negative organism highlights the mechanistic conservation of transcription attenuation despite the absence of primary sequence conservation.IMPORTANCE This study analyzes the regulation of the biosynthesis of branched-chain amino acids (leucine, valine, and isoleucine) in Vibrio alginolyticus, a marine bacterium that is pathogenic to fish and humans. The results highlight the conservation of the main regulatory mechanism with that of the enterobacterium Escherichia coli, suggesting that such a mechanism appeared early during the evolution of Gram-negative bacteria, allowing adaptation to a wide range of environments.

Metabolic engineering of l-leucine production in Escherichia coli and Corynebacterium glutamicum: a review.

l-Leucine, as an essential branched-chain amino acid for humans and animals, has recently been attracting much attention because of its potential for a fast-growing market demand. The applicability ranges from flavor enhancers, animal feed additives and ingredients in cosmetic to specialty nutrients in pharmaceutical and medical fields. Microbial fermentation is the major method for producing l-leucine by using Escherichia coli and Corynebacterium glutamicum as host bacteria. This review gives an overview of the metabolic pathway of l-leucine (i.e. production, import and export systems) and highlights the main regulatory mechanisms of operons in E. coli and C. glutamicum l-leucine biosynthesis. We summarize here the current trends in metabolic engineering techniques and strategies for manipulating l-leucine producing strains. Finally, future perspectives to construct industrially advantageous strains are considered with respect to recent advances in biology.

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.

Complete Genome Sequence of Streptomyces olivoreticuli ATCC 31159 Which can Produce Anticancer Bestatin and Show Diverse Secondary Metabolic Potentials.

Because of its competitive inhibitor activity against aminopeptidase B, bestatin isolated from the broth of Streptomyces olivoreticuli ATCC 31159 is famous and currently used as an approved therapeutic agent for cancer and bacterial infections. It can be used alone or in combination with other antibiotics or anticancer drugs as adjuvant therapy drug for chemotherapy and radiotherapy. Due to the therapeutic importance of bestatin, mining of its biosynthetic mechanism is imperative. Genome mining, one of the bioinformatics-based approaches for the discovery of novel natural product, has been developed and applied widely. Herein, we reported the complete genome of Streptomyces olivoreticuli ATCC 31159 obtained from American Type Culture Collection (ATCC). It consists of 8,809,793 base pairs with a linear chromosome, GC content of 71.1%, 7520 protein-coding genes, 75 tRNA operons, 21 rRNA operons, 63 sRNAs. In addition, predictive analysis showed that at least 37 putative biosynthetic gene clusters (BGCs) of the secondary metabolites were obtained, 18 new BGCs with low similarity (< 25%) were included. The availability of novel and abundant gene clusters not only will provide clues for cracking the biosynthetic mechanism of bestatin, but also will provide valuable insight for mining the diverse bioactive compounds based on rational strategies.

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.