High throughput 13C-metabolic flux analysis of 3-hydroxypropionic acid producing Pichia pastoris reveals limited availability of acetyl-CoA and ATP due to tight control of the glycolytic flux.

BACKGROUND: Production of 3-hydroxypropionic acid (3-HP) through the malonyl-CoA pathway has yielded promising results in Pichia pastoris (Komagataella phaffii), demonstrating the potential of this cell factory to produce this platform chemical and other acetyl-CoA-derived products using glycerol as a carbon source. However, further metabolic engineering of the original P. pastoris 3-HP-producing strains resulted in unexpected outcomes, e.g., significantly lower product yield and/or growth rate. To gain an understanding on the metabolic constraints underlying these observations, the fluxome (metabolic flux phenotype) of ten 3-HP-producing P. pastoris strains has been characterized using a high throughput 13C-metabolic flux analysis platform. Such platform enabled the operation of an optimised workflow to obtain comprehensive maps of the carbon flux distribution in the central carbon metabolism in a parallel-automated manner, thereby accelerating the time-consuming strain characterization step in the design-build-test-learn cycle for metabolic engineering of P. pastoris. RESULTS: We generated detailed maps of the carbon fluxes in the central carbon metabolism of the 3-HP producing strain series, revealing the metabolic consequences of different metabolic engineering strategies aimed at improving NADPH regeneration, enhancing conversion of pyruvate into cytosolic acetyl-CoA, or eliminating by-product (arabitol) formation. Results indicate that the expression of the POS5 NADH kinase leads to a reduction in the fluxes of the pentose phosphate pathway reactions, whereas an increase in the pentose phosphate pathway fluxes was observed when the cytosolic acetyl-CoA synthesis pathway was overexpressed. Results also show that the tight control of the glycolytic flux hampers cell growth due to limited acetyl-CoA biosynthesis. When the cytosolic acetyl-CoA synthesis pathway was overexpressed, the cell growth increased, but the product yield decreased due to higher growth-associated ATP costs. Finally, the six most relevant strains were also cultured at pH 3.5 to assess the effect of a lower pH on their fluxome. Notably, similar metabolic fluxes were observed at pH 3.5 compared to the reference condition at pH 5. CONCLUSIONS: This study shows that existing fluoxomics workflows for high-throughput analyses of metabolic phenotypes can be adapted to investigate P. pastoris, providing valuable information on the impact of genetic manipulations on the metabolic phenotype of this yeast. Specifically, our results highlight the metabolic robustness of P. pastoris's central carbon metabolism when genetic modifications are made to increase the availability of NADPH and cytosolic acetyl-CoA. Such knowledge can guide further metabolic engineering of these strains. Moreover, insights into the metabolic adaptation of P. pastoris to an acidic pH have also been obtained, showing the capability of the fluoxomics workflow to assess the metabolic impact of environmental changes.

ScalaFlux: A scalable approach to quantify fluxes in metabolic subnetworks.

13C-metabolic flux analysis (13C-MFA) allows metabolic fluxes to be quantified in living organisms and is a major tool in biotechnology and systems biology. Current 13C-MFA approaches model label propagation starting from the extracellular 13C-labeled nutrient(s), which limits their applicability to the analysis of pathways close to this metabolic entry point. Here, we propose a new approach to quantify fluxes through any metabolic subnetwork of interest by modeling label propagation directly from the metabolic precursor(s) of this subnetwork. The flux calculations are thus purely based on information from within the subnetwork of interest, and no additional knowledge about the surrounding network (such as atom transitions in upstream reactions or the labeling of the extracellular nutrient) is required. This approach, termed ScalaFlux for SCALAble metabolic FLUX analysis, can be scaled up from individual reactions to pathways to sets of pathways. ScalaFlux has several benefits compared with current 13C-MFA approaches: greater network coverage, lower data requirements, independence from cell physiology, robustness to gaps in data and network information, better computational efficiency, applicability to rich media, and enhanced flux identifiability. We validated ScalaFlux using a theoretical network and simulated data. We also used the approach to quantify fluxes through the prenyl pyrophosphate pathway of Saccharomyces cerevisiae mutants engineered to produce phytoene, using a dataset for which fluxes could not be calculated using existing approaches. A broad range of metabolic systems can be targeted with minimal cost and effort, making ScalaFlux a valuable tool for the analysis of metabolic fluxes.

MetExplore: collaborative edition and exploration of metabolic networks.

Metabolism of an organism is composed of hundreds to thousands of interconnected biochemical reactions responding to environmental or genetic constraints. This metabolic network provides a rich knowledge to contextualize omics data and to elaborate hypotheses on metabolic modulations. Nevertheless, performing this kind of integrative analysis is challenging for end users with not sufficiently advanced computer skills since it requires the use of various tools and web servers. MetExplore offers an all-in-one online solution composed of interactive tools for metabolic network curation, network exploration and omics data analysis. In particular, it is possible to curate and annotate metabolic networks in a collaborative environment. The network exploration is also facilitated in MetExplore by a system of interactive tables connected to a powerful network visualization module. Finally, the contextualization of metabolic elements in the network and the calculation of over-representation statistics make it possible to interpret any kind of omics data. MetExplore is a sustainable project maintained since 2010 freely available at https://metexplore.toulouse.inra.fr/metexplore2/.

Methanol-Essential Growth of Corynebacterium glutamicum: Adaptive Laboratory Evolution Overcomes Limitation due to Methanethiol Assimilation Pathway.

Methanol is a sustainable substrate for biotechnology. In addition to natural methylotrophs, metabolic engineering has gained attention for transfer of methylotrophy. Here, we engineered Corynebacterium glutamicum for methanol-dependent growth with a sugar co-substrate. Heterologous expression of genes for methanol dehydrogenase from Bacillus methanolicus and of ribulose monophosphate pathway genes for hexulose phosphate synthase and isomerase from Bacillus subtilis enabled methanol-dependent growth of mutants carrying one of two independent metabolic cut-offs, i.e., either lacking ribose-5-phosphate isomerase or ribulose-5-phosphate epimerase. Whole genome sequencing of strains selected by adaptive laboratory evolution (ALE) for faster methanol-dependent growth was performed. Subsequently, three mutations were identified that caused improved methanol-dependent growth by (1) increased plasmid copy numbers, (2) enhanced riboflavin supply and (3) reduced formation of the methionine-analogue O-methyl-homoserine in the methanethiol pathway. Our findings serve as a foundation for the engineering of C. glutamicum to unleash the full potential of methanol as a carbon source in biotechnological processes.

Chemical and Metabolic Controls on Dihydroxyacetone Metabolism Lead to Suboptimal Growth of Escherichia coli.

In this work, we shed light on the metabolism of dihydroxyacetone (DHA), a versatile, ubiquitous, and important intermediate for various chemicals in industry, by analyzing its metabolism at the system level in Escherichia coli Using constraint-based modeling, we show that the growth of E. coli on DHA is suboptimal and identify the potential causes. Nuclear magnetic resonance analysis shows that DHA is degraded nonenzymatically into substrates known to be unfavorable to high growth rates. Transcriptomic analysis reveals that DHA promotes genes involved in biofilm formation, which may reduce the bacterial growth rate. Functional analysis of the genes involved in DHA metabolism proves that under the aerobic conditions used in this study, DHA is mainly assimilated via the dihydroxyacetone kinase pathway. In addition, these results show that the alternative routes of DHA assimilation (i.e., the glycerol and fructose-6-phosphate aldolase pathways) are not fully activated under our conditions because of anaerobically mediated hierarchical control. These pathways are therefore certainly unable to sustain fluxes as high as the ones predicted in silico for optimal aerobic growth on DHA. Overexpressing some of the genes in these pathways releases these constraints and restores the predicted optimal growth on DHA.IMPORTANCE DHA is an attractive triose molecule with a wide range of applications, notably in cosmetics and the food and pharmaceutical industries. DHA is found in many species, from microorganisms to humans, and can be used by Escherichia coli as a growth substrate. However, knowledge about the mechanisms and regulation of this process is currently lacking, motivating our investigation of DHA metabolism in E. coli We show that under aerobic conditions, E. coli growth on DHA is far from optimal and is hindered by chemical, hierarchical, and possibly allosteric constraints. We show that optimal growth on DHA can be restored by releasing the hierarchical constraint. These results improve our understanding of DHA metabolism and are likely to help unlock biotechnological applications involving DHA as an intermediate, such as the bioconversion of glycerol or C1 substrates into value-added chemicals.

Functional analysis of isoprenoid precursors biosynthesis by quantitative metabolomics and isotopologue profiling.

INTRODUCTION: Isoprenoids are amongst the most abundant and diverse biological molecules and are involved in a broad range of biological functions. Functional understanding of their biosynthesis is thus key in many fundamental and applicative fields, including systems biology, medicine and biotechnology. However, available methods do not yet allow accurate quantification and tracing of stable isotopes incorporation for all the isoprenoids precursors. OBJECTIVES: We developed and validated a complete methodology for quantitative metabolomics and isotopologue profiling of isoprenoid precursors in the yeast Saccharomyces cerevisiae. METHODS: This workflow covers all the experimental and computational steps from sample collection and preparation to data acquisition and processing. It also includes a novel quantification method based on liquid chromatography coupled to high-resolution mass spectrometry. Method validation followed the Metabolomics Standards Initiative guidelines. RESULTS: This workflow ensures accurate absolute quantification (RSD < 20%) of all mevalonate and prenyl pyrophosphates intermediates with a high sensitivity over a large linear range (from 0.1 to 50 pmol). In addition, we demonstrate that this workflow brings crucial information to design more efficient phytoene producers. Results indicate stable turnover rates of prenyl pyrophosphate intermediates in the constructed strains and provide quantitative information on the change of the biosynthetic flux of phytoene precursors. CONCLUSION: This methodology fills one of the last technical gaps for functional studies of isoprenoids biosynthesis and should be applicable to other eukaryotic and prokaryotic (micro)organisms after adaptation of some organism-dependent steps. This methodology also opens the way to 13C-metabolic flux analysis of isoprenoid biosynthesis.

Engineering of Escherichia coli for Krebs cycle-dependent production of malic acid.

BACKGROUND: Malate is a C4-dicarboxylic acid widely used as an acidulant in the food and beverage industry. Rational engineering has been performed in the past for the development of microbial strains capable of efficient production of this metabolite. However, as malate can be a precursor for specialty chemicals, such as 2,4-dihydroxybutyric acid, that require additional cofactors NADP(H) and ATP, we set out to reengineer Escherichia coli for Krebs cycle-dependent production of malic acid that can satisfy these requirements. RESULTS: We found that significant malate production required at least simultaneous deletion of all malic enzymes and dehydrogenases, and concomitant expression of a malate-insensitive PEP carboxylase. Metabolic flux analysis using 13C-labeled glucose indicated that malate-producing strains had a very high flux over the glyoxylate shunt with almost no flux passing through the isocitrate dehydrogenase reaction. The highest malate yield of 0.82 mol/mol was obtained with E. coli Δmdh Δmqo ΔmaeAB ΔiclR ΔarcA which expressed malate-insensitive PEP carboxylase PpcK620S and NADH-insensitive citrate synthase GltAR164L. We also showed that inactivation of the dicarboxylic acid transporter DcuA strongly reduced malate production arguing for a pivotal role of this permease in malate export. CONCLUSIONS: Since more NAD(P)H and ATP cofactors are generated in the Krebs cycle-dependent malate production when compared to pathways which depend on the function of anaplerotic PEP carboxylase or PEP carboxykinase enzymes, the engineered strain developed in this study can serve as a platform to increase biosynthesis of malate-derived metabolites such as 2,4-dihydroxybutyric acid.

Functional characterization of a gene locus from an uncultured gut Bacteroides conferring xylo-oligosaccharides utilization to Escherichia coli.

In prominent gut Bacteroides strains, sophisticated strategies have been evolved to achieve the complete degradation of dietary polysaccharides such as xylan, which is one of the major components of the plant cell wall. Polysaccharide Utilization Loci (PULs) consist of gene clusters encoding different proteins with a vast arsenal of functions, including carbohydrate binding, transport and hydrolysis. Transport is often attributed to TonB-dependent transporters, although major facilitator superfamily (MFS) transporters have also been identified in some PULs. However, until now, few of these transporters have been biochemically characterized. Here, we targeted a PUL-like system from an uncultivated Bacteroides species that is highly prevalent in the human gut metagenome. It encodes three glycoside-hydrolases specific for xylo-oligosaccharides, a SusC/SusD tandem homolog and a MFS transporter. We combined PUL rational engineering, metabolic and transcriptional analysis in Escherichia coli to functionally characterize this genomic locus. We demonstrated that the SusC and the MFS transporters are specific for internalization of linear xylo-oligosaccharides of polymerization degree up to 3 and 4 respectively. These results were strengthened by the study of growth dynamics and transcriptional analyses in response to XOS induction of the PUL in the native strain, Bacteroides vulgatus.

Quantitative metabolomics of the thermophilic methylotroph Bacillus methanolicus.

BACKGROUND: The gram-positive bacterium Bacillus methanolicus MGA3 is a promising candidate for methanol-based biotechnologies. Accurate determination of intracellular metabolites is crucial for engineering this bacteria into an efficient microbial cell factory. Due to the diversity of chemical and cell properties, an experimental protocol validated on B. methanolicus is needed. Here a systematic evaluation of different techniques for establishing a reliable basis for metabolome investigations is presented. RESULTS: Metabolome analysis was focused on metabolites closely linked with B. methanolicus central methanol metabolism. As an alternative to cold solvent based procedures, a solvent-free quenching strategy using stainless steel beads cooled to -20 °C was assessed. The precision, the consistency of the measurements, and the extent of metabolite leakage from quenched cells were evaluated in procedures with and without cell separation. The most accurate and reliable performance was provided by the method without cell separation, as significant metabolite leakage occurred in the procedures based on fast filtration. As a biological test case, the best protocol was used to assess the metabolome of B. methanolicus grown in chemostat on methanol at two different growth rates and its validity was demonstrated. CONCLUSION: The presented protocol is a first and helpful step towards developing reliable metabolomics data for thermophilic methylotroph B. methanolicus. This will definitely help for designing an efficient methylotrophic cell factory.

FindPath: a Matlab solution for in silico design of synthetic metabolic pathways.

SUMMARY: Several methods and computational tools have been developed to design novel metabolic pathways. A major challenge is evaluating the metabolic efficiency of the designed pathways in the host organism. Here we present FindPath, a unified system to predict and rank possible pathways according to their metabolic efficiency in the cellular system. This tool uses a chemical reaction database to generate possible metabolic pathways and exploits constraint-based models (CBMs) to identify the most efficient synthetic pathway to achieve the desired metabolic function in a given host microorganism. FindPath can be used with common tools for CBM manipulation and uses the standard SBML format for both input and output files. AVAILABILITY AND IMPLEMENTATION: http://metasys.insa-toulouse.fr/software/findpath/. CONTACT: heux@insa-toulouse.fr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Engineering Escherichia coli for methanol conversion.

Methylotrophic bacteria utilize methanol and other reduced one-carbon compounds as their sole source of carbon and energy. For this purpose, these bacteria evolved a number of specialized enzymes and pathways. Here, we used a synthetic biology approach to select and introduce a set of "methylotrophy genes" into Escherichia coli based on in silico considerations and flux balance analysis to enable methanol dissimilation and assimilation. We determined that the most promising approach allowing the utilization of methanol was the implementation of NAD-dependent methanol dehydrogenase and the establishment of the ribulose monophosphate cycle by expressing the genes for hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi). To test for the best-performing enzymes in the heterologous host, a number of enzyme candidates from different donor organisms were selected and systematically analyzed for their in vitro and in vivo activities in E. coli. Among these, Mdh2, Hps and Phi originating from Bacillus methanolicus were found to be the most effective. Labeling experiments using (13)C methanol with E. coli producing these enzymes showed up to 40% incorporation of methanol into central metabolites. The presence of the endogenous glutathione-dependent formaldehyde oxidation pathway of E. coli did not adversely affect the methanol conversion rate. Taken together, the results of this study represent a major advancement towards establishing synthetic methylotrophs by gene transfer.

Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate.

Methanol, a one-carbon compound, can be utilized by a variety of bacteria and other organisms as carbon and energy source and is regarded as a promising substrate for biotechnological production. In this study, a strain of non-methylotrophic Corynebacterium glutamicum, which was able to produce the polyamide building block cadaverine as non-native product, was engineered for co-utilization of methanol. Expression of the gene encoding NAD+-dependent methanol dehydrogenase (Mdh) from the natural methylotroph Bacillus methanolicus increased methanol oxidation. Deletion of the endogenous aldehyde dehydrogenase genes ald and fadH prevented methanol oxidation to carbon dioxide and formaldehyde detoxification via the linear formaldehyde dissimilation pathway. Heterologous expression of genes for the key enzymes hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase of the ribulose monophosphate (RuMP) pathway in this strain restored growth in the presence of methanol or formaldehyde, which suggested efficient formaldehyde detoxification involving RuMP key enzymes. While growth with methanol as sole carbon source was not observed, the fate of 13C-methanol added as co-substrate to sugars was followed and the isotopologue distribution indicated incorporation into central metabolites and in vivo activity of the RuMP pathway. In addition, 13C-label from methanol was traced to the secreted product cadaverine. Thus, this synthetic biology approach led to a C. glutamicum strain that converted the non-natural carbon substrate methanol at least partially to the non-native product cadaverine.

A novel platform for automated high-throughput fluxome profiling of metabolic variants.

Advances in metabolic engineering are enabling the creation of a large number of cell factories. However, high-throughput platforms do not yet exist for rapidly analyzing the metabolic network of the engineered cells. To fill the gap, we developed an integrated solution for fluxome profiling of large sets of biological systems and conditions. This platform combines a robotic system for (13)C-labelling experiments and sampling of labelled material with NMR-based isotopic fingerprinting and automated data interpretation. As a proof-of-concept, this workflow was applied to discriminate between Escherichia coli mutants with gradual expression of the glucose-6-phosphate dehydrogenase. Metabolic variants were clearly discriminated while pathways that support metabolic flexibility towards modulation of a single enzyme were elucidating. By directly connecting the data flow between cell cultivation and flux quantification, considerable advances in throughput, robustness, release of resources and screening capacity were achieved. This will undoubtedly facilitate the development of efficient cell factories.

From a Hetero- to a Methylotrophic Lifestyle: Flash Back on the Engineering Strategies to Create Synthetic Methanol-User Strains.

Engineering microorganisms to grow on alternative feedstocks is crucial not just because of the indisputable biotechnological applications but also to deepen our understanding of microbial metabolism. One-carbon (C1) substrate metabolism has been the focus of extensive research for the prominent role of C1 compounds in establishing a circular bioeconomy. Methanol in particular holds great promise as it can be produced directly from greenhouse gases methane and carbon dioxide using renewable resources. Synthetic methylotrophy, i.e. introducing a non-native methanol utilization pathway into a model host, has therefore been the focus of long-time efforts and is perhaps the pinnacle of metabolic engineering. It entails completely changing a microorganism's lifestyle, from breaking up multi-carbon nutrients for growth to building C-C bonds from a single-carbon molecule to obtain all metabolites necessary to biomass formation as well as energy. The frontiers of synthetic methylotrophy have been pushed further than ever before and in this review, we outline the advances that paved the way for the more recent accomplishments. These include optimizing the host's metabolism, "copy and pasting" naturally existing methylotrophic pathways, "mixing and matching" enzymes to build new pathways, and even creating novel enzymatic functions to obtain strains that are able to grow solely on methanol. Finally, new approaches are contemplated to further advance the field and succeed in obtaining a strain that efficiently grows on methanol and allows C1-based production of added-value compounds.

Corrigendum: Combining metabolic engineering and multiplexed screening methods for 3-hydroxypropionic acid production in Pichia pastoris.

[This corrects the article DOI: 10.3389/fbioe.2022.942304.].

Aerobic Utilization of Methanol for Microbial Growth and Production.

Methanol is a reduced one-carbon (C1) compound. It supports growth of aerobic methylotrophs that gain ATP from reduced redox equivalents by respiratory phosphorylation in their electron transport chains. Notably, linear oxidation of methanol to carbon dioxide may yield three reduced redox equivalents if methanol oxidation is NAD-dependent as, e.g., in Bacillus methanolicus. Methanol has a higher degree of reduction per carbon than glucose (6 vs. 4), and thus, lends itself as an ideal carbon source for microbial production of reduced target compounds. However, C-C bond formation in the RuMP or serine cycle, a prerequisite for production of larger molecules, requires ATP and/or reduced redox equivalents. Moreover, heat dissipation and a high demand for oxygen during catabolic oxidation of methanol may pose challenges for fermentation processes. In this chapter, we summarize metabolic pathways for aerobic methanol utilization, aerobic methylotrophs as industrial production hosts, strain engineering, and methanol bioreactor processes. In addition, we provide technological and market outlooks.

Combining Metabolic Engineering and Multiplexed Screening Methods for 3-Hydroxypropionic Acid Production in Pichia pastoris.

Production of 3-hydroxypropionic acid (3-HP) in Pichia pastoris (syn. Komagataella phaffii) via the malonyl-CoA pathway has been recently demonstrated using glycerol as a carbon source, but the reported metrics were not commercially relevant. The flux through the heterologous pathway from malonyl-CoA to 3-HP was hypothesized as the main bottleneck. In the present study, different metabolic engineering approaches have been combined to improve the productivity of the original 3-HP producing strains. To do so, an additional copy of the gene encoding for the potential rate-limiting step of the pathway, i.e., the C-terminal domain of the malonyl-CoA reductase, was introduced. In addition, a variant of the endogenous acetyl-CoA carboxylase (ACC1 S1132A ) was overexpressed with the aim to increase the delivery of malonyl-CoA. Furthermore, the genes encoding for the pyruvate decarboxylase, aldehyde dehydrogenase and acetyl-CoA synthase, respectively, were overexpressed to enhance conversion of pyruvate into cytosolic acetyl-CoA, and the main gene responsible for the production of the by-product D-arabitol was deleted. Three different screening conditions were used to classify the performance of the different strains: 24-deep-well plates batch cultures, small-scale cultures in falcon tubes using FeedBeads® (i.e., slow release of glycerol over time), and mini bioreactor batch cultures. The best two strains from the FeedBeads® screening, PpHP8 and PpHP18, were tested in bioreactor fed-batch cultures using a pre-fixed exponentially increasing feeding rate. The strain PpHP18 produced up to 37.05 g L-1 of 3-HP at 0.712 g L-1 h-1 with a final product yield on glycerol of 0.194 Cmol-1 in fed-batch cultures. Remarkably, PpHP18 did not rank among the 2-top producer strains in small scale batch cultivations in deep-well plates and mini bioreactors, highlighting the importance of multiplexed screening conditions for adequate assessment of metabolic engineering strategies. These results represent a 50% increase in the product yield and final concentration, as well as over 30% increase in volumetric productivity compared to the previously obtained metrics for P. pastoris. Overall, the combination of glycerol as carbon source and a metabolically engineered P. pastoris strain resulted in the highest 3-HP concentration and productivity reported so far in yeast.

High-throughput workflow for monitoring and mining bioprocess data and its application to inferring the physiological response of Escherichia coli to perturbations.

Miniaturization and high-throughput screening are currently the focus of emerging research areas such as systems biology and systems biotechnology. A fluorescence-based screening assay for the online monitoring of oxygen and pH and a numerical method to mine the resulting online process data are described. The assay employs commercial phosphorescent oxygen- and pH-sensitive probes in standard 48- or 96-well plates on a plate reader equipped with a shaker. In addition to dual parametric analysis of both pH and oxygen in a single well, the assay allows monitoring of growth, as measured by absorbance. Validation of the assay is presented and compared with commercially available plates equipped with optical sensors for oxygen and pH. By using model-free fitting to the readily available online measurements, the length and rate of each phase such as the duration of lag and transition phase or acidification, growth, and oxygen consumption rates are automatically detected. In total, nine physiological descriptors, which can be used for further statistical and comparison analysis, are extracted from the pH, oxygen partial pressure (pO(2)), and optical density (OD) profiles. The combination of a simple mix-and-measure procedure with an automatic data mining method allows high sample throughput and good reproducibility while providing a physiological state identification and characterization of test cells. As a proof of concept, the utility of the workflow in assessing the physiological response of Escherichia coli to environmental and genetic perturbations is demonstrated.

Evaluation of Heterologous Biosynthetic Pathways for Methanol-Based 5-Aminovalerate Production by Thermophilic Bacillus methanolicus.

The use of methanol as carbon source for biotechnological processes has recently attracted great interest due to its relatively low price, high abundance, high purity, and the fact that it is a non-food raw material. In this study, methanol-based production of 5-aminovalerate (5AVA) was established using recombinant Bacillus methanolicus strains. 5AVA is a building block of polyamides and a candidate to become the C5 platform chemical for the production of, among others, δ-valerolactam, 5-hydroxy-valerate, glutarate, and 1,5-pentanediol. In this study, we test five different 5AVA biosynthesis pathways, whereof two directly convert L-lysine to 5AVA and three use cadaverine as an intermediate. The conversion of L-lysine to 5AVA employs lysine 2-monooxygenase (DavB) and 5-aminovaleramidase (DavA), encoded by the well-known Pseudomonas putida cluster davBA, among others, or lysine α-oxidase (RaiP) in the presence of hydrogen peroxide. Cadaverine is converted either to γ-glutamine-cadaverine by glutamine synthetase (SpuI) or to 5-aminopentanal through activity of putrescine oxidase (Puo) or putrescine transaminase (PatA). Our efforts resulted in proof-of-concept 5AVA production from methanol at 50°C, enabled by two pathways out of the five tested with the highest titer of 0.02 g l-1. To our knowledge, this is the first report of 5AVA production from methanol in methylotrophic bacteria, and the recombinant strains and knowledge generated should represent a valuable basis for further improved 5AVA production from methanol.

Exploring the Glucose Fluxotype of the E. coli y-ome Using High-Resolution Fluxomics.

We have developed a robust workflow to measure high-resolution fluxotypes (metabolic flux phenotypes) for large strain libraries under fully controlled growth conditions. This was achieved by optimizing and automating the whole high-throughput fluxomics process and integrating all relevant software tools. This workflow allowed us to obtain highly detailed maps of carbon fluxes in the central carbon metabolism in a fully automated manner. It was applied to investigate the glucose fluxotypes of 180 Escherichia coli strains deleted for y-genes. Since the products of these y-genes potentially play a role in a variety of metabolic processes, the experiments were designed to be agnostic as to their potential metabolic impact. The obtained data highlight the robustness of E. coli's central metabolism to y-gene deletion. For two y-genes, deletion resulted in significant changes in carbon and energy fluxes, demonstrating the involvement of the corresponding y-gene products in metabolic function or regulation. This work also introduces novel metrics to measure the actual scope and quality of high-throughput fluxomics investigations.