Adaptive laboratory evolution of Clostridium autoethanogenum to metabolize CO2 and H2 enhances growth rates in chemostat and unravels proteome and metabolome alterations.

Gas fermentation of CO2 and H2 is an attractive means to sustainably produce fuels and chemicals. Clostridium autoethanogenum is a model organism for industrial CO to ethanol and presents an opportunity for CO2-to-ethanol processes. As we have previously characterized its CO2/H2 chemostat growth, here we use adaptive laboratory evolution (ALE) with the aim of improving growth with CO2/H2. Seven ALE lineages were generated, all with improved specific growth rates. ALE conducted in the presence of 2% CO along with CO2/H2 generated Evolved lineage D, which showed the highest ethanol titres amongst all the ALE lineages during the fermentation of CO2/H2. Chemostat comparison against the parental strain shows no change in acetate or ethanol production, while Evolved D could achieve a higher maximum dilution rate. Multi-omics analyses at steady state revealed that Evolved D has widespread proteome and intracellular metabolome changes. However, the uptake and production rates and titres remain unaltered until investigating their maximum dilution rate. Yet, we provide numerous insights into CO2/H2 metabolism via these multi-omics data and link these results to mutations, suggesting novel targets for metabolic engineering in this bacterium.

Role of the substrate on Ni inhibition in biological sulfate reduction.

In treating mine-impacted waters using sulfate-reducing bacteria (SRB), metal inhibition and substrate selection are important factors affecting the efficiency of the bioprocess. This work investigated the role of the substrate (i.e. lactate, formate, glycerol and glucose) on Ni inhibition to SRB with sulfate-reducing activity tests at initial pH 5, 7 and 9 and 100 mg/L of Ni. Results indicated that the type of substrate was a significant factor affecting Ni inhibition in SRB, which was the most negligible in the lactate system, followed by glycerol, glucose, and formate. Although less significant, Ni inhibition also varied with the pH, leading for instance, to a reduction of 77% in the sulfate reducing activity for the formate system, but only of 28% for lactate at pH 5. The added substrate also influenced the precipitation kinetics and the characteristics of the precipitates, reaching Ni precipitation extents above 95%, except for glucose (83.2%).

Multi-omic characterisation of Streptomyces hygroscopicus NRRL 30439: detailed assessment of its secondary metabolic potential.

The emergence of multidrug-resistant pathogenic bacteria creates a demand for novel antibiotics with distinct mechanisms of action. Advances in next-generation genome sequencing promised a paradigm shift in the quest to find new bioactive secondary metabolites. Genome mining has proven successful for predicting putative biosynthetic elements in secondary metabolite superproducers such as Streptomycetes. However, genome mining approaches do not inform whether biosynthetic gene clusters are dormant or active under given culture conditions. Here we show that using a multi-omics approach in combination with antiSMASH, it is possible to assess the secondary metabolic potential of a Streptomyces strain capable of producing mannopeptimycin, an important cyclic peptide effective against Gram-positive infections. The genome of Streptomyces hygroscopicus NRRL 30439 was first sequenced using PacBio RSII to obtain a closed genome. A chemically defined medium was then used to elicit a nutrient stress response in S. hygroscopicus NRRL 30439. Detailed extracellular metabolomics and intracellular proteomics were used to profile and segregate primary and secondary metabolism. Our results demonstrate that the combination of genomics, proteomics and metabolomics enables rapid evaluation of a strain's performance in bioreactors for industrial production of secondary metabolites.

Network Analyses Predict Small RNAs That Might Modulate Gene Expression in the Testis and Epididymis of Bos indicus Bulls.

Spermatogenesis relies on complex molecular mechanisms, essential for the genesis and differentiation of the male gamete. Germ cell differentiation starts at the testicular parenchyma and finishes in the epididymis, which has three main regions: head, body, and tail. RNA-sequencing data of the testicular parenchyma (TP), head epididymis (HE), and tail epididymis (TE) from four bulls (three biopsies per bull: 12 samples) were subjected to differential expression analyses, functional enrichment analyses, and co-expression analyses. The aim was to investigate the co-expression and infer possible regulatory roles for transcripts involved in the spermatogenesis of Bos indicus bulls. Across the three pairwise comparisons, 3,826 differentially expressed (DE) transcripts were identified, of which 384 are small RNAs. Functional enrichment analysis pointed to gene ontology (GO) terms related to ion channel activity, detoxification of copper, neuroactive receptors, and spermatogenesis. Using the regulatory impact factor (RIF) algorithm, we detected 70 DE small RNAs likely to regulate the DE transcripts considering all pairwise comparisons among tissues. The pattern of small RNA co-expression suggested that these elements are involved in spermatogenesis regulation. The 3,826 DE transcripts (mRNAs and small RNAs) were further subjected to co-expression analyses using the partial correlation and information theory (PCIT) algorithm for network prediction. Significant correlations underpinned the co-expression network, which had 2,216 transcripts connected by 158,807 predicted interactions. The larger network cluster was enriched for male gamete generation and had 15 miRNAs with significant RIF. The miRNA bta-mir-2886 showed the highest number of connections (601) and was predicted to down-regulate ELOVL3, FEZF2, and HOXA13 (negative co-expression correlations and confirmed with TargetScan). In short, we suggest that bta-mir-2886 and other small RNAs might modulate gene expression in the testis and epididymis, in Bos indicus cattle.

Time-course transcriptomics reveals that amino acids catabolism plays a key role in toxinogenesis and morphology in Clostridium tetani.

Tetanus is a fatal disease caused by Clostridium tetani infections. To prevent infections, a toxoid vaccine, developed almost a century ago, is routinely used in humans and animals. The vaccine is listed in the World Health Organisation list of Essential Medicines and can be produced and administered very cheaply in the developing world for less than one US Dollar per dose. Recent developments in both analytical tools and frameworks for systems biology provide industry with an opportunity to gain a deeper understanding of the parameters that determine C. tetani virulence and physiological behaviour in bioreactors. Here, we compared a traditional fermentation process with a fermentation medium supplemented with five heavily consumed amino acids. The experiment demonstrated that amino acid catabolism plays a key role in the virulence of C. tetani. The addition of the five amino acids favoured growth, decreased toxin production and changed C. tetani morphology. Using time-course transcriptomics, we created a "fermentation map", which shows that the tetanus toxin transcriptional regulator BotR, P21 and the tetanus toxin gene was downregulated. Moreover, this in-depth analysis revealed potential genes that might be involved in C. tetani virulence regulation. We observed differential expression of genes related to cell separation, surface/cell adhesion, pyrimidine biosynthesis and salvage, flagellar motility, and prophage genes. Overall, the fermentation map shows that, mediated by free amino acid concentrations, virulence in C. tetani is regulated at the transcriptional level and affects a plethora of metabolic functions.

A Pan-Genome Guided Metabolic Network Reconstruction of Five Propionibacterium Species Reveals Extensive Metabolic Diversity.

Propionibacteria have been studied extensively since the early 1930s due to their relevance to industry and importance as human pathogens. Still, their unique metabolism is far from fully understood. This is partly due to their signature high GC content, which has previously hampered the acquisition of quality sequence data, the accurate annotation of the available genomes, and the functional characterization of genes. The recent completion of the genome sequences for several species has led researchers to reassess the taxonomical classification of the genus Propionibacterium, which has been divided into several new genres. Such data also enable a comparative genomic approach to annotation and provide a new opportunity to revisit our understanding of their metabolism. Using pan-genome analysis combined with the reconstruction of the first high-quality Propionibacterium genome-scale metabolic model and a pan-metabolic model of current and former members of the genus Propionibacterium, we demonstrate that despite sharing unique metabolic traits, these organisms have an unexpected diversity in central carbon metabolism and a hidden layer of metabolic complexity. This combined approach gave us new insights into the evolution of Propionibacterium metabolism and led us to propose a novel, putative ferredoxin-linked energy conservation strategy. The pan-genomic approach highlighted key differences in Propionibacterium metabolism that reflect adaptation to their environment. Results were mathematically captured in genome-scale metabolic reconstructions that can be used to further explore metabolism using metabolic modeling techniques. Overall, the data provide a platform to explore Propionibacterium metabolism and a tool for the rational design of strains.

A snapshot of microbial diversity and function in an undisturbed sugarcane bagasse pile.

BACKGROUND: Sugarcane bagasse is a major source of lignocellulosic biomass, yet its economic potential is not fully realised. To add value to bagasse, processing is needed to gain access to the embodied recalcitrant biomaterials. When bagasse is stored in piles in the open for long periods it is colonised by microbes originating from the sugarcane, the soil nearby or spores in the environment. For these microorganisms to proliferate they must digest the bagasse to access carbon for growth. The microbial community in bagasse piles is thus a potential resource for the discovery of useful and novel microbes and industrial enzymes. We used culturing and metabarcoding to understand the diversity of microorganisms found in a uniquely undisturbed bagasse storage pile and screened the cultured organisms for fibre-degrading enzymes. RESULTS: Samples collected from 60 to 80 cm deep in the bagasse pile showed hemicellulose and partial lignin degradation. One hundred and four microbes were cultured from different layers and included a high proportion of oleaginous yeast and biomass-degrading fungi. Overall, 70, 67, 70 and 57% of the microbes showed carboxy-methyl cellulase, xylanase, laccase and peroxidase activity, respectively. These percentages were higher in microbes selectively cultured from deep layers, with all four activities found for 44% of these organisms. Culturing and amplicon sequencing showed that there was less diversity and therefore more selection in the deeper layers, which were dominated by thermophiles and acid tolerant organisms, compared with the top of pile. Amplicon sequencing indicated that novel fungi were present in the pile. CONCLUSIONS: A combination of culture-dependent and independent methods was successful in exploring the diversity in the bagasse pile. The variety of species that was found and that are known for biomass degradation shows that the bagasse pile was a valuable selective environment for the identification of new microbes and enzymes with biotechnological potential. In particular, lignin-modifying activities have not been reported previously for many of the species that were identified, suggesting future studies are warranted.

A TetR-Family Protein (CAETHG_0459) Activates Transcription From a New Promoter Motif Associated With Essential Genes for Autotrophic Growth in Acetogens.

Acetogens can fix carbon (CO or CO2) into acetyl-CoA via the Wood-Ljungdahl pathway (WLP) that also makes them attractive cell factories for the production of fuels and chemicals from waste feedstocks. Although most biochemical details of the WLP are well understood and systems-level characterization of acetogen metabolism has recently improved, key transcriptional features such as promoter motifs and transcriptional regulators are still unknown in acetogens. Here, we use differential RNA-sequencing to identify a previously undescribed promoter motif associated with essential genes for autotrophic growth of the model-acetogen Clostridium autoethanogenum. RNA polymerase was shown to bind to the new promoter motif using a DNA-binding protein assay and proteomics enabled the discovery of four candidates to potentially function directly in control of transcription of the WLP and other key genes of C1 fixation metabolism. Next, in vivo experiments showed that a TetR-family transcriptional regulator (CAETHG_0459) and the housekeeping sigma factor (σA) activate expression of a reporter protein (GFP) in-frame with the new promoter motif from a fusion vector in Escherichia coli. Lastly, a protein-protein interaction assay with the RNA polymerase (RNAP) shows that CAETHG_0459 directly binds to the RNAP. Together, the data presented here advance the fundamental understanding of transcriptional regulation of C1 fixation in acetogens and provide a strategy for improving the performance of gas-fermenting bacteria by genetic engineering.

Systems-level engineering and characterisation of Clostridium autoethanogenum through heterologous production of poly-3-hydroxybutyrate (PHB).

Gas fermentation is emerging as an economically attractive option for the sustainable production of fuels and chemicals from gaseous waste feedstocks. Clostridium autoethanogenum can use CO and/or CO2 + H2 as its sole carbon and energy sources. Fermentation of C. autoethanogenum is currently being deployed on a commercial scale for ethanol production. Expanding the product spectrum of acetogens will enhance the economics of gas fermentation. To achieve efficient heterologous product synthesis, limitations in redox and energy metabolism must be overcome. Here, we engineered and characterised at a systems-level, a recombinant poly-3-hydroxybutyrate (PHB)-producing strain of C. autoethanogenum. Cells were grown in CO-limited steady-state chemostats on two gas mixtures, one resembling syngas (20% H2) and the other steel mill off-gas (2% H2). Results were characterised using metabolomics and transcriptomics, and then integrated using a genome-scale metabolic model reconstruction. PHB-producing cells had an increased expression of the Rnf complex, suggesting energy limitations for heterologous production. Subsequent optimisation of the bioprocess led to a 12-fold increase in the cellular PHB content. The data suggest that the cellular redox state, rather than the acetyl-CoA pool, was limiting PHB production. Integration of the data into the genome-scale metabolic model showed that ATP availability limits PHB production. Altogether, the data presented here advances the fundamental understanding of heterologous product synthesis in gas-fermenting acetogens.

From reconstruction to C4 metabolic engineering: A case study for overproduction of polyhydroxybutyrate in bioenergy grasses.

The compartmentalization of C4 plants increases photosynthetic efficiency, while constraining how material and energy must flow in leaf tissues. To capture this metabolic phenomenon, a generic plant metabolic reconstruction was replicated into four connected spatiotemporal compartments, namely bundle sheath (B) and mesophyll (M) across the day and night cycle. The C4 leaf model was used to explore how amenable polyhydroxybutyrate (PHB) production is with these four compartments working cooperatively. A strategic pattern of metabolite conversion and exchange emerged from a systems-level network that has very few constraints imposed; mainly the sequential two-step carbon capture in mesophyll, then bundle sheath and photosynthesis during the day only. The building of starch reserves during the day and their mobilization during the night connects day and night metabolism. Flux simulations revealed that PHB production did not require rerouting of metabolic pathways beyond what is already utilised for growth. PHB yield was sensitive to photoassimilation capacity, availability of carbon reserves, ATP maintenance, relative photosynthetic activity of B and M, and type of metabolites exchanged in the plasmodesmata, but not sensitive towards compartmentalization. Hence, the compartmentalization issues currently encountered are likely to be kinetic or thermodynamic limitations rather than stoichiometric.

RNA-Seq Highlights High Clonal Variation in Monoclonal Antibody Producing CHO Cells.

The development of next-generation sequencing technologies has opened new opportunities to better characterize complex eukaryotic cells. Chinese hamster ovary (CHO) cells play a primary role in therapeutic protein production, with currently five of the top ten blockbuster drugs produced in CHO. However, engineering superior CHO cells with improved production features has had limited success to date and cell lines are still developed through the generation and screening of large strain pools. Here, we applied RNA sequencing to contrast a high and a low monoclonal antibody producing cell line. Rigorous experimental design achieved high reproducibility between biological replicates, remarkably reducing variation to less than 10%. More than 14 000 gene-transcripts are identified and surprisingly 58% are classified as differentially expressed, including 2900 with a fold change higher than 1.5. The largest differences are found for gene-transcripts belonging to regulation of apoptosis, cell death, and protein intracellular transport GO term classifications, which are found to be significantly enriched in the high producing cell line. RNA sequencing is also performed on subclones, where down-regulation of genes encoding secreted glycoproteins is found to be the most significant change. The large number of significant differences even between subclones challenges the notion of identifying and manipulating a few key genes to generate high production CHO cell lines.

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris.

Pichia pastoris (syn. Komagataella phaffii) is one of the most common eukaryotic expression systems for heterologous protein production. Expression cassettes are typically integrated in the genome to obtain stable expression strains. In contrast to Saccharomyces cerevisiae, where short overhangs are sufficient to target highly specific integration, long overhangs are more efficient in P. pastoris and ectopic integration of foreign DNA can occur. Here, we aimed to elucidate the influence of ectopic integration by high-throughput screening of >700 transformants and whole-genome sequencing of 27 transformants. Different vector designs and linearization approaches were used to mimic the most common integration events targeted in P. pastoris Fluorescence of an enhanced green fluorescent protein (eGFP) reporter protein was highly uniform among transformants when the expression cassettes were correctly integrated in the targeted locus. Surprisingly, most nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression. However, a few clones (<10%) harboring ectopically integrated cassettes showed a greater variation spanning a 25-fold range, surpassing specifically integrated reference strains up to 6-fold. High-expression strains showed a correlation between increased gene copy numbers and high reporter protein fluorescence levels. Our results suggest that for comparing expression levels between strains, the integration locus can be neglected as long as a sufficient numbers of transformed strains are compared. For expression optimization of highly expressible proteins, increasing copy number appears to be the dominant positive influence rather than the integration locus, genomic rearrangements, deletions, or single-nucleotide polymorphisms (SNPs).IMPORTANCE Yeasts are commonly used as biotechnological production hosts for proteins and metabolites. In the yeast Saccharomyces cerevisiae, expression cassettes carrying foreign genes integrate highly specifically at the targeted sites in the genome. In contrast, cassettes often integrate at random genomic positions in nonconventional yeasts, such as Pichia pastoris (syn. Komagataella phaffii). Hence, cells from the same transformation event often behave differently, with significant clonal variation necessitating the screening of large numbers of strains. The importance of this study is that we systematically investigated the influence of integration events in more than 700 strains. Our findings provide novel insight into clonal variation in P. pastoris and, thus, how to avoid pitfalls and obtain reliable results. The underlying mechanisms may also play a role in other yeasts and hence could be generally relevant for recombinant yeast protein production strains.

Maintenance of ATP Homeostasis Triggers Metabolic Shifts in Gas-Fermenting Acetogens.

Acetogens are promising cell factories for producing fuels and chemicals from waste feedstocks via gas fermentation, but quantitative characterization of carbon, energy, and redox metabolism is required to guide their rational metabolic engineering. Here, we explore acetogen gas fermentation using physiological, metabolomics, and transcriptomics data for Clostridium autoethanogenum steady-state chemostat cultures grown on syngas at various gas-liquid mass transfer rates. We observe that C. autoethanogenum shifts from acetate to ethanol production to maintain ATP homeostasis at higher biomass concentrations but reaches a limit at a molar acetate/ethanol ratio of ∼1. This regulatory mechanism eventually leads to depletion of the intracellular acetyl-CoA pool and collapse of metabolism. We accurately predict growth phenotypes using a genome-scale metabolic model. Modeling revealed that the methylene-THF reductase reaction was ferredoxin reducing. This work provides a reference dataset to advance the understanding and engineering of arguably the first carbon fixation pathway on Earth.

Improved production of propionic acid using genome shuffling.

Traditionally derived from fossil fuels, biological production of propionic acid has recently gained interest. Propionibacterium species produce propionic acid as their main fermentation product. Production of other organic acids reduces propionic acid yield and productivity, pointing to by-products gene-knockout strategies as a logical solution to increase yield. However, removing by-product formation has seen limited success due to our inability to genetically engineer the best producing strains (i.e. Propionibacterium acidipropionici). To overcome this limitation, random mutagenesis continues to be the best path towards improving strains for biological propionic acid production. Recent advances in next generation sequencing opened new avenues to understand improved strains. In this work, we use genome shuffling on two wild type strains to generate a better propionic acid producing strain. Using next generation sequencing, we mapped the genomic changes leading to the improved phenotype. The best strain produced 25% more propionic acid than the wild type strain. Sequencing of the strains showed that genomic changes were restricted to single point mutations and gene duplications in well-conserved regions in the genomes. Such results confirm the involvement of gene conversion in genome shuffling as opposed to long genomic insertions.

Metabolic Reconstruction of Setaria italica: A Systems Biology Approach for Integrating Tissue-Specific Omics and Pathway Analysis of Bioenergy Grasses.

The urgent need for major gains in industrial crops productivity and in biofuel production from bioenergy grasses have reinforced attention on understanding C4 photosynthesis. Systems biology studies of C4 model plants may reveal important features of C4 metabolism. Here we chose foxtail millet (Setaria italica), as a C4 model plant and developed protocols to perform systems biology studies. As part of the systems approach, we have developed and used a genome-scale metabolic reconstruction in combination with the use of multi-omics technologies to gain more insights into the metabolism of S. italica. mRNA, protein, and metabolite abundances, were measured in mature and immature stem/leaf phytomers, and the multi-omics data were integrated into the metabolic reconstruction framework to capture key metabolic features in different developmental stages of the plant. RNA-Seq reads were mapped to the S. italica resulting for 83% coverage of the protein coding genes of S. italica. Besides revealing similarities and differences in central metabolism of mature and immature tissues, transcriptome analysis indicates significant gene expression of two malic enzyme isoforms (NADP- ME and NAD-ME). Although much greater expression levels of NADP-ME genes are observed and confirmed by the correspondent protein abundances in the samples, the expression of multiple genes combined to the significant abundance of metabolites that participates in C4 metabolism of NAD-ME and NADP-ME subtypes suggest that S. italica may use mixed decarboxylation modes of C4 photosynthetic pathways under different plant developmental stages. The overall analysis also indicates different levels of regulation in mature and immature tissues in carbon fixation, glycolysis, TCA cycle, amino acids, fatty acids, lignin, and cellulose syntheses. Altogether, the multi-omics analysis reveals different biological entities and their interrelation and regulation over plant development. With this study, we demonstrated that this systems approach is powerful enough to complement the functional metabolic annotation of bioenergy grasses.

Genomic characterization of the uncultured Bacteroidales family S24-7 inhabiting the guts of homeothermic animals.

BACKGROUND: Our view of host-associated microbiota remains incomplete due to the presence of as yet uncultured constituents. The Bacteroidales family S24-7 is a prominent example of one of these groups. Marker gene surveys indicate that members of this family are highly localized to the gastrointestinal tracts of homeothermic animals and are increasingly being recognized as a numerically predominant member of the gut microbiota; however, little is known about the nature of their interactions with the host. RESULTS: Here, we provide the first whole genome exploration of this family, for which we propose the name "Candidatus Homeothermaceae," using 30 population genomes extracted from fecal samples of four different animal hosts: human, mouse, koala, and guinea pig. We infer the core metabolism of "Ca. Homeothermaceae" to be that of fermentative or nanaerobic bacteria, resembling that of related Bacteroidales families. In addition, we describe three trophic guilds within the family, plant glycan (hemicellulose and pectin), host glycan, and α-glucan, each broadly defined by increased abundance of enzymes involved in the degradation of particular carbohydrates. CONCLUSIONS: "Ca. Homeothermaceae" representatives constitute a substantial component of the murine gut microbiota, as well as being present within the human gut, and this study provides important first insights into the nature of their residency. The presence of trophic guilds within the family indicates the potential for niche partitioning and specific roles for each guild in gut health and dysbiosis.

Systems biology and metabolic modelling unveils limitations to polyhydroxybutyrate accumulation in sugarcane leaves; lessons for C4 engineering.

In planta production of the bioplastic polyhydroxybutyrate (PHB) is one important way in which plant biotechnology can address environmental problems and emerging issues related to peak oil. However, high biomass C4 plants such as maize, switch grass and sugarcane develop adverse phenotypes including stunting, chlorosis and reduced biomass as PHB levels in leaves increase. In this study, we explore limitations to PHB accumulation in sugarcane chloroplasts using a systems biology approach, coupled with a metabolic model of C4 photosynthesis. Decreased assimilation was evident in high PHB-producing sugarcane plants, which also showed a dramatic decrease in sucrose and starch content of leaves. A subtle decrease in the C/N ratio was found which was not associated with a decrease in total protein content. An increase in amino acids used for nitrogen recapture was also observed. Based on the accumulation of substrates of ATP-dependent reactions, we hypothesized ATP starvation in bundle sheath chloroplasts. This was supported by mRNA differential expression patterns. The disruption in ATP supply in bundle sheath cells appears to be linked to the physical presence of the PHB polymer which may disrupt photosynthesis by scattering photosynthetically active radiation and/or physically disrupting thylakoid membranes.

Evolutionary Engineering Improves Tolerance for Replacement Jet Fuels in Saccharomyces cerevisiae.

Monoterpenes are liquid hydrocarbons with applications ranging from flavor and fragrance to replacement jet fuel. Their toxicity, however, presents a major challenge for microbial synthesis. Here we evolved limonene-tolerant Saccharomyces cerevisiae strains and sequenced six strains across the 200-generation evolutionary time course. Mutations were found in the tricalbin proteins Tcb2p and Tcb3p. Genomic reconstruction in the parent strain showed that truncation of a single protein (tTcb3p(1-989)), but not its complete deletion, was sufficient to recover the evolved phenotype improving limonene fitness 9-fold. tTcb3p(1-989) increased tolerance toward two other monoterpenes (ß-pinene and myrcene) 11- and 8-fold, respectively, and tolerance toward the biojet fuel blend AMJ-700t (10% cymene, 50% limonene, 40% farnesene) 4-fold. tTcb3p(1-989) is the first example of successful engineering of phase tolerance and creates opportunities for production of the highly toxic C10 alkenes in yeast.

Plant genome-scale modeling and implementation.

Considerable progress has been made in plant genome-scale metabolic reconstruction and modeling in recent years. Such reconstructions made it possible to explore metabolic phenotypes through appropriate model formulation and optimization methods. As a result, plant genome-scale modeling has increasingly attracted interest from the plant research community. In this chapter, the first generation of plant genome-scale metabolic reconstructions is presented, along with the important concepts behind model and constraint formulation. A brief protocol describing the use of constraint-based reconstruction and analysis (COBRA) Toolbox in flux simulation and model modification is provided. This is followed by a presentation of metabolic constraints required to generate fluxes in AraGEM using COBRA that describe photosynthesis, photorespiration, and respiration, respectively. Overall, plant genome-scale modeling is a powerful approach that is accessible and readily adopted.

Re-annotation of the Saccharopolyspora erythraea genome using a systems biology approach.

BACKGROUND: Accurate bacterial genome annotations provide a framework to understanding cellular functions, behavior and pathogenicity and are essential for metabolic engineering. Annotations based only on in silico predictions are inaccurate, particularly for large, high G + C content genomes due to the lack of similarities in gene length and gene organization to model organisms. RESULTS: Here we describe a 2D systems biology driven re-annotation of the Saccharopolyspora erythraea genome using proteogenomics, a genome-scale metabolic reconstruction, RNA-sequencing and small-RNA-sequencing. We observed transcription of more than 300 intergenic regions, detected 59 peptides in intergenic regions, confirmed 164 open reading frames previously annotated as hypothetical proteins and reassigned function to open reading frames using the genome-scale metabolic reconstruction. Finally, we present a novel way of mapping ribosomal binding sites across the genome by sequencing small RNAs. CONCLUSIONS: The work presented here describes a novel framework for annotation of the Saccharopolyspora erythraea genome. Based on experimental observations, the 2D annotation framework greatly reduces errors that are commonly made when annotating large-high G + C content genomes using computational prediction algorithms.