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Abstract Background The wasabi receptor, also known as the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel, is a potential target for development of repellents for insects, like the pine weevil (Hylobius abietis) feeding on conifer seedlings and causing damage in forestry. Heterologous expression of TRPA1 from pine weevil in the yeast Pichia pastoris can potentially provide protein for structural and functional studies. Here we take advantage of the Green Fluorescent Protein (GFP) tag to examine the various steps of heterologous expression, to get more insight in clone selection, expression and isolation of the intact purified protein. Results The sequence of HaTRPA1 is reported and GFP-tagged constructs were made of the full-length protein and a truncated version (Δ1-708 HaTRPA1), lacking the N-terminal ankyrin repeat domain. Clones were screened on GFP expression plates, induced in small liquid cultures and in fed-batch cultures, and evaluated by flow cytometry and fluorescence microscopy. The screening on plates successfully identifies low-expression clones, but fails to predict the ranking of the best performing clones in small-scale liquid cultures. The two constructs differ in their cellular localization. Δ1-708 HaTRPA1 is found in a ring at the perimeter of cell, whereas HaTRPA1 is forming highly fluorescent speckles in interior regions of the cell. The pattern is consistent in different clones of the same construct and persists in fed-batch culture. The expression of Δ1-708 HaTRPA1 decreases the viability more than HaTRPA1, and in fed-batch culture it is clear that intact cells first express Δ1-708 HaTRPA1 and then become damaged. Purifications show that both constructs suffer from degradation of the expressed protein, but especially the HaTRPA1 construct. Conclusions The GFP tag makes it possible to follow expression by flow cytometry and fluorescence microscopy. Analyses of localization, cell viability and expression show that the former two parameters are specific for each of the two evaluated constructs, whereas the relative expression of the constructs varies with the cultivation method. High expression is not all that matters, so taking damaged cells into account, something that may be linked to protein degradation, is important when picking the most suitable construct, clone, and expression scheme.

Fatty acids are produced by eukaryotes like baker's yeast Saccharomyces cerevisiae mainly using a large multifunctional type I fatty acid synthase (FASI) where seven catalytic steps and a carrier domain are shared between one or two protein subunits. While this system may offer efficiency in catalysis, only a narrow range of fatty acids are produced. Prokaryotes, chloroplasts and mitochondria rely instead on a FAS type II (FASII) where each catalytic step is carried out by a monofunctional enzyme encoded by a separate gene. FASII is more flexible and capable of producing a wider range of fatty acid structures, such as the direct production of unsaturated fatty acids. An efficient FASII in the preferred industrial organism S. cerevisiae could provide a platform for developing sustainable production of specialized fatty acids. We functionally replaced either yeast FASI genes (FAS1 or FAS2) with a FASII consisting of nine genes from Escherichia coli (acpP, acpS and fab -A, -B, -D, -F, -G, -H, -Z) as well as three from Arabidopsis thaliana (MOD1, FATA1 and FATB). The genes were expressed from an autonomously replicating multicopy vector assembled using the Yeast Pathway Kit for in-vivo assembly in yeast. Two rounds of adaptation led to a strain with a maximum growth rate (μmax) of 0.19 h−1 without exogenous fatty acids, twice the growth rate previously reported for a comparable strain. Additional copies of the MOD1 or fabH genes resulted in cultures with higher final cell densities and three times higher lipid content compared to the control.

In Saccharomyces cerevisiae, pH homeostasis is reliant on ATP due to the use of proton-translocating ATPase (H+-ATPase) which constitutes a major drain within cellular ATP supply. Here, an exogenous proton-translocating pyrophosphatase (H+-PPase) from Arabidopsis thaliana, which uses inorganic pyrophosphate (PPi) rather than ATP, was evaluated for its effect on reducing the ATP burden. The H+-Ppase was localized to the vacuolar membrane or to the cell membrane, and their impact was studied under acetate stress at a low pH. Biosensors (pHluorin and mQueen-2m) were used to observe changes in intracellular pH (pHi) and ATP levels during growth on either glucose or xylose. A significant improvement of 35% in the growth rate at a pH of 3.7 and 6 g·L−1 acetic acid stress was observed in the vacuolar membrane H+-PPase strain compared to the parent strain. ATP levels were elevated in the same strain during anaerobic glucose and xylose fermentations. During anaerobic xylose fermentations, co-expression of pHluorin and a vacuolar membrane H+-PPase improved the growth characteristics by means of an improved growth rate (11.4%) and elongated logarithmic growth duration. Our study identified a potential method for improving productivity in the use of S. cerevisiae as a cell factory under the harsh conditions present in industry.

The physiological effects of oxygen on Limosilactobacillus reuteri DSM 17938 during cultivation and the ensuing properties of the freeze-dried probiotic product was investigated. On-line flow cytometry and k-means clustering gating was used to follow growth and viability in real time during cultivation. The bacterium tolerated aeration at 500 mL/min, with a growth rate of 0.74 ± 0.13 h−1 which demonstrated that low levels of oxygen did not influence the growth kinetics of the bacterium. Modulation of the redox metabolism was, however, seen already at non-inhibitory oxygen levels by 1.5-fold higher production of acetate and 1.5-fold lower ethanol production. A significantly higher survival rate in the freeze-dried product was observed for cells cultivated in presence of oxygen compared to absence of oxygen (61.8% ± 2.4% vs. 11.5% ± 4.3%), coinciding with a higher degree of unsaturated fatty acids (UFA:SFA ratio of 10 for air sparged vs. 3.59 for N2 sparged conditions.). Oxygen also resulted in improved bile tolerance and boosted 5′nucleotidase activity (370 U/L vs. 240 U/L in N2 sparged conditions) but lower tolerance to acidic conditions compared bacteria grown under complete anaerobic conditions which survived up to 90 min of exposure at pH 2. Overall, our results indicate the controlled supply of oxygen during production may be used as means for probiotic activity optimization of L. reuteri DSM 17938.

Abstract Background Capsaicinoids are produced by plants in the Capsicum genus and are the main reason for the pungency of chili pepper fruits. They are strong agonists of TRPV1 (the transient receptor potential cation channel subfamily V member 1) and used as active ingredients in pharmaceuticals for the treatment of pain. The use of bioengineered microorganisms in a fermentation process may be an efficient route for their preparation, as well as for the discovery of (bio-)synthetic capsaicinoids with improved or novel bioactivities. Results Saccharomyces cerevisiae was engineered to over-express a selection of amide-forming N-acyltransferase and CoA-ligase enzyme cascades using a combinatorial gene assembly method, and was screened for nonivamide production from supplemented vanillylamine and nonanoic acid. Data from this work demonstrate that Tyramine N-hydroxycinnamoyl transferase from Capsicum annuum (CaAT) was most efficient for nonivamide formation in yeast, outcompeting the other candidates including AT3 (Pun1) from Capsicum spp. The CoA-ligase partner with highest activity from the ones evaluated here were from Petunia hybrida (PhCL) and Spingomonas sp. Ibu-2 (IpfF). A yeast strain expressing CaAT and IpfF produced 10.6 mg L−1 nonivamide in a controlled bioreactor setup, demonstrating nonivamide biosynthesis by S. cerevisiae for the first time. Conclusions Baker’s yeast was engineered for production of nonivamide as a model capsaicinoid, by expressing N-acyltransferases and CoA-ligases of plant and bacterial origin. The constructed yeast platform holds potential for in vivo biocatalytic formation of capsaicinoids and could be a useful tool for the discovery of novel drugs.

We report on the development of a method based on recombinant yeast Saccharomyces cerevisiae to produce nonivamide, a capsaicinoid and potent agonist of the pain receptor TRPV1. Nonivamide was produced in a two-step batch process where yeast was i) grown aerobically on glucose and ii) used to produce nonivamide from vanillin and non-anoic acid by bioconversion. The yeast was engineered to express multiple copies of an amine transaminase from Chromobacterium violaceum (CvTA), along with an NADH-dependent alanine dehydrogenase from Bacillus subtilis (BsAlaDH) to enable efficient reductive amination of vanillin. Oxygen-limited conditions and the use of ethanol as a co-substrate to regenerate NADH were identified to favour amination over the formation of the by-products vanillic alcohol and vanillic acid. The native alcohol dehydrogenase ADH6 was deleted to further reduce the formation of vanillic alcohol. A two-enzyme system consisting of an N-acyltransferase from Capsicum annuum (CaAT), and a CoA ligase from Sphingomonas sp. Ibu-2 (IpfF) was co-expressed to produce the amide. This study provides proof of concept for yeast-based production of non-ivamide by combined transamination and amidation of vanillin.

Abstract Optimisation of cultivation conditions in the industrial production of probiotics is crucial to reach a high-quality product with retained probiotic functionality. Flow cytometry-based descriptors of bacterial morphology may be used as markers to estimate physiological fitness during cultivation, and can be applied for online monitoring to avoid suboptimal growth. In the current study, the effects of temperature, initial pH and oxygen levels on cell growth and cell size distributions of Limosilactobacillus reuteri DSM 17938 were measured using multivariate flow cytometry. A pleomorphic behaviour was evident from the measurements of light scatter and pulse width distributions. A pattern of high growth yielding smaller cells and less heterogeneous populations could be observed. Analysis of pulse width distributions revealed significant morphological heterogeneities within the bacterial cell population under non-optimal growth conditions, and pointed towards low temperature, high initial pH, and high oxygen levels all being triggers for changes in morphology towards cell chain formation. However, cell size did not correlate to survivability after freeze-thaw or freeze-drying stress, indicating that it is not a key determinant for physical stress tolerance. The fact that L. reuteri morphology varies depending on cultivation conditions suggests that it can be used as marker for estimating physiological fitness and responses to its environment.

Summary Whole‐cell bioconversion of technical lignins using Pseudomonas putida strains overexpressing amine transaminases (ATAs) has the potential to become an eco‐efficient route to produce phenolic amines. Here, a novel cell growth‐based screening method to evaluate the in vivo activity of recombinant ATAs towards vanillylamine in P. putida KT2440 was developed. It allowed the identification of the native enzyme Pp‐SpuC‐II and ATA from Chromobacterium violaceum (Cv‐ATA) as highly active towards vanillylamine in vivo. Overexpression of Pp‐SpuC‐II and Cv‐ATA in the strain GN442ΔPP_2426, previously engineered for reduced vanillin assimilation, resulted in 94‐ and 92‐fold increased specific transaminase activity, respectively. Whole‐cell bioconversion of vanillin yielded 0.70 ± 0.20 mM and 0.92 ± 0.30 mM vanillylamine, for Pp‐SpuC‐II and Cv‐ATA, respectively. Still, amine production was limited by a substantial re‐assimilation of the product and formation of the by‐products vanillic acid and vanillyl alcohol. Concomitant overexpression of Cv‐ATA and alanine dehydrogenase from Bacillus subtilis increased the production of vanillylamine with ammonium as the only nitrogen source and a reduction in the amount of amine product re‐assimilation. Identification and deletion of additional native genes encoding oxidoreductases acting on vanillin are crucial engineering targets for further improvement.

The commercial production of bioethanol from lignocellulosic biomass such as wheat straw requires utilizing a microorganism that can withstand all the stressors encountered in the process while fermenting all the sugars in the biomass. Therefore, it is essential to develop tools for monitoring and controlling the cellular fitness during both cell propagation and sugar fermentation to ethanol. In the present study, on-line flow cytometry was adopted to assess the response of the biosensor TRX2p-yEGFP for redox imbalance in an industrial xylose-fermenting strain of Saccharomyces cerevisiae during cell propagation and the following fermentation of wheat-straw hydrolysate. Rapid and transient induction of the sensor was recorded upon exposure to furfural and wheat straw hydrolysate containing up to 3.8 g/L furfural. During the fermentation step, the induction rate of the sensor was also found to correlate to the initial ethanol production rate, highlighting the relevance of redox monitoring and the potential of the presented tool to assess the ethanol production rate in hydrolysates. Three different propagation strategies were also compared, and it was confirmed that pre-exposure to hydrolysate during propagation remains the most efficient method for high ethanol productivity in the following wheat-straw hydrolysate fermentations.

Transcription factor-based biosensors represent promising tools in the construction and evaluation of efficient cell factories for the sustainable production of fuels, chemicals and pharmaceuticals. They can notably be designed to follow the production of a target compound or to monitor key cellular properties, such as stress or starvation. In most cases, the biosensors are built with fluorescent protein (FP) genes as reporter genes because of the direct correlation between promoter activity and fluorescence level that can be measured using, for instance, flow cytometry or fluorometry. The expansion of available FPs offers the possibility of using several FPs - and biosensors – in parallel in one host, with simultaneous detection using multicolor flow cytometry. However, the technique is currently limited by the unavailability of combinations of FP whose genes can be successfully expressed in the host and whose fluorescence can be efficiently distinguished from each other.In the present study, the broad collection of available FPs was explored and four different FPs were successfully expressed in the yeast Saccharomyces cerevisiae: yEGFP, mEGFP, CyOFP1opt and mBeRFPopt. After studying their fluorescence signals, population heterogeneity and possible interactions, we recommend two original combinations of FPs for bi-color flow cytometry: mEGFP together with either CyOFP1opt or mBeRFPopt, as well as the combination of all three FPs mEGFP, CyOFP1opt and mBeRFPopt for tri-color flow cytometry. These combinations will allow to perform different types of bi-color or possibly tri-color flow cytometry and FACS experiments with yeast, such as phenotype evaluation, screening or sorting, by single-laser excitation with a standard 488 nm blue laser.

Lignin is an abundant cell wall component, and it has been used mainly for generating steam and electricity. Nevertheless, lignin valorization, i.e. the conversion of lignin into high value-added fuels, chemicals, or materials, is crucial for the full implementation of cost-effective lignocellulosic biorefineries. From this perspective, rapid screening methods are crucial for time- and resource-efficient development of novel microbial strains and enzymes with applications in the lignin biorefinery. The present review gives an overview of recent developments and applications of a vast arsenal of activity and sequence-based methodologies for uncovering novel microbial strains with ligninolytic potential, novel enzymes for lignin depolymerization and for unraveling the main metabolic routes during growth on lignin. Finally, perspectives on the use of each of the presented methods and their respective advantages and disadvantages are discussed.

Abstract Background Production of second-generation bioethanol and other bulk chemicals by yeast fermentation requires cells that tolerate inhibitory lignocellulosic compounds at low pH. Saccharomyces cerevisiae displays high plasticity with regard to inhibitor tolerance, and adaptation of cell populations to process conditions is essential for reaching efficient and robust fermentations. Results In this study, we assessed responses of isogenic yeast cell populations in different physiological states to combinations of acetic acid, vanillin and furfural at low pH. We found that cells in early stationary phase (ESP) exhibited significantly increased tolerance compared to cells in logarithmic phase, and had a similar ability to initiate growth in the presence of inhibitors as pre-adapted cells. The ESP cultures consisted of subpopulations with different buoyant cell densities which were isolated with flotation and analysed separately. These so-called quiescent (Q) and non-quiescent (NQ) cells were found to possess similar abilities to initiate growth in the presence of lignocellulosic inhibitors at pH 3.7, and had similar viabilities under static conditions. Therefore, differentiation into Q-cells was not the cause for increased tolerance of ESP cultures. Flow cytometry analysis of cell viability, intracellular pH and reactive oxygen species levels revealed that tolerant cell populations had a characteristic response upon inhibitor perturbations. Growth in the presence of a combination of inhibitors at low pH correlated with pre-cultures having a high frequency of cells with low pHi and low ROS levels. Furthermore, only a subpopulation of ESP cultures was able to tolerate lignocellulosic inhibitors at low pH, while pre-adapted cell populations displayed an almost uniform high tolerance to the adverse condition. This was in stark contrast to cell populations growing exponentially in non-inhibitory medium that were uniformly sensitive to the inhibitors at low pH. Conclusions ESP cultures of S. cerevisiae were found to have high tolerance to lignocellulosic inhibitors at low pH, and were able to initiate growth to the same degree as cells that were pre-adapted to inhibitors at a slightly acidic pH. Carbon starvation may thus be a potential strategy to prepare cell populations for adjacent stressful environments which may be beneficial from a process perspective for fermentation of non-detoxified lignocellulosic substrates at low pH. Furthermore, flow cytometry analysis of pHi and ROS level distributions in ESP cultures revealed responses that were characteristic for populations with high tolerance to lignocellulosic inhibitors. Measurement of population distribution responses as described herein may be applied to predict the outcome of environmental perturbations and thus can function as feedback for process control of yeast fitness during lignocellulosic fermentation.

Microbial cells in bioprocesses are usually described with averaged parameters. But in fact, single cells within populations vary greatly in characteristics such as stress resistance, especially in response to carbon source gradients. Our aim was to introduce tools to quantify population heterogeneity in bioprocesses using a combination of reporter strains, flow cytometry, and easily comprehensible parameters. We calculated mean, mode, peak width, and coefficient of variance to describe distribution characteristics and temporal shifts in fluorescence intensity. The skewness and the slope of cumulative distribution function plots illustrated differences in distribution shape. These parameters are person-independent and precise. We demonstrated this by quantifying growth-related population heterogeneity of Saccharomyces cerevisiae and Escherichia coli reporter strains in steady-state of aerobic glucose-limited chemostat cultures at different dilution rates and in response to glucose pulses. Generally, slow-growing cells showed stronger responses to glucose excess than fast-growing cells. Cell robustness, measured as membrane integrity after exposure to freeze-thaw treatment, of fast-growing cells was strongly affected in subpopulations of low membrane robustness. Glucose pulses protected subpopulations of fast-growing but not slower-growing yeast cells against membrane damage. Our parameters could successfully describe population heterogeneity, thereby revealing physiological characteristics that might have been overlooked during traditional averaged analysis.

A control point for keeping redox homeostasis in Saccharomyces cerevisiae during fermentative growth is the dynamic regulation of transcription for the glycerol-3-phosphate dehydrogenase 2 (GPD2) gene. In this study, the possibility to steer the activity of the GPD2 promoter was investigated by placing it in strains with different ability to reoxidise NADH, and applying different environmental conditions. Flow cytometric analysis of reporter strains expressing green fluorescent protein (GFP) under the control of the GPD2 promoter was used to determine the promoter activity at the single-cell level. When placed in a gpd1Δgpd2Δ strain background, the GPD2 promoter displayed a 2-fold higher activity as compared to the strong constitutive glyceraldehyde-3-phosphate dehydrogenase (TDH3). In contrast, the GPD2 promoter was found to be inactive when cells were cultivated in continuous mode at a growth rate of 0.3 h−1 and in conditions with excess oxygen (i.e. with an aeration of 2.5 vvm, and a stirring of 800 rpm). In addition, a clear window of operation where the gpd1Δgpd2Δ strain can be grown with the same efficiency as wild type yeast was identified. In conclusion, the flow cytometry mapping revealed conditions where the GPD2 promoter was either completely inactive or hyperactive, which has implications for its implementation in future biotechnological applications such as for process control of heterologous gene expression.