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Biotechnology & bioengineering 120(5), 1269-1287 (2023). doi:10.1002/bit.28334, Published by Wiley, New York, NY [u.a.]

Chemie - Ingenieur - Technik : CIT 95(4), 485-490 (2022). doi:10.1002/cite.202200169 special issue: "Special Issue:Prof. Dr.‐Ing. Christian Wandrey zum 80. Geburtstag gewidmet / Issue Edited by: Andreas Liese, Michael Müller, Thomas Noll, Ralf Takors", Published by Wiley-VCH Verl., Weinheim

D-Xylose, a major constituent of plant biomass and second most abundant sugar on Earth, holds a considerable potential as a substrate for sustainable bio-production. Pseudomonas putida KT2440 is an attractive bacterial host for valorizing biogenic feedstocks but lacks a xylose utilization pathway. While several attempts to engineer P. putida for growth on xylose have been reported, a comprehensive understanding of xylose metabolism in this bacterium is lacking, hindering its further improvement and rational tailoring for specific biotechnological purposes. In this study, we elucidated the xylose metabolism in the genome-reduced P. putida strain, EM42, endowed with xylose isomerase pathway (xylAB) and transporter (xylE) from Escherichia coli and used the obtained knowledge in combination with adaptive laboratory evolution to accelerate the bacterium’s growth on the pentose sugar. Carbon flux analyses, targeted gene knock-outs, and in vitro enzyme assays portrayed xylose assimilation in P. putida and confirmed a partially cyclic upper xylose metabolism. Deletion of the local transcriptional regulator gene hexR de-repressed genes of several key catabolic enzymes and reduced the lag phase on xylose. Guided by metabolic modeling, we augmented P. putida with additional heterologous pentose phosphate pathway genes and subjected rationally prepared strains to adaptive laboratory evolution (ALE) on xylose. The descendants showed accelerated growth and reduced growth lag. Genomic and proteomic analysis of engineered and evolved mutants revealed the importance of a large genomic re-arrangement, transaldolase overexpression, and balancing gene expression in the synthetic xylABE operon. Importantly, omics analyses found that similar growth characteristics of two superior mutants were achieved through distinct evolutionary paths. This work provides a unique insight into how cell metabolism adjusts to a non-native substrate; it highlights the remarkable genomic and metabolic plasticity of P. putida and demonstrates the power of combining knowledge-driven engineering with ALE in generating desirable microbial phenotypes.HighlightsElucidated xylose catabolism via exogenous isomerase pathway inP. putidaEM42.Deletion of transcriptional regulator HexR improved growth on xylose.Knowledge-guided interventions and adaptive evolution accelerated growth.Omics analyses of selected mutants highlighted the genomic and metabolic plasticity ofP. putida.Two mutants with superior characteristics emerged from distinct evolutionary paths.

The black-yeast-like Aureobasidium is discussed as a versatile cell factory for many biotechnological applications. This article describes the 25.05-Mb draft genome sequence of Aureobasidium pullulans NRRL 62031, which was isolated in Thailand. The genome sequence provides evidence for a plethora of synthesis pathways for valuable secondary metabolites.

The field of metabolic modelling at the genomescale continues to grow with more models being created and curated. This comes with an increasing demand for adopting common principles regarding transparency and versioning, in addition to standardisation efforts regarding file formats, annotation and testing. Here, we present a standardised template for git-based and GitHub-hosted genome-scale metabolic models (GEMs) supporting both new models and curated ones, following FAIR principles (findability, accessibility, interoperability, and reusability), and incorporating bestpractices.standard-GEMfacilitates the reuse of GEMs across web services and platforms in the metabolic modelling field and enables automatic validation of GEMs. The use of this template for new models, and its adoption for existing ones, paves the way for increasing model quality, openness, and accessibility with minimal effort.Availabilitystandard-GEMis available fromgithub.com/MetabolicAtlas/standard-GEMunder the conditions of the CC BY 4.0 licence along with additional supporting material.

Poly-γ-glutamic acid (γ-PGA) is an emerging biopolymer produced by several Bacillus species. To improve γ-PGA synthesis, metabolic engineering of the production host B. subtilis poses great potential and is facilitated by the convenient genetical amenability of the organism. In this study, a 3.7-fold increase in γ-PGA production using a bdhA, alsSD, pta, yvmC, and cypX deletion mutant with blocked by-product synthesis pathways was obtained. A detailed analysis of intracellular metabolites for reference strains and the γ-PGA-producing deletion strain identified the accumulation of pyruvate and acetyl-CoA in deletion mutants, highlighting the citrate synthase activity as an important metabolic engineering target for further metabolic flux optimization towards γ-PGA synthesis. An in-depth analysis of growth and γ-PGA production with on-line measurement techniques revealed significant variations across cultivations with deletion mutants that are likely caused by culture acidification due to pyruvate accumulation. Despite the observed acidification, the by-product deletion mutants outperformed the reference strains independent of the promoter controlling the PGA synthetase expression. The constructed deletion strains exhibit high γ-PGA production in minimal medium with glucose as sole carbon source as well as in modified Medium E reaching γ-PGA concentrations of 0.57 gL-1 and 14.46 gL-1, respectively. The results presented in this work broaden the understanding of the microbial metabolism during γ-PGA production and will be useful to guide future metabolic engineering for improved γ-PGA production.

Biosurfactants are amphiphilic compounds of microbial origin with a wide range of industrial applications. Rhamnolipids (RLs) offer a broad range of potential applications as biosurfactants in industry and agriculture. Several studies report a high capacity for controlling different plant pests and pathogens and consider RLs as promising candidates for bio-based plant protection agents. RLs are a class of glycolipids consisting of one (mRLs) or two (dRLs) rhamnose moieties linked to a single or branched β-fatty acid. Due to the diversity of RLs, so far little is known about the relation between molecular structure and biological activity.Engineering the synthesis pathway allowed us to differentiate between the activities of mixtures of pure mRL and of pure dRL congeners and elaborated HPLC techniques further enabled to analyse the activity of single congeners. In a model system with the plantArabidopsis thalianaand the plant-parasitic nematodeHeterodera schachtiiwe demonstrate that RLs can significantly reduce infection, whereas their impact on the host plant varies depending on their molecular structure. While mRLs reduced plant growth even at low concentration, dRLs showed no or a beneficial impact on plant development. In addition, the effect of RLs on plant H2O2production was measured as an indicator of plant defense activity. Treatment with mRLs or dRLs at a concentration of 50 ppm increased H2O2production. Lower concentrations of up to 10 ppm were used to stimulate plants prior to a treatment with water or flagellin (flg22), a bacterial inducer of plant defense responses. At 10 ppm both mRLs and dRLs fostered an increased response to flg22. However, mRLs also led to an increased response to water, the non-inducing negative control, indicating a generally elevated stress level. Neither mRLs nor dRLs induced expression of plant defense marker genes of salicylic acid, jasmonic acid and ethylene pathways within a 1 hour and 48 hours treatment.Due to the negative effect of mRLs on plants further studies were concentrated on dRLs. Treatment of pre-parasitic infective juveniles ofH. schachtiirevealed that dRLs did not increase mortality even at a very high concentration of 755 ppm. In order to analyse the effect of single dRL congeners nematode infection assays were performed. While dRL congeners with a C10-C8 acyl chain increased nematode infection, dRLs with C10-C12 and C10-C12:1 acyl chains reduced nematode infection even at concentrations below 2 ppm. Plant growth was not reduced by C10-C8 dRLs, but by C10-C12 and C10-C12:1 dRLs at concentrations of 8.3 ppm. H2O2production was increased compared to the water control upon treatment with C10-C8 dRLs at a concentration of 200 ppm, while C10-C12 and C10-C12:1 dRLs triggered the same effect already at 50 ppm.Our experiments show a clear structure-effect relation. In conclusion, functional assessment and analysis of mode of action of RLs in plants require careful consideration of their molecular structure and composition.

Methyl ketones are of interest for the application as biofuels. To achieve a sustainable production of methyl ketones, the fatty acid metabolism of different microbes has been rearranged. The biofuel properties and possible further chemical modifications of these methyl ketones are influenced by their chain length as well as their degree of saturation and the corresponding double bond position.In order to determine the double bond position of methyl ketones, a method based on gas chromatography-electron ionization mass spectrometry was used. Derivatization by dimethyl disulfide and an iodine catalyst enabled activation of the double bonds for selective fragmentation during electron ionization. The cleavage led to key fragments in the Orbitrap high resolution mass spectrum and allowed the unequivocal localization of the double bond position of the respective monounsaturated methyl ketone.The double bond position of several medium chain length methyl ketones originating from the gram-negative bacterium Pseudomonas taiwanensis VLB120 was determined. The dimethyl disulfide derivatives of methyl ketones can yield isobaric fragment ions for different possible double bond positions, which can only be distinguished by high resolution mass spectrometry. The double bond position of all methyl ketones deriving from Pseudomonas taiwanensis VLB120 was the same, counting from the end of the aliphatic chain, and was determined as ω-7.The derivatization of medium chain length monounsaturated methyl ketones with dimethyl disulfide allowed the determination of the corresponding double bond position via gas chromatography-electron ionization mass spectrometry. High resolution mass spectrometry is needed to differentiate possible double bond positions that yield isobaric fragment ions.

Applied Microbiology 3(1), 175-198 (2023). doi:10.3390/applmicrobiol3010013, Published by MDPI, Basel, Switzerland

Plastic mulch films and biofertilizers (processed sewage sludge, compost or manure) have helped to increase crop yields. However, there is increasing evidence that these practices significantly contribute to microplastic contamination in agricultural soils, affecting biodiversity and soil health. Here, we draw attention to the use of hydrolase enzymes that depolymerize polyester-based plastics as a bioremediation technique for agricultural soils (in situ), biofertilizers and irrigation water (ex situ), and discuss the need for fully biodegradable plastic mulches. We also highlight the need for ecotoxicological assessment of the proposed approach and its effects on different soil organisms. Enzymes should be optimized to work effectively and efficiently under the conditions found in natural soils (typically, moist solids at an ambient temperature with low salinity). Such optimization is also necessary to ensure that already distressed ecosystems are not disrupted any further.

Discover Chemical Engineering 3(1), 2 (2023). doi:10.1007/s43938-023-00018-5, Published by Springer International Publishing, [Cham]

Bile acid (BA) metabolism is a complex system that includes a wide variety of primary and secondary, as well as conjugated and unconjugated BAs that undergo continuous enterohepatic circulation (EHC). Alterations in both composition and dynamics of BAs have been associated with various diseases. However, a mechanistic understanding of the relationship between altered BA metabolism and related diseases is lacking. Computational modeling may support functional analyses of the physiological processes involved in the EHC of BAs along the gut-liver axis. In this study, we developed a physiologically-based model of murine BA metabolism describing synthesis, conjugation, microbial transformations, systemic distribution, excretion and EHC of BAs at the whole-body level. For model development, BA metabolism of specific pathogen-free (SPF) mice was characterized in vivo by measuring BA levels and composition in various organs, expression of transporters along the gut and cecal microbiota composition. We found significantly different BA levels between male and female mice that could only be explained by adjusted expression of the hepatic enzymes and transporters in the model. Of note, this finding was in agreement with experimental observations. The model for SPF mice could also describe equivalent experimental data in germ-free mice by specifically switching of microbial activity in the intestine. The here presented model can therefore facilitate and guide functional analyses of BA metabolism in mice, e.g., the effect of pathophysiological alterations on BA metabolism and translation of results from mouse studies to a clinically relevant context through cross-species extrapolation.

The novel multiphase loop reactor is a modified airlift reactor with an internal loop enabling continuous in situ liquid-liquid extraction. In this study, the reactor is applied for a microbial production of biosurfactants. The obligate aerobic bacterium Pseudomonas putida KT2440 was engineered for rhamnolipid production. Rhamnolipids are biosurfactants with strong foaming capabilities making cultivations in an aerated stirred tank fermenter challenging. The continuous removal of rhamnolipids via in situ liquid-liquid extraction can remedy this foam challenge, and thereby supports long-term cultivation and production. The initially designed multiphase loop reactor had an oxygen transfer rate, which was too low to meet the oxygen demand of the whole-cell biocatalyst, resulting in inefficient growth and production. A re-design of the sparger via 3D-printing enabled a raise in oxygen supply allowed rhamnolipid production at key performance indicators that matched stirred-tank reactor cultivations, but with the advantage of enabling continuous cultivation in the future. Concluding, we present the successful use of the multiphase loop reactor for rhamnolipid synthesis, highlighting its potential to become a new platform technology for intensified bioprocessing.

In recent years, it was shown that itaconic acid can be produced from glucose with

We present a generic workflow combining physiology-based computational modeling and in vitro data to assess the clinical cholestatic risk of different drugs systematically. Changes in expression levels of genes involved in the enterohepatic circulation of bile acids were obtained from an in vitro assay mimicking 14 days of repeated drug administration for 10 marketed drugs. These changes in gene expression over time were contextualized in a physiology-based bile acid model of glycochenodeoxycholic acid. The simulated drug-induced response in bile acid concentrations was then scaled with the applied drug doses to calculate the cholestatic potential for each compound. A ranking of the cholestatic potential correlated very well with the clinical cholestasis risk obtained from medical literature. The proposed workflow allows benchmarking the cholestatic risk of novel drug candidates. We expect the application of our workflow to significantly contribute to the stratification of the cholestatic potential of new drugs and to support animal-free testing in future drug development.

Many wild type Pseudomonas strains have the potential to contribute to the valorization of lignin in future biorefineries. Through a robust aromatic catabolism, i.e., biofunneling capacity, they can ease the inherent aromatic heterogeneity found in lignin hydrolysates and accumulate naturally marketable biopolymers like mcl-polyhydroxyalkanoate (mcl-PHA) under nitrogen limitation. Besides a comparative strain evaluation, we present fundamental research on the funneling of aromatic mixtures under specific bioprocess conditions to improve biocatalytic lignin valorization. For the most robust and best performing strain, P. putida KT2440, we improve the mcl-PHA accumulation from a defined aromatic mixture of p-coumarate, ferulate, and benzoate under technically relevant conditions by up to 40% by tailoring the nitrogen and oxygen supply. The highest mcl-PHA concentration (582 +/- 41 mg L-1) was obtained for a C/N ratio of 60 for oxygen-unlimited conditions (oxygen transfer rate >20 mmol L-1 h(-1)). In contrast, aromatic intermediates accumulated under oxygen-limited conditions at oxygen transfer rates below 10 mmol L-1 h(-1). The experimental conditions were scalable into a 1L stirred tank bioreactor. This study contributes to deepening our understanding of the biocatalytic capability of promising Pseudomonas strains toward downstream microbial conversions of lignin aromatics for future biorefinery applications.

Poly-γ-glutamic acid (γ-PGA) is a biopolymer with a wide range of applications, mainly produced using Bacillus strains. The formation and concomitant secretion of γ-PGA increases the culture broth viscosity, while enzymatic depolymerisation and degradation of γ-PGA decreases the culture broth viscosity. In this study, the recently published ViMOS (Viscosity Monitoring Online System) is applied for optical online measurements of broth viscosity in eight parallel shake flasks. It is shown that the ViMOS is suitable to monitor γ-PGA production and degradation online in shake flasks. This online monitoring enables the detailed analysis of the P

Since 2008, legislative initiatives for climate protection and reduced dependency on fossil resource imports led to the introduction of biofuels as CO2-reduced alternatives in the heating oil sector. In the case of biodiesel, the oil industry or its customers were confronted with accelerated and escalating microbial contaminations during heating oil storage. Since then, other fuel alternatives, like hydrogenated vegetable oils, gas-to-liquid products (GtL), or Oxymethylenether (OME) have been or will be developed and potentially introduced to the market. In this study, we use online monitoring of microbial CO2 production and the simulation of onset of microbial contamination to investigate the contamination potential of fuel alternatives during storage. As reference and blends, fossil heating oils of various refineries, in the course of this from various crude oils, and refinery processes reveal considerable variation in potential microbial activity. Oxymethylene ethers have an antimicrobial effect, while various forms of biodiesel confirm the promotion of microbial activity and diversity. The paraffinic Fischer-Tropsch products and biogenic hydrogenation products demonstrate high resistance to microbial contamination despite allowing microbial diversity. Through an array of analytics, including advanced chromatography coupled mass spectrometry, elemental analysis, and microbial sequencing, we can discuss critical fuel properties that promote or inhibit microbial contaminations. In summary, novel, non-fossil heating oils show different strengths and weaknesses for long-term storage. Designing blends for microbial activity reduced long-term storage might be an option. While being niche products, these fuels will contribute to the rapid reduction of fossil resource use.

The marine bacterium Alcanivorax borkumensis produces a surface-active glycine-glucolipid during growth with long-chain alkanes. A high-performance liquid chromatography (HPLC) method was developed for absolute quantification. This method is based on the conversion of the glycine-glucolipid to phenacyl esters with subsequent measurement by HPLC with diode array detection (HPLC-DAD). Different molecular species were separated by HPLC and identified as glucosyl-tetra(3-hydroxy-acyl)-glycine with varying numbers of 3-hydroxy-decanoic acid or 3-hydroxy-octanoic acid groups via mass spectrometry. The growth rate of A. borkumensis cells with pyruvate as the sole carbon source was elevated compared to hexadecane as recorded by the increase in cell density as well as oxygen/carbon dioxide transfer rates. The amount of the glycine-glucolipid produced per cell during growth on hexadecane was higher compared with growth on pyruvate. The glycine-glucolipid from pyruvate-grown cells contained considerable amounts of 3-hydroxy-octanoic acid, in contrast to hexadecane-grown cells, which almost exclusively incorporated 3-hydroxy-decanoic acid into the glycine-glucolipid. The predominant proportion of the glycine-glucolipid was found in the cell pellet, while only minute amounts were present in the cell-free supernatant. The glycine-glucolipid isolated from the bacterial cell broth, cell pellet, or cell-free supernatant showed the same structure containing a glycine residue, in contrast to previous reports, which suggested that a glycine-free form of the glucolipid exists which is secreted into the supernatant. In conclusion, the glycine-glucolipid of A. borkumensis is resident to the cell wall and enables the bacterium to bind and solubilize alkanes at the lipid-water interface. IMPORTANCE Alcanivorax borkumensis is one of the most abundant marine bacteria found in areas of oil spills, where it degrades alkanes. The production of a glycine-glucolipid is considered an essential element for alkane degradation. We developed a quantitative method and determined the structure of the A. borkumensis glycine-glucolipid in different fractions of the cultures after growth in various media. Our results show that the amount of the glycine-glucolipid in the cells by far exceeds the amount measured in the supernatant, confirming the proposed cell wall localization. These results support the scenario that the surface hydrophobicity of A. borkumensis cells increases by producing the glycine-glucolipid, allowing the cells to attach to the alkane-water interface and form a biofilm. We found no evidence for a glycine-free form of the glucolipid.

Triterpenoids, one of the most diverse classes of natural products, have been used for centuries as active ingredients in essential oils and Chinese medicines and are of interest for many industrial applications ranging from low-calorie sweeteners to cosmetic ingredients and vaccine adjuvants. However, not only can the extraction from plant material be cumbersome due to low concentrations of the specific triterpenoid, but concerns are also increasing regarding the sustainability of wild plant harvest while meeting market demands. The alternative is to produce triterpenoids with engineered microbes. Here, we present a generally applicable strategy for triterpenoid production in the yeast Saccharomyces cerevisiae based on a modified oxidosqualene cyclase Erg7. The modification reduces the flux into the sterol pathway while increasing the precursor supply for triterpenoid production. The minimally engineered strain was exploited for the exemplary production of the lupane triterpenoids betulin, betulin aldehyde, and betulinic acid at a total titer above 6 g/L, the highest reported so far. To further highlight the chassis concept, squalene, oleanane- and dammarane-type triterpenoids were synthesized to titers at a similar gram scale. We propose the developed baker’s yeast as a host for the thousands of triterpenoid synthesis pathways from plants, reducing the pressure on the natural resources.

Physiologically based pharmacokinetic (PBPK) models have been proposed as a tool for more accurate individual pharmacokinetic (PK) predictions and model‐informed precision dosing, but their application in clinical practice is still rare. This study systematically assesses the benefit of using individual patient information to improve PK predictions. A PBPK model of caffeine was stepwise personalized by using individual data on (1) demography, (2) physiology, and (3) cytochrome P450 (CYP) 1A2 phenotype of 48 healthy volunteers participating in a single‐dose clinical study. Model performance was benchmarked against a caffeine base model simulated with parameters of an average individual. In the first step, virtual twins were generated based on the study subjects' demography (height, weight, age, sex), which implicated the rescaling of average organ volumes and blood flows. The accuracy of PK simulations improved compared with the base model. The percentage of predictions within 0.8‐fold to 1.25‐fold of the observed values increased from 45.8% (base model) to 57.8% (Step 1). However, setting physiological parameters (liver blood flow determined by magnetic resonance imaging, glomerular filtration rate, hematocrit) to measured values in the second step did not further improve the simulation result (59.1% in the 1.25‐fold range). In the third step, virtual twins matching individual demography, physiology, and CYP1A2 activity considerably improved the simulation results. The percentage of data within the 1.25‐fold range was 66.15%. This case study shows that individual PK profiles can be predicted more accurately by considering individual attributes and that personalized PBPK models could be a valuable tool for model‐informed precision dosing approaches in the future.

Soft matter 17(11), 3191-3206 (2021). doi:10.1039/D0SM01934H, Published by Royal Society of Chemistry, London

We present an integrated process for in situ CO2 hydrogenation during bioethanol production combining bio- and chemo-catalysis. The biphasic catalytic system comprises an aqueous whole-cell fermentation broth and a tailored organometallic catalyst in an organic phase. Glucose is converted to ethanol and the by-product CO2 is simultaneously upgraded to formic acid in a single reactor unit. Under optimized conditions, 26% of the generated CO2 was hydrogenated directly.

Lignin is a ubiquitously available and sustainable feedstock that is underused as its depolymerization yields a range of aromatic monomers that are challenging substrates for microbes. In this study, we investigated the growth of

Volatile organic compounds play an essential role in every domain of life, with diverse functions. In this study, we use novel secondary electrospray ionisation high-resolution Orbitrap mass spectrometry (SESI-Orbitrap MS) to monitor the complete yeast volatilome every 2.3 s. Over 200 metabolites were identified during growth in shake flasks and bioreactor cultivations, all with their unique intensity profile. Special attention was paid to ethanol as biotech largest product and to acetaldehyde as an example of a low-abundance but highly-volatile metabolite. While HPLC and Orbitrap measurements show a high agreement for ethanol, acetaldehyde could be measured five hours earlier in the SESI-Orbitrap MS. Volatilome shifts are visible, e.g. after glucose depletion, fatty acids are converted to ethyl esters in a detoxification mechanism after stopped fatty acid biosynthesis. This work showcases the SESI-Orbitrap MS system for tracking microbial physiology without the need for sampling and for time-resolved discoveries during metabolic transitions.

Allicin is the main flavour component of crushed raw garlic. This plant defence molecule has strong antibiotic properties. While measurements in the liquid phase using LC-MS are established, accessing reactive organosulfur compounds in the gas phase is still a challenge due to heat-degradation in the gas chromatograph. Using a gentle secondary electrospray ionisation coupled Orbitrap mass spectrometry procedure (SESI-Orbitrap MS), we measured gas phase concentrations of allicin evaporating from a pure solution. Despite the mild conditions, two quantitatively major allicin-derived breakdown products were found. The SESI-Orbitrap MS technique was used to follow the known chemistry of alliin, isoallin and methiin conversion in garlic, onion and ramsons. Allicin and its metabolites were also measured over two hours in human breath after garlic consumption. These results demonstrate the utility of SESI-Orbitrap MS for analysis of sulfur-containing volatiles from plants in the genus Allium and potentially for capturing volatilomes of foodstuffs in general.

Biotechnology for biofuels 13(1), 1-18 (2020). doi:10.1186/s13068-020-01835-4, Published by BioMed Central, London

The industrially relevant biopolymer poly-γ-glutamic acid (γ-PGA) is commonly synthesized using glycerol, citrate, and glutamic acid as carbon sources. In this study, two strains capable of utilizing glucose as sole carbon source for γ-PGA synthesis were constructed. Efficient γ-PGA production was achieved with derivatives of the well-investigated laboratory strain Bacillus subtilis 168, by replacing the native promoter of the PGA synthetase operon with the strong constitutive promoter Pveg or with the xylose-inducible promoter Pxyl. The carbon yield for γ-PGA increased by 129% to 0.131 C-mol C-mol−1 when using glucose as the sole substrate compared to the conventional carbon source mixture glycerol, citrate, and glutamic acid. The characterization of the produced γ-PGA demonstrated a time-dependent molecular weight of 1180–1850 kDa and a d -glutamic acid monomer content of 49–62%. To elucidate the consequences of γ-PGA production, we characterized the engineered strain by metabolomics. While the metabolite concentrations in the TCA cycle leading up to 2-oxoglutarate decreased in γ-PGA producer strains, the glutamic acid concentration was constant, despite the drastic increase in glutamic acid demand. The results are discussed in the context of metabolic regulation and future metabolic engineering strategies to enhance precursor supply for γ-PGA synthesis from glucose.

Supplementary information is available for this paper at https://doi.org/10.1038/s41587-020-0446-y, Reconstructing metabolic reaction networks enables the development of testable hypotheses of an organisms metabolism under different conditions1. State-of-the-art genome-scale metabolic models (GEMs) can include thousands of metabolites and reactions that are assigned to subcellular locations. Geneproteinreaction (GPR) rules and annotations using database information can add meta-information to GEMs. GEMs with metadata can be built using standard reconstruction protocols2, and guidelines have been put in place for tracking provenance and enabling interoperability, but a standardized means of quality control for GEMs is lacking3. Here we report a community effort to develop a test suite named MEMOTE (for metabolic model tests) to assess GEM quality., We acknowledge D. Dannaher and A. Lopez for their supporting work on the Angular parts of MEMOTE; resources and support from the DTU Computing Center; J. Cardoso, S. Gudmundsson, K. Jensen and D. Lappa for their feedback on conceptual details; and P. D. Karp and I. Thiele for critically reviewing the manuscript. We thank J. Daniel, T. Kristjánsdóttir, J. Saez-Saez, S. Sulheim, and P. Tubergen for being early adopters of MEMOTE and for providing written testimonials. J.O.V. received the Research Council of Norway grants 244164 (GenoSysFat), 248792 (DigiSal) and 248810 (Digital Life Norway); M.Z. received the Research Council of Norway grant 244164 (GenoSysFat); C.L. received funding from the Innovation Fund Denmark (project “Environmentally Friendly Protein Production (EFPro2)”); C.L., A.K., N. S., M.B., M.A., D.M., P.M, B.J.S., P.V., K.R.P. and M.H. received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 686070 (DD-DeCaF); B.G.O., F.T.B. and A.D. acknowledge funding from the US National Institutes of Health (NIH, grant number 2R01GM070923-13); A.D. was supported by infrastructural funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), Cluster of Excellence EXC 2124 Controlling Microbes to Fight Infections; N.E.L. received funding from NIGMS R35 GM119850, Novo Nordisk Foundation NNF10CC1016517 and the Keck Foundation; A.R. received a Lilly Innovation Fellowship Award; B.G.-J. and J. Nogales received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no 686585 for the project LIAR, and the Spanish Ministry of Economy and Competitivity through the RobDcode grant (BIO2014-59528-JIN); L.M.B. has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 633962 for project P4SB; R.F. received funding from the US Department of Energy, Offices of Advanced Scientific Computing Research and the Biological and Environmental Research as part of the Scientific Discovery Through Advanced Computing program, grant DE-SC0010429; A.M., C.Z., S.L. and J. Nielsen received funding from The Knut and Alice Wallenberg Foundation, Advanced Computing program, grant #DE-SC0010429; S.K.’s work was in part supported by the German Federal Ministry of Education and Research (de.NBI partner project “ModSim” (FKZ: 031L104B)); E.K. and J.A.H.W. were supported by the German Federal Ministry of Education and Research (project “SysToxChip”, FKZ 031A303A); M.K. is supported by the Federal Ministry of Education and Research (BMBF, Germany) within the research network Systems Medicine of the Liver (LiSyM, grant number 031L0054); J.A.P. and G.L.M. acknowledge funding from US National Institutes of Health (T32-LM012416, R01-AT010253, R01-GM108501) and the Wagner Foundation; G.L.M. acknowledges funding from a Grand Challenges Exploration Phase I grant (OPP1211869) from the Bill & Melinda Gates Foundation; H.H. and R.S.M.S. received funding from the Biotechnology and Biological Sciences Research Council MultiMod (BB/N019482/1); H.U.K. and S.Y.L. received funding from the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (grants NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) from the Ministry of Science and ICT through the National Research Foundation (NRF) of Korea; H.U.K. received funding from the Bio & Medical Technology Development Program of the NRF, the Ministry of Science and ICT (NRF-2018M3A9H3020459); P.B., B.J.S., Z.K., B.O.P., C.L., M.B., N.S., M.H. and A.F. received funding through Novo Nordisk Foundation through the Center for Biosustainability at the Technical University of Denmark (NNF10CC1016517); D.-Y.L. received funding from the Next-Generation BioGreen 21 Program (SSAC, PJ01334605), Rural Development Administration, Republic of Korea; G.F. was supported by the RobustYeast within ERA net project via SystemsX.ch; V.H. received funding from the ETH Domain and Swiss National Science Foundation; M.P. acknowledges Oxford Brookes University; J.C.X. received support via European Research Council (666053) to W.F. Martin; B.E.E. acknowledges funding through the CSIRO-UQ Synthetic Biology Alliance; C.D. is supported by a Washington Research Foundation Distinguished Investigator Award. I.N. received funding from National Institutes of Health (NIH)/National Institute of General Medical Sciences (NIGMS) (grant P20GM125503)., info:eu-repo/semantics/publishedVersion

A key aspect of the transformation of the economic sector towards a sustainable bioeconomy is the development of environmentally friendly alternatives for hitherto used chemicals, which have negative impacts on environmental health. However, the implementation of an ecotoxicological hazard assessment at early steps of product development to elaborate the most promising candidates of lowest harm is scarce in industry practice. The present article introduces the interdisciplinary proof-of-concept project GreenToxiConomy, which shows the successful application of a Green Toxicology strategy for biosurfactants and a novel microgel-based pesticide release system. Both groups are promising candidates for industrial and agricultural applications and the ecotoxicological characterization is yet missing important information. An iterative substance- and application-oriented bioassay battery for acute and mechanism-specific toxicity within aquatic and terrestrial model species is introduced for both potentially hazardous materials getting into contact with humans and ending up in the environment. By applying in silico QSAR-based models on genotoxicity, endocrine disruption, skin sensitization and acute toxicity to algae, daphnids and fish individual biosurfactants resulted in deviating toxicity, suggesting a pre-ranking of the compounds. Experimental toxicity assessment will further complement the predicted toxicity to elaborate the most promising candidates in an efficient pre-screening of new substances.