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2,4-dihydroxybutyric acid (DHB) and 2-keto-4-hydroxybutyrate (OHB) are non-natural molecules obtained through synthetic pathways from renewable carbon source. As they are structurally similar to lactate and pyruvate respectively, they could possibly interfere with the metabolic network of Escherichia coli. In fact, we showed that DHB can be easily oxidized by the membrane associated L and D-lactate dehydrogenases encoded by lldD, dld and ykgF into OHB, and the latter being cleaved into pyruvate and formaldehyde by several pyruvate-dependent aldolases, with YagE being the most effective. While formaldehyde was readily detoxified into formate, Escherichia coli K12 MG1655 strain failed to grow on DHB despite of the production of pyruvate. To find out the reason for this failure, we constructed a mutant strain whose growth was rendered dependent on DHB and subjected this strain to adaptive evolution. Genome sequencing of the adapted strain revealed an essential role for ygbI encoding a transcriptional repressor of the threonate operon in this DHB-dependent growth. This critical function was attributed to the derepression of ygbN encoding a putative threonate transporter, which was found to exclusively transport the D form of DHB. A subsequent laboratory evolution was carried out with E. coli K12 MG1655 deleted for ΔygbI to adapt for growth on DHB as sole carbon source. Remarkably, only two additional mutations were disclosed in the adapted strain, which were demonstrated by reverse engineering to be necessary and sufficient for robust growth on DHB. One mutation was in nanR encoding the transcription repressor of sialic acid metabolic genes, causing 140-fold increase in expression of nanA encoding N-acetyl neuraminic acid lyase, a pyruvate-dependent aldolase, and the other was in the promoter of dld leading to 14-fold increase in D-lactate dehydrogenase activity on DHB. Taken together, this work illustrates the importance of promiscuous enzymes in underground metabolism and moreover, in the frame of synthetic pathways aiming at producing non-natural products, these underground reactions could potentially penalize yield and title of these bio-based products., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The study of microorganism interactions is important for understanding the organization and functioning of microbial consortia. Additionally, the interaction between yeast and bacteria is of interest in the field of health and nutrition area for the development of probiotics. To investigate these microbial interactions at the cellular and molecular levels, a simple, reliable, and quantitative method is proposed. We demonstrated that flow cytometry enables the measurement of interactions at a single-cell level by detecting and counting yeast cells with bound fluorescent bacteria. Imaging flow cytometry revealed that the number of bacteria attached to yeast followed a Gaussian distribution whose maximum reached 14 bacterial cells using a clinical Escherichia coli strain E22 and the laboratory yeast strain BY4741. We found that the dynamics of adhesion resemble a Langmuir adsorption model, albeit it is a rapid and almost irreversible process. This adhesion is dependent on the mannose-specific type 1 fimbriae, as E. coli mutants lacking these appendages no longer adhere to yeast. However, this type 1 fimbriae-dependent adhesion could involve additional yeast cell wall factors, since the interaction between bacteria and yeast mutants with altered mannan content remained comparable to that of wild-type yeast. In summary, flow cytometry is an appropriate method for studying bacteria-yeast adhesion, as well as for the high-throughput screening of candidate molecules likely to promote or counteract this interaction., (© 2024. The Author(s).)

The genetic stability and metabolic robustness of production strains is one of the key criteria for the production of bio-based products by microbial fermentation on an industrial scale. These criteria were here explored in an industrial ethanol-producer strain of Saccharomyces cerevisiae able to co-ferment D-xylose and L-arabinose with glucose through the chromosomal integration of several copies of pivotal genes for the use of these pentose (C5) sugars. Using batch sequential cultures in a controlled bioreactor that mimics long-term fermentation in an industrial setting, this strain was found to exhibit significant fluctuations in D-xylose and L-arabinose consumption as early as the 50th generation and beyond. These fluctuations seem not related to the few low-consumption C5 sugar clones that appeared throughout the sequential batch cultures at a frequency lower than 1.5% and that were due to the reduction in the number of copies of transgenes coding for C5 sugar assimilation enzymes. Also, subpopulations enriched with low or high RAD52 expression, whose expression level was reported to be proportional to homologous recombination rate did not exhibit defect in C5-sugar assimilation, arguing that other mechanisms may be responsible for copy number variation of transgenes. Overall, this work highlighted the existence of genetic and metabolic instabilities in an industrial yeast which, although modest in our conditions, could be more deleterious in harsher industrial conditions, leading to reduced production performance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Duperray, Delvenne, François, Delvigne and Capp.)

The essential sulphur-containing amino acid, methionine, is becoming a mass-commodity product with an annual production that exceeded 1,500,000 tons in 2018. This amino acid is today almost exclusively produced by chemical process from fossil resources. The environmental problems caused by this industrial process, and the expected scarcity of oil resources in the coming years, have recently accelerated the development of bioprocesses for producing methionine from renewable carbon feedstock. After a brief description of the chemical process and the techno-economic context that still justify the production of methionine by petrochemical processes, this review will present the current state of the art of biobased alternatives aiming at a sustainable production of this amino acid and its hydroxyl analogues from renewable carbon feedstock. In particular, this review will focus on three bio-based processes, namely a purely fermentative process based on the metabolic engineering of the natural methionine pathway, a mixed process combining the production of the O-acetyl/O-succinyl homoserine intermediate of this pathway by fermentation followed by an enzyme-based conversion of this intermediate into L-methionine and lately, a hybrid process in which the non-natural chemical synthon, 2,4-dihydroxybutyric acid, obtained by fermentation of sugars is converted by chemo-catalysis into hydroxyl methionine analogues. The industrial potential of these three bioprocesses, as well as the major technical and economic obstacles that remain to be overcome to reach industrial maturity are discussed. This review concludes by bringing up the assets of these bioprocesses to meet the challenge of the "green transition", with the accomplishment of the objective "zero carbon" by 2050 and how they can be part of a model of Bioeconomy enhancing local resources., Competing Interests: Declaration of Competing Interest The author declare that he has no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Dermatophytosis is a superficial fungal infection with an ever-increasing number of patients. Culture-based mycology remains the most commonly used diagnosis, but it takes around four weeks to identify the causative agent. Therefore, routine clinical laboratories need rapid, high throughput, and accurate species-specific analytical methods for diagnosis and therapeutic management. Based on these requirements, we investigated the feasibility of DendrisCHIP ® technology as an innovative molecular diagnostic method for the identification of a subset of 13 pathogens potentially responsible for dermatophytosis infections in clinical samples. This technology is based on DNA microarray, which potentially enables the detection and discrimination of several germs in a single sample. A major originality of DendrisCHIP ® technology is the use of a decision algorithm for probability presence or absence of pathogens based on machine learning methods. In this study, the diagnosis of dermatophyte infection was carried out on more than 284 isolates by conventional microbial culture and DendrisCHIP ® DP, which correspond to the DendrisCHIP ® carrying oligoprobes of the targeted pathogens implicated in dermatophytosis. While convergence ranging from 75 to 86% depending on the sampling procedure was obtained with both methods, the DendrisCHIP ® DP proved to identify more isolates with pathogens that escaped the culture method. These results were confirmed at 86% by a third method, which was either a specific RT-PCR or genome sequencing. In addition, diagnostic results with DendrisCHIP ® DP can be obtained within a day. This faster and more accurate identification of fungal pathogens with DendrisCHIP ® DP enables the clinician to quickly and successfully implement appropriate antifungal treatment to prevent the spread and elimination of dermatophyte infection. Taken together, these results demonstrate that this technology is a very promising method for routine diagnosis of dermatophytosis.

Non-conventional yeast species, or non- Saccharomyces yeasts, are increasingly recognized for their involvement in fermented foods. Many of them exhibit probiotic characteristics that are mainly due to direct contacts with other cell types through various molecular components of their cell wall. The biochemical composition and/or the molecular structure of the cell wall components are currently considered the primary determinant of their probiotic properties. Here we first present the techniques that are used to extract and analyze the cell wall components of food industry-related non- Saccharomyces yeasts. We then review the current understanding of the cell wall composition and structure of each polysaccharide from these yeasts. Finally, the data exploring the potential beneficial role of their cell wall components, which could be a source of innovative functional ingredients, are discussed. Such research would allow the development of high value-added products and provide the food industry with novel inputs beyond the well-established S. cerevisiae ., Competing Interests: MS is an employee of Lallemand SAS., (© 2023 The Authors.)

Knr4/Smi1 proteins are specific to the fungal kingdom and their deletion in the model yeast Saccharomyces cerevisiae and the human pathogen Candida albicans results in hypersensitivity to specific antifungal agents and a wide range of parietal stresses. In S. cerevisiae, Knr4 is located at the crossroads of several signalling pathways, including the conserved cell wall integrity and calcineurin pathways. Knr4 interacts genetically and physically with several protein members of those pathways. Its sequence suggests that it contains large intrinsically disordered regions. Here, a combination of small-angle X-ray scattering (SAXS) and crystallographic analysis led to a comprehensive structural view of Knr4. This experimental work unambiguously showed that Knr4 comprises two large intrinsically disordered regions flanking a central globular domain whose structure has been established. The structured domain is itself interrupted by a disordered loop. Using the CRISPR/Cas9 genome editing technique, strains expressing KNR4 genes deleted from different domains were constructed. The N-terminal domain and the loop are essential for optimal resistance to cell wall-binding stressors. The C-terminal disordered domain, on the other hand, acts as a negative regulator of this function of Knr4. The identification of molecular recognition features, the possible presence of secondary structure in these disordered domains and the functional importance of the disordered domains revealed here designate these domains as putative interacting spots with partners in either pathway. Targeting these interacting regions is a promising route to the discovery of inhibitory molecules that could increase the susceptibility of pathogens to the antifungals currently in clinical use., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

2-Phenylethanol is an aromatic compound commonly used in the food, cosmetic, and pharmaceutical industries. Due to increasing demand for natural products by consumers, the production of this flavor by microbial fermentation is gaining interest, as a sustainable alternative to chemical synthesis or expensive plant extraction, both processes relying on the use of fossil resources. However, the drawback of the fermentation process is the high toxicity of 2-phenylethanol to the producing microorganism. The aim of this study was to obtain a 2-phenylethanol-resistant Saccharomyces cerevisiae strain by in vivo evolutionary engineering and characterize the adapted yeast at the genomic, transcriptomic and metabolic levels. For this purpose, the tolerance to 2-phenylethanol was developed by gradually increasing the concentration of this flavor compound through successive batch cultivations, leading to an adapted strain that could tolerate 3.4 g/L of 2-phenylethanol, which was about 3-times better than the reference strain. Genome sequencing of the adapted strain identified point mutations in several genes, notably in HOG1 that encodes the Mitogen-Activated Kinase of the high-osmolarity signaling pathway. As this mutation is localized in the phosphorylation lip of this protein, it likely resulted in a hyperactive protein kinase. Transcriptomic analysis of the adapted strain supported this suggestion by revealing a large set of upregulated stress-responsive genes that could be explained in great part by HOG1 -dependent activation of the Msn2/Msn4 transcription factor. Another relevant mutation was found in PDE2 encoding the low affinity cAMP phosphodiesterase, the missense mutation of which may lead to hyperactivation of this enzyme and thereby enhance the stressful state of the 2-phenylethanol adapted strain. In addition, the mutation in CRH1 that encodes a chitin transglycosylase implicated in cell wall remodeling could account for the increased resistance of the adapted strain to the cell wall-degrading enzyme lyticase. Finally, the potent upregulation of ALD3 and ALD4 encoding NAD +  -dependent aldehyde dehydrogenase together with the observed phenylacetate resistance of the evolved strain suggest a resistance mechanism involving conversion of 2-phenylethanol into phenylacetaldehyde and phenylacetate implicating these dehydrogenases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Holyavkin, Turanlı-Yıldız, Yılmaz, Alkım, Arslan, Topaloğlu, Kısakesen, de Billerbeck, François and Çakar.)

The bacterial toxin-antitoxin systems are each composed of a toxin, which severely inhibits bacterial cells growth, and a specific neutralizing antitoxin. Some toxin-antitoxin systems are functional when expressed in the yeast Saccharomyces cerevisiae. For instance, the expression of the relE toxin gene leads to a strong growth defect in yeast, whereas the expression of the relB antitoxin gene restores growth. Nevertheless, there is no available data regarding the required expression levels of each component of the relBE system leading to these growth phenotypes, neither their effects on cell viability. Here we used a double inducible plasmid-based system to independently modulate the relative amounts of relB and relE, and performed growth and gene expression analyses. These results allow us to correlate growth phenotypes to the expression levels of the toxin and the antitoxin, and to determine the levels necessary to observe either a strong growth inhibition or a normal growth. We also showed that the relE expression produces cell cycle progression defect without affecting cell viability. These results provide a detailed characterization of the functioning of the relBE system in S. cerevisiae, and open applicative perspectives of yeast growth control by bacterial toxin-antitoxin systems., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

L-homoserine is a pivotal intermediate in the carbon and nitrogen metabolism of E. coli. However, this non-canonical amino acid cannot be used as a nitrogen source for growth. Furthermore, growth of this bacterium in a synthetic media is potently inhibited by L-homoserine. To understand this dual effect, an adapted laboratory evolution (ALE) was applied, which allowed the isolation of a strain able to grow with L-homoserine as the nitrogen source and was, at the same time, desensitized to growth inhibition by this amino acid. Sequencing of this evolved strain identified only four genomic modifications, including a 49 bp truncation starting from the stop codon of thrL . This mutation resulted in a modified thrL locus carrying a thrL* allele encoding a polypeptide 9 amino acids longer than the thrL encoded leader peptide. Remarkably, the replacement of thrL with thrL* in the original strain MG1655 alleviated L-homoserine inhibition to the same extent as strain 4E, but did not allow growth with this amino acid as a nitrogen source. The loss of L-homoserine toxic effect could be explained by the rapid conversion of L-homoserine into threonine via the thrL*- dependent transcriptional activation of the threonine operon thrABC . On the other hand, the growth of E. coli on a mineral medium with L-homoserine required an activation of the threonine degradation pathway II and glycine cleavage system, resulting in the release of ammonium ions that were likely recaptured by NAD(P)-dependent glutamate dehydrogenase. To infer about the direct molecular targets of L-homoserine toxicity, a transcriptomic analysis of wild-type MG1655 in the presence of 10 mM L-homoserine was performed, which notably identified a potent repression of locomotion-motility-chemotaxis process and of branched-chain amino acids synthesis. Since the magnitude of these effects was lower in a ΔthrL mutant, concomitant with a twofold lower sensitivity of this mutant to L-homoserine, it could be argued that growth inhibition by L-homoserine is due to the repression of these biological processes. In addition, L-homoserine induced a strong upregulation of genes in the sulfate reductive assimilation pathway, including those encoding its transport. How this non-canonical amino acid triggers these transcriptomic changes is discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Alkim, Farias, Fredonnet, Serrano-Bataille, Herviou, Picot, Slama, Dejean, Morin, Enjalbert and François.)

α-unsaturated esters are fruity-aromatic compounds which are largely spread in the volatilome of many different fruits, but they are rarely found in the volatilome of yeasts. The yeast S. suaveolens has been recently shown to produce relatively high amounts of α-unsaturated esters and it appears to be an interesting model for the production of these compounds. This study aimed to isolate new α-unsaturated ester-producing yeasts by focusing on strains displaying a similar metabolism to S. suaveolens . While the production of α-unsaturated esters by S. suaveolens is believed to be closely related to its ability to grow on media containing branched-chain amino acids (isoleucine, leucine and valine) as the sole carbon source (ILV + phenotype), in this study, an original screening method was developed that selects for yeast strains displaying ILV + phenotypes and is able to produce α-unsaturated esters. Among the 119 yeast strains isolated from the feces of 42 different South African wild animal species, 43 isolates showed the ILV + phenotype, among which 12 strains were able to produce α-unsaturated esters. Two interesting α-unsaturated esters were detected in two freshly isolated strains, both identified as Galactomyces candidus . These new esters were detected neither in the volatilome of the reference strain S. suaveolens , nor in any other yeast species previously studied for their aroma production. This work demonstrated the efficiency of an original method to rapidly screen for α-unsaturated ester-producing yeasts. In addition, it demonstrated that wild animal feces are interesting resources to isolate novel strains producing compounds with original aromas.

Osteoarticular infections are major disabling diseases that can occur after orthopedic implant surgery in patients. The management of these infections is very complex and painful, requiring surgical intervention in combination with long-term antibiotic treatment. Therefore, early and accurate diagnosis of the causal pathogens is essential before formulating chemotherapeutic regimens. Although culture-based microbiology remains the most common diagnosis of osteoarticular infections, its regular failure to identify the causative pathogen as well as its long-term modus operandi motivates the development of rapid, accurate, and sufficiently comprehensive bacterial species-specific diagnostics that must be easy to use by routine clinical laboratories. Based on these criteria, we reported on the feasibility of our DendrisCHIP ® technology using DendrisCHIP ® OA as an innovative molecular diagnostic method to diagnose pathogen bacteria implicated in osteoarticular infections. This technology is based on the principle of microarrays in which the hybridization signals between oligoprobes and complementary labeled DNA fragments from isolates queries a database of hybridization signatures corresponding to a list of pre-established bacteria implicated in osteoarticular infections by a decision algorithm based on machine learning methods. In this way, this technology combines the advantages of a PCR-based method and next-generation sequencing (NGS) while reducing the limitations and constraints of the two latter technologies. On the one hand, DendrisCHIP ® OA is more comprehensive than multiplex PCR tests as it is able to detect many more germs on a single sample. On the other hand, this method is not affected by the large number of nonclinically relevant bacteria or false positives that characterize NGS, as our DendrisCHIP ® OA has been designed to date to target only a subset of 20 bacteria potentially responsible for osteoarticular infections. DendrisCHIP ® OA has been compared with microbial culture on more than 300 isolates and a 40% discrepancy between the two methods was found, which could be due in part but not solely to the absence or poor identification of germs detected by microbial culture. We also demonstrated the reliability of our technology in correctly identifying bacteria in isolates by showing a convergence (i.e., same bacteria identified) with NGS superior to 55% while this convergence was only 32% between NGS and microbial culture data. Finally, we showed that our technology can provide a diagnostic result in less than one day (technically, 5 h), which is comparatively faster and less labor intensive than microbial cultures and NGS.

Background: In Belgium, the first COVID-19 death was reported on 10 March 2020. Nursing home (NH) residents are particularly vulnerable for COVID-19, making it essential to follow-up the spread of COVID-19 in this setting. This manuscript describes the methodology of surveillance and epidemiology of COVID-19 cases, hospitalizations and deaths in Belgian NHs., Methods: A COVID-19 surveillance in all Belgian NHs (n = 1542) was set up by the regional health authorities and Sciensano. Aggregated data on possible/confirmed COVID-19 cases and hospitalizations and case-based data on deaths were reported by NHs at least once a week. The study period covered April-December 2020. Weekly incidence/prevalence data were calculated per 1000 residents or staff members., Results: This surveillance has been launched within 14 days after the first COVID-19 death in Belgium. Automatic data cleaning was installed using different validation rules. More than 99% of NHs participated at least once, with a median weekly participation rate of 95%. The cumulative incidence of possible/confirmed COVID-19 cases among residents was 206/1000 in the first wave and 367/1000 in the second wave. Most NHs (82%) reported cases in both waves and 74% registered ≥10 possible/confirmed cases among residents at one point in time. In 51% of NHs, at least 10% of staff was absent due to COVID-19 at one point. Between 11 March 2020 and 3 January 2021, 11,329 COVID-19 deaths among NH residents were reported, comprising 57% of all COVID-19 deaths in Belgium in that period., Conclusions: This surveillance was crucial in mapping COVID-19 in this vulnerable setting and guiding public health interventions, despite limitations of aggregated data and necessary changes in protocol over time. Belgian NHs were severely hit by COVID-19 with many fatal cases. The measure of not allowing visitors, implemented in the beginning of the pandemic, could not avoid the spread of SARS-CoV-2 in the NHs during the first wave. The virus was probably often introduced by staff. Once the virus was introduced, it was difficult to prevent healthcare-associated outbreaks. Although, in contrast to the first wave, personal protective equipment was available in the second wave, again a high number of cases were reported., (© 2022. The Author(s).)

The yeast Saccharomyces cerevisiae has a remarkable ability to adapt its lifestyle to fluctuating or hostile environmental conditions. This adaptation most often involves morphological changes such as pseudofilaments, biofilm formation, or cell aggregation in the form of flocs. A prerequisite for these phenotypic changes is the ability to self-adhere and to adhere to abiotic surfaces. This ability is conferred by specialized surface proteins called flocculins, which are encoded by the FLO genes family in this yeast species. This mini-review focuses on the flocculin encoded by FLO11 , which differs significantly from other flocculins in domain sequence and mode of genetic and epigenetic regulation, giving it an impressive plasticity that enables yeast cells to swiftly adapt to hostile environments or into new ecological niches. Furthermore, the common features of Flo11p with those of adhesins from pathogenic yeasts make FLO11 a good model to study the molecular mechanism underlying cell adhesion and biofilm formation, which are part of the initial step leading to fungal infections.

Rapeseed meal (RSM) is a major by-product of oil extraction from rapeseed, consists mainly of proteins and phenolic compounds. The use of RSM as protein feedstock for microbial fermentation is always hampered by phenolic compounds, which have antioxidant property with health-promoting benefits but inhibit bacterial growth. However, there is still not any good process that simultaneously improve extraction efficiency of phenolic compounds with conversion efficiency of protein residue into microbial production. Here we established a two-step strategy including fungal pretreatment followed by extraction of phenolic compounds. This could not only increase extraction efficiency and antioxidant property of phenolic compounds by about 2-fold, but also improve conversion efficiency of protein residue into iturin A production by Bacillus amyloliquefaciens CX-20 by about 33%. The antioxidant and antibacterial activities of phenolic extracts were influenced by both total phenolic content and profile, while microbial feedstock value of residue was greatly improved because protein content was increased by ∼5% and phenolic content was decreased by ∼60%. Moreover, this two-step process resulted in isolating more proteins from RSM, bringing iturin A production to 1.95 g/L. In conclusion, high-value-added and graded utilization of phenolic extract and protein residue from RSM with zero waste is realized by a two-step strategy, which combines both benefits of fungal pretreatment and phenolic extraction procedures., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Yang, François, Wan, Deng, Feng, Deng, Chen, Huang, Chen and Gong.)

Fungal adhesins (Als) or flocculins are family of cell surface proteins that mediate adhesion to diverse biotic and abiotic surfaces. A striking characteristic of Als proteins originally identified in the pathogenic Candida albicans is to form functional amyloids that mediate cis- interaction leading to the formation of adhesin nanodomains and trans -interaction between amyloid sequences of opposing cells. In this report, we show that flocculins encoded by FLO11 in Saccharomyces cerevisiae behave like adhesins in C. albicans . To do so, we show that the formation of nanodomains under an external physical force requires a threshold number of amyloid-forming sequences in the Flo11 protein. Then, using a genome editing approach, we constructed strains expressing variants of the Flo11 protein under the endogenous FLO11 promoter, leading to the demonstration that the loss of amyloid-forming sequences strongly reduces cell-cell interaction but has no effect on either plastic adherence or invasive growth in agar, both phenotypes being dependent on the N- and C-terminal ends of Flo11p. Finally, we show that the location of Flo11 is not altered either by the absence of amyloid-forming sequences or by the removal of the N- or C-terminus of the protein., Competing Interests: CB, MS, MT, ED, NS, AJ, JF none, (© 2021, Bouyx et al.)

Fruity beers can be promoted through production of flavoring compounds during fermentation by partial replacement of brewing yeast by non-conventional-yeasts with high aroma production abilities. We evaluated here the use of a wild Saprochaete suaveolens strain, producing atypical aroma compounds, to produce new natural fruity beer, while keeping classical production conditions used in brewing industry. S. suaveolens was inoculated as starter of culture during beer fermentation and the fermentation performance was evaluated through measurement of several physicochemical parameters. The aroma profile of the engineered beers was monitored using HS-SPME GC/MS. The results showed that high fruity aroma and low-ethanol content beers were obtained through single-fermentation using S. suaveolens. We also demonstrated that during mixed-fermentation, S. suaveolens maintained high metabolic activity and allowed production of beer enriched with fruity aroma. Production of high or low ethanol content fruity beer could be achieved by varying the composition of the starter of culture., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Yeasts are becoming popular as novel ingredients in fish feeds because of their potential to support better growth and concomitantly ensure good fish health. Here, three species of yeasts (Cyberlindnera jadinii, Blastobotrys adeninivorans and Wickerhamomyces anomalus), grown on wood sugars and hydrolysates of chicken were subjected to two down-stream processes, either direct heat-inactivation or autolysis, and the feed potential of the resulting yeast preparations was assessed through a feeding trial with Atlantic salmon fry. Histological examination of distal intestine based on widening of lamina propria, showed that autolyzed W. anomalus was effective in alleviating mild intestinal enteritis, while only limited effects were observed for other yeasts. Our results showed that the functionality of yeast in counteracting intestinal enteritis in Atlantic salmon was dependent on both the type of yeast and the down-stream processing method, and demonstrated that C. jadinii and W. anomalus have promising effects on gut health of Atlantic salmon.

Single-cell variability of growth is a biological phenomenon that has attracted growing interest in recent years. Important progress has been made in the knowledge of the origin of cell-to-cell heterogeneity of growth, especially in microbial cells. To better understand the origins of such heterogeneity at the single-cell level, we developed a new methodological pipeline that coupled cytometry-based cell sorting with automatized microscopy and image analysis to score the growth rate of thousands of single cells. This allowed investigating the influence of the initial amount of proteins of interest on the subsequent growth of the microcolony. As a preliminary step to validate this experimental setup, we referred to previous findings in yeast where the expression level of Tsl1, a member of the Trehalose Phosphate Synthase (TPS) complex, negatively correlated with cell division rate. We unfortunately could not find any influence of the initial TSL1 expression level on the growth rate of the microcolonies. We also analyzed the effect of the natural variations of trehalose-6-phosphate synthase ( TPS1 ) expression on cell-to-cell growth heterogeneity, but we did not find any correlation. However, due to the already known altered growth of the tps1 Δ mutants, we tested this strain at the single-cell level on a permissive carbon source. This mutant showed an outstanding lack of reproducibility of growth rate distributions as compared to the wild-type strain, with variable proportions of non-growing cells between cultivations and more heterogeneous microcolonies in terms of individual growth rates. Interestingly, this variable behavior at the single-cell level was reminiscent to the high variability that is also stochastically suffered at the population level when cultivating this tps1 Δ strain, even when using controlled bioreactors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Arabaciyan, Saint-Antoine, Maugis-Rabusseau, François, Singh, Parrou and Capp.)

Sugarcane Distillery Spent Wash (DSW) is among the most pollutant industrial effluents, generally characterized by high Chemical Oxygen Demand (COD), high mineral matters and acidic pH, causing strong environmental impacts. Bioremediation is considered to be a good and cheap alternative to DSW treatment. In this study, 37 strains of yeasts and filamentous fungi were performed to assess their potential to significantly reduce four parameters characterizing the organic load of vinasses (COD, pH, minerals and OD 475nm ). In all cases, a pH increase (until a final pH higher than 8.5, being an increase superior to 3.5 units, as compared to initial pH) and a COD and minerals removal could be observed, respectively (until 76.53% using Aspergillus terreus var. africanus and 77.57% using Aspergillus niger ). Depending on the microorganism, the OD 475nm could decrease (generally when filamentous fungi were used) or increase (generally when yeasts were used). Among the strains tested, the species from Aspergillus and Trametes genus offered the best results in the depollution of DSW. Concomitant with the pollutant load removal, fungal biomass, with yields exceeding 20 g·L -1 , was produced.

Chromatin structure clearly modulates gene expression noise, but the reverse influence has never been investigated, namely how the cell-to-cell expression heterogeneity of chromatin modifiers may generate variable rates of epigenetic modification. Sir2 is a well-characterized histone deacetylase of the Sirtuin family. It strongly influences chromatin silencing, especially at telomeres, subtelomeres and rDNA. This ability to influence epigenetic landscapes makes it a good model to study the largely unexplored interplay between gene expression noise and other epigenetic processes leading to phenotypic diversification. Here, we addressed this question by investigating whether noise in the expression of SIR2 was associated with cell-to-cell heterogeneity in the frequency of epigenetic silencing at subtelomeres in Saccharomyces cerevisiae Using cell sorting to isolate subpopulations with various expression levels, we found that heterogeneity in the cellular concentration of Sir2 does not lead to heterogeneity in the epigenetic silencing of subtelomeric URA3 between these subpopulations. We also noticed that SIR2 expression noise can generate cell-to-cell variability in viability, with lower levels being associated with better viability. This work shows that SIR2 expression fluctuations are not sufficient to generate cell-to-cell heterogeneity in the epigenetic silencing of URA3 at subtelomeres in Saccharomyces cerevisiae but can strongly affect cellular viability., (Copyright © 2020 Liu et al.)

The global warming conjugated with our reliance to petrol derived processes and products have raised strong concern about the future of our planet, asking urgently to find sustainable substitute solutions to decrease this reliance and annihilate this climate change mainly due to excess of CO 2 emission. In this regard, the exploitation of microorganisms as microbial cell factories able to convert non-edible but renewable carbon sources into biofuels and commodity chemicals appears as an attractive solution. However, there is still a long way to go to make this solution economically viable and to introduce the use of microorganisms as one of the motor of the forthcoming bio-based economy. In this review, we address a scientific issue that must be challenged in order to improve the value of microbial organisms as cell factories. This issue is related to the capability of microbial systems to optimize carbon conservation during their metabolic processes. This initiative, which can be addressed nowadays using the advances in Synthetic Biology, should lead to an increase in products yield per carbon assimilated which is a key performance indice in biotechnological processes, as well as to indirectly contribute to a reduction of CO 2 emission., (Copyright © 2020 François, Lachaux and Morin.)

Glycolic acid is a two-carbon α-hydroxy acid with many applications in industrial sectors including packaging, fine chemistry, cosmetics, and pharmaceutics. Currently, glycolic acid is chemically manufactured from fossil resources. This chemical mode of production is raising some concerns regarding its use in health for personal care. Microbial production of GA stands as a remarkable challenge to meet these concerns, while responding to the increasing demand to produce bio-sourced products from renewable carbon resources. We here report on the design and expression of a novel non-natural pathway of glycolic acid in E. coli . The originality of this new pathway, termed "glycoptimus" relies on two pillars. On the one hand, it requires the overexpression of three naturally occurring E. coli genes, namely kdsD encoding a D-arabinose-5-P isomerase, fsaA encoding a class 1 aldolase that cleaves D-arabinose-5-P into glyceraldehyde-3-P and glycolaldehyde, and aldA coding for an aldehyde dehydrogenase that oxidizes glycoladehyde in glycolate. These three genes constitute the "glycoptimus module." On the other hand, the expression of these genes together with a reshaping of the central carbon metabolism should enable a production of glycolic acid from pentose and hexose at a molar ratio of 2.5 and 3, respectively, which corresponds to 50% increase as compared to the existing pathways. We demonstrated the ' in vivo ' potentiality of this pathway using an E. coli strain, which constitutively expressed the glycoptimus module and whose carbon flow in glycolysis was blocked at the level of glyceraldehyde-3-P dehydrogenase reaction step. This engineered strain was cultivated on a permissive medium containing malate and D-glucose. Upon exhaustion of malate, addition of either D-glucose, D-xylose or L-arabinose led to the production of glycolic acid reaching about 30% of the maximum molar yield. Further improvements at the level of enzymes, strains and bioprocess engineering are awaited to increase yield and titer, rendering the microbial production of glycolic acid affordable for a cost-effective industrial process., (Copyright © 2019 Lachaux, Frazao, Krauβer, Morin, Walther and François.)

In this work, we describe the construction of a synthetic metabolic pathway enabling direct biosynthesis of 1,3-propanediol (PDO) from glucose via the Krebs cycle intermediate malate. This non-natural pathway extends a previously published synthetic pathway for the synthesis of (L)-2,4-dihydroxybutyrate (L-DHB) from malate by three additional reaction steps catalyzed respectively, by a DHB dehydrogenase, a 2-keto-4-hydroxybutyrate (OHB) dehydrogenase and a PDO oxidoreductase. Screening and structure-guided protein engineering provided a (L)-DHB dehydrogenase from the membrane-associated (L)-lactate dehydrogenase of E. coli and OHB decarboxylase variants derived from the branched-chain keto-acid decarboxylase encoded by kdcA from Lactococcus lactis or pyruvate decarboxylase from Zymomonas mobilis. The simultaneous overexpression of the genes encoding these enzymes together with the endogenous ydhD-encoded aldehyde reductase enabled PDO biosynthesis from (L)-DHB. While the simultaneous expression of the six enzymatic activities in a single engineered E. coli strain resulted in a low production of 0.1 mM PDO from 110 mM glucose, a 40-fold increased PDO titer was obtained by co-cultivation of an E. coli strain expressing the malate-DHB pathway with another strain harboring the DHB-to-PDO pathway.

The yeast cell wall is composed of mannoproteins, β-1,3/β-1, 6-glucans and chitin. Each of these components has technological properties that are relevant for industrial and medical applications. To address issues related to cell wall structure and alteration in response to stress or conditioning processes, AFM dendritips were functionalized with biomolecules that are specific for each of the wall components, which was wheat germ agglutinin (WGA) for chitin, concanavalin A (ConA) for mannans and anti-β-1,3/anti-β-1,6-glucan antibodies for β-1,3/β-1,6-glucans. Binding specificity of these biomolecules were validated using penta- N -acetylchitopentaose, α-mannans, laminarin (short β-1,3-glucan chain) and gentiobiose (2 glucose units linked in β 1→6) immobilized on epoxy glass slides. Dynamic force spectroscopy was employed to obtain kinetic and thermodynamic information on the intermolecular interaction of the binary complexes using the model of Friddle-Noy-de Yoreo. Using this model, transition state distance x t , dissociate rate k off and the lowest force ( f eq ) required to break the intermolecular bond of the complexes were approximated. These functionalized dendritips were then used to probe the yeast cell surface treated with a bacterial protease. As expected, this treatment, which removed the outer layer of the cell wall, gave accessibility to the inner layer composed of β-glucans. Likewise, bud scars were nicely localized using AFM dendritip bearing the WGA probe. To conclude, these functionalized AFM dendritips constitute a new toolbox that can be used to investigate cell surface structure and organization in response to a wide arrays of cultures and process conditions., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2019 The Author(s).)

Variation in gene expression among genetically identical individual cells (called gene expression noise) directly contributes to phenotypic diversity. Whether such variation can impact genome stability and lead to variation in genotype remains poorly explored. We addressed this question by investigating whether noise in the expression of genes affecting homologous recombination (HR) activity either directly ( RAD52 ) or indirectly ( RAD27 ) confers cell-to-cell heterogeneity in HR rate in Saccharomyces cerevisiae . Using cell sorting to isolate subpopulations with various expression levels, we show that spontaneous HR rate is highly heterogeneous from cell-to-cell in clonal populations depending on the cellular amount of proteins affecting HR activity. Phleomycin-induced HR is even more heterogeneous, showing that RAD27 expression variation strongly affects the rate of recombination from cell-to-cell. Strong variations in HR rate between subpopulations are not correlated to strong changes in cell cycle stage. Moreover, this heterogeneity occurs even when simultaneously sorting cells at equal expression level of another gene involved in DNA damage response ( BMH1 ) that is upregulated by DNA damage, showing that the initiating DNA damage is not responsible for the observed heterogeneity in HR rate. Thus gene expression noise seems mainly responsible for this phenomenon. Finally, HR rate non-linearly scales with Rad27 levels showing that total amount of HR cannot be explained solely by the time- or population-averaged Rad27 expression. Altogether, our data reveal interplay between heterogeneity at the gene expression and genetic levels in the production of phenotypic diversity with evolutionary consequences from microbial to cancer cell populations.

Simultaneous detection of 1 H and 31 P NMR signals through a dual-detection scheme with two receivers allows monitoring of both the signals of a molecule and the pH of the solution through the resonance of the inorganic phosphate. We evaluate here the method in terms of sensitivity and ease of implementation and show that the additional information obtained without any loss of information or increase in measuring time can be of practical importance in a number of biochemical systems.

Inside the human host, Leishmania infection starts with phagocytosis of infective promastigotes by macrophages. In order to survive, Leishmania has developed several strategies to manipulate macrophage functions. Among these strategies, Leishmania as a source of bioactive lipids has been poorly explored. Herein, we assessed the biosynthesis of polyunsaturated fatty acid metabolites by infective and noninfective stages of Leishmania and further explored the role of these metabolites in macrophage polarization. The concentration of docosahexaenoic acid metabolites, precursors of proresolving lipid mediators, was increased in the infective stage of the parasite compared with the noninfective stage, and cytochrome P450-like proteins were shown to be implicated in the biosynthesis of these metabolites. The treatment of macrophages with lipids extracted from the infective forms of the parasite led to M2 macrophage polarization and blocked the differentiation into the M1 phenotype induced by IFN-γ. In conclusion, Leishmania polyunsaturated fatty acid metabolites, produced by cytochrome P450-like protein activity, are implicated in parasite/host interactions by promoting the polarization of macrophages into a proresolving M2 phenotype., (Copyright © 2019 Paloque et al.)

Background: Research on filamentous fungi emphasized the remarkable redundancy in genes encoding hydrolytic enzymes, the similarities but also the large differences in their expression, especially through the role of the XlnR/XYR1 transcriptional activator. The purpose of this study was to evaluate the specificities of the industrial fungus Talaromyces versatilis, getting clues into the role of XlnR and the importance of glucose repression at the transcriptional level, to provide further levers for cocktail production., Results: By studying a set of 62 redundant genes representative of several categories of enzymes, our results underlined the huge plasticity of transcriptional responses when changing nutritional status. As a general trend, the more heterogeneous the substrate, the more efficient to trigger activation. Genetic modifications of xlnR led to significant reorganisation of transcriptional patterns. Just a minimal set of genes actually fitted in a simplistic model of regulation by a transcriptional activator, and this under specific substrates. On the contrary, the diversity of xlnR + versus ΔxlnR responses illustrated the existence of complex and unpredicted patterns of co-regulated genes that were highly dependent on the culture condition, even between genes that encode members of a functional category of enzymes. They notably revealed a dual, substrate-dependant repressor-activator role of XlnR, with counter-intuitive transcripts regulations that targeted specific genes. About glucose, it appeared as a formal repressive sugar as we observed a massive repression of most genes upon glucose addition to the mycelium grown on wheat straw. However, we also noticed a positive role of this sugar on the basal expression of a few genes, (notably those encoding cellulases), showing again the strong dependence of these regulatory mechanisms upon promoter and nutritional contexts., Conclusions: The diversity of transcriptional patterns appeared to be the rule, while common and stable behaviour, both within gene families and with fungal literature, the exception. The setup of a new biotechnological process to reach optimized, if not customized expression patterns of enzymes, hence appeared tricky just relying on published data that can lead, in the best scenario, to approximate trends. We instead encourage preliminary experimental assays, carried out in the context of interest to reassess gene responses, as a mandatory step before thinking in (genetic) strategies for the improvement of enzyme production in fungi.

A synthetic pathway for the production of 2,4-dihydroxybutyric acid from homoserine (HMS), composed of two consecutive enzymatic reaction steps has been recently reported. An important step in this pathway consists in the reduction in 2-keto-4-hydroxybutyrate (OHB) into (l)-dihydroxybutyrate (DHB), by an enzyme with OHB reductase activity. In the present study, we used a rational approach to engineer an OHB reductase by using the cytosolic (l)-malate dehydrogenase from Escherichia coli (Ec-Mdh) as the template enzyme. Structural analysis of (l)-malate dehydrogenase and (l)-lactate dehydrogenase enzymes acting on sterically cognate substrates revealed key residues in the substrate and co-substrate-binding sites responsible for substrate discrimination. Accordingly, amino acid changes were introduced in a stepwise manner into these regions of the protein. This rational engineering led to the production of an Ec-Mdh-5E variant (I12V/R81A/M85E/G179D/D86S) with a turnover number ( k cat ) on OHB that was increased by more than 2000-fold (from 0.03 up to 65.0 s -1 ), which turned out to be 7-fold higher than that on its natural substrate oxaloacetate. Further kinetic analysis revealed the engineered enzyme to possess comparable catalytic efficiencies ( k cat / K m ) between natural and synthetic OHB substrates (84 and 31 s -1  mM -1 , respectively). Shake-flask cultivation of a HMS-overproducing E. coli strain expressing this improved OHB reductase together with a transaminase encoded by aspC able to convert HMS to OHB resulted in 89% increased DHB production as compared with our previous report using a E. coli host strain expressing an OHB reductase derived from the lactate dehydrogenase A of Lactococcus lactis ., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Clinical microbiology is experiencing the emergence of the syndromic approach of diagnosis. This paradigm shift will require innovative technologies to detect rapidly, and in a single sample, multiple pathogens associated with an infectious disease. Here, we report on a multiplex technology based on DNA-microarray that allows detecting and discriminating 11 bacteria implicated in respiratory tract infection. The process requires a PCR amplification of bacterial 16S rDNA, a 30 min hybridization step on species-specific oligoprobes covalently linked on dendrimers coated glass slides (DendriChips ® ) and a reading of the slides by a dedicated laser scanner. A diagnostic result is delivered in about 4 h as a predictive value of presence/absence of pathogens using a decision algorithm based on machine-learning method, which was constructed from hybridization profiles of known bacterial and clinical isolated samples and which can be regularly enriched with hybridization profiles from clinical samples. We demonstrated that our technology converged in more than 95% of cases with the microbiological culture for bacteria detection and identification.

The yeast trehalose-6-phosphate synthase (Tps1) catalyzes the formation of trehalose-6-phosphate (T6P) in trehalose synthesis. Besides, Tps1 plays a key role in carbon and energy homeostasis in this microbial cell, as shown by the well documented loss of ATP and hyper accumulation of sugar phosphates in response to glucose addition in a mutant defective in this protein. The inability of a Saccharomyces cerevisiae tps1 mutant to cope with fermentable sugars is still a matter of debate. We reexamined this question through a quantitative analysis of the capability of TPS1 homologues from different origins to complement phenotypic defects of this mutant. Our results allowed to classify this complementation in three groups. A first group enclosed TPS1 of Klyveromyces lactis with that of S. cerevisiae as their expression in Sctps1 cells fully recovered wild type metabolic patterns and fermentation capacity in response to glucose. At the opposite was the group with TPS1 homologues from the bacteria Escherichia coli and Ralstonia solanacearum , the plant Arabidopsis thaliana and the insect Drosophila melanogaster whose metabolic profiles were comparable to those of a tps1 mutant, notably with almost no accumulation of T6P, strong impairment of ATP recovery and potent reduction of fermentation capacity, albeit these homologous genes were able to rescue growth of Sc tps1 on glucose. In between was a group consisting of TPS1 homologues from other yeast species and filamentous fungi characterized by 5 to 10 times lower accumulation of T6P, a weaker recovery of ATP and a 3-times lower fermentation capacity than wild type. Finally, we found that glucose repression of gluconeogenic genes was strongly dependent on T6P. Altogether, our results suggest that the TPS protein is indispensable for growth on fermentable sugars, and points to a critical role of T6P as a sensing molecule that promotes sugar fermentation and glucose repression., Competing Interests: Conflict of interest: The authors declare that they have no competing interests.

Higher noise in the expression of stress-related genes was previously shown to confer better resistance in selective conditions. Thus, evolving the promoter of such genes toward higher transcriptional noise appears to be an attractive strategy to engineer microbial strains with enhanced stress resistance. Here we generated hundreds of promoter variants of the GRX1 gene involved in oxidative stress resistance in Saccharomyces cerevisiae and created a yeast library by replacing the native GRX1 promoter by these variants at the native locus. An outlier clone with very strong increase in noise (6-times) at the same mean expression level as the native strain was identified whereas the other noisiest clones were only 3-times increased. This variant provides constitutive bimodal expression and consists in 3 repeated but differently mutated copies of the GRX1 promoter. In spite of the multi-factorial oxidative stress-response in yeast, replacement of the native promoter by this variant is sufficient alone to confer strongly enhanced resistance to H 2 O 2 and cumene hydroperoxide. New replacement of this variant by the native promoter in the resistant strain suppresses the resistance. This work shows that increasing noise of target genes in a relevant strategy to engineer microbial strains toward better stress resistance. Multiple promoter replacement could synergize the effect observed here with the sole GRX1 promoter replacement. Finally this work suggests that combining several mutated copies of the target promoter could allow enhancing transcriptional-mediated noise at higher levels than mutating a single copy by providing constitutive bimodal and highly heterogeneous expression distribution.

We have developed a fluorescence-based metabolite sensor enabling in vivo detection of various aldehydes of biotechnological interest in Escherichia coli . YqhC is a transcriptional regulator that is known to be involved in the upregulation of the yqhD-dgkA operon in the presence of aldehydes. We took advantage of this property by constructing a bi-modular biosensor, in which a sensing module constitutively expresses yqhC while a reporter module drives the expression of the syfp2 reporter gene that is put under control of the yqhD promoter. The sensitivity of the sensor has been optimized by engineering the 5'-UTRs of both the sensing and the reporter modules resulting in a 70-fold gain of fluorescence in response to the model compound glycolaldehyde at 5 mM. The optimized sensor further responded to other aldehydes when supplemented to the cultivation medium at concentrations of 1-10 mM. We furthermore showed that this metabolite sensor was functional in vivo as it responded to the presence of glycoladehyde that is specifically produced upon induction of a synthetic xylulose-1-phosphate pathway expressed in E. coli . This bi-modular sensor can therefore be employed as an exquisite tool for FACS-based ultra-high-throughput screening of aldehyde (over) producing enzymes.

Background: Malate is a C4-dicarboxylic acid widely used as an acidulant in the food and beverage industry. Rational engineering has been performed in the past for the development of microbial strains capable of efficient production of this metabolite. However, as malate can be a precursor for specialty chemicals, such as 2,4-dihydroxybutyric acid, that require additional cofactors NADP(H) and ATP, we set out to reengineer Escherichia coli for Krebs cycle-dependent production of malic acid that can satisfy these requirements., Results: We found that significant malate production required at least simultaneous deletion of all malic enzymes and dehydrogenases, and concomitant expression of a malate-insensitive PEP carboxylase. Metabolic flux analysis using 13 C-labeled glucose indicated that malate-producing strains had a very high flux over the glyoxylate shunt with almost no flux passing through the isocitrate dehydrogenase reaction. The highest malate yield of 0.82 mol/mol was obtained with E. coli Δmdh Δmqo ΔmaeAB ΔiclR ΔarcA which expressed malate-insensitive PEP carboxylase Ppc K620S and NADH-insensitive citrate synthase GltA R164L . We also showed that inactivation of the dicarboxylic acid transporter DcuA strongly reduced malate production arguing for a pivotal role of this permease in malate export., Conclusions: Since more NAD(P)H and ATP cofactors are generated in the Krebs cycle-dependent malate production when compared to pathways which depend on the function of anaplerotic PEP carboxylase or PEP carboxykinase enzymes, the engineered strain developed in this study can serve as a platform to increase biosynthesis of malate-derived metabolites such as 2,4-dihydroxybutyric acid.

Bimodality in gene expression is thought to provide a high phenotypic heterogeneity that can be favourable for adaptation or unfavourable notably in industrial processes that require stable and homogeneous properties. Whether this property is produced or suppressed in different conditions has been understudied. Here we identified tens of Saccharomyces cerevisiae genomic fragments conferring bimodal yEGFP expression on centromeric plasmid and studied some of these promoters in different DNA contexts, inducing conditions or strain backgrounds. First, we observed that the bimodal behaviour identified on plasmid is generally suppressed at the genomic level. Second, an inducible promoter such as the copper-regulated CUP1 promoter can produce bimodal expression in a time- and dose-dependent fashion. For a given copper sulphate concentration, a constant proportion of the subpopulation is induced and only the induction level of this subpopulation changed with induction duration, while for a same induction time, higher copper sulphate concentrations induced more cells at higher levels. Third, we showed that bimodality conferred by the CUP1 promoter in expression profile is strain background dependent, revealing epistasis in the generation of bimodality. The influence of these parameters on bimodality has to be taken into account when considering transgene expression for industrial microbial productions.

An end-point ADP/NAD+ acid/alkali assay procedure, directly applicable to library screening of any type of ATP-utilising/ADP producing enzyme activity, was implemented. Typically, ADP production is coupled to NAD+ co-enzyme formation by the conventional addition of pyruvate kinase and lactate dehydrogenase. Transformation of enzymatically generated NAD+ into a photometrically active alkali derivative product is then achieved through the successive application of acidic/alkali treatment steps. The assay was successfully miniaturized to search for malate kinase activity in a structurally-guided library of LysC aspartate kinase variants comprising 6,700 clones. The screening procedure enabled the isolation of nine positive variants showing novel kinase activity on (L)-malate, the best mutant, LysC V115A:E119S:E434V exhibited strong substrate selectivity for (L)-malate compared to (L)-aspartate with a (kcat/Km)malate/(kcat/Km)aspartate ratio of 86. Double mutants V115A:E119S, V115A:E119C and E119S:E434V were constructed to further probe the origins of stabilising substrate binding energy gains for (L)-malate due to mutation. The introduction of less sterically hindering side-chains in engineered enzymes carrying E119S and V115A mutations increases the effective volume available for substrate binding in the catalytic pocket. Improved binding of the (L)-malate substrate may be assisted by less hindered movement of the Phe184 aromatic side-chain. Additional favourable long-range electostatic effects on binding arising from the E434V surface mutation are conditionally dependent upon the presence of the V115A mutation close to Phe184 in the active-site.

2,4-dihydroxybutyrate (DHB) is a precursor for the chemical synthesis of the methionine analogue 2-hydroxy-4-(methylthio)butyrate. Since no annotated metabolic pathway exists for its microbial production from sugar, we have conceived a two-step synthetic metabolic pathway which converts the natural amino acid homoserine to DHB. The pathway proceeds through the homoserine transaminase-catalyzed deamination of homoserine to obtain 2-oxo-4-hydroxybutyrate (OHB), and continues with the reduction of OHB to DHB, which is catalyzed by an OHB reductase enzyme. We identified homoserine transaminase and OHB reductase activity in several candidate enzymes which act on sterically cognate substrates, and improved OHB reductase activity of lactate dehydrogenase A of Lactococcus lactis by structure-based enzyme engineering. Fed-batch cultivation of a homoserine-overproducing Escherichia coli strain which expressed homoserine transaminase and OHB reductase enzymes resulted in the production of 5.3g/L DHB at a yield of 0.1g/g., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

In this study, a total of 30 yeast strains belonging to the genera Dipodascus, Galactomyces, Geotrichum, Magnusiomyces and Saprochaete were investigated for volatile organic compound production using HS-SPME-GC/MS analysis. The resulting flavour profiles, including 36 esters and 6 alcohols compounds, were statistically evaluated by cluster and PCA analysis. Two main groups of strains were extracted from this analysis, namely a group with a low ability to produce flavour and a group producing mainly alcohols. Two other minor groups of strains including Saprochaete suaveolens, Geotrichum marinum and Saprochaete gigas were diverging significantly from the main groups precisely because they showed a good ability to produce a large diversity of esters. In particular, we found that the Saprochaete genus (and their closed relatives) was characterized by a high production of unsaturated esters arising from partial catabolism of branched chain amino-acids. These esters were produced by eight phylogenetically related strains of Saprochaete genus., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The yeast cell is surrounded by a cell wall conferring protection and resistance to environmental conditions that can be harmful. Identify the molecular cues (genes) which shape the biochemical composition and the nanomechanical properties of the cell wall and the links between these two parameters represent a major issue in the understanding of the biogenesis and the molecular assembly of this essential cellular structure, which may have consequences in diverse biotechnological applications. We addressed this question in two ways. Firstly, we compared the biochemical and biophysical properties using atomic force microscopy (AFM) methods of 4 industrial strains with the laboratory sequenced strain BY4743 and used transcriptome data of these strains to infer biological hypothesis about differences of these properties between strains. This comparative approach showed a 4-6-fold higher hydrophobicity of industrial strains that was correlated to higher expression of genes encoding adhesin and adhesin-like proteins and not to their higher mannans content. The second approach was to employ a multivariate statistical analysis to identify highly correlated variables among biochemical, biophysical and genes expression data. Accordingly, we found a tight association between hydrophobicity and adhesion events that positively correlated with a set of 22 genes in which the main enriched GO function was the sterol metabolic process. We also identified a strong association of β-1,3-glucans with contour length that corresponds to the extension of mannans chains upon pulling the mannosyl units with the lectin-coated AFM tips. This association was positively correlated with a group of 27 genes in which the seripauperin multigene family was highly documented and negatively connected with a set of 23 genes whose main GO biological process was sulfur assimilation/cysteine biosynthetic process. On the other hand, the elasticity modulus was found weakly associated with levels of β-1,6-glucans, and this biophysical variable was positively correlated with a set of genes implicated in microtubules polymerization, tubulin folding and mitotic organization.

Microbial ethanol production is an important alternative energy resource to replace fossil fuels, but at high level, this product is highly toxic, which hampers its efficient production. Towards increasing ethanol-tolerance of Saccharomyces cerevisiae, the so far best industrial ethanol-producer, we evaluated an in vivo evolutionary engineering strategy based on batch selection under both constant (5%, v v -1 ) and gradually increasing (5-11.4%, v v -1 ) ethanol concentrations. Selection under increasing ethanol levels yielded evolved clones that could tolerate up to 12% (v v -1 ) ethanol and had cross-resistance to other stresses. Quite surprisingly, diploidization of the yeast population took place already at 7% (v v -1 ) ethanol level during evolutionary engineering, and this event was abolished by the loss of MKT1, a gene previously identified as being implicated in ethanol tolerance (Swinnen et al., Genome Res., 22, 975-984, 2012). Transcriptomic analysis confirmed diploidization of the evolved clones with strong down-regulation in mating process, and in several haploid-specific genes. We selected two clones exhibiting the highest viability on 12% ethanol, and found productivity and titer of ethanol significantly higher than those of the reference strain under aerated fed-batch cultivation conditions. This higher fermentation performance could be related with a higher abundance of glycolytic and ribosomal proteins and with a relatively lower respiratory capacity of the evolved strain, as revealed by a comparative transcriptomic and proteomic analysis between the evolved and the reference strains. Altogether, these results emphasize the efficiency of the in vivo evolutionary engineering strategy for improving ethanol tolerance, and the link between ethanol tolerance and diploidization., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

2,4-Dihydroxybutyric acid (DHB) is a molecule with considerable potential as a versatile chemical synthon. Notably, it may serve as a precursor for chemical synthesis of the methionine analogue 2-hydroxy-4-(methylthio)butyrate, thus, targeting a considerable market in animal nutrition. However, no natural metabolic pathway exists for the biosynthesis of DHB. Here we have therefore conceived a three-step metabolic pathway for the synthesis of DHB starting from the natural metabolite malate. The pathway employs previously unreported malate kinase, malate semialdehyde dehydrogenase and malate semialdehyde reductase activities. The kinase and semialdehyde dehydrogenase activities were obtained by rational design based on structural and mechanistic knowledge of candidate enzymes acting on sterically cognate substrates. Malate semialdehyde reductase activity was identified from an initial screening of several natural enzymes, and was further improved by rational design. The pathway was expressed in a minimally engineered Escherichia coli strain and produces 1.8 g l -1 DHB with a molar yield of 0.15.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of Marie-Ange Teste, Isabelle Léger-Silvestre, Jean M François and Jean-Luc Parrou. Marjorie Petitjean could not be reached. The corresponding author identified major issues and brought them to the attention of the Journal. These issues span from significant errors in the Material and Methods section of the article and major flaws in cytometry data analysis to data fabrication on the part of one of the authors. Given these errors, the retracting authors state that the only responsible course of action would be to retract the article, to respect scientific integrity and maintain the standards and rigor of literature from the retracting authors' group as well as the Journal. The retracting authors sincerely apologize to the readers and editors., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)