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Cyanobacteria are photosynthetic prokaryotes with strong potential to be used for industrial terpenoid production. However, the key enzymes forming the principal terpenoid building blocks, called short-chain prenyltransferases (SPTs), are insufficiently characterized. Here, we examined SPTs in the model cyanobacteria Synechococcus elongatus sp. PCC 7942 and Synechocystis sp. PCC 6803. Each species has a single putative SPT (SeCrtE and SyCrtE, respectively). Sequence analysis identified these as type-II geranylgeranyl pyrophosphate synthases (GGPPSs) with high homology to GGPPSs found in the plastids of green plants and other photosynthetic organisms. In vitro analysis demonstrated that SyCrtE is multifunctional, producing geranylgeranyl pyrophosphate (GGPP; C

Summary The yeast Saccharomyces cerevisiae uses the pyruvate dehydrogenase‐bypass for acetyl‐CoA biosynthesis. This relatively inefficient pathway limits production potential for acetyl‐CoA‐derived biochemical due to carbon loss and the cost of two high‐energy phosphate bonds per molecule of acetyl‐CoA. Here, we attempted to improve acetyl‐CoA production efficiency by introducing heterologous acetylating aldehyde dehydrogenase and phosphoketolase pathways for acetyl‐CoA synthesis to enhance production of the sesquiterpene trans‐nerolidol. In addition, we introduced auxin‐mediated degradation of the glucose‐dependent repressor Mig1p to allow induced expression of GAL promoters on glucose so that production potential on glucose could be examined. The novel genes that we used to reconstruct the heterologous acetyl‐CoA pathways did not sufficiently complement the loss of endogenous acetyl‐CoA pathways, indicating that superior heterologous enzymes are necessary to establish fully functional synthetic acetyl‐CoA pathways and properly explore their potential for nerolidol synthesis. Notwithstanding this, nerolidol production was improved twofold to a titre of ˜ 900 mg l−1 in flask cultivation using a combination of heterologous acetyl‐CoA pathways and Mig1p degradation. Conditional Mig1p depletion is presented as a valuable strategy to improve the productivities in the strains engineered with GAL promoters‐controlled pathways when growing on glucose., We attempted to improve acetyl‐CoA production efficiency by introducing heterologous acetylating aldehyde dehydrogenase and phosphoketolase pathways for acetyl‐CoA synthesis to enhance production of the sesquiterpene nerolidol. Conditional Mig1p depletion is presented as a valuable strategy to improve the productivities in the strains engineered with GAL promoters‐controlled pathways when growing on glucose.

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.

Isoprenoids, also known as terpenes or terpenoids, are compounds made of one or more isoprene (C

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

The strigolactone (SL) class of phytohormones shows broad chemical diversity, the functional importance of which remains to be fully elucidated, along with the enzymes responsible for the diversification of the SL structure. Here we explore the functional evolution of the highly conserved CYP711A P450 family, members of which catalyze several key monooxygenation reactions in the strigolactone pathway. Ancestral sequence reconstruction was utilized to infer ancestral CYP711A sequences based on a comprehensive set of extant CYP711 sequences. Eleven ancestral enzymes, corresponding to key points in the CYP711A phylogenetic tree, were resurrected and their activity was characterized towards the native substrate carlactone and the pure enantiomers of the synthetic strigolactone analogue, GR24. The ancestral and extant CYP711As tested accepted GR24 as a substrate and catalyzed several diversifying oxidation reactions on the structure. Evidence was obtained for functional divergence in the CYP711A family. The monocot group 3 ancestor, arising from gene duplication events within monocot grasses, showed both increased catalytic activity towards GR24 and high stereoselectivity towards the GR24 isomer resembling strigol-type SLs. These results are consistent with a role for CYP711As in strigolactone diversification in early land plants, which may have extended to the diversification of strigol-type SLs.

mSystems 6(2), (2021). doi:10.1128/mSystems.00625-20, Published by American Society for Microbiology, Washington, DC

Frontiers in genetics 11, 637738 (2021). doi:10.3389/fgene.2020.637738, Published by Frontiers Media, Lausanne

In metabolic engineering, loss-of-function experiments are used to understand and optimise metabolism. A conditional gene inactivation tool is required when gene deletion is lethal or detrimental to growth. Here, we exploit auxin-inducible protein degradation as a metabolic engineering approach in yeast. We demonstrate its effectiveness using terpenoid production. First, we target an essential prenyl-pyrophosphate metabolism protein, farnesyl pyrophosphate synthase (Erg20p). Degradation successfully redirects metabolic flux toward monoterpene (C10) production. Second, depleting hexokinase-2, a key protein in glucose signalling transduction, lifts glucose repression and boosts production of sesquiterpene (C15) nerolidol to 3.5 g L−1 in flask cultivation. Third, depleting acetyl-CoA carboxylase (Acc1p), another essential protein, delivers growth arrest without diminishing production capacity in nerolidol-producing yeast, providing a strategy to decouple growth and production. These studies demonstrate auxin-mediated protein degradation as an advanced tool for metabolic engineering. It also has potential for broader metabolic perturbation studies to better understand metabolism., Loss-of-function experiments are used in metabolic engineering to understand and optimise metabolism. Here, the authors apply auxin inducible protein degradation to test different metabolic engineering strategies for improved terpenoid production in yeast.

Methyl ketones present a group of highly reduced platform chemicals industrially produced from petroleum-derived hydrocarbons. They find applications in the fragrance, flavor, pharmacological, and agrochemical industries, and are further discussed as biodiesel blends. In recent years, intense research has been carried out to achieve sustainable production of these molecules by re-arranging the fatty acid metabolism of various microbes. One challenge in the development of a highly productive microbe is the high demand for reducing power. Here, we engineeredPseudomonas taiwanensisVLB120 for methyl ketone production as this microbe has been shown to sustain exceptionally high NAD(P)H regeneration rates. The implementation of published strategies resulted in 2.1 g Laq-1methyl ketones in fed-batch fermentation. We further increased the production by eliminating competing reactions suggested by metabolic analyses. These efforts resulted in the production of 9.8 g Laq-1methyl ketones (corresponding to 69.3 g Lorg-1in thein situextraction phase) at 53 % of the maximum theoretical yield. This represents a 4-fold improvement in product titer compared to the initial production strain and the highest titer of recombinantly produced methyl ketones reported to date. Accordingly, this study underlines the high potential ofP. taiwanensisVLB120 to produce methyl ketones and emphasizes model-driven metabolic engineering to rationalize and accelerate strain optimization efforts.

Proteins have generally been recognized to constitute the key cellular component in shaping microbial phenotypes. Due to limited cellular resources and space, optimal allocation of proteins is crucial for microbes to facilitate maximum proliferation rates while allowing a flexible response to environmental changes. Regulatory patterns of protein allocation were utilized to account for the condition-dependent proteome in a genome-scale metabolic reconstruction of Escherichia coli by linearly linking mass concentrations of protein sectors and single metabolic enzymes to flux variables. The resulting protein allocation model (PAM) correctly approximates wildtype phenotypes and flux distributions for various substrates, even under data scarcity. Moreover, we showed the ability of the PAM to predict metabolic responses of single gene deletion mutants by additionally assuming growth-limiting, transcriptional restrictions. Thus, we promote the integration of protein allocation constraints into classical constraint-based models to foster their predictive capabilities and application for strain analysis and metabolic engineering purposes.

Obligate aerobic organisms rely on a functional electron transport chain for energy conservation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions. We here investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the generated single or double knockout strains displayed no, or only a weak, phenotype. Only the mutant deficient in both type-2 dehydrogenases showed a clear phenotype with biphasic growth behavior and a strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants including quantitative physiological experiments, transcript analysis, proteomics, and enzyme activity assays revealed distinct responses to type-2 and type-1 dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes, likely by re-routing of metabolic fluxes. This metabolic adaptability has implications for biotechnological applications. While the phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, the possible versatile redirecting of carbon fluxes upon genetic interventions can thwart metabolic engineering efforts.Importance While Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and, consequently, competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 presented here, provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. The observed great metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse towards a host with controlled and efficient supply of redox cofactors for product synthesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper., (undefined), info:eu-repo/semantics/publishedVersion

Methyl ketones present a group of highly reduced platform chemicals industrially produced from petroleum-derived hydrocarbons. They find applications in the fragrance, flavor, pharmacological, and agrochemical industries, and are further discussed as biodiesel blends. In recent years, intense research has been carried out to achieve sustainable production of these molecules by re-arranging the fatty acid metabolism of various microbes. One challenge in the development of a highly productive microbe is the high demand for reducing power. Here, we engineered Pseudomonas taiwanensis VLB120 for methyl ketone production as this microbe has been shown to sustain exceptionally high NAD(P)H regeneration rates. The implementation of published strategies resulted in 2.1 g Laq−1 methyl ketones in fed-batch fermentation. We further increased the production by eliminating competing reactions suggested by metabolic analyses. These efforts resulted in the production of 9.8 g Laq−1 methyl ketones (corresponding to 69.3 g Lorg−1 in the in situ extraction phase) at 53% of the maximum theoretical yield. This represents a 4-fold improvement in product titer compared to the initial production strain and the highest titer of recombinantly produced methyl ketones reported to date. Accordingly, this study underlines the high potential of P. taiwanensis VLB120 to produce methyl ketones and emphasizes model-driven metabolic engineering to rationalize and accelerate strain optimization efforts.

While yeast is one of the most studied organisms, its intricate biology remains to be fully mapped and understood. This is especially the case when it comes to capture rapid, in vivo fluctuations of metabolite levels. Secondary electrospray ionization-high resolution mass spectrometry SESI-HRMS is introduced here as a sensitive and noninvasive analytical technique for online monitoring of microbial metabolic activity. The power of this technique is exemplarily shown for baker’s yeast fermentation, for which the time-resolved abundance of about 300 metabolites is demonstrated. The results suggest that a large number of metabolites produced by yeast from glucose neither are reported in the literature nor are their biochemical origins deciphered. With the technique demonstrated here, researchers interested in distant disciplines such as yeast physiology and food quality will gain new insights into the biochemical capability of this simple eukaryote., Scientific Reports, 7 (1), ISSN:2045-2322

Microbial production of plant derived, biologically active compounds has the potential to provide economic and ecologic alternatives to existing low productive, plant-based processes. Current production of the pharmacologically active cyclic triterpenoid betulinic acid is realized by extraction from the bark of plane tree or birch. Here, we reengineered the reported betulinic acid pathway into S. cerevisiae and used this novel strain to develop efficient fermentation and product purification methods. Fed-batch cultivations with ethanol excess, using either an ethanol-pulse feed or controlling a constant ethanol concentration in the fermentation medium, significantly enhanced production of betulinic acid and its triterpenoid precursors. The beneficial effect of excess ethanol was further exploited in nitrogen-limited resting cell fermentations, yielding betulinic acid concentrations of 182 mg/L and total triterpenoid concentrations of 854 mg/L, the highest concentrations reported so far. Purification of lupane-type triterpenoids with high selectivity and yield was achieved by solid-liquid extraction without prior cell disruption using polar aprotic solvents such as acetone or ethyl acetate and subsequent precipitation with strong acids This study highlights the potential of microbial production of plant derived triterpenoids in S. cerevisiae by combining metabolic and process engineering. This article is protected by copyright. All rights reserved.

Frontiers in Bioengineering and Biotechnology 7, 458 (2020). doi:10.3389/fbioe.2019.00458, Published by Frontiers Media, Lausanne

BMC bioinformatics 20(1), 447 (2019). doi:10.1186/s12859-019-2946-7, Published by Springer, Heidelberg

Exploring the dynamic behavior of cellular metabolism requires a standard laboratory method that guarantees rapid sampling and extraction of the cellular content. We propose a versatile sampling technique applicable to cells with different cell wall and cell membrane properties. The technique is based on irreversible electroporation with simultaneous quenching and extraction by using a microfluidic device. By application of electric pulses in the millisecond range, permanent lethal pores are formed in the cell membrane of Escherichia coli and Saccharomyces cerevisiae, facilitating the release of the cellular contents, here demonstrated by the measurement of glucose-6-phosphate and the activity of the enzyme glucose-6-phosphate dehydrogenase. The successful application of this device was demonstrated by pulsed electric field treatment in a flow-through configuration of the microfluidic chip in combination with sampling, inactivation, and extraction of the intracellular content in a few seconds. Minimum electric field strengths of 10 kV/cm for E. coli and 7.5 kV/cm for yeast S. cerevisiae were required for successful cell lysis. The results are discussed in the context of applications in industrial biotechnology, where metabolomics analyses are important.

BMC microbiology 19(1), 100 (2019). doi:10.1186/s12866-019-1453-3, Published by Springer, Heidelberg

Engineering in life sciences 19(10), 711-720 (2019). doi:10.1002/elsc.201900080, Published by Wiley-VCH, Weinheim

Microbial carbon dioxide assimilation and conversion to chemical platform molecules has the potential to be developed as economic, sustainable processes. The carbon dioxide assimilation can proceed by a variety of natural pathways and recently even synthetic CO2 fixation routes have been designed. Early assessment of the performance of the different carbon fixation alternatives within biotechnological processes is desirable to evaluate their potential. Here we applied stoichiometric metabolic modeling based on physiological and process data to evaluate different process variants for the conversion of C1 carbon compounds to the industrial relevant platform chemical succinic acid. We computationally analyzed the performance of cyanobacteria, acetogens, methylotrophs, and synthetic CO2 fixation pathways in Saccharomyces cerevisiae in terms of production rates, product yields, and the optimization potential. This analysis provided insight into the economic feasibility and allowed to estimate the future industrial applicability by estimating overall production costs. With reported, or estimated data of engineered or wild type strains, none of the simulated microbial succinate production processes showed a performance allowing competitive production. The main limiting factors were identified as gas and photon transfer and metabolic activities whereas metabolic network structure was not restricting. In simulations with optimized parameters most process alternatives reached economically interesting values, hence, represent promising alternatives to sugar-based fermentations., Graphical abstract fx1, Highlights • Biocatalytic processes have the potential to economically convert carbon dioxide to succinate. • Carbon and photon uptake rates limit productivity of cyanobacterial production routes. • Cyanobacterial and acetogenic CO2 fixation require technical and genetic tuning. • Major challenge for methanol conversion is a competitive CO2 to methanol conversion.

Pseudomonas putida efficiently utilizes many different carbon sources without the formation of byproducts even under conditions of stress. This implies a high degree of flexibility to cope with conditions that require a significantly altered distribution of carbon to either biomass or energy in the form of NADH. In the literature, co‐feeding of the reduced C1 compound formate to Escherichia coli heterologously expressing the NAD(+)‐dependent formate dehydrogenase of the yeast Candida boidinii was demonstrated to boost various NADH‐demanding applications. Pseudomonas putida as emerging biotechnological workhorse is inherently equipped with an NAD(+)‐dependent formate dehydrogenase encouraging us to investigate the use of formate and its effect on P. putida’s metabolism. Hence, this study provides a detailed insight into the co‐utilization of formate and glucose by P. putida. Our results show that the addition of formate leads to a high increase in the NADH regeneration rate resulting in a very high biomass yield on glucose. Metabolic flux analysis revealed a significant flux rerouting from catabolism to anabolism. These metabolic insights argue further for P. putida as a host for redox cofactor demanding bioprocesses.

Heterologous synthesis of triterpenoids in Saccharomyces cerevisiae from its native metabolite squalene has been reported to offer an alternative to chemical synthesis and extraction from plant material if productivities can be increased.Here, we physiologically characterized a squalene overproducing S. cerevisiae CEN.PK strain to elucidate the effect of cultivation conditions on the production of this central triterpenoid precursor. The maximum achievable squalene concentration was substantially influenced by nutritional conditions, medium composition and cultivation mode. Batch growth on glucose resulted in minimal squalene accumulation, while squalene only significantly accumulated during ethanol consumption (up to 59 mg/gCDW), probably due to increased acetyl-CoA supply on this carbon source. Likewise, low squalene concentrations were observed in glucose-limited chemostat cultivations and improved up to 8-fold upon increasing the ethanol fraction in the feed. In those experiments, a constant, growth-rate-independent specific squalene accumulation rate (2.2 mg/gCDW/h) was recorded resulting in a maximal squalene loading of 30 mg/gCDW at low dilution rates with longer residence times. Coenzyme A availability was identified as possible bottleneck as increased vitamin concentrations, including the Coenzyme A precursor pantothenate, improved squalene titers in batch and chemostat cultivations. This analysis demonstrates that thorough physiologic characterization of production strains is valuable for the identification of bottlenecks already in early stages of strain development and for guiding further optimization efforts.

Metabolites : open access journal 8(2), 33 (2018). doi:10.18154/RWTH-2018-225568 special issue: "Special Issue "Metabolism and Systems Biology Volume 2" / Guest Editor Prof. Dr. Frank J. Bruggeman", Published by MDPI, Basel

Rational strain engineering requires solid testing of phenotypes including productivity and ideally contributes thereby directly to our understanding of the genotype-phenotype relationship. Actually, the test step of the strain engineering cycle becomes the limiting step, as ever advancing tools for generating genetic diversity exist. Here, we briefly define the challenge one faces in quantifying phenotypes and summarize existing analytical techniques that partially overcome this challenge. We argue that the evolution of volatile metabolites can be used as proxy for cellular metabolism. In the simplest case, the product of interest is a volatile (e.g., from bulk alcohols to special fragrances) that is directly quantified over time. But also nonvolatile products (e.g., from bulk long-chain fatty acids to natural products) require major flux rerouting that result potentially in altered volatile production. While alternative techniques for volatile determination exist, rather few can be envisaged for medium to high-throughput analysis required for phenotype testing. Here, we contribute a detailed protocol for an ion mobility spectrometry (IMS) analysis that allows volatile metabolite quantification down to the ppb range. The sensitivity can be exploited for small-scale fermentation monitoring. The insights shared might contribute to a more frequent use of IMS in biotechnology, while the experimental aspects are of general use for researchers interested in volatile monitoring.

Volatile organic compounds (VOCs) are small molecular mass substances, which exhibit low boiling points and high-vapour pressures. They are ubiquitous in nature and produced by almost any organism of all kingdoms of life. VOCs are involved in many inter- and intraspecies interactions ranging from antimicrobial or fungal effects to plant growth promotion and human taste perception of fermentation products. VOC profiles further reflect the metabolic or phenotypic state of the living organism that produces them. Hence, they can be exploited for non-invasive medicinal diagnoses or industrial fermentation control. Here, we introduce the reader to these diverse applications associated with the monitoring and analysis of VOC emissions. We also present our vision of real-time VOC analysis enabled by newly developed analytical techniques, which will further broaden the use of VOCs in even wider applications. Hence, we foresee a bright future for VOC research and its associated fields of applications.