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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.

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.

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.

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.

Fatty alcohols are widely used in various applications within a diverse set of industries, such as the soap and detergent industry, the personal care, and cosmetics industry, as well as the food industry. The total world production of fatty alcohols is over 2 million tons with approximately equal parts derived from fossil oil and from plant oils or animal fats. Due to the environmental impact of these production methods, there is an interest in alternative methods for fatty alcohol production via microbial fermentation using cheap renewable feedstocks. In this study, we aimed to obtain a better understanding of how fatty alcohol biosynthesis impacts the host organism, baker’s yeast Saccharomyces cerevisiae or oleaginous yeast Yarrowia lipolytica. Producing and non-producing strains were compared in growth and nitrogen-depletion cultivation phases. The multi-omics analysis included physiological characterization, transcriptome analysis by RNAseq, 13Cmetabolic flux analysis, and intracellular metabolomics. Both species accumulated fatty alcohols under nitrogen-depletion conditions but not during growth. The fatty alcohol–producing Y. lipolytica strain had a higher fatty alcohol production rate than an analogous S. cerevisiae strain. Nitrogen-depletion phase was associated with lower glucose uptake rates and a decrease in the intracellular concentration of acetyl–CoA in both yeast species, as well as increased organic acid secretion rates in Y. lipolytica. Expression of the fatty alcohol–producing enzyme fatty acyl–CoA reductase alleviated the growth defect caused by deletion of hexadecenal dehydrogenase encoding genes (HFD1 and HFD4) in Y. lipolytica. RNAseq analysis showed that fatty alcohol production triggered a cell wall stress response in S. cerevisiae. RNAseq analysis also showed that both nitrogen-depletion and fatty alcohol production have substantial effects on the expression of transporter encoding genes in Y. lipolytica. In conclusion, through this multi-omics study, we uncovered some effects of fatty alcohol production on the host metabolism. This knowledge can be used as guidance for further strain improvement towards the production of fatty alcohols.

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.

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.

Background In the future, oil- and gas-derived polymers may be replaced with bio-based polymers, produced from renewable feedstocks using engineered cell factories. Acrylic acid and acrylic esters with an estimated world annual production of approximately 6 million tons by 2017 can be derived from 3-hydroxypropionic acid (3HP), which can be produced by microbial fermentation. For an economically viable process 3HP must be produced at high titer, rate and yield and preferably at low pH to minimize downstream processing costs. Results Here we describe the metabolic engineering of baker’s yeast Saccharomyces cerevisiae for biosynthesis of 3HP via a malonyl-CoA reductase (MCR)-dependent pathway. Integration of multiple copies of MCR from Chloroflexus aurantiacus and of phosphorylation-deficient acetyl-CoA carboxylase ACC1 genes into the genome of yeast increased 3HP titer fivefold in comparison with single integration. Furthermore we optimized the supply of acetyl-CoA by overexpressing native pyruvate decarboxylase PDC1, aldehyde dehydrogenase ALD6, and acetyl-CoA synthase from Salmonella entericaSEacsL641P. Finally we engineered the cofactor specificity of the glyceraldehyde-3-phosphate dehydrogenase to increase the intracellular production of NADPH at the expense of NADH and thus improve 3HP production and reduce formation of glycerol as by-product. The final strain produced 9.8 ± 0.4 g L−1 3HP with a yield of 13 % C-mol C-mol−1 glucose after 100 h in carbon-limited fed-batch cultivation at pH 5. The 3HP-producing strain was characterized by 13C metabolic flux analysis and by transcriptome analysis, which revealed some unexpected consequences of the undertaken metabolic engineering strategy, and based on this data, future metabolic engineering directions are proposed. Conclusions In this study, S. cerevisiae was engineered for high-level production of 3HP by increasing the copy numbers of biosynthetic genes and improving flux towards precursors and redox cofactors. This strain represents a good platform for further optimization of 3HP production and hence an important step towards potential commercial bio-based production of 3HP. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0451-5) contains supplementary material, which is available to authorized users.

Background Copper is an essential chemical element for life as it is a part of prosthetic groups of enzymes including super oxide dismutase and cytochrome c oxidase; however, it is also toxic at high concentrations. Here, we present the trade-off of copper availability and growth inhibition of a common host used for copper-dependent protein production, Pichia pastoris. Results At copper concentrations ranging from 0.1 mM (6.35 mg/L) to 2 mM (127 mg/L), growth rates of 0.25 h−1 to 0.16 h−1 were observed with copper uptake of as high as 20 mgcopper/gCDW. The intracellular copper content was estimated by subtracting the copper adsorbed on the cell wall from the total copper concentration in the biomass. Higher copper concentrations led to stronger cell growth retardation and, at 10 mM (635 mg/L) and above, to growth inhibition. To test the determined copper concentration range for optimal recombinant protein production, a laccase gene from Aspergillus clavatus [EMBL: EAW07265.1] was cloned under the control of the constitutive glyceraldehyde-3-phosphate (GAP) dehydrogenase promoter for expression in P. pastoris. Notably, in the presence of copper, laccase expression improved the specific growth rate of P. pastoris. Although copper concentrations of 0.1 mM and 0.2 mM augmented laccase expression 4 times up to 3 U/mL compared to the control (0.75 U/mL), while higher copper concentrations resulted in reduced laccase production. An intracellular copper content between 1 and 2 mgcopper/gCDW was sufficient for increased laccase activity. The physiology of the yeast could be excluded as a reason for the stop of laccase production at moderate copper concentrations as no flux redistribution could be observed by 13C-metabolic flux analysis. Conclusion Copper and its pivotal role to sustain cellular functions is noteworthy. However, knowledge on its cellular accumulation, availability and distribution for recombinant protein production is limited. This study attempts to address one such challenge, which revealed the fact that intracellular copper accumulation influenced laccase production and should be considered for high protein expression of copper-dependent enzymes when using P. pastoris. The results are discussed in the context of P. pastoris as a general host for copper -dependent enzyme production. Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0251-3) contains supplementary material, which is available to authorized users.

Whole-cell biocatalysis utilizes native or recombinant enzymes produced by cellular metabolism to perform synthetically interesting reactions. Besides hydrolases, oxidoreductases represent the most applied enzyme class in industry. Oxidoreductases are attributed a high future potential, especially for applications in the chemical and pharmaceutical industries, as they enable highly interesting chemistry (e.g., the selective oxyfunctionalization of unactivated C-H bonds). Redox reactions are characterized by electron transfer steps that often depend on redox cofactors as additional substrates. Their regeneration typically is accomplished via the metabolism of whole-cell catalysts. Traditionally, studies towards productive redox biocatalysis focused on the biocatalytic enzyme, its activity, selectivity, and specificity, and several successful examples of such processes are running commercially. However, redox cofactor regeneration by host metabolism was hardly considered for the optimization of biocatalytic rate, yield, and/or titer. This article reviews molecular mechanisms of oxidoreductases with synthetic potential and the host redox metabolism that fuels biocatalytic reactions with redox equivalents. The tools discussed in this review for investigating redox metabolism provide the basis for studies aiming at a deeper understanding of the interplay between synthetically active enzymes and metabolic networks. The ultimate goal of rational whole-cell biocatalyst engineering and use for fine chemical production is discussed.

A key limitation of whole-cell redox biocatalysis for the production of valuable, specifically functionalized products is substrate/product toxicity, which can potentially be overcome by using solvent-tolerant micro-organisms. To investigate the inter-relationship of solvent tolerance and energy-dependent biocatalysis, we established a model system for biocatalysis in the presence of toxic low logPow solvents: recombinant solvent-tolerant Pseudomonas putida DOT-T1E catalyzing the stereospecific epoxidation of styrene in an aqueous/octanol two-liquid phase reaction medium. Using 13C tracer based metabolic flux analysis, we investigated the central carbon and energy metabolism and quantified the NAD(P)H regeneration rate in the presence of toxic solvents and during redox biocatalysis, which both drastically increased the energy demands of solvent-tolerant P. putida. According to the driven by demand concept, the NAD(P)H regeneration rate was increased up to eightfold by two mechanisms: (a) an increase in glucose uptake rate without secretion of metabolic side products, and (b) reduced biomass formation. However, in the presence of octanol, only ∼ 1% of the maximally observed NAD(P)H regeneration rate could be exploited for styrene epoxidation, of which the rate was more than threefold lower compared with operation with a non-toxic solvent. This points to a high energy and redox cofactor demand for cell maintenance, which limits redox biocatalysis in the presence of octanol. An estimated upper bound for the NAD(P)H regeneration rate available for biocatalysis suggests that cofactor availability does not limit redox biocatalysis under optimized conditions, for example, in the absence of toxic solvent, and illustrates the high metabolic capacity of solvent-tolerant P. putida. This study shows that solvent-tolerant P. putida have the remarkable ability to compensate for high energy demands by boosting their energy metabolism to levels up to an order of magnitude higher than those observed during unlimited growth.

Metabolites 4(3), 751-774 (2014). doi:10.3390/metabo4030751, Published by MDPI, Basel

Adenosine phosphate and NAD cofactors play a vital role in the operation of cell metabolism, and their levels and ratios are carefully regulated in tight ranges. Perturbations of the consumption of these metabolites might have a great impact on cell metabolism and physiology. Here, we investigated the impact of increased ATP hydrolysis and NADH oxidation rates on the metabolism of Pseudomonas putida KT2440 by titration of 2,4-dinitrophenol (DNP) and overproduction of a water-forming NADH oxidase, respectively. Both perturbations resulted in a reduction of the biomass yield and, as a consequence of the uncoupling of catabolic and anabolic activities, in an amplification of the net NADH regeneration rate. However, a stimulation of the specific carbon uptake rate was observed only when P. putida was challenged with very high 2,4-dinitrophenol concentrations and was comparatively unaffected by recombinant NADH oxidase activity. This behavior contrasts with the comparably sensitive performance described, for example, for Escherichia coli or Saccharomyces cerevisiae . The apparent robustness of P. putida metabolism indicates that it possesses a certain buffering capacity and a high flexibility to adapt to and counteract different stresses without showing a distinct phenotype. These findings are important, e.g., for the development of whole-cell redox biocatalytic processes that impose equivalent burdens on the cell metabolism: stoichiometric consumption of (reduced) redox cofactors and increased energy expenditures, due to the toxicity of the biocatalytic compounds.

The limited supply of fossil fuels and the establishment of new environmental policies shifted research in industry and academia towards sustainable production of the 2ndgeneration of biofuels, with Methyl Ethyl Ketone (MEK) being one promising fuel candidate. MEK is a commercially valuable petrochemical with an extensive application as a solvent. However, as of today, a sustainable and economically viable production of MEK has not yet been achieved despite several attempts of introducing biosynthetic pathways in industrial microorganisms. We used BNICE.ch as a retrobiosynthesis tool to discover all novel pathways around MEK. Out of 1’325 identified compounds connecting to MEK with one reaction step, we selected 3-oxopentanoate, but-3-en-2-one, but-1-en-2-olate, butylamine, and 2-hydroxy-2-methyl-butanenitrile for further study. We reconstructed 3’679’610 novel biosynthetic pathways towards these 5 compounds. We then embedded these pathways into the genome-scale model ofE. coli, and a set of 18’622 were found to be most biologically feasible ones based on thermodynamics and their yields. For each novel reaction in the viable pathways, we proposed the most similar KEGG reactions, with their gene and protein sequences, as candidates for either a direct experimental implementation or as a basis for enzyme engineering. Through pathway similarity analysis we classified the pathways and identified the enzymes and precursors that were indispensable for the production of the target molecules. These retrobiosynthesis studies demonstrate the potential of BNICE.ch for discovery, systematic evaluation, and analysis of novel pathways in synthetic biology and metabolic engineering studies.Graphical abstract