Amino Acid Biosynthesis Pathways Research Articles

Urea Is Both a Carbon and Nitrogen Source for Microcystis aeruginosa: Tracking 13C Incorporation at Bloom pH Conditions.

The use of urea as a nitrogenous fertilizer has increased over the past two decades, with urea itself being readily detected at high concentrations in many lakes. Urea has been linked to cyanobacterial blooms as it is a readily assimilated nitrogen (N) - source for cyanobacteria that possess the enzyme urease. We tested the hypothesis that urea may also act as a carbon (C) source to supplemental growth requirements during the alkaline conditions created by dense cyanobacterial blooms, when concentrations of dissolved CO2 are vanishingly low. High rates of photosynthesis markedly reduce dissolved CO2 concentrations and drive up pH. This was observed in Lake Erie during the largest bloom on record (2015) over long periods (months) and short periods (days) of time, suggesting blooms experience periods of CO2-limitation on a seasonal and daily basis. We used 13C-urea to demonstrate that axenic cultures of the model toxic cyanobacterium, Microcystis aeruginosa NIES843, assimilated C at varying environmentally relevant pH conditions directly into a spectrum of metabolic pools during urea hydrolysis. Primarily, 13C from urea was assimilated into central C metabolism and amino acid biosynthesis pathways, including those important for the production of the hepatotoxin, microcystin, and incorporation into these pathways was at a higher percentage during growth at higher pH. This corresponded to increased growth rates on urea as the sole N source with increasing pH. We propose this ability to incorporate C from urea represents yet another competitive advantage for this cyanobacterium during dense algal blooms.

Quantitative Proteomic and Transcriptomic Analyses of Metabolic Regulation of Adult Reproductive Diapause in Drosophila suzukii (Diptera: Drosophilidae) Females.

Diapause is a form of dormancy used by many insects to survive adverse environmental conditions, which can occur in specific developmental stages in different species. Drosophila suzukii is a serious economic pest and we determined the conditions for adult reproductive diapause by the females in our previous studies. In this study, we combined RNA-Seq transcriptomic and quantitative proteomic analyses to identify adult reproductive diapause-related genes and proteins. According to the transcriptomic analysis, among 242 annotated differentially expressed genes in non-diapause and diapause females, 129 and 113 genes were up- and down-regulated, respectively. In addition, among the 2,375 proteins quantified, 39 and 23 proteins were up- and down-regulated, respectively. The gene expression patterns in diapause- and non-diapause were confirmed by qRT-PCR or western blot analysis. The overall analysis of robustly regulated genes at the protein and mRNA levels found four genes that overlapped in the up-regulated group and six genes in the down-regulated group, and thus these proteins/genes may regulate adult reproductive diapause. These differentially expressed proteins/genes act in the citrate cycle, insulin signaling pathway, PI3K-Akt signaling pathway, and amino acid biosynthesis pathways. These results provide the basis for further studies of the molecular regulation of reproductive diapause in this species.

Transcriptome profiling of Gossypium arboreum during fiber initiation and the genome-wide identification of trihelix transcription factors

Cotton fiber initiation is the first step in fiber development, and it determines the yield. Here, genome-wide transcriptome profiling of Gossypium arboreum was performed to determine the molecular basis of cotton fiber initiation. A comparison of the transcriptomes of fiber-bearing ovules at −0.5, 0, 0.5, 1, 1.5, 2, 2.5 and 3 d post-anthesis detected 12,049 differentially expressed genes that mainly participated in ribosome, carbon metabolism and amino acid biosynthesis pathways. Genes encoding alcohol dehydrogenase 1 and hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase, involving in fatty acid degradation and flavonoid biosynthesis, were enriched. Furthermore, 1049 differentially expressed transcription factors were identified. Among these, 17 were trihelix family transcription factors, which play important roles in plant development and responses to biotic and abiotic stresses. In total, 52 full-length trihelix genes, named as GaGTs, were identified in G. arboreum and located in 12 of the 13 cotton chromosomes. Transcriptomic data and a quantitative real-time PCR analysis indicated that several GaGTs were significantly induced during fiber initiation in G. arboreum. Thus, the genome-wide comprehensive analysis of gene expression in G. arboreum fiber initiation will serve as a useful resource for unraveling the functions of specific genes. The phylogenetic relationships and expression analyses of the G. arboreum trihelix genes established a solid foundation for future comprehensive functional analyses of the GaGTs.

Key Maize Drought-Responsive Genes and Pathways Revealed by Comparative Transcriptome and Physiological Analyses of Contrasting Inbred Lines.

To unravel the molecular mechanisms underpinning maize (Zea mays L.) drought stress tolerance, we conducted comprehensive comparative transcriptome and physiological analyses of drought-tolerant YE8112 and drought-sensitive MO17 inbred line seedlings that had been exposed to drought treatment for seven days. Resultantly, YE8112 seedlings maintained comparatively higher leaf relative water and proline contents, greatly increased peroxidase activity, but decreased malondialdehyde content, than MO17 seedlings. Using an RNA sequencing (RNA-seq)-based approach, we identified a total of 10,612 differentially expressed genes (DEGs). From these, we mined out four critical sets of drought responsive DEGs, including 80 specific to YE8112, 5140 shared between the two lines after drought treatment (SD_TD), five DEGs of YE8112 also regulated in SD_TD, and four overlapping DEGs between the two lines. Drought-stressed YE8112 DEGs were primarily associated with nitrogen metabolism and amino-acid biosynthesis pathways, whereas MO17 DEGs were enriched in the ribosome pathway. Additionally, our physiological analyses results were consistent with the predicted RNA-seq-based findings. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) analysis and the RNA-seq results of twenty representative DEGs were highly correlated (R2 = 98.86%). Crucially, tolerant line YE8112 drought-responsive genes were predominantly implicated in stress signal transduction; cellular redox homeostasis maintenance; MYB, NAC, WRKY, and PLATZ transcriptional factor modulated; carbohydrate synthesis and cell-wall remodeling; amino acid biosynthesis; and protein ubiquitination processes. Our findings offer insights into the molecular networks mediating maize drought stress tolerance.

Metabolomic and Immunological Profiling of Respiratory Syncytial Virus Infection after Intranasal Immunization with a Subunit Vaccine Candidate.

Respiratory syncytial virus (RSV) is a significant cause of mortality and morbidity in infants, the elderly, immunocompromised individuals, and patients with congenital heart diseases. Despite extensive efforts, a vaccine against RSV is still not available. We have previously reported the development of a subunit vaccine (ΔF/TriAdj) composed of a truncated version of the fusion protein (ΔF) and a polymer-based combination adjuvant (TriAdj). We compared inflammatory responses of ΔF/TriAdj-vaccinated and unvaccinated mice following intranasal challenge with RSV. Rapid and early inflammatory responses were observed in lung samples from both groups but modulated in the vaccinated group 7 days after the viral challenge. The underlying mechanism of action of ΔF/TriAdj was further studied through LC-MS-based metabolomic profiling by using 12C- or 13C-dansyl labeling for the amine/phenol submetabolome. RSV infection predominantly affected the amino acid biosynthesis pathways and urea cycle, whereas ΔF/TriAdj modulated the concentrations of almost all of the altered metabolites. Tryptophan metabolites were significantly affected, including indole, l-kynurenine, xanthurenic acid, serotonin, 5-hydroxyindoleacetic acid, and 6-hydroxymelatonin. The results from the present study provide further mechanistic insights into the mode of action of this RSV vaccine candidate and have important implications in the design of metabolic therapeutic interventions.

The unique composition of Indian gut microbiome, gene catalogue, and associated fecal metabolome deciphered using multi-omics approaches.

BackgroundMetagenomic studies carried out in the past decade have led to an enhanced understanding of the gut microbiome in human health; however, the Indian gut microbiome has not been well explored. We analyzed the gut microbiome of 110 healthy individuals from two distinct locations (North-Central and Southern) in India using multi-omics approaches, including 16S rRNA gene amplicon sequencing, whole-genome shotgun metagenomic sequencing, and metabolomic profiling of fecal and serum samples.ResultsThe gene catalogue established in this study emphasizes the uniqueness of the Indian gut microbiome in comparison to other populations. The gut microbiome of the cohort from North-Central India, which was primarily consuming a plant-based diet, was found to be associated with Prevotella and also showed an enrichment of branched chain amino acid (BCAA) and lipopolysaccharide biosynthesis pathways. In contrast, the gut microbiome of the cohort from Southern India, which was consuming an omnivorous diet, showed associations with Bacteroides, Ruminococcus, and Faecalibacterium and had an enrichment of short chain fatty acid biosynthesis pathway and BCAA transporters. This corroborated well with the metabolomics results, which showed higher concentration of BCAAs in the serum metabolome of the North-Central cohort and an association with Prevotella. In contrast, the concentration of BCAAs was found to be higher in the fecal metabolome of the Southern-India cohort and showed a positive correlation with the higher abundance of BCAA transporters.ConclusionsThe study reveals the unique composition of the Indian gut microbiome, establishes the Indian gut microbial gene catalogue, and compares it with the gut microbiome of other populations. The functional associations revealed using metagenomic and metabolomic approaches provide novel insights on the gut-microbe-metabolic axis, which will be useful for future epidemiological and translational researches.

Discovery and evaluation of novel Mycobacterium tuberculosis ketol-acid reductoisomerase inhibitors as therapeutic drug leads.

Tuberculosis (TB) remains a major threat to human health. This due to the fact that current drug treatments are less than optimal and the increasing occurrence of multi drug-resistant strains of etiological agent, Mycobacterium tuberculosis (Mt). Given the wide-spread significance of this disease, we have undertaken a design and evaluation program to discover new anti-TB drug leads. Here, we focused on ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid biosynthesis pathway. Importantly, this enzyme is present in bacteria but not in humans, making it an attractive proposition for drug discovery. In the present work, we used molecular docking to identify seventeen potential inhibitors of KARI using an in-house database. Compounds were selected based on docking scores, which were assigned as the result of favourable interactions between the compound and the active site of KARI. The inhibitory constant values for two leads, compounds 14 and 16 are 3.71 and 3.06µM respectively. To assess the mode of binding, 100ns molecular dynamics simulations for these two compounds in association with Mt KARI were performed and showed that the complex was stable with an average root mean square deviation of less than 3.5Å for all atoms. Furthermore, compound 16 showed a minimum inhibitory concentration of 2.06 ± 0.91µM and a 1.9 fold logarithmic reduction in the growth of Mt in an infected macrophage model. The two compounds exhibited low toxicity against RAW 264.7 cell lines. Thus, both compounds are promising candidates for development as an anti-TB drug leads.

Allosteric Feedback Inhibition Enables Robust Amino Acid Biosynthesis in E.coli by Enforcing Enzyme Overabundance.

SummaryMicrobes must ensure robust amino acid metabolism in the face of external and internal perturbations. This robustness is thought to emerge from regulatory interactions in metabolic and genetic networks. Here, we explored the consequences of removing allosteric feedback inhibition in seven amino acid biosynthesis pathways in Escherichia coli (arginine, histidine, tryptophan, leucine, isoleucine, threonine, and proline). Proteome data revealed that enzyme levels decreased in five of the seven dysregulated pathways. Despite that, flux through the dysregulated pathways was not limited, indicating that enzyme levels are higher than absolutely needed in wild-type cells. We showed that such enzyme overabundance renders the arginine, histidine, and tryptophan pathways robust against perturbations of gene expression, using a metabolic model and CRISPR interference experiments. The results suggested a sensitive interaction between allosteric feedback inhibition and enzyme-level regulation that ensures robust yet efficient biosynthesis of histidine, arginine, and tryptophan in E. coli.

Chloroplast proteome analysis of Nicotiana tabacum overexpressing TERF1 under drought stress condition

BackgroundChloroplast is indispensable for plant response to environmental stresses, growth and development, whose function is regulated by different plant hormones. The chloroplast proteome is encoded by chloroplast genome and nuclear genome, which play essential roles in plant photosynthesis, metabolism and other biological processes. Ethylene response factors (ERFs) are key transcription factors in activating the ethylene signaling pathway and plant response to abiotic stress. But we know little about how ethylene regulates plastid function under drought stress condition. In this study we utilized tobacco overexpressing tomato ethylene responsive factor 1 (TERF1), an ERF transcription factor isolated from tomato, to investigate its effects on the plastid proteome under drought stress condition by method of iTRAQ technology.ResultsResults show that TERF1 represses the genes encoding the photosynthetic apparatus at both transcriptional and translational level, but the genes involved in carbon fixation are significantly induced by TERF1. TERF1 regulates multiple retrograde signaling pathways, providing a new mechanism for regulating nuclear gene expression. TERF1 also regulates plant utilization of phosphorus (Pi) and nitrogen (N). We find that several metabolic and signaling pathways related with Pi are significantly repressed and gene expression analysis shows that TERF1 significantly represses the Pi transport from root to shoot. However, the N metabolism is upregulated by TERF1 as shown by the activation of different amino acids biosynthesis pathways due to the induction of glutamine synthetase and stabilization of nitrate reductase although the root-to-shoot N transport is also reduced. TERF1 also regulates other core metabolic pathways and secondary metabolic pathways that are important for plant growth, development and response to environmental stresses. Gene set linkage analysis was applied for the upregulated proteins by TERF1, showing some new potential for regulating plant response to drought stress by TERF1.ConclusionsOur research reveals effects of ethylene signaling on plastid proteome related with two key biological processes, including photosynthesis and nutrition utilization. We also provide a new mechanism to regulate nuclear gene expression by ERF1 transcription factor through retrograde signals in chloroplast. These results can enrich our knowledge about ERF1 transcription factor and function of ethylene signaling pathway.

Lifestyle of Lactobacillus hordei isolated from water kefir based on genomic, proteomic and physiological characterization

Water kefir is a traditional fermented beverage made from sucrose, water, kefir granules, dried or fresh fruits. In our water kefir granules, Lactobacillus (L.) hordei is one of the predominant lactic acid bacteria (LAB) species of this presumed symbiotic consortium. It faces abundant sucrose versus limitation of amino- and fatty acids in an acidic environment. Sequencing of the genome of L. hordei TMW 1.1822 revealed one chromosome plus three plasmids. The size of the chromosome was 2.42 Mbp with a GC content of 35% GC and 2461 predicted coding sequences. Furthermore, we identified 1474 proteins upon growth on water kefir medium. Metabolic prediction revealed all enzymes required for the glycolytic Embden-Meyerhof (EMP) and phosphoketolase (PKP) pathways. Genes encoding all enzymes involved in citrate, pyruvate and mannitol metabolism are present.Moreover, it was confirmed that L. hordei is prototrophic for 11 amino acids and auxotrophic for 6 amino acids when combining putative biosynthesis pathways for amino acids with physiological characterization. Still, for glycine, serine and methionine no sure auxotype could be determined. The OppABCDF peptide transport system is complete, and 13 genes encoding peptidases are present. The arginine deiminase system, was predicted to be complete except for carbamate kinase, thus enabling neutralization reactions via ammonium formation but no additional energy generation. Taken together our findings enable prediction of the L. hordei lifestyle in water kefir: Abundant sucrose is consumed directly via parallel EMP and PK pathways and is also extracellularly converted to dextran and fructose by a glucansucrase, leaving fructose as additional carbon source. Essential amino acids (in the form of peptides) and citrate are acquired from fruits. In the lack of FabB unsaturated fatty acids are synthesized by predicted alternative enzymes. Formation of acetoin and diacetyl as well as arginine conversion reactions enable acidification limitation. Other members of the water kefir consortium (yeasts, acetic acid bacteria) likely facilitate or support growth of L. hordei by delivering gluconate, mannitol, amino- and fatty acids and vitamins.

Genome-scale fitness profile of Caulobacter crescentus grown in natural freshwater.

Bacterial genomes evolve in complex ecosystems and are best understood in this natural context, but replicating such conditions in the lab is challenging. We used transposon sequencing to define the fitness consequences of gene disruption in the bacterium Caulobacter crescentus grown in natural freshwater, compared with axenic growth in common laboratory media. Gene disruptions in amino-acid and nucleotide sugarbiosynthesis pathways and in metabolic substrate transport machinery impaired fitness in both lake water and defined minimal medium relative to complex peptone broth. Fitness in lake water was enhanced by insertions in genes required for flagellum biosynthesis and reduced by insertions in genes involved in biosynthesis of the holdfast surface adhesin. We further uncovered numerous hypothetical and uncharacterized genes for which disruption impaired fitness in lake water, defined minimal medium, or both. At the genome scale, the fitness profile of mutants cultivated in lake water was more similar to that in complex peptone broth than in defined minimal medium. Microfiltration of lake water did not significantly affect the terminal cell density or the fitness profile of the transposon mutant pool, suggesting that Caulobacter does not strongly interact with other microbes in this ecosystem on the measured timescale. Fitness of select mutants with defects in cell surface biosynthesis and environmental sensing were significantly more variable across daysin lake water than in defined medium, presumably owing to day-to-day heterogeneity in the lake environment. This study reveals genetic interactions between Caulobacter and a natural freshwater environment, and provides a new avenue to study gene function in complex ecosystems.

High-Resolution Metabolomics Study of Occupational Exposure to Trichloroethylene

Introduction: Trichloroethylene (TCE) is a common water contaminant and continues to be used in the workplaces of many developing countries. It causes kidney cancer and possibly non-Hodgkin lymphoma and liver cancer. However, the underlying pathophysiology of TCE-induced cancer is not well understood. To evaluate biological responses to TCE, we conducted a cross-sectional molecular epidemiology study of workers exposed to TCE and unexposed controls and applied non-targeted metabolomics analysis.Methods: We studied 80 healthy workers that used TCE and 95 comparable unexposed controls in Guangdong, China. The TCE-exposed workers had a median 8-hour time weighted average personal air exposure of 12 ppm (range: 0.4 to 230 ppm), with almost all workers having exposure under the current U.S. OSHA Permissible Exposure Limit of 100 ppm. Metabolomics analysis of plasma samples was conducted by ultra-high resolution mass spectrometryResults: Metabolic features associated with TCE exposure included known TCE metabolites and additional unidentified chlorinated compounds. TCE exposure was also associated with multiple alterations in endogenous metabolism, including changes in purine catabolism and decreases in sulphur amino acid and bile acid biosynthesis pathways. Specific metabolite associations with TCE exposure included uric acid, glutamine, cystine, methylthioadenosine, taurine and chenodeoxycholic acid, consistent with immunosuppressive, hepatotoxic and nephrotoxic effects that have been related to TCE. There were also correlations between multiple metabolites and additional exposure, immunologic, and nephrotoxic biomarkers that had been measured previously.Conclusion: Untargeted high-resolution metabolomics correlated occupational exposure to TCE with internal dose and metabolic responses, providing new insights into molecular mechanisms of TCE-related disease.

Exploration of the Regulatory Mechanism of Secondary Metabolism by Comparative Transcriptomics in Aspergillus flavus.

Mycotoxins cause a huge threaten to agriculture, food safety, and human and animal life. Among them, aflatoxins (AFs) have always been considered the most potent carcinogens, and filamentous fungi from Aspergillus genus are their major producers, especially A. flavus. Although the biosynthesis path of these chemicals had been well-identified, the regulatory mechanisms controlling expression of AF gene cluster were poorly understood. In this report, genome-wide transcriptome profiles of A. flavus from AF conducing [yeast sucrose media (YES)] and non-conducing [yeast peptone media (YEP)] conditions were compared by using deep RNA sequencing (RNA-seq), and the results revealed that AF biosynthesis pathway and biosynthesis of amino acids were significantly upregulated in YES vs. YEP. Further, a novel LaeA-like methyltransferase AFLA_121330 (Lael1) was identified for the first time, to play a specific role in the regulation of AF biosynthesis. Contrary to LaeA, which gene deletion reduced the level, lael1 deletion resulted in a significant increase in AF production. Further, co-expression network analysis revealed that mitochondrial pyruvate transport and signal peptide processing were potentially involved in AF synthesis for the first time, as well as biological processes of ribosome, branched-chain amino acid biosynthetic process and translation were co-regulated by AfRafA and AfStuA. To sum up, our analyses could provide novel insights into the molecular mechanism for controlling the AF and other secondary metabolite synthesis, adding novel targets for plant breeding and making fungicides.

Phenotype and RNA-seq-Based transcriptome profiling of Staphylococcus aureus biofilms in response to tea tree oil

Staphylococcus aureus (S. aureus) is a Gram-positive bacterium that causes a wide range of diseases, including food poisoning. Tea tree oil (TTO), an essential oil distilled from Melaleuca alternifolia, is well-known for its antibacterial activities. TTO effectively inhibited all 19 tested strains of S. aureus biofilm and planktonic cells. Phenotype analyses of S. aureus biofilm cells exposed to TTO were performed by biofilm adhesion assays, eDNA detection and PIA release. RNA sequencing (RNA-seq) was used in our study to elucidate the mechanism of TTO as a potential antibacterial agent to evaluate differentially expressed genes (DEGs) and the functional network in S. aureus ATCC 29213 biofilms. TTO significantly changed (greater than a 2- or less than a 2-fold change) the expression of 304 genes in S. aureus contained in biofilms. The levels of genes related to the glycine, serine and threonine metabolism pathway, purine metabolism pathway, pyrimidine metabolism pathway and amino acid biosynthesis pathway were dramatically changed in the biofilm exposed to TTO. Furthermore, the expression changes identified by RNA-seq analysis were verified by real-time RT-PCR. To the best of our knowledge, this research is the first study to report the phenotype and expression profiles of S. aureus in biofilms exposed to TTO.

Metabolic engineering of Escherichia coli W3110 strain by incorporating genome-level modifications and synthetic plasmid modules to enhance L-Dopa production from glycerol

L-Tyrosine which is one of the terminal metabolites of highly regulated aromatic amino-acid biosynthesis pathway in Escherichia coli is a precursor for synthesis of L-Dopa. In this study, we report over production of L-Dopa by enhancing expression of rate limiting isoenzyme of shikimate kinase (aroL), chorismate synthase (aroC), aromatic-amino-acid aminotransferase (tyrB) and 3-phosphoshikimate 1-carboxyvinyltransferase (aroA) form a plasmid module harboring five enzymes under two inducible promoters converting shikimate to tyrosine. 4-hydroxyphenylacetate-3-hydrolase (hpaBC) which converts L-Tyrosine to L-Dopa was expressed constitutively from a separate plasmid module. Feedback deregulated expression of 3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (aroG*) replacing wild type aroG under its natural promoter led to enhancement of L-Dopa production. Deletion of transcriptional repressor tyrR and links to other competing pathways improved titers of L-Dopa. We focused on having a balanced flux by constitutive expression of pathway enzymes from plasmid constructs rather than achieving higher amounts of catalytic protein by induction. We observed glycerol when used as a carbon source for the final strain led to low acid production. The best performing strain led to decoupling of acid production and product formation in bioreactor. Fed batch analysis of the final strain led to 12.5 g/L of L-Dopa produced in bioreactor.

Enumerating all possible biosynthetic pathways in metabolic networks

Exhaustive identification of all possible alternate pathways that exist in metabolic networks can provide valuable insights into cellular metabolism. With the growing number of metabolic reconstructions, there is a need for an efficient method to enumerate pathways, which can also scale well to large metabolic networks, such as those corresponding to microbial communities. We developed MetQuest, an efficient graph-theoretic algorithm to enumerate all possible pathways of a particular size between a given set of source and target molecules. Our algorithm employs a guided breadth-first search to identify all feasible reactions based on the availability of the precursor molecules, followed by a novel dynamic-programming based enumeration, which assembles these reactions into pathways of a specified size producing the target from the source. We demonstrate several interesting applications of our algorithm, ranging from identifying amino acid biosynthesis pathways to identifying the most diverse pathways involved in degradation of complex molecules. We also illustrate the scalability of our algorithm, by studying large graphs such as those corresponding to microbial communities, and identify several metabolic interactions happening therein. MetQuest is available as a Python package, and the source codes can be found at https://github.com/RamanLab/metquest.

Comparative transcription profiling of two fermentation cultures of Xanthomonas campestris pv. campestris B100 sampled in the growth and in the stationary phase.

The ɣ-proteobacterium Xanthomonas campestris pv. campestris (Xcc) is the producer of the biopolymer xanthan, a polysaccharide which is used as a thickener in numerous industrial applications. In this study, we present a global transcriptome profiling of two Xcc strain B100 cultures obtained from fermentation during the growth phase and the subsequent stationary phase associated with xanthan biosynthesis. During the xanthan production phase, highly abundant transcripts belonged to genes encoding for small RNAs, glycogen biosynthesis, and xanthan export. A total of 1850 (40%) genes were differentially transcribed during the stationary phase where 924 were transcriptionally up-regulated and 926 genes were down-regulated. An overview of differentially transcribed genes includes a significant down-regulation of genes involved in transcription, translation, and amino acid biosynthesis pathways. A group of up-regulated genes was involved in cellular response against oxidative stress, such as those coding for superoxide dismutase and catalase. Genes encoding enzymes involved in nucleotide sugar precursor synthesis of xanthan biosynthesis, such as xanA, galU, and ugd, exhibited a transcription pattern that did not change during the growth and stationary phase. Regarding the transcription pattern of the gum gene cluster that govern xanthan biosynthesis, a significant up-regulation of the genes gumB, gumC, and gumD was observed, while the transcript pools of the genes gumG, gumH, gumI, and gumJ were reduced and those of genes gumE, gumF, gumK, gumL, and gumM remained un-changed during the stationary phase compared to the growth phase. The obtained data represents the first analysis of gene expression patterns under xanthan production conditions and provides the bases for future studies aiming at enhancing xanthan yield.

Establishing an innovative carbohydrate metabolic pathway for efficient production of 2-keto-l-gulonic acid in Ketogulonicigenium robustum initiated by intronic promoters

Background2-Keto-l-gulonic acid (2-KGA), the precursor of vitamin C, is currently produced by two-step fermentation. In the second step, l-sorbose is transformed into 2-KGA by the symbiosis system composed of Ketogulonicigenium vulgare and Bacillus megaterium. Due to the different nutrient requirements and the uncertain ratio of the two strains, the symbiosis system significantly limits strain improvement and fermentation optimization.ResultsIn this study, Ketogulonicigenium robustum SPU_B003 was reported for its capability to grow well independently and to produce more 2-KGA than that of K. vulgare in a mono-culture system. The complete genome of K. robustum SPU_B003 was sequenced, and the metabolic characteristics were analyzed. Compared to the four reported K. vulgare genomes, K. robustum SPU_B003 contained more tRNAs, rRNAs, NAD and NADP biosynthetic genes, as well as regulation- and cell signaling-related genes. Moreover, the amino acid biosynthesis pathways were more complete. Two species-specific internal promoters, P1 (orf_01408 promoter) and P2 (orf_02221 promoter), were predicted and validated by detecting their initiation activity. To efficiently produce 2-KGA with decreased CO2 release, an innovative acetyl-CoA biosynthetic pathway (XFP-PTA pathway) was introduced into K. robustum SPU_B003 by expressing heterologous phosphoketolase (xfp) and phosphotransacetylase (pta) initiated by internal promoters. After gene optimization, the recombinant strain K. robustum/pBBR-P1_xfp2502-P2_pta2145 enhanced acetyl-CoA approximately 2.4-fold and increased 2-KGA production by 22.27% compared to the control strain K. robustum/pBBR1MCS-2. Accordingly, the transcriptional level of the 6-phosphogluconate dehydrogenase (pgd) and pyruvate dehydrogenase genes (pdh) decreased by 24.33 ± 6.67 and 8.67 ± 5.51%, respectively. The key genes responsible for 2-KGA biosynthesis, sorbose dehydrogenase gene (sdh) and sorbosone dehydrogenase gene (sndh), were up-regulated to different degrees in the recombinant strain.ConclusionsThe genome-based functional analysis of K. robustum SPU_B003 provided a new understanding of the specific metabolic characteristics. The new XFP-PTA pathway was an efficient route to enhance acetyl-CoA levels and to therefore promote 2-KGA production.

Structural insights into the mechanism of inhibition of AHAS by herbicides

Acetohydroxyacid synthase (AHAS), the first enzyme in the branched amino acid biosynthesis pathway, is present only in plants and microorganisms, and it is the target of >50 commercial herbicides. Penoxsulam (PS), which is a highly effective broad-spectrum AHAS-inhibiting herbicide, is used extensively to control weed growth in rice crops. However, the molecular basis for its inhibition of AHAS is poorly understood. This is despite the availability of structural data for all other classes of AHAS-inhibiting herbicides. Here, crystallographic data for Saccharomyces cerevisiae AHAS (2.3 Å) and Arabidopsis thaliana AHAS (2.5 Å) in complex with PS reveal the extraordinary molecular mechanisms that underpin its inhibitory activity. The structures show that inhibition of AHAS by PS triggers expulsion of two molecules of oxygen bound in the active site, releasing them as substrates for an oxygenase side reaction of the enzyme. The structures also show that PS either stabilizes the thiamin diphosphate (ThDP)-peracetate adduct, a product of this oxygenase reaction, or traps within the active site an intact molecule of peracetate in the presence of a degraded form of ThDP: thiamine aminoethenethiol diphosphate. Kinetic analysis shows that PS inhibits AHAS by a combination of events involving FAD oxidation and chemical alteration of ThDP. With the emergence of increasing levels of resistance toward front-line herbicides and the need to optimize the use of arable land, these data suggest strategies for next generation herbicide design.

Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes

Amino acid biosynthesis pathways observed in nature typically require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine: serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (CysK/CysM). To solve this chicken-and-egg problem, we substituted alternate amino acids in CysE, CysK and CysM for cysteine and methionine, which are the only two sulfur-containing proteinogenic amino acids. Using a cysteine-dependent auxotrophic E. coli strain, CysE function was rescued by cysteine-free and methionine-deficient enzymes, and CysM function was rescued by cysteine-free enzymes. CysK function, however, was not rescued in either case. Enzymatic assays showed that the enzymes responsible for rescuing the function in CysE and CysM also retained their activities in vitro. Additionally, substitution of the two highly conserved methionines in CysM decreased but did not eliminate overall activity. Engineering amino acid biosynthetic enzymes to lack the so-produced amino acids can provide insights into, and perhaps eventually fully recapitulate via a synthetic approach, the biogenesis of biotic amino acids.