•A method for determining maximum Kinetically Accessible Yield (KAY) is presented.•KAY is bounded by the instability point when additional flux is drawn from the system.•A full E. coli model is used to predict isobutanol yield in 3 genotypes.•Predicted KAY's match reported results in the literature.The product formation yield (product formed per unit substrate consumed) is often the most important performance indicator in metabolic engineering. Until now, the actual yield cannot be predicted, but it can be bounded by its maximum theoretical value. The maximum theoretical yield is calculated by considering the stoichiometry of the pathways and cofactor regeneration involved. Here we found that in many cases, dynamic stability becomes an issue when excessive pathway flux is drawn to a product. This constraint reduces the yield and renders the maximal theoretical yield too loose to be predictive. We propose a more realistic quantity, defined as the kinetically accessible yield (KAY) to predict the maximum accessible yield for a given flux alteration. KAY is either determined by the point of instability, beyond which steady states become unstable and disappear, or a local maximum before becoming unstable. Thus, KAY is the maximum flux that can be redirected for a given metabolic engineering strategy without losing stability. Strictly speaking, calculation of KAY requires complete kinetic information. With limited or no kinetic information, an Ensemble Modeling strategy can be used to determine a range of likely values for KAY, including an average prediction. We first apply the KAY concept with a toy model to demonstrate the principle of kinetic limitations on yield. We then used a full-scale E. coli model (193 reactions, 153 metabolites) and this approach was successful in E. coli for predicting production of isobutanol: the calculated KAY values are consistent with experimental data for three genotypes previously published.

•This paper is the first report of an OPLSR-based metabolomics approach for strain improvement of 1-butanol producing E.coli.•Resolving acetyl-CoA to acetoacetyl-CoA bottleneck reaction improved 1-butanol productivity in the highest producing strain.•OPLSR-based metabolomics approach also indicated a CoA limitation in the highest producing strain.Metabolomics is the comprehensive analysis of metabolites in biological systems that uses multivariate analyses such as principal component analysis (PCA) or partial least squares/projections to latent structures regression (PLSR) to understand the metabolome state and extract important information from biological systems. In this study, orthogonal PLSR (OPLSR) model-based metabolomics approach was applied to 1-butanol producing Escherichia coli to facilitate in strain improvement strategies. Here, metabolite data obtained by liquid chromatography/mass spectrometry (LC/MS) was used to construct an OPLSR model to correlate metabolite changes with 1-butanol production and rationally identify gene targets for strain improvement. Using this approach, acetyl-CoA was determined as the rate-limiting step of the pathway while free CoA was found to be insufficient for 1-butanol production. By resolving the problems addressed by the OPLSR model, higher 1-butanol productivity was achieved. In this study, the usefulness of OPLSR-based metabolomics approach for understanding the whole metabolome state and determining the most relevant metabolites was demonstrated. Moreover, it was able to provide valuable insights for selection of rational gene targets for strain improvement.

Methanol is an important intermediate in the utilization of natural gas for synthesizing other feedstock chemicals. Typically, chemical approaches for building C–C bonds from methanol require high temperature and pressure. Biological conversion of methanol to longer carbon chain compounds is feasible; however, the natural biological pathways for methanol utilization involve carbon dioxide loss or ATP expenditure. Here we demonstrated a biocatalytic pathway, termed the methanol condensation cycle (MCC), by combining the nonoxidative glycolysis with the ribulose monophosphate pathway to convert methanol to higher-chain alcohols or other acetyl-CoA derivatives using enzymatic reactions in a carbon-conserved and ATP-independent system. We investigated the robustness of MCC and identified operational regions. We confirmed that the pathway forms a catalytic cycle through 13C-carbon labeling. With a cell-free system, we demonstrated the conversion of methanol to ethanol or n-butanol. The high carbon efficiency and low operating temperature are attractive for transforming natural gas-derived methanol to longer-chain liquid fuels and other chemical derivatives.

Glycogen synthesis initiated by glucose-1-phosphate adenylyltransferase (glgC) represents a major carbon storage route in cyanobacteria which could divert a significant portion of assimilated carbon. Significant growth retardation in cyanobacteria with glgC knocked out (ΔglgC) has been reported in high light conditions. Here, we knocked out the glgC gene and analyzed its effects on carbon distribution in an isobutanol-producing strain of Synechococcus elongatus PCC7942 and its parental wild-type strain. We showed that isobutanol production was able to partially rescue the growth of ΔglgC mutant where the growth rescue effect positively correlated with the rate of isobutanol production. Using NaH14CO3 incorporation analysis, we observed a 28 % loss of total carbon fixation rate in the ΔglgC mutant compared to the wild-type. Upon expression of the isobutanol production pathway in ΔglgC mutant, the total carbon fixation rate was restored to the wild-type level. Furthermore, we showed that 52 % of the total carbon fixed was redirected into isobutanol biosynthesis in the ΔglgC mutant expressing enzymes for isobutanol production, which is 2.5 times higher than that of the wild-type expressing the same enzymes. These results suggest that biosynthesis of non-native product such as isobutanol can serve as a metabolic sink for replacing glycogen to rescue growth and restore carbon fixation rate. The rescue effect may further serve as a platform for cyanobacteria energy and carbon metabolism study.

To achieve sustainable growth of human society, fossil fuels must eventually be replaced with renewable resources. Ultimately, the energy and carbon in fuels and chemicals synthesized must come from the sun and CO2 directly. Biological systems hold the promise to catalyze the synthesis of such fuels or chemicals. This article discusses recent advances in developing biofuel production processes from CO2, which include photosynthetic processes using algae and cyanobacteria and the non-photosynthetic “electrofuel” processes using Ralstonia eutropha and other lithoautotrophic microorganisms. Each of these processes involves strengths and weaknesses. While none of these processes have achieved industrial success, the challenges involved may point the direction for further improvement within the limit of theoretical possibility. Finally, all biological processes produce cell mass rich in protein. Regenerating ammonium by deamination of hydrolyzed proteins may close the loop of the global nitrogen cycle, which is also one of the major challenges in large scale biological processes.

Metabolic engineering of photosynthetic microorganisms such as cyanobacteria for the production of fuels or chemicals is challenging, particularly when the pathway involves oxygen-sensitive enzymes. We have previously designed a coenzyme A (CoA) dependent n-butanol biosynthesis pathway tailored to the metabolic physiology of the cyanobacterium Synechococcus elongatus PCC 7942 by incorporating an ATP driving force and a kinetically irreversible trap. However, one of the enzymes involved, CoA-acylating butyraldehyde dehydrogenase (Bldh) is oxygen sensitive, therefore hindering efficient n-butanol synthesis in cyanobacteria. To overcome this obstacle of n-butanol biosynthesis, we characterized six oxygen tolerant CoA-acylating aldehyde dehydrogenases (PduP) from the 1,2-propandiol degradation pathway for their activity toward acyl-CoA. We showed that PduP catalyzes the reversible reduction of a broad range of acyl-CoAs (C2 to C12) into corresponding aldehydes. In particular, PduP from Salmonella enterica has the highest catalytic efficiency (kcat/Km) of 292 s−1 mM−1 for butyryl-CoA, which is about 7 times higher than that for acetyl-CoA. Finally, replacing Bldh with PduP in the n-butanol synthesis pathway resulted in n-butanol production to a cumulative titer of 404 mg L−1 with peak productivity of 51 mg per L per day, exceeding the base strain by 20 fold. Thus, the oxygen sensitivity of CoA-acylating aldehyde dehydrogenase appears to be a key limiting factor for cyanobacteria to produce alcohols through the CoA-dependent route.

Direct conversion of CO2 to bio-based fuels and chemicals has emerged as a significant thrust to address the energy and environmental concerns caused by over-reliance on fossil fuels and the increasing level of atmospheric CO2. Here we report the first photosynthetic production of 2-methyl-1-butanol (2MB), an energy-dense fuel molecule, from CO2 in the genetically engineered cyanobacterium Synechococcus elongatus PCC7942. 2MB is synthesized through the isoleucine pathway by decarboxylation of 2-keto-3-methylvalerate followed by reduction and has been produced from glucose by recombinant Escherichia coli with 1-propanol and isobutanol as the major by-products. However, direct photosynthetic production of 2MB from CO2 has not been reported. In this work, introduction of a ketoacid decarboxylase (Kivd), an alcohol dehydrogenase (YqhD), and the citramalate pathway, which produces the isoleucine precursor 2-ketobutyrate (2KB), in S. elongatus PCC7942 successfully redirected the flux to 2MB biosynthesis with significant productivity (an average of 20 mg per L per day). Interestingly, the native isoleucine pathway activity was able to compete with the overexpressed Kivd activity for the same substrate 2KB, such that 1-propanol formation was minimal. Kinetic analysis of the key enzyme in the isoleucine pathway, acetohydroxyacid synthase (AHAS) from S. elongatus PCC7942, yielded a Vmax(2KB) of 1.21 ± 0.03 U mg−1 and a Km(2KB) of 1.9 ± 0.3 mM using the purified protein and demonstrated preferential selectivity towards 2KB. The final titer of 2MB reached 200 mg L−1 in 12 days with minor accumulation of other alcohols. The high in vivo activity of the native S. elongatus PCC7942 AHAS suggests the advantage of utilizing branched-chain amino acid pathways in this organism for the production of fuels and chemicals.

Pentanol isomers such as 2-methyl-1-butanol and 3-methyl-1-butanol are a useful class of chemicals with a potential application as biofuels. They are found as natural by-products of microbial fermentations from amino acid substrates. However, the production titer and yield of the natural processes are too low to be considered for practical applications. Through metabolic engineering, microbial strains for the production of these isomers have been developed, as well as that for 1-pentanol and pentenol. Although the current production levels are still too low for immediate industrial applications, the approach holds significant promise for major breakthroughs in production efficiency.

The dramatic increase in healthcare cost has become a significant burden to the world. Many patients are denied the accessibility of medication because of the high price of drugs. Total biosynthesis of chiral drug intermediates is an environmentally friendly approach that helps provide more affordable pharmaceuticals. Here we have expanded the natural metabolic capability to biosynthesize a nonnatural amino acid L-homoalanine, which is a chiral precursor of levetiracetam, brivaracetam, and ethambutol. We developed a selection strategy and altered the substrate specificity of ammonium-assimilating enzyme glutamate dehydrogenase. The specificity constant k_cat/K_m of the best mutant towards 2-ketobutyrate is 50-fold higher than that towards the natural substrate 2-ketoglutarate. Compared to transaminase IlvE and NADH-dependent valine dehydrogenases, the evolved glutamate dehydrogenase increased the conversion yield of 2-ketobutyrate to L-homoalanine by over 300% in aerobic condition. As a result of overexpressing the mutant glutamate dehydrogenase and Bacillus subtilis threonine dehydratase in a modified threonine-hyperproducing Escherichia coli strain (ATCC98082, ΔrhtA), 5.4 g/L L-homoalanine was produced from 30 g/L glucose (0.18 g/g glucose yield, 26% of the theoretical maximum). This work opens the possibility of total biosynthesis of other nonnatural chiral compounds that could be useful pharmaceutical intermediates.

We characterized the activity and stereospecificity of four secondary alcohol dehydrogenases (sADHs) towards acetoin reduction and constructed synthetic pathways in E. coli to produce enantiomerically pure (R,R)-2,3-butanediol (2,3-BDO) from glucose with a titer of 6.1 g/L (enantio purity >99%), and yield of 0.31 g product/g glucose (62% of theoretical maximum).

The depleting petroleum storage and increasing environmental deterioration are threatening the sustainable development of human societies. As such, biofuels and chemical feedstocks generated from renewable sources are becoming increasingly important. Although previous efforts led to great success in bio-ethanol production, higher alcohols, fatty acid derivatives including biodiesels, alkanes, and alkenes offer additional advantages because of their compatibility with existing infrastructure. In addition, some of these compounds are useful chemical feedstocks. Since native organisms do not naturally produce these compounds in high quantities, metabolic engineering becomes essential in constructing producing organisms. In this article, we briefly review the four major metabolic systems, the coenzyme-A mediated pathways, the keto acid pathways, the fatty acid pathway, and the isoprenoid pathways, that allow production of these fuel-grade chemicals.

Recent progress has been made in the production of higher alcohols by harnessing the power of natural amino acid biosynthetic pathways. Here, we describe the first strain of Escherichia coli developed to produce the higher alcohol and potential new biofuel 2-methyl-1-butanol (2MB). To accomplish this, we explored the biodiversity of enzymes catalyzing key parts of the isoleucine biosynthetic pathway, finding that AHAS II (ilvGM) from Salmonella typhimurium and threonine deaminase (ilvA) from Corynebacterium glutamicum improve 2MB production the most. Overexpression of the native threonine biosynthetic operon (thrABC) on plasmid without the native transcription regulation also improved 2MB production in E. coli. Finally, we knocked out competing pathways upstream of threonine production (ΔmetA, Δtdh) to increase its availability for further improvement of 2MB production. This work led to a strain of E. coli that produces 1.25 g/L 2MB in 24 h, a total alcohol content of 3 g/L, and with yields of up to 0.17 g 2MB/g glucose.

Nitric oxide (NO) is used by mammalian immune systems to counter microbial invasions and is produced by bacteria during denitrification. As a defense, microorganisms possess a complex network to cope with NO. Here we report a combined transcriptomic, chemical, and phenotypic approach to identify direct NO targets and construct the biochemical response network. In particular, network component analysis was used to identify transcription factors that are perturbed by NO. Such information was screened with potential NO reaction mechanisms and phenotypic data from genetic knockouts to identify active chemistry and direct NO targets in Escherichia coli. This approach identified the comprehensive E. coli NO response network and evinced that NO halts bacterial growth via inhibition of the branched-chain amino acid biosynthesis enzyme dihydroxyacid dehydratase. Because mammals do not synthesize branched-chain amino acids, inhibition of dihydroxyacid dehydratase may have served to foster the role of NO in the immune arsenal.

Autonomous oscillations found in gene expression and metabolic, cardiac and neuronal systems1, 2, 3, 4 have attracted significant attention both because of their obvious biological roles and their intriguing dynamics. In addition, de novo designed5, 6, 7, 8, 9, 10, 11, 12 oscillators13, 14 have been demonstrated, using components that are not part of the natural oscillators. Such oscillators are useful in testing the design principles and in exploring potential applications not limited by natural cellular behaviour15. To achieve transcriptional and metabolic integration characteristic of natural oscillators, here we designed and constructed a synthetic circuit in Escherichia coli K12, using glycolytic flux to generate oscillation through the signalling metabolite acetyl phosphate. If two metabolite pools are interconverted by two enzymes that are placed under the transcriptional control of acetyl phosphate, the system oscillates when the glycolytic rate exceeds a critical value. We used bifurcation analysis to identify the boundaries of oscillation, and verified these experimentally. This work demonstrates the possibility of using metabolic flux as a control factor in system-wide oscillation, as well as the predictability of a de novo gene–metabolic circuit designed using nonlinear dynamic analysis.

Artificial transcriptional networks have been used to achieve novel, nonnative behavior in bacteria. Typically, these artificial circuits are isolated from cellular metabolism and are designed to function without intercellular communication. To attain concerted biological behavior in a population, synchronization through intercellular communication is highly desirable. Here we demonstrate the design and construction of a gene-metabolic circuit that uses a common metabolite to achieve tunable artificial cell–cell communication. This circuit uses a threshold concentration of acetate to induce gene expression by acetate kinase and part of the nitrogen-regulation two-component system. As one application of the cell–cell communication circuit we created an artificial quorum sensor. Engineering of carbon metabolism in Escherichia coli made acetate secretion proportional to cell density and independent of oxygen availability. In these cells the circuit induced gene expression in response to a threshold cell density. This threshold can be tuned effectively by controlling ΔpH over the cell membrane, which determines the partition of acetate between medium and cells. Mutagenesis of the enhancer sequence of the glnAp2 promoter produced variants of the circuit with changed sensitivity demonstrating tunability of the circuit by engineering of its components. The behavior of the circuit shows remarkable predictability based on a mathematical design model.

Cells adjust gene expression profiles in response to environmental and physiological changes through a series of signal transduction pathways. Upon activation or deactivation, the terminal regulators bind to or dissociate from DNA, respectively, and modulate transcriptional activities on particular promoters. Traditionally, individual reporter genes have been used to detect the activity of the transcription factors. This approach works well for simple, non-overlapping transcription pathways. For complex transcriptional networks, more sophisticated tools are required to deconvolute the contribution of each regulator. Here, we demonstrate the utility of network component analysis in determining multiple transcription factor activities based on transcriptome profiles and available connectivity information regarding network connectivity. We used Escherichia coli carbon source transition from glucose to acetate as a model system. Key results from this analysis were either consistent with physiology or verified by using independent measurements.

The homeostasis of nitric oxide (NO) is attained through a balance between its production and consumption. Shifts in NO bioavailability have been linked to a variety of diseases. Although the regulation of NO production has been well documented, its consumption is largely thought to be unregulated. Here, we have demonstrated that under hypoxic conditions, NO accelerates its own consumption by increasing its entry into RBCs. When RBCs were exposed to NO (1:400 NO/heme ratio) under hypoxic conditions to form HbFe(II)NO, the consumption rate of NO increased significantly. This increase in NO consumption converted the bioactivity of serotonin from a vasodilator to a vasoconstrictor in isolated coronary arterioles. We identified HbFe(II)NO as a potential mediator of accelerated NO consumption. Accelerated NO consumption by HbFe(II)NO-bearing RBCs may contribute to hypoxic pulmonary vasoconstriction and the rebound effect seen on termination of NO inhalation therapy. Furthermore, accelerated NO consumption may exacerbate ischemia-mediated vasospasm and nitrate tolerance. Finally, this phenomenon may be an evolved mechanism to stabilize the vasculature in sepsis.

High-dimensional data sets generated by high-throughput technologies, such as DNA microarray, are often the outputs of complex networked systems driven by hidden regulatory signals. Traditional statistical methods for computing low-dimensional or hidden representations of these data sets, such as principal component analysis and independent component analysis, ignore the underlying network structures and provide decompositions based purely on a priori statistical constraints on the computed component signals. The resulting decomposition thus provides a phenomenological model for the observed data and does not necessarily contain physically or biologically meaningful signals. Here, we develop a method, called network component analysis, for uncovering hidden regulatory signals from outputs of networked systems, when only a partial knowledge of the underlying network topology is available. The a priori network structure information is first tested for compliance with a set of identifiability criteria. For networks that satisfy the criteria, the signals from the regulatory nodes and their strengths of influence on each output node can be faithfully reconstructed. This method is first validated experimentally by using the absorbance spectra of a network of various hemoglobin species. The method is then applied to microarray data generated from yeast Saccharamyces cerevisiae and the activities of various transcription factors during cell cycle are reconstructed by using recently discovered connectivity information for the underlying transcriptional regulatory networks.

Nitric oxide (NO) activates soluble guanylyl cyclase in smooth muscle cells to induce vasodilation in the vasculature. However, as hemoglobin (Hb) is an effective scavenger of NO and is present in high concentrations inside the red blood cell (RBC), the bioavailability of NO would be too low to elicit soluble guanylyl cyclase activation in the presence of blood. Therefore, NO bioactivity must be preserved. Here we present evidence suggesting that the RBC participates in the preservation of NO bioactivity by reducing NO influx. The NO uptake by RBCs was increased and decreased by altering the degree of band 3 binding to the cytoskeleton. Methemoglobin and denatured hemoglobin binding to the RBC membrane or cytoskeleton also were shown to contribute to reducing the NO uptake rate of the RBC. These alterations in NO uptake by the RBC, hence the NO bioavailability, were determined to correlate with the vasodilation of isolated blood vessels. Our observations suggest that RBC membrane and cytoskeleton associated NO-inert proteins provide a barrier for NO diffusion and thus account for the reduction in the NO uptake rate of RBCs.

Compared to ethanol, butanol offers many advantages as a substitute for gasoline because of higher energy content and higher hydrophobicity. Typically, 1-butanol is produced by Clostridium in a mixed-product fermentation. To facilitate strain improvement for specificity and productivity, we engineered a synthetic pathway in Escherichia coli and demonstrated the production of 1-butanol from this non-native user-friendly host. Alternative genes and competing pathway deletions were evaluated for 1-butanol production. Results show promise for using E. coli for 1-butanol production.

Nitrosylhemoglobin (HbFeIINO) has been detected in vivo, and its role in NO transport and preservation has been discussed. To gain insight into the potential role of HbFeIINO, we performed in vitro experiments to determine the effect of oxygenated red blood cells (RBCs) on the dissociation of cell-free HbFeIINO, using carboxyhemoglobin (HbFeIICO) as a comparison. Results show that the apparent half-life of the cell-free HbFeIICO was reduced significantly in the presence of RBCs at 1% hematocrit. In contrast, RBC did not change the apparent half-life of extracellular HbFeIINO, but caused a shift in the HbFeIINO dissociation product from methemoglobin (metHbFeIII) to oxyhemoglobin (HbFeIIO2). Extracellular hemoglobin was able to extract CO from HbFeIICO-containing RBC, but not NO from HbFeIINO-containing RBC. Although these results appear to suggest some unusual interactions between HbFeIINO and RBC, the data are explainable by simple HbFeIINO dissociation and hemoglobin oxidation with known rate constants. A kinetic model consisting of these reactions shows that (i) deoxyhemoglobin is an intermediate in the reaction of HbFeIINO oxidation to metHbFeIII, (ii) the rate-limiting step of HbFeIINO decay is the dissociation of NO from HbFeIINO, (iii) the magnitude of NO diffusion rate constant into RBC is estimated to be ∼104 M−1 s−1, consistent with previous results determined from a competition assay, and (iv) no additional chemical reactions are required to explain these data.

•Recent developments of mathematical modeling have facilitated strain design.•Robustness for stability has emerged as an important criterion for strain design.•Data acquisition and managing techniques are in need in modern systems biology.Techniques for modeling microbial bioproduction systems have evolved over many decades. Here, we survey recent literature and focus on modeling approaches for improving bioproduction. These techniques from systems biology are based on different methodologies, starting from stoichiometry only to various stoichiometry with kinetics approaches that address different issues in metabolic systems. Techniques to overcome unknown kinetic parameters using random sampling have emerged to address meaningful questions. Among those questions, pathway robustness seems to be an important issue for metabolic engineering. We also discuss the increasing significance of databases in biology and their potential impact for biotechnology.

The liver plays a central role in maintaining whole body metabolic and energy homeostasis by consuming and producing glucose and fatty acids. Glucose and fatty acids compete for hepatic substrate oxidation with regulation ensuring glucose is oxidized preferentially. Increasing fatty acid oxidation is expected to decrease lipid storage in the liver and avoid lipid-induced insulin-resistance. To increase hepatic lipid oxidation in the presence of glucose, we previously engineered a synthetic glyoxylate shunt into human hepatocyte cultures and a mouse model and showed that this synthetic pathway increases free fatty acid β-oxidation and confers resistance to diet-induced obesity in the mouse model. Here we used ensemble modeling to decipher the effects of perturbations to the hepatic metabolic network on fatty acid oxidation and glucose uptake. Despite sampling of kinetic parameters using the most fundamental elementary reaction models, the models based on current metabolic regulation did not readily describe the phenotype generated by glyoxylate shunt expression. Although not conclusive, this initial negative result prompted us to probe unknown regulations, and malate was identified as inhibitor of hexokinase 2 expression either through direct or indirect actions. This regulation allows the explanation of observed phenotypes (increased fatty acid degradation and decreased glucose consumption). Moreover, the result is a function of pyruvate-carboxylase, mitochondrial pyruvate transporter, citrate transporter protein, and citrate synthase activities. Some subsets of these flux ratios predict increases in fatty acid and decreases in glucose uptake after glyoxylate expression, whereas others predict no change. Altogether, this work defines the possible biochemical space where the synthetic shunt will produce the desired phenotype and demonstrates the efficacy of ensemble modeling for synthetic pathway design.

We characterized the activity and stereospecificity of four secondary alcohol dehydrogenases (sADHs) towards acetoin reduction and constructed synthetic pathways in E. coli to produce enantiomerically pure (R,R)-2,3-butanediol (2,3-BDO) from glucose with a titer of 6.1 g/L (enantio purity >99%), and yield of 0.31 g product/g glucose (62% of theoretical maximum).