Metabolomics-Driven Identification of the Rate-Limiting Steps in 1-Propanol Production.

The concerted effort for bioproduction of higher alcohols and other commodity chemicals has yielded a consortium of metabolic engineering techniques to identify targets to enhance performance of engineered microbial strains. Here, we demonstrate the use of metabolomics as a tool to systematically identify targets for improved production phenotypes in Escherichia coli. Gas chromatography/mass spectrometry (GC/MS) and ion-pair LC-MS/MS were performed to investigate metabolic perturbations in various 1-propanol producing strains. Two initial strains were compared that differ in the expression of the citramalate and threonine pathways, which hold a synergistic relationship to maximize production yields. While this results in increased productivity, no change in titer was observed when the threonine pathway was overexpressed beyond native levels. Metabolomics revealed accumulation of upstream byproducts, norvaline and 2-aminobutyrate, both of which are derived from 2-ketobutyrate (2KB). Eliminating the competing pathway by gene knockouts or improving flux through overexpression of glycolysis gene effectively increased the intracellular 2KB pool. However, the increase in 2KB intracellular concentration yielded decreased production titers, indicating toxicity caused by 2KB and an insufficient turnover rate of 2KB to 1-propanol. Optimization of alcohol dehydrogenase YqhD activity using an ribosome binding site (RBS) library improved 1-propanol titer (g/L) and yield (g/g of glucose) by 38 and 29% in 72 h compared to the base strain, respectively. This study demonstrates the use of metabolomics as a powerful tool to aid systematic strain improvement for metabolically engineered organisms.

Factors for the Variability of Three Acceptable Maximal Expiratory Flow-Volume Curves in Chronic Obstructive Pulmonary Disease.

BACKGROUND: Generally, the maximal expiratory flow-volume (MEFV) curve must be measured for the diagnosis and staging of chronic obstructive pulmonary disease (COPD). As this test is effort dependent, international guidelines recommend that three acceptable trials are required for each test. However, no study has examined the magnitude and factors for the variability in parameters among three acceptable trials. METHODS: We evaluated the intra-individual variations in several parameters among three acceptable MEFV curves obtained at one-time point in patients with COPD (n = 28, stage 1; n = 36, stage 2; n = 21, stages 3-4). Next, the factors for such variations were examined using forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC). RESULTS: The averages of coefficient of variation (CV) for FEV1 and FVC were 2.0% (range: 1.0-3.0%) and 1.6% (0.9-2.2%), respectively. Both parameters were significantly better than peak expiratory flow rate, forced expiratory flow at 50% of expired FVC, and forced expiratory flow at 75% of expired FVC (CVs: 5.0-6.9%). A higher spirometric stage was significantly associated with higher CVs for FVC and FEV1, and older age was significantly correlated with a higher variation in FEV1 alone. Furthermore, a significantly inverse association was observed between emphysema severity, and the CVs for FEV1, but not that for FVC, regardless of spirometric stage. CONCLUSION: Both FVC and FEV1 are highly reproducible; nevertheless, older age, lower FEV1 at baseline, and non-emphysema phenotype are factors for a higher variability in FEV1 in patients with COPD.

Investigation of the effects of actinorhodin biosynthetic gene cluster expression and a rpoB point mutation on the metabolome of Streptomyces coelicolor M1146.

The previously reported Streptomyces coelicolor M1146 is commonly used as a host strain for engineering of secondary metabolite production. In this study, absolute quantification of intracellular and extracellular metabolites of M1146 was performed in mid-log phase and stationary phase to observe major metabolites and the changes that occurred during growth. Decreased levels of central carbon metabolites (glycolysis, TCA cycle, and pentose phosphate pathway) and increased levels of amino acids were observed in stationary phase compared to mid-log phase. Furthermore, comparative metabolome analyses of M1146 upon expression of the actinorhodin biosynthetic gene cluster (M1146+ACT), a point mutation on the rpoB gene encoding RNA polymerase beta-subunit (M1152), and both expression of actinorhodin biosynthetic gene cluster and a rpoB point mutation (M1152+ACT) were performed. M1146+ACT showed higher levels of important cofactors, such as ATP, NADPH, and FMN while M1152 led to higher levels of intracellular S-adenosyl-methionine, acyl-CoAs, and extracellular nucleosides compared to M1146. M1152+ACT exhibited the highest levels of actinorhodin with elevated bases, nucleosides, and nucleotides, such as intracellular PRPP (phosphoribosyl phosphate), ATP, along with extracellular inosine, uridine, and guanine compared to the other three strains, which were considered to be combined effects of actinorhodin gene cluster expression and a rpoB point mutation. Metabolites analysis by means of absolute quantification demonstrated changes in precursors of secondary metabolites before and after phosphate depletion in M1146. Comparative metabolome analysis provided further insights into the effects of actinorhodin gene cluster expression along with a rpoB point mutation on the metabolome of S. coelicolor.

Multi-Omics Analysis of the Effect of cAMP on Actinorhodin Production in Streptomyces coelicolor.

Cyclic adenosine monophosphate (cAMP) has been known to play an important role in regulating morphological development and antibiotic production in Streptomyces coelicolor. However, the functional connection between cAMP levels and antibiotic production and the mechanism by which cAMP regulates antibiotic production remain unclear. In this study, metabolomics- and transcriptomics-based multi-omics analysis was applied to S. coelicolor strains that either produce the secondary metabolite actinorhodin (Act) or lack most secondary metabolite biosynthesis pathways including Act. Comparative multi-omics analysis of the two strains revealed that intracellular and extracellular cAMP abundance was strongly correlated with actinorhodin production. Notably, supplementation of cAMP improved cell growth and antibiotic production. Further multi-omics analysis of cAMP-supplemented S. coelicolor cultures showed an increase of guanine and the expression level of purine metabolism genes. Based on this phenomenon, supplementation with 7-methylguanine, a competitive inhibitor of reactions utilizing guanine, with or without additional cAMP supplementation, was performed. This experiment revealed that the reactions inhibited by 7-methylguanine are mediating the positive effect on growth and antibiotic production, which may occur downstream of cAMP supplementation.

Comparison of metabolic profiles of yeasts based on the difference of the Crabtree positive and negative.

The Crabtree effect involves energy management in which yeasts utilize glycolysis as the terminal electron acceptor instead of oxygen, despite the presence of sufficient dissolved oxygen, when oxygen concentrations exceed a certain limit. The Crabtree effect is detrimental to bakery yeast production, because it results in lower cellular glucose yields. Batch culture of Saccharomyces cerevisiae, a Crabtree positive yeast, decreased the cell yield of glucose and produced large amounts of ethanol despite a high specific glucose consumption rate compared to Candida utilis, a Crabtree negative yeast. This study investigated the effect of these characteristics on metabolite levels. We performed metabolome analysis of both yeasts during each growth phase of batch culture using liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. Principle component analysis of metabolome data indicated that the Crabtree effect affected metabolites related to NADH synthesis in central metabolism. The amount of these metabolites in S. cerevisiae was lower than that in C. utilis. However, to maintain the specific glucose consumption rate at high levels, yeasts must avoid depletion of NAD+, which is essential for glucose utilization. Our results indicated that NADH was oxidized by converting acetaldehyde to ethanol in S. cerevisiae, which is in accordance with previous reports. Therefore, the specific NADH production rates of S. cerevisiae and C. utilis did not show a difference. This study suggested that NAD+/NADH ratio is disrupted by the Crabtree effect, which in turn influenced central metabolism and that S. cerevisiae maintained the NAD+/NADH ratio by producing ethanol.

Metabolome analysis revealed the knockout of glyoxylate shunt as an effective strategy for improvement of 1-butanol production in transgenic Escherichia coli.

High 1-butanol titer has been achieved in a transgenic Escherichia coli strain JCL299FT with a heterologous 1-butanol pathway by deleting competing pathways, balancing of cofactor and resolving free CoA imbalance. However, further improvement of 1-butanol production is still possible in the highest producing strain JCL299FT as indicated by the accumulation of acetate, a major undesired by-product during bio-production by microorganisms that competes with 1-butanol production for the available acetyl-CoA and inhibits protein synthesis resulting in poor growth. In this study, liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based metabolome analysis was performed to identify new rate limiting steps in the 1-butanol production pathway of E. coli strain JCL299FT. The results of metabolome analysis showed increased amounts of glyoxylate in JCL299FT compared to the previous highest-producing strain JCL299F. Knocking out aceA successfully decreased the amount of glyoxylate and reduced acetate accumulation, resulting in the increased levels of TCA cycle and 1-butanol pathway metabolites. These observations indicated that there was a redirection of flux from acetate to TCA cycle and 1-butanol producing pathway, which led to better growth of the 1-butanol producing strain. Consequently, 1-butanol production titer was improved by 39% and the production yield was improved by 12% in M9 medium supplemented with yeast extract. This study is the first report of using the knockout of aceA, the first gene in the glyoxylate shunt that encodes isocitrate lyase, as an effective strategy to reduce acetate overflow in 1-butanol producing E. coli.

Identifying metabolic elements that contribute to productivity of 1-propanol bioproduction using metabolomic analysis.

INTRODUCTION: Previously constructed Escherichia coli strains that produce 1-propanol use the native threonine pathway, or a heterologous citramalate pathway. However, based on the energy and cofactor requirements of each pathway, a combination of the two pathways produces synergistic effects that increase the theoretical maximum yield with a simultaneous unexplained increase in productivity. OBJECTIVE: Identification of key factors that contribute to synergistic effect leading to 1-propanol yield and productivity improvement in E. coli with native threonine pathway and heterologous citramalate pathway. METHOD: A combination of snapshot metabolomic profiling and dynamic metabolic turnover analysis were used to identify system-wide perturbations that contribute to the productivity improvement. RESULT AND CONCLUSION: In the presence of both pathways, increased glucose consumption and elevated levels of glycolytic intermediates are attributed to an elevated phosphoenolpyruvate (PEP)/pyruvate ratio that is known to increase the function of the native phosphotransferase. Turnover analysis of nitrogen containing byproducts reveals that ammonia assimilation, required for the threonine pathway, is streamlined when provided with an NAD(P)H surplus in the presence of the citramalate pathway. Our study illustrates the application of metabolomics in identification of factors that alter cellular physiology for improvement of 1-propanol bioproduction.

Orthogonal partial least squares/projections to latent structures regression-based metabolomics approach for identification of gene targets for improvement of 1-butanol production in Escherichia coli.

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.