Applied Microbiology and Biotechnology

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/666879v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@9139c3org.highwire.dtl.DTLVardef@673bc1org.highwire.dtl.DTLVardef@1842210org.highwire.dtl.DTLVardef@1d86903_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG Therapeutic proteins are produced frequently by mammalian cells in large-scale bioreactors. As a result, producer cells are exposed to a chemically (nutrients, gas exchange, target protein overexpression) and physically (shear due to mixing) stressful environment, which can lead to loss of proteostasis and endoplasmic reticulum (ER) stress. In response, cells activate the unfolded protein response (UPR). The UPR includes activation of autophagy and proteasomes, both of which target unfolded/misfolded proteins for degradation. To investigate the impacts of autophagy and proteasome activity on secreted protein production in ER-stressed cells, we used HeLa and MDA-MB-231 cells transfected to express Gaussia luciferase (as a model for therapeutic protein production) and exposed to tunicamycin (TM) (to activate ER stress). As expected, TM exposure decreased protein production and secretion. Inhibiting autophagy improved secretion in stressed cells as expected. However, counterintuitively, increasing proteasomal degradation improved secretion while inhibiting proteasomal activity decreased secretion, that is proteasomal activity was directly correlated to secretion. Taken together, our results demonstrate that protein secretion can be improved through control of autophagy and proteasomal activity, providing insight into strategies for improving yield from protein production bioprocesses. Key PointsO_LITunicamycin induced ER stress reduced protein production. C_LIO_LIAutophagy inhibition improved secretion in ER stressed cells. C_LIO_LIActivation of proteasomal degradation improved secretion in ER stressed cells. C_LI

There is an ever-increasing demand for reduction of unit operations and a growing interest in the physiology of yeasts used in beer fermentation. In this context, cell immobilization is an interesting alternative, since it reduces steps to separate biomass from fermented broth. Yet, physiological alterations in yeast metabolism caused by immobilization are still to be fully described. Thus, the main objective of this work was to evaluate the physiology of three brewers S. cerevisiae yeast strains (SY025, SY067 and SY001) immobilized on a porous cellulose-based support. Batch fermentations in malt extract 12 {degrees}P were carried out for all strains both in free and immobilized forms in order to compare kinetic parameters obtained from distinct process conditions. Mathematical modeling was performed following two viewpoints: modeling of fermentation kinetics by parameter estimation from experimental data and application of a reaction-diffusion model for estimation of substrate concentration gradient inside the immobilization support. Moreover, fermentations with different initial substrate and biomass concentrations were carried out using strain SY025, aiming to evaluate their influence over flavor compounds, using statistical models. Compared to free cells, immobilized yeasts showed both higher glycerol yield (SY025, 40%; SY067, 53%; SY001, 19%) and biomass yield in the system (SY025, 67%; SY067, 78%; SY001, 56%). On the other hand, free cells presented higher ethanol yields when compared to immobilized ones (SY025, 9%; SY067, 9%; and SY001, 13%). According to the model developed, a substrate gradient inside the support was predicted, but with low mass transfer limitations. KEY POINTSO_LIYeast immobilization not always hinder biomass growth, here it was stimulated. C_LIO_LIA classic kinetic model describes accurately immobilized yeast fermentations. C_LIO_LIPhysiology changes occur in immobilization even with low mass transfer limitations. C_LI

Both hexose- and pentose-fermenting yeasts are commercially available. The aim of this study was to test several yeast strains for their ability to ferment lignocellulosic feedstock and to evaluate their usability in a bioethanol production process based on the Cellunolix(R) concept. The Cellunolix(R) bioethanol demonstration plant of St1 uses sawdust to produce second-generation lignocellulosic ethanol. The study was performed in collaboration with yeast providers using two types of pretreated and filtered lignocellulosic ethanol hydrolysate originating from pine and willow. Ten pentose- and three hexose-fermenting yeast strains were tested. They all performed well under industrial conditions but differed in the rate of detoxification and profile for utilization of different sugars. A satisfactory 82-97% fermentation yield of a multi-sugar hydrolysate containing glucose, mannose, galactose, arabinose and xylose was achieved within 48 h. The results indicate significant potential for the usability of pentose-fermenting strains with real industrial hydrolysates and settings.

Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) and other microorganisms have attracted considerable interest due to their structural diversity and physicochemical properties, which makes them valuable across various industrial applications. To achieve high cell densities and maximize EPS yields, microorganisms are typically cultivated in nutrient-rich media containing yeast extract. However, yeast extract may contain high molecular weight polysaccharides that are not metabolized by the bacteria. This can lead to an overestimation of EPS yields and contamination of the bacterial EPS, potentially resulting in misinterpretation of their structure and biological activity. In this study, we demonstrate the presence of high molecular weight -mannan and {beta}-glucan in yeast extract in EPS isolates using both ultrafiltration and the commonly used trichloroacetic acid/ethanol (TCA/EtOH) precipitation method. These polysaccharides were characterized by size-exclusion chromatography, high-performance anion-exchange chromatography, and nuclear magnetic resonance spectroscopy. Their abundances were estimated to range from 10 to 50 mg/L in MRS medium, depending on the supplier of the yeast extract. The main contaminant identified was yeast -mannan. By cultivating L. rhamnosus GG (ATCC 53103) and L. pentosus KW1 and isolating their respective EPS, we illustrate how these yeast extract contaminants affect the structural interpretation of the EPS and that the contaminants can be completely removed by ultrafiltration of the growth medium prior to bacterial cultivation. In conclusion, we emphasize the necessity of stringent controls in the production and purification of microbial EPS, with particular attention to the chemical purity of medium constituents.

Norwegian kveik are a recently described family of domesticated Saccharomyces cerevisiae brewing yeasts used by farmhouse brewers in western Norway for generations to produce traditional Norwegian farmhouse ale. Kveik ale yeasts have been domesticated by farmhouse brewers through serial repitching of the yeast in warm wort (>30{degrees}C) punctuated by long periods of dry storage. Kveik yeasts are alcohol tolerant, flocculant, capable of utilizing maltose/maltotriose, phenolic off flavour negative, and exhibit elevated thermotolerance when compared to other modern brewers yeasts belonging to the Beer 1 clade. However, the optimal fermentation and growth temperatures (Topt) for kveik ale yeasts and the influence of fermentation temperature of the production of flavour-active metabolites like fusel alcohols and sulfur compounds (H2S, SO2) are not known. Here we show that kveik ale yeasts have an elevated optimal fermentation temperature (Topt) when compared to commercial American Ale yeast (SafAle US-05) and that they produce fewer off-flavours at high temperatures (>30{degrees}C) when compared to commercial American Ale yeasts. The tested kveik yeasts show significantly higher maximum fermentation rates than American Ale yeast not only at elevated temperatures (>30{degrees}C), but also at typical ale fermentation temperatures (20{degrees}C-25{degrees}C). Finally, we demonstrate that kveik ale yeasts are heterogeneous in their Topt and that they attenuate standard wort robustly above their Topt unlike our control American Ale yeast which showed very poor apparent attenuation in our standard wort at temperatures >> Topt. Our results provide further support that kveik yeasts may possess favourable fermentation kinetics and sensory properties compared to American Ale yeasts. The observations here provide a roadmap for brewers to fine tune their commercial fermentations using kveik ale yeasts for optimal performance and/or flavour impact.

Background3-hydroxypropionic acid (3-HP) and succinic acid (SA) were announced as two of the top twelve value-added platform chemicals from biomass out of a group of over 300 potential compounds that could be made from biomass in a government report in 2004 (Werpy and Petersen, 2004) and in an updated report in 2010 (Bozell and Petersen, 2010). The screening criteria used in the report classified 3-HP and SA as direct petroleum replacement building block chemicals. 3-HP is a precursor to several high-value compounds, such as acrylic acid, 1,3-propanediol, acrylamide, and methyl acrylates, that ultimately end up in products such as fibers, contact lenses, diapers, fabric coatings, and other super absorbent polymers (SAPs). SA is a high-value platform chemical used in polyester production and a precursor for nylon and other bioplastics. Additionally, these reports identified pathways to building block compounds from sugars. Yeast fermentations were identified in these reports as a preferred potential pathway to 3-HP and SA production from sugars because of yeasts natural low pH tolerance. ResultsThe laboratory strain Saccharomyces cerevisiae BY4741 was engineered to produce either 3-HP or SA. These yeasts can convert fermentable sugars from glucose-rich lignocellulosic hardwood feedstocks into organic acid products such as 3-HP and SA under low pH conditions using exponential fed-batch cultivation strategies. Glucose-rich wood sugars provided a better growth environment for the engineered yeast strains, increasing production titers by 6.1 and 6.5 times for SA and 3-HP, respectively. ConclusionsThis study shows the potential of locally produced glucose-rich wood sugars to increase the production of platform chemicals necessary in the production of biobased polymers by engineered yeast cell factories.

Carboxylates like volatile fatty acids (VFAs) can be produced by acidogenic fermentation (AF) of dairy wastes like cheese whey, a massive residue produced at 160.67 million m3 of which 42% are not valorized and impact the environment. In mixed-culture fermentations, selection pressures are needed to favor AF and halt methanogenesis. Inoculum pre-treatment was studied here as selective pressure for AF demineralized cheese whey in batch processes. Alkaline (NaOH, pH 8.0, 6 h) and thermal (90{degrees}C for 5 min, ice-bath until 23{degrees}C) pre-treatments, were tested together with batch operations run at initial pH 7.0 and 9.0, food-to-microorganism (F/M) ratios of 0.5 to 4.0 g COD g-1 VS, and under pressurized and non-pressurized headspace, in experiments duplicated in two institutes. Acetic acid was highly produced (1.36 and 1.40 g CODAcOH L-1) at the expense of methanogenesis by combining a thermal pre-treatment of inoculum with a non-pressurized batch operation started at pH 9.0. Microbial communities comprised of VFAs and alcohol producers, such as Clostridium, Fonticella, and Intestinimonas, and fermenters such as Longilinea and Leptolinea. Communities also presented the lipid-accumulating and bulk and foaming Candidatus Microthrix and the metanogenic Methanosaeta regardless of no methane production. An F/M ratio of 0.5 g COD g-1 VS led to the best VFA production of 1,769.38 mg L-1. Overall, inoculum thermal pre-treatment, initial pH 9.0, and non-pressurized headspace acted as a selective pressure for halting methanogen and producing VFAs, valorizing cheese whey via batch acidogenic fermentation.

BackgroundYeast research in the context of food/beverage production and industrial biotechnology faces a dilemma: to use real industrial media or to use fully defined laboratory media? While the former option might lead to experiments closer to industrial conditions, the latter has the advantage of allowing for reproducibility and comparability of results among different laboratories, as well as being suitable for the investigation of how different individual components affect microbial or process performance. It is undoubtable that the development of a synthetic must a few decades ago led to important advances in wine yeast research. ResultsWe developed a fully defined medium that mimics sugarcane molasses, a frequently used medium in different industrial processes where yeast is cultivated. The medium, named 2SMol, builds upon a previously published semi-defined formulation and is conveniently prepared from some stock solutions: C-source, organic N, inorganic N, organic acids, trace elements, vitamins, Mg+K, and Ca. We validated the 2SMol recipe in a scaled-down sugarcane biorefinery model, comparing the performance of different yeast strains in different real molasses-based media. We also showcase the flexibility of the medium by investigating the effect of nitrogen availability on the ethanol yield during fermentation. ConclusionsHere we present in detail the development of a fully defined synthetic molasses medium, and we hope the 2SMol formulation will be valuable to researchers both in academia and industry to obtain new insights and developments in industrial yeast biotechnology.

SUMMARY/ ABSTRACTHigh societies consumption, elevated residues generation and environmental awareness strengthen alternatives solutions for bioprocess residues. This study investigated the production of volatile acids from a complex substrate, which intends to be replaceable in the future by vinasse of sugar cane, in anaerobic reactors operated in triplicates at 35 {degrees}C. Two different inoculum were studied: Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii ATCC 25752. The nutrient medium had as carbon source a complex substrate containing sucrose without addition of vitamins, buffer solution and micronutrients. The experiment was conducted in the variation of F/M-ratio (food-to-microorganisms) by increasing substrate concentration. The concentration of sucrose in the complex substrate were 5.2 g{middle dot}L-1 (conditions of 10,000 mg O2{middle dot} L-1 in terms of COD) and 10.5 g{middle dot} L-1 (conditions of 20,000 mg O2{middle dot}L-1 in terms of COD), keeping the initial concentration of inoculum in 500 mg SVT{middle dot}L-1. Cultures C. acetobutylicum and C. beijerinckii resulted in high lactic acid production. Concentrations of COD of 10,000 mg O2{middle dot} L-1 produced optimum lactic acid of 3,331 mg{middle dot}L-1 and 5,709 mg{middle dot}L-1 with respectively C. acetobutylicum and C. beijerinckii. Moreover, cultures C. acetobutylicum and C. beijerinckii with 20,000 mg O2{middle dot} L-1 concentrations in terms of COD produced optimum lactic acid of 6,417 mg{middle dot} L-1 and 7.136 mg{middle dot}L-1 respectively. There was repeatability in the reactors when considering level of significance of 0.05, independent of the concentration and inoculum used.

Developing intranasal vaccines against pandemics and devastating airborne infectious diseases is imperative. The superiority of intranasal vaccines over injectable systemic vaccines is evident, but the challenge in developing effective intranasal vaccines is more substantial. Fusing a protein antigen with the catalytic domain of cholera toxin (CTA1) and the two-domain D of staphylococcal protein A (DD) has significant potential for intranasal vaccines. In the present study, we constructed two fusion proteins containing CTA1, tandem repeat linear epitopes of the SARS-CoV-2 spike protein (S14P5 or S21P2), and DD. The in silico characteristics and solubility of the fusion proteins CTA1-(S14P5)4-DD and CTA1-(S21P2)4-DD were analyzed when overexpressed in Escherichia coli. Structural predictions indicated that each component of the fusion proteins was compatible with its origin. Both fusion proteins were predicted by computational tools to be soluble when overexpressed in E. coli. Contrary to these predictions, the constructs exhibited limited solubility. The solubility did not improve even after lowering the cultivation temperature from 37{degrees}C to 18{degrees}C. Induction with IPTG at the early log phase, instead of the usual mid-log phase growth, significantly increased soluble CTA1-(S21P2)4-DD but not CTA1-(S14P5)4-DD. The solubility of overexpressed fusion proteins significantly increased when a non-denaturing detergent (Nonidet P40, Triton X100, or Tween 20) was added to the extraction buffer. In a scale-up purification experiment, the yields were low, only 1-2 mg/L of culture, due to substantial losses during the purification stages, indicating the need for further optimization of the purification process.

AO_SCPLOWBSTRACTC_SCPLOWSimultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers an advance towards more cost-effective second-generation (2G) ethanol production. As xylan is one of the most abundant polysaccharides present in lignocellulosic residues, the transport and breakdown of XOS in an intracellular environment might bring a competitive advantage for recombinant strains in competition with contaminating microbes, which are always present in fermentation tanks; furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing NADPH-linked xylose reductase (XR), NAD+-linked xylitol dehydrogenase (XDH) (for xylose assimilation), as well as NADH-linked acetylating acetaldehyde dehydrogenase (AADH) and acetyl-CoA synthetase (ACS) (for an NADH-dependent acetate reduction pathway), was used as the host for expressing of two {beta}-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered strain SR8A6S3-CDT2-GH432/7. Both {beta}-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, which catalyse steps in xylitol production. Xylitol accumulation during xylose fermentation is a problem for 2G ethanol production since it reduces final ethanol yield. The engineered strain, SR8A6S3-CDT2-GH432/7, produced ethanol through simultaneous co-utilization of XOS, xylose, and acetate. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan compared with the parental strain. The consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production. HighlightsO_LIIntegration of XOS pathway in an acetate-xylose-consuming S. cerevisiae strain; C_LIO_LIIntracellular fermentation of XOS, acetate and xylose improved ethanol production; C_LIO_LIDeletion of both sor1{Delta} and gre3{Delta} reduced xylitol production. C_LI

BACKGROUNDSugarcane hemicellulosic material is a compelling source of usually neglected xylose that could figure as feedstock to produce chemical building blocks of high economic value, such as xylitol. In this context, Saccharomyces cerevisiae strains typically used in the Brazilian bioethanol industry are a robust chassis for genetic engineering, given their robustness towards harsh operational conditions and outstanding fermentation performance. Nevertheless, there are no reports on the use of these strains for xylitol production using sugarcane hydrolysate. RESULTSPotential single-guided RNA off-targets were analyzed in two preeminent industrial strains (PE-2 and SA-1), providing a database of 5-NGG 20 nt sequences, and guidelines for the fast and cost-effective CRISPR-editing of such strains. After genomic integration of a NADPH-preferring xylose reductase (XR), FMYX (SA-1 ho{Delta}::xyl1) and CENPKX (CEN.PK-122 ho{Delta}::xyl1) were tested in varying cultivation conditions for xylitol productivity to infer influence of the genetic background. Near-theoretical yields were achieved for all strains, however the industrial consistently outperformed the laboratory strain. Batch fermentation of raw sugarcane bagasse hydrolysate with remaining solid particles represented a challenge for xylose metabolization and 3.65 {+/-} 0.16 g/L xylitol titre was achieved by FMYX. Finally, quantification of NADPH - cofactor implied in XR activity - revealed that FMYX has 33% more available cofactors than CENPKX. CONCLUSIONSAlthough widely used in several S. cerevisiae strains, this is the first report of CRISPR-Cas9 editing major yeast of the Brazilian bioethanol industry. Fermentative assays of xylose consumption revealed that NADPH availability is closely related to mutant strains performance. We also pioneer the use of sugarcane bagasse as a substrate for xylitol production. Finally, we demonstrate how industrial background SA-1 is a compelling chassis for the second-generation industry, given its inhibitor tolerance and better redox environment that may favor production of reduced sugars.

The primary challenge in utilizing palm kernel meal (PKM, an agricultural by-product) as non- ruminant livestock feed is its high fibre content, predominantly in the form of mannan. Microbial fermentation offers an economically favourable alternative to enzyme supplementation for breaking down fibre in lignocellulosic biomass. In a recent study, we have isolated and characterized an undomesticated strain (Bacillus subtilis F6) that is able to secrete mannanase. In this work, the mannanase production was substantially improved by optimizing multiple regulatory elements controlling the mannanase expression. Mannanase GmuG, sourced from B. subtilis F6 and verified for its hydrolytic activity on PKM fibre, was expressed using a replicative plasmid (pBE-S). The recombinant strain of B. subtilis F6 exhibited 1.9-fold increase in the mannanase activity during solid-state fermentation. Optimization of signal peptide and ribosome binding site further enhanced mannanase activity by 3.1-fold. Subsequently, promoter screening based on highly transcribed genes in B. subtilis F6 resulted in a significant 5.4-fold improvement in mannanase activity under the nprE promoter. The nprE promoter was further refined by eliminating specific transcription factor binding sites, enhancing the mannanase activity further by 1.8-fold. Notably, a substantial 35-40% reduction in PKM fibre content was observed after 30 h of fermentation using the recombinant strains. Lastly, the highest mannanase-producing strain was examined for scaled-up fermentation. The impacts of fermentation on fibre and protein contents, as well as the surface morphology of PKM, were analysed. The outcomes of this study offer an efficient method for robust mannanase expression in B. subtilis and its potential application in the biotransformation of PKM and other mannan-rich bioresources for improved feed utilization. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/602432v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@10fbb9corg.highwire.dtl.DTLVardef@1e619fborg.highwire.dtl.DTLVardef@1b3bc0corg.highwire.dtl.DTLVardef@fec816_HPS_FORMAT_FIGEXP M_FIG C_FIG

Prior to the introduction of novel food ingredients into the food supply, safety risk assessments are required, and numerous prediction models have been developed and validated to evaluate safety. The allergenic risk potential of Helaina recombinant human lactoferrin (rhLF, Effera), produced in Komagataella phaffii (K. phaffii) was assessed by literature search, bioinformatics sequence comparisons to known allergens, glycan allergenicity assessment, and a simulated pepsin digestion model. The literature search identified no allergenic risk for Helaina rhLF, K. phaffii, or its glycans. Bioinformatics search strategies showed no significant risk for cross-reactivity or allergenicity between rhLF or the 36 residual host proteins and known human allergens. Helaina rhLF was also rapidly digested in simulated gastric fluid and its digestibility profile was comparable to human milk lactoferrin (hmLF), further demonstrating a low allergenic risk and similarity to the hmLF protein. Collectively, these results demonstrate a low allergenic risk potential of Helaina rhLF and do not indicate the need for further clinical testing or serum IgE binding to evaluate Helaina rhLF for risk of food allergy prior to introduction into the food supply.

Municipal solid waste (MSW) represents tonnes of material that, for the most part, is relegated to landfill. Synthetic biology proposes solutions to many of the challenges faced by humanity today, but many approaches are confined to use in classical chassis organisms. In MSW there are a variety of potentially toxic materials such as glues, dyes, and preservatives that could pose a challenge to its capitalisation when using these commonplace chassis. We have isolated a bank of strains that utilise paper and cardboard waste from a relevant waste environment. From these we have identified three strains that are capable of utilising cellulose as a sole carbon source. We have analysed how they utilise cellulose and hemicelluloses, both alone and in coculture. This revealed insights to how they might be used in synthetic consortia which were then produced under laboratory conditions. Production of complete genome sequences of these strains provides genetic insight to how these processes may be occurring at the metabolic level, and how they could be augmented using synthetic biology. To this end, we have produced protocols for transforming plasmids into these strains and have produced high value metabolites from this material. HighlightsO_LIFully annotated genomes were produced from novel mesophilic aerobic strains isolated from lignocellulosic solid waste C_LIO_LILycopene was produced directly from relevant solid waste substrates by genetically modified variants of these strains C_LIO_LIOptimised carbon source blends influence coculture compositions of specific strains C_LI

A plethora of studies have focused on improvements of xylitol production. The challenges of establishing a biotechnological route for the industrial production of this sugar have been explored using different microorganisms and renewable feedstock. Nevertheless, sugarcane biomass has been neglected as the pentose source for xylitol production using Saccharomyces cerevisiae. Therefore, here we investigate the use of an industrial S. cerevisiae strain for xylitol production in batch fermentation of non-detoxified sugarcane straw hydrolysate, envisioning the diversification of the current infrastructure used for second-generation bioethanol production from the same lignocellulosic material. In order to optimize the xylose conversion in a non-fed cultivation system, guidelines in cell inoculum and medium supplementation are suggested, as well as the first attempt to use electro-fermentation for this purpose. Accordingly, our results show that the increase in initial cell density and hydrolysate supplementation allows a xylitol production of 19.24 {+/-} 0.68 g/L, representing 0,132 g/L.h productivity.

The yeasts, Saccharomyces pastorianus, are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus and have acquired traits from the combined parental genomes such as ability to ferment a range of sugars at low temperatures and to produce aromatic flavour compounds, allowing for the production of lager beers with crisp, clean flavours. The polyploid strains are sterile and have reached an evolutionary bottleneck for genetic variation. Here we describe an accelerated evolution approach to obtain lager yeasts with enhanced flavour profiles. As the relative expression of orthologous alleles is a significant contributor to the transcriptome during fermentation, we aimed to induce genetic variation by altering the S. cerevisiae to S. eubayanus chromosome ratio. Aneuploidy was induced through the temporary inhibition of the cells stress response and strains with increased production of aromatic amino acids via the Shikimate pathway were selected by resistance to amino acid analogues. Genomic changes such as gross chromosomal rearrangements, chromosome loss and chromosome gain were detected in the characterised mutants, as were Single Nucleotide Polymorphisms in ARO4, encoding for DAHP synthase, the catalytic enzyme in the first step of the Shikimate pathway. Transcriptome analysis confirmed the upregulation of genes encoding enzymes in the Ehrlich pathway and the concomitant increase in the production of higher alcohols and esters such as 2-phenylethanol, 2-phenylethyl acetate, tryptophol, and tyrosol. We propose that the plasticity of polyploid S. pastorianus genomes is an advantageous trait supporting opportunities for genetic diversity in otherwise sterile strains. Significance StatementLager beer is the product of fermentations conducted with Saccharomyces pastorianus, which are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus. A quintessential property of lager beers is the distinctive flavours produced during fermentation. Hybrids are sterile and have reached an evolutionary bottleneck. Finding ways to introduce genetic variation as a means of enhancing the flavour profiles is a challenge. Here, we describe an approach to introduce genetic variation by inducing aneuploidy through the temporary inhibition of the cells stress response. Strains with an enhanced flavour production were selected by resistance to amino acid analogues. We identified genomic changes and transcriptome analysis confirmed the upregulation of genes in the Ehrlich pathway which is responsible for the production of flavour compounds.

Lignocellulosic biomass (LCB) captures a major fraction of agro-industrial wastes that are mostly valorised for the production of second-generation biofuels. The extensive pre-treatment followed by saccharification of LCB restricts its usability for the production of a wide array of bioproducts. This study highlights the performance comparison of sono-assisted alkaline pre-treatment versus microbial pre-treatment of sugarcane bagasse by a no. of analytical techniques such as FTIR, XRD, CHNS, and SEM. Moreover, simultaneous delignification, saccharification and fermentation (SDSF) in one pot is highly desirable for the cost effective and environment friendly production of microbial products. In the present study SDSF was carried out by a cellulolytic bacterium Cellulomonas flavigena, for the production of extracellular polymeric substance (EPS), which is, by chemical nature a biopolymer made up of carbohydrate and protein subunits. The biopolymer was found to have an overall positive charge as analysed by ion exchange chromatography. Two major fractions of molecular weight 237 KDa and 29 KDa were obtained from gel filtration chromatography, in addition to that, the EPS was found to be composed of monosaccharides, D (+) mannosamine and D (+) xylose as identified from high pressure liquid chromatography (HPLC).

Polyethylene glycol is commonly used in fermentation as an anti-foam for preventing the rise of foam to the top plate of the bioreactor, which increases contamination risk. However, its potential toxicity to growth of various microorganisms is not well understood at the species and strain level. Hence, the objective of this study was to understand the impact of different concentrations of polyethylene glycol at the 1, 5 and 10 g/L level on the aerobic growth of Escherichia coli DH5 and Bacillus subtilis NRS-762 in LB Lennox medium in shake flasks. Experiment results revealed that polyethylene glycol (PEG) (molecular weight [~]8000 Da), at all concentrations tested, did not affect biomass formation and metabolism in E. coli DH5 at 37 {degrees}C. This came about through the observation of similar maximal optical density obtained during growth of E. coli DH5 under differing concentrations of PEG. Furthermore, the anti-foam did not affect the pH profile. On the other hand, PEG did exhibit some toxicity towards the growth of B. subtilis NRS-762 in LB Lennox medium. Specifically, maximal optical density obtained decline with higher exposure to PEG in a concentration dependent manner, up to a threshold concentration of 5 g/L. For example, maximal optical density obtained in B. subtilis NRS-762 without addition of PEG was 4.4, but the value obtained with 1 g/L of the anti-foam decreased to 4.1 and a further 3.8 on exposure to 5 g/L and 10 g/L PEG. pH variation in culture broth, however, told a different story, where the profiles for exposure to PEG at all concentrations coincide with each other and was similar to the one without exposure to the anti-foam; thereby, suggesting that metabolic processes in B. subtilis NRS-762 were not significantly affected by exposure to PEG. Collectively, PEG anti-foam exerted species-specific toxicity effect on biomass formation, and possibly metabolism. The latter may not be sufficiently significant to affect the types of metabolites secreted by the bacterium, and thus, be detected by measurement of pH of culture broth. E. coli DH5 was better able to cope with PEG at all concentrations compared to B. subtilis NRS-762, which showed dose-dependent toxicity effect on biomass formation. HighlightsO_LIPolyethylene glycol (molecular weight [~] 8000 Da) did not affect aerobic growth of Escherichia coli DH5 at 37 {degrees}C in LB medium at all concentrations tested: 0, 1, 5, 10 g/L. C_LIO_LIGrowth curves of the bacterium at different concentrations of polyethylene glycol (PEG) coincided with each other. C_LIO_LISimilar pH profiles were also obtained for E. coli DH5 growth in LB medium with different PEG concentrations. C_LIO_LIHowever, PEG exerted toxicity effect on Bacillus subtilis NRS-762 during growth in LB medium at 30 {degrees}C, with reduction of biomass formation in a dose dependent manner. C_LIO_LISimilar to the case for E. coli DH5. pH profiles of B. subtilis NRS-762 coincided with each other irrespective of the concentrations of PEG used. C_LI

Debaryomyces hansenii is a yeast with considerable biotechnological potential as an osmotolerant, stress tolerant oleaginous microbe. However, targeted genome modification tools are limited and require a strain with auxotrophic markers. Gene targeting by homologous recombination has been reported to be inefficient but here we describe a set of reagents and a method that allows gene targeting at high efficiency in wild type isolates. It uses a simple PCR-based amplification that extends a completely heterologous selectable marker with 50 bp flanks identical to the target site in the genome. Transformants integrate the PCR product through homologous recombination at high frequency (>75%). We illustrate the potential of this method by disrupting genes at high efficiency and by expressing a heterologous protein from a safe chromosomal harbour site. These methods should stimulate and facilitate further analysis of D. hansenii strains and open the way to engineer strains for biotechnology.