Large-scale (13)C-flux analysis reveals mechanistic principles of metabolic network robustness to null mutations in yeast

BACKGROUND: Quantification of intracellular metabolite fluxes by (13)C-tracer experiments is maturing into a routine higher-throughput analysis. The question now arises as to which mutants should be analyzed. Here we identify key experiments in a systems biology approach with a genome-scale model of Saccharomyces cerevisiae metabolism, thereby reducing the workload for experimental network analyses and functional genomics. RESULTS: Genome-scale (13)C flux analysis revealed that about half of the 745 biochemical reactions were active during growth on glucose, but that alternative pathways exist for only 51 gene-encoded reactions with significant flux. These flexible reactions identified in silico are key targets for experimental flux analysis, and we present the first large-scale metabolic flux data for yeast, covering half of these mutants during growth on glucose. The metabolic lesions were often counteracted by flux rerouting, but knockout of cofactor-dependent reactions, as in the adh1, ald6, cox5A, fum1, mdh1, pda1, and zwf1 mutations, caused flux responses in more distant parts of the network. By integrating computational analyses, flux data, and physiological phenotypes of all mutants in active reactions, we quantified the relative importance of 'genetic buffering' through alternative pathways and network redundancy through duplicate genes for genetic robustness of the network. CONCLUSIONS: The apparent dispensability of knockout mutants with metabolic function is explained by gene inactivity under a particular condition in about half of the cases. For the remaining 207 viable mutants of active reactions, network redundancy through duplicate genes was the major (75%) and alternative pathways the minor (25%) molecular mechanism of genetic network robustness in S. cerevisiae

Approach to splitting an academic group into project teams

A detection of communities in a group of people allows researchers to study the modular organizationof the network and use this information for various applications. The purpose of the study is to develop anapproach of uniform split the academic group on project teams. The project team is a group of people who areable to act in concert and collectively to achieve a common goal. In our experiments, we formed a social networkbased on reciprocal nomination. Using methods of social network analysis in the designed social network, project teams have been identified. Programming language R and library igraph were used to simulation

Designer rhamnolipid production

Rhamnolipids are biosurfactants featuring surface-active properties that render them suitable for a broad range of applications, e.g., in detergents, food, bioremediation, medicine/pharmacology, and crop science. These properties include their emulsification and foaming capacities and their ability to lower the surface tension. Further, aspects like biocompatibility and environmental friendliness, both features of rhamnolipids [1] are becoming increasingly important. Rhamnolipids thus constitute suitable substitutes for synthetic surfactants produced from fossil resources. Native producers of rhamnolipids are mainly pathogenic bacteria like Pseudomonas aeruginosa. We previously designed and constructed a recombinant Pseudomonas putida KT2440, which synthesizes rhamnolipids by decoupling production from host-intrinsic regulations and cell growth [2]. As most biosurfactants, rhamnolipids are synthesized in mixtures. We here show our approach to alter the native mixture of surfactant molecules to produce specific new-to-nature combinations. The molecular structure (Figure 1) can on the one hand be altered in the hydrophilic moiety by changing the number of rhamnose molecules. We achieved this by using only distinct genes from the native rhamnolipid synthesis pathway. On the other hand, we were also able to change the length of the fatty acids in the hydrophobic part. This chain length is determined by the acyl-transferase (RhlA). Using rhlA genes from different organisms enables our microbial cell factory to synthesize molecules with different chain lengths [3]. The different molecular structures have further been shown to feature diverse physico-chemical properties [4]. Exploiting the natural structural diversity will thus allow for the synthesis of designer rhamnolipids tailormade for specific applications. We thus present a novel approach to use biochemical engineering to create tailormade products for a more sustainable future. Please click Additional Files below to see the full abstract

Genome scale model reconstruction of the methylotrophic yeast Ogataea polymorpha

Ogataea polymorpha is a thermotolerant, methylotrophic yeast with significant industrial applications. It is a promising host to generate platform chemicals from methanol, derived e.g. from carbon capture and utilization streams. Full development of the organism into a production strain requires additional strain design, supported by metabolic modeling on the basis of a genome-scale metabolic model. However, to date, no genome-scale metabolic model is available for O. polymorpha. To overcome this limitation, we used a published reconstruction of the closely related yeast Pichia pastoris as reference and corrected reactions based on KEGG annotations. Additionally, we conducted phenotype microarray experiments to test O. polymorpha’s metabolic capabilities to grown on or respire 192 different carbon sources. Over three-quarter of the substrate usage was correctly reproduced by the model. However, O. polymorpha failed to metabolize eight substrates and gained 38 new substrates compared to the P. pastoris reference model. To enable the usage of these compounds, metabolic pathways were inferred from literature and database searches and potential enzymes and genes assigned by conducting BLAST searches. To facilitate strain engineering and identify beneficial mutants, gene-protein-reaction relationships need to be included in the model. Again, we used the P. pastoris model as reference to extend the O. polymorpha model with this relevant information. The final metabolic model of O. polymorpha supports the engineering of synthetic metabolic capabilities and enabling the optimization of production processes, thereby supporting a sustainable future methanol econom

Model-guided metabolic engineering of Pseudomonas taiwanensis VLB120 for the production of methyl ketones

Aliphatic methyl ketones are discussed as promising novel diesel blendstocks because of their favorable cetane numbers. To achieve sustainable production of these compounds, bio-based production in engineered microbes is followed and successful synthesis in Escherichia coli1,2,3 and Pseudomonas putida4 has recently been shown. In this presentation, we report on the metabolic engineering of Pseudomonas taiwanensis VLB1205 for the production of saturated and monounsaturated medium chain methyl ketones (C11, C13, C15, C17). Major arguments for the use of this microbe are its metabolic versatility, high tolerance of organic solvents5 and ease of cultivation. P. taiwanensis VLB120 can grow on various carbon sources besides glucose such as glycerol, an important by-product of biodiesel production, as well as on major components of biomass hydrolysate such as xylose, organic acids and aromatic compounds, e.g., 4-hydroxybenzoate4. Further, its superior redox cofactor regeneration capability6 might benefit the synthesis of the reduced, aliphatic target compounds. The transformation of P. taiwanensis VLB120 into a microbial cell factory for methyl ketone production was achieved by: (i) overproduction of the fatty acyl-CoA synthetase FadB to increase acyl-CoA availability, (ii) oxidation of acyl-CoA to a trans-2-enoyl-CoA by a heterologously expressed acyl-CoA oxidase from Micrococcus luteus, (iii) conversion of this intermediate to β-hydroxyacyl-CoA and further oxidation to a β -ketoacyl-CoA by overexpression of the native fadB gene, (iv) increased thioesterase activity by overexpression of fadM to form free β -keto acids, which spontaneously decarboxylate to methyl ketones. The 1st generation production strain yielded 550 mg L-1aq methyl ketones in a batch fermentation with in situ product extraction into a second organic layer of n-decane. Further strain optimization was guided by metabolic modeling, which suggested an additional deletion of the acyl-CoA thioesterase II (tesB). TesB hydrolyzes acyl-CoA to free fatty acids, hence, reverses the desired FadD reaction. In a simple batch fermentation, the proposed gene deletion resulted in a 2.5-fold increased product titer of 1.4 g L-1aq while 9.4 g L-1aq were reached in fed-batch cultivations. Additional, successful strategies tested in parallel were the deletion of the pha operon, responsible for polyhydroxyalkanoate synthesis and deletion of a fadA homologue in the 1st generation production strain, with the later resulting in an even 4-fold improvement of the product titer. While the production of 9.4 g L-1aq is already the highest reported titer of recombinantly produced methyl ketones so far, consolidation of all successfully tested engineering strategies holds great promise to significantly boost methyl ketone production in P. taiwanensis VLB120 to even higher titers. Overall, the results of this study underline the high potential of P. taiwanensis VLB120 for the production of methyl ketones and highlight model-guided metabolic engineering as a means to rationalize and accelerate strain optimization efforts. 1Dong et al. 2018: doi:10.1101/496497 2Goh et al. 2012: doi: 10.1128/AEM.06785-11 3Goh et al. 2014: doi: 10.1016/j.ymben.2014.09.003. 4Goh et al. 2018: doi: 10.1002/bit.26558. 5Rühl et al. 2009: doi: 10.1128/AEM.00225-09 6Blank et al. 2008: doi: 10.1111/j.1742-4658.2008.06648.x

Metabolic Engineering of Pseudomonas putida KT2440 for enhanced rhamnolipid production

The production of chemicals and fuels is mainly based on fossil resources. The reduced availability of these resources and thus the increasing prices for crude oil as well as the resulting pollution of the environment require alternative strategies to be developed. One approach is the employment of microorganisms for the production of platform molecules using renewable resources as substrate. Biosurfactants, such as rhamnolipids, are an example for such products as they can be naturally produced by microorganisms and are biodegradable in contrast to chemical surfactants. The bio-based production of chemicals has to be efficient and sustainable to become competitive on the market. Several strategies can be applied to increase the efficiency of a microbial cell factory, e.g., streamlining the chassis. Here, we show the heterologous production of rhamnolipids with the non-pathogenic Pseudomonas putida KT2440 with the aim of increasing the yield. P. putida KT2440 is a well-characterized microorganism and its genome is sequenced and well annotated. Thus, the targeted removal of genes is possible and can lead to a reduction of the metabolic burden and by-product formation, which can result in a higher yield. Furthermore, the efficient supply of precursors is an important factor for optimized production processes. Rhamnolipids are amphiphilic molecules containing rhamnose and ß-hydroxy fatty acids. These precursors are synthesized by two pathways, the fatty acid de novo synthesis and the rhamnose pathway. We performed gene deletions to avoid the synthesis of by-products, like pyoverdine, exopolysaccharides, and large surface proteins and energy consuming devices as the flagellum. Most of the genome-reduced mutants reached a higher yield compared to the strain with wildtype background. With the best chassis, the yield could be increased by 35%. Furthermore, we conducted the overexpression of genes for precursor supply, either plasmid-based or genomically integrated. In this regard, the genes for the phosphoglucomutase, the complete rhamnose-synthesis pathway operon, and different enzymes in the pathway for acetyl-CoA synthesis were targeted. Various combinations were tested, and the highest yield reached was 51% higher compared to the initial rhamnolipid producer. Finally, a genome-reduced mutant was equipped with the overexpression modules and the rhamnolipid titer was increased from approximately 590 mg/L for the wildtype background to 960 mg/L, which represents a 63% increase. In conclusion, we were able to enhance the yield of rhamnolipids per glucose using metabolic engineering

The inflection point hypothesis: The relationship between the temperature dependence of enzyme-catalyzed reaction rates and microbial growth rates.

The temperature dependence of biological rates at different scales (from individual enzymes to isolated organisms to ecosystem processes such as soil respiration and photosynthesis) is the subject of much historical and contemporary research. The precise relationship between the temperature dependence of enzyme rates and those at larger scales is not well understood. We have developed macromolecular rate theory (MMRT) to describe the temperature dependence of biological processes at all scales. Here we formalize the scaling relationship by investigating MMRT both at the molecular scale (constituent enzymes) and for growth of the parent organism. We demonstrate that the inflection point (ᵢₙ) for the temperature dependence of individual metabolic enzymes coincides with the optimal growth temperature for the parent organism, and we rationalize this concordance in terms of the necessity for linearly correlated rates for metabolic enzymes over fluctuating environmental temperatures to maintain homeostasis. Indeed, ᵢₙ is likely to be under strong selection pressure to maintain coordinated rates across environmental temperature ranges. At temperatures at which rates become uncorrelated, we postulate a regulatory catastrophe and organism growth rates precipitously decline at temperatures where this occurs. We show that the curvature in the plots of the natural log of the rate versus temperature for individual enzymes determines the curvature for the metabolic process overall and the curvature for the temperature dependence of the growth of the organism. We have called this "the inflection point hypothesis", and this hypothesis suggests many avenues for future investigation, including avenues for engineering the thermal tolerance of organisms

Customized Woven Carbon Fiber Electrodes for Bioelectrochemical Systems—A Study of Structural Parameters

Commercial carbon fiber (CF) fabrics are popular electrode materials for bioelectrochemical systems (BES), but are usually not optimized for the specific application. This study investigates BES-relevant material characteristics on fabric level, such as weave types and weave parameters. The two contrasting weave types plain and leno weave were characterized with respect to their envisaged application types: 1) BES with mainly advective flow regimes and 2) stirred systems, which could benefit from fluid flow through a fabric electrode. Experiments with batch and continuously fed pure cultures of Geobacter sulfurreducens PCA and Shewanella oneidensis MR-1 reveal that µm-scale electrode topologies are of limited use for the thick biofilms of G. sulfurreducens , but can boost S. oneidensis ’ current generation especially in batch and fed-batch reactors. For advective flow regimes, deeper layers of biofilm inside microporous electrodes are often mass transport limited, even with thin biofilms of S. oneidensis . Therefore, low porosity plain weave electrodes for advective flow operation as in wastewater treating BES should be thin and flat. A trade-off between maximized current density and electrode material utilization exists, which is optimized exemplarily for an advective flow operation. For stirred BES of biotechnological applications, a flow-through of electrolyte is desired. For this, leno weave fabrics with pores at cm-scale are produced from 100% CF for the first time. In a preliminary evaluation, they outperform plain weave fabrics. Mass transfer investigations in stirred BES demonstrate that the large pores enable efficient electrode utilization at lower power input in terms of stirring speed