Academic literature on the topic 'Increased fermentation fitness'

ABSTRACTWith a completely reengineered and humanized glycosylation pathway, glycoengineeredPichia pastorishas emerged as a promising production host for the manufacture of therapeutic glycoproteins. However, the extensive genetic modifications have also negatively affected the overall fitness levels of the glycoengineered host cells. To make glycoengineeredPichiastrains more compatible with a scalable industrial fermentation process, we sought to identify genetic solutions to broadly improve cell robustness during fermentation. In this study, we report that mutations within thePichia pastorisATT1(PpATT1) gene (a homolog of theSaccharomyces cerevisiaeGAL4[ScGAL4] transcriptional activator) dramatically increased the cellular fitness levels of glycoengineeredPichiastrains. We demonstrate that deletion of thePpATT1gene enabled glycoengineeredPichiastrains to improve their thermal tolerance levels, reduce their cell lysis defects, and greatly improve fermentation robustness. The extension of the duration of fermentation enabled thePpATT1-modified glycoengineeredPichiastrains to increase their product yields significantly without any sacrifice in product quality. Because theATT1gene could be deleted from anyPichiastrains, including empty hosts and protein-expressing production strains alike, we suggest that the findings described in this study are broadly applicable to anyPichiastrains used for the production of therapeutic proteins, including monoclonal antibodies, Fc fusions, peptides, hormones, and growth factors.

BackgroundMany bacteria are facultative anaerobes, and can proliferate in both anoxic and oxic environments. Under anaerobic conditions, fermentation is the primary means of energy generation in contrast to respiration. Furthermore, the rates and spectra of spontaneous mutations that arise during anaerobic growth differ to those under aerobic growth. A long-term selection experiment was undertaken to investigate the genetic changes that underpin how the facultative anaerobe,Escherichia coli, adapts to anaerobic environments.MethodsTwenty-one populations ofE. coliREL4536, an aerobically evolved 10,000th generation descendent of theE. coliB strain, REL606, were established from a clonal ancestral culture. These were serially sub-cultured for 2,000 generations in a defined minimal glucose medium in strict aerobic and strict anaerobic environments, as well as in a treatment that fluctuated between the two environments. The competitive fitness of the evolving lineages was assessed at approximately 0, 1,000 and 2,000 generations, in both the environment of selection and the alternative environment. Whole genome re-sequencing was performed on random colonies from all lineages after 2,000-generations. Mutations were identified relative to the ancestral genome, and based on the extent of parallelism, traits that were likely to have contributed towards adaptation were inferred.ResultsThere were increases in fitness relative to the ancestor among anaerobically evolved lineages when tested in the anaerobic environment, but no increases were found in the aerobic environment. For lineages that had evolved under the fluctuating regime, relative fitness increased significantly in the anaerobic environment, but did not increase in the aerobic environment. The aerobically-evolved lineages did not increase in fitness when tested in either the aerobic or anaerobic environments. The strictly anaerobic lineages adapted more rapidly to the anaerobic environment than did the fluctuating lineages. Two main strategies appeared to predominate during adaptation to the anaerobic environment: modification of energy generation pathways, and inactivation of non-essential functions. Fermentation pathways appeared to alter through selection for mutations in genes such asnadR, adhE, dcuS/R, andpflB. Mutations were frequently identified in genes for presumably dispensable functions such as toxin-antitoxin systems, prophages, virulence and amino acid transport. Adaptation of the fluctuating lineages to the anaerobic environments involved mutations affecting traits similar to those observed in the anaerobically evolved lineages.DiscussionThere appeared to be strong selective pressure for activities that conferred cell yield advantages during anaerobic growth, which include restoring activities that had previously been inactivated under long-term continuous aerobic evolution of the ancestor.

Upon entering the human gastrointestinal tract, foodborne bacterial enteropathogens encounter, among numerous other stress conditions, nutrient competition with the host organism and the commensal microbiota. The main carbon, nitrogen and energy sources exploited by pathogens during proliferation in, and colonization of, the gut have, however, not been identified completely. In recent years, a huge body of literature has provided evidence that most enteropathogens are equipped with a large set of specific metabolic pathways to overcome nutritional limitations in vivo, thus increasing bacterial fitness during infection. These adaptations include the degradation of myo-inositol, ethanolamine cleaved from phospholipids, fucose derived from mucosal glycoconjugates, 1,2-propanediol as the fermentation product of fucose or rhamnose and several other metabolites not accessible for commensal bacteria or present in competition-free microenvironments. Interestingly, the data reviewed here point to common metabolic strategies of enteric pathogens allowing the exploitation of nutrient sources that not only are present in the gut lumen, the mucosa or epithelial cells, but also are abundant in food. An increased knowledge of the metabolic strategies developed by enteropathogens is therefore a key factor to better control foodborne diseases.

Human gut microbiota is able to influence the host physiology by regulating multiple processes, including nutrient absorption, inflammation, oxidative stress, immune function, and anabolic balance. Aging is associated with reduced microbiota biodiversity, increased inter-individual variability, and over-representation of pathobionts, and these phenomena may have great relevance for skeletal muscle mass and function. For this reason, the presence of a gut-muscle axis regulating the onset and progression of age-related physical frailty and sarcopenia has been recently hypothesized. In this narrative review, we summarize the studies supporting a possible association between gut microbiota-related parameters with measures of muscle mass, muscle function, and physical performance in animal models and humans. Reduced muscle mass has been associated with distinct microbiota composition and reduced fermentative capacity in mice, and the administration of probiotics or butyrate to mouse models of muscle wasting has been associated with improved muscle mass. However, no studies have targeted the human microbiome associated with sarcopenia. Limited evidence from human studies shows an association between microbiota composition, involving key taxa such as Faecalibacterium and Bifidobacterium, and grip strength. Similarly, few studies conducted on patients with parkinsonism showed a trend towards a different microbiota composition in those with reduced gait speed. No studies have assessed the association of fecal microbiota with other measures of physical performance. However, several studies, mainly with a cross-sectional design, suggest an association between microbiota composition and frailty, mostly assessed according to the deficit accumulation model. Namely, frailty was associated with reduced microbiota biodiversity, and lower representation of butyrate-producing bacteria. Therefore, we conclude that the causal link between microbiota and physical fitness is still uncertain due to the lack of targeted studies and the influence of a large number of covariates, including diet, exercise, multimorbidity, and polypharmacy, on both microbiota composition and physical function in older age. However, the relationship between gut microbiota and physical function remains a very promising area of research for the future.

ABSTRACTDaqu is a spontaneous solid-state cereal fermentation used as saccharification and starter culture in Chinese vinegar and liquor production. The evolution of microbiota in this spontaneous fermentation is controlled by the temperature profile, which reaches temperatures from 50 to 65°C for several days. Despite these high temperatures, mesophilicEnterobacteriaceae(includingCronobacter) and bacilli are present throughout Daqu fermentation. This study aimed to determine whether Daqu spontaneous solid-state fermentation selects for heat-resistant variants of these organisms. Heat resistance inEnterobacteriaceaeis mediated by the locus of heat resistance (LHR). One LHR-positive strain ofKosakonia cowaniiwas identified in Daqu, and it exhibited higher heat resistance than the LHR-negativeK. cowaniiisolated from malted oats. Heat resistance inBacillusendospores is mediated by thespoVA2moboperon. Out of 10 Daqu isolates of the speciesBacillus licheniformis,Brevibacillus parabrevis,Bacillus subtilis,Bacillus amyloliquefaciens, andBacillus velezensis, 5 did not containspoVA2mob, 3 contained one copy, and 2 contained two copies. The presence and copy number of thespoVA2moboperon increased the resistance of spores to treatment with 110°C. To confirm the selection of LHR- andspoVA2mob-positive strains during Daqu fermentation, the copy numbers of these genetic elements in Daqu samples were quantified by quantitative PCR (qPCR). The abundance of LHR and thespoVA2moboperon in community DNA relative to that of total bacterial 16S rRNA genes increased 3-fold and 5-fold, respectively, during processing. In conclusion, culture-dependent and culture-independent analyses suggest that Daqu fermentation selects for heat-resistantEnterobacteriaceaeand bacilli.IMPORTANCEDaqu fermentations select for mobile genetic elements conferring heat resistance inEnterobacteriaceaeand bacilli. The locus of heat resistance (LHR), a genomic island conferring heat resistance inEnterobacteriaceae, and thespoVA2moboperon, conferring heat resistance on bacterial endospores, were enriched 3- to 5-fold during Daqu fermentation and maturation. It is therefore remarkable that the LHR and thespoVA2moboperon are accumulated in the same food fermentation. The presence of heat-resistantKosakoniaspp. andBacillusspp. in Daqu is not of concern for food safety; however, both genomic islands are mobile and transferable to pathogenic bacteria or toxin-producing bacteria by horizontal gene transfer. The identification of the LHR and thespoVA2moboperon as indicators of fitness ofEnterobacteriaceaeand bacilli in Daqu fermentation provides insights into environmental sources of heat-resistant organisms that may contaminate the food supply.

ABSTRACT The intestines house a diverse microbiota that must compete for nutrients to survive, but the specific limiting nutrients that control pathogen colonization are not clearly defined. Clostridioides difficile colonization typically requires prior disruption of the microbiota, suggesting that outcompeting commensals for resources is critical to establishing C. difficile infection (CDI). The immune protein calprotectin (CP) is released into the gut lumen during CDI to chelate zinc (Zn) and other essential nutrient metals. Yet, the impact of Zn limitation on C. difficile colonization is unknown. To define C. difficile responses to Zn limitation, we performed RNA sequencing on C. difficile exposed to CP. In medium containing CP, C. difficile upregulated genes involved in metal homeostasis and amino acid metabolism. To identify CP-responsive genes important during infection, we measured the abundance of select C. difficile transcripts in a mouse CDI model relative to expression in vitro. Gene transcripts involved in selenium (Se)-dependent proline fermentation increased during infection and in response to CP. Increased proline fermentation gene transcription was dependent on CP Zn binding and proline availability, yet proline fermentation was only enhanced when Se was supplemented. CP-deficient mice could not restrain C. difficile proline fermentation-dependent growth, suggesting that CP-mediated Zn sequestration along with limited Se restricts C. difficile proline fermentation. Overall, these results highlight how C. difficile colonization depends on the availability of multiple nutrients whose abundances are dynamically influenced by the host response. IMPORTANCE Clostridioides difficile infection (CDI) is the leading cause of postantibiotic nosocomial infection. Antibiotic therapy can be successful, yet up to one-third of individuals suffer from recurrent infections. Understanding the mechanisms controlling C. difficile colonization is paramount in designing novel treatments for primary and recurrent CDI. Here, we found that limiting nutrients control C. difficile metabolism during CDI and influence overall pathogen fitness. Specifically, the immune protein CP limits Zn availability and increases transcription of C. difficile genes necessary for proline fermentation. Paradoxically, this leads to reduced C. difficile proline fermentation. This reduced fermentation is due to limited availability of another nutrient required for proline fermentation, Se. Therefore, CP-mediated Zn limitation combined with low Se levels overall reduce C. difficile fitness in the intestines. These results emphasize the complexities of how nutrient availability influences C. difficile colonization and provide insight into critical metabolic processes that drive the pathogen’s growth.

ABSTRACT How microbial metabolism is translated into cellular reproduction under energy-limited settings below the seafloor over long timescales is poorly understood. Here, we show that microbial abundance increases an order of magnitude over a 5 million-year-long sequence in anoxic subseafloor clay of the abyssal North Atlantic Ocean. This increase in biomass correlated with an increased number of transcribed protein-encoding genes that included those involved in cytokinesis, demonstrating that active microbial reproduction outpaces cell death in these ancient sediments. Metagenomes, metatranscriptomes, and 16S rRNA gene sequencing all show that the actively reproducing community was dominated by the candidate phylum “Candidatus Atribacteria,” which exhibited patterns of gene expression consistent with fermentative, and potentially acetogenic, metabolism. “Ca. Atribacteria” dominated throughout the 8 million-year-old cored sequence, despite the detection limit for gene expression being reached in 5 million-year-old sediments. The subseafloor reproducing “Ca. Atribacteria” also expressed genes encoding a bacterial microcompartment that has potential to assist in secondary fermentation by recycling aldehydes and, thereby, harness additional power to reduce ferredoxin and NAD+. Expression of genes encoding the Rnf complex for generation of chemiosmotic ATP synthesis were also detected from the subseafloor “Ca. Atribacteria,” as well as the Wood-Ljungdahl pathway that could potentially have an anabolic or catabolic function. The correlation of this metabolism with cytokinesis gene expression and a net increase in biomass over the million-year-old sampled interval indicates that the “Ca. Atribacteria” can perform the necessary catabolic and anabolic functions necessary for cellular reproduction, even under energy limitation in millions-of-years-old anoxic sediments. IMPORTANCE The deep subseafloor sedimentary biosphere is one of the largest ecosystems on Earth, where microbes subsist under energy-limited conditions over long timescales. It remains poorly understood how mechanisms of microbial metabolism promote increased fitness in these settings. We discovered that the candidate bacterial phylum “Candidatus Atribacteria” dominated a deep-sea subseafloor ecosystem, where it exhibited increased transcription of genes associated with acetogenic fermentation and reproduction in million-year-old sediment. We attribute its improved fitness after burial in the seabed to its capabilities to derive energy from increasingly oxidized metabolites via a bacterial microcompartment and utilize a potentially reversible Wood-Ljungdahl pathway to help meet anabolic and catabolic requirements for growth. Our findings show that “Ca. Atribacteria” can perform all the necessary catabolic and anabolic functions necessary for cellular reproduction, even under energy limitation in anoxic sediments that are millions of years old.

A novel rapid ex vivo assay was developed as part of a concept to determine potential tailor-made combinations of pre- and probiotics for individual farms. Sow faecal slurries from 20 German pig farms were anaerobically incubated with pre- and probiotics or their combinations together with pathogenic strains that are of interest in pig production. Aliquots of these slurries were then incubated with media containing antibiotic mixtures allowing only growth of the specific pathogen. Growth was monitored and lag time was used to determine the residual fitness of the pathogenic strains. The background growth could be inhibited for an Escherichia coli- and a Clostridium difficile- but not for a Clostridium perfringens strain. The prebiotic fructo-oligosaccharides (FOS) and its combination with probiotics reduced the residual fitness of the E. coli strain in some farms. However, notable exceptions occurred in other farms where FOS increased the fitness of the E. coli strain. Generally, combinations of pre- and probiotics did not show additive effects on fitness for E. coli but displayed farm dependent differences. The effects of pre- and probiotics on the residual fitness of the C. difficile strain were less pronounced, but distinct differences between single application of prebiotics and their combination with probiotics were observed. It was concluded that the initial composition of the microbiota in the samples was more determinative for incubations with the C. difficile strain than for incubations with the E. coli strain, as the presumed fermentation of prebiotic products showed less influence on the fitness of the C. difficile strain. Farm dependent differences were pronounced for both pathogenic strains and therefore, this novel screening method offers a promising approach for pre-selecting pre- and probiotics for individual farms. However, evaluation of farm metadata (husbandry, feed, management) will be crucial in future studies to determine a tailor-made solution for combinations of pre- and probiotics for individual farms. Also, refinement of the ex vivo assay in terms of on-farm processing of samples and validation of unambiguous growth for pathogenic strains from individual farms should be addressed.

AbstractCurrent industrial bioethanol production by yeast through fermentation generates carbon dioxide. Carbon neutral bioethanol production by cyanobacteria uses biological fixation (photosynthesis) of carbon dioxide or other waste inorganic carbon sources, whilst being sustainable and renewable. The first ethanologenic cyanobacterial process was developed over two decades ago using Synechococcus elongatus PCC 7942, by incorporating the recombinant pdc and adh genes from Zymomonas mobilis. Further engineering has increased bioethanol titres 24-fold, yet current levels are far below what is required for industrial application. At the heart of the problem is that the rate of carbon fixation cannot be drastically accelerated and carbon partitioning towards bioethanol production impacts on cell fitness. Key progress has been achieved by increasing the precursor pyruvate levels intracellularly, upregulating synthetic genes and knocking out pathways competing for pyruvate. Studies have shown that cyanobacteria accumulate high proportions of carbon reserves that are mobilised under specific environmental stresses or through pathway engineering to increase ethanol production. When used in conjunction with specific genetic knockouts, they supply significantly more carbon for ethanol production. This review will discuss the progress in generating ethanologenic cyanobacteria through chassis engineering, and exploring the impact of environmental stresses on increasing carbon flux towards ethanol production.

Abstract Background Tetragenococcus (T.) halophilus is a common member of the microbial consortia of food fermented under high salt conditions. These comprises salty condiments based on soy or lupine beans, fish sauce, shrimp paste and brined anchovies. Within these fermentations this lactic acid bacterium (LAB) is responsible for the formation of lactic and other short chain acids that contribute to the flavor and lower the pH of the product. In this study, we investigated the transcriptomic profile of the two T. halophilus strains TMW 2.2254 and TMW 2.2256 in a lupine moromi model medium supplied with galactose. To get further insights into which genomic trait is important, we used a setup with two strains. That way we can determine if strain dependent pathways contribute to the overall fitness. These strains differ in the ability to utilize L-arginine, L-aspartate, L-arabinose, D-sorbitol, glycerol, D-lactose or D-melibiose. The lupine moromi model medium is an adapted version of the regular MRS medium supplied with lupine peptone instead of casein peptone and meat extract, to simulate the amino acid availabilities in lupine moromi. Results The transcriptomic profiles of the T. halophilus strains TMW 2.2254 and TMW 2.2256 in a lupine peptone-based model media supplied with galactose, used as simulation media for a lupine seasoning sauce fermentation, were compared to the determine potentially important traits. Both strains, have a great overlap in their response to the culture conditions but some strain specific features such as the utilization of glycerol, sorbitol and arginine contribute to the overall fitness of the strain TMW 2.2256. Interestingly, although both strains have two non-identical copies of the tagatose-6P pathway and the Leloir pathway increased under the same conditions, TMW 2.2256 prefers the degradation via the tagatose-6P pathway while TMW 2.2254 does not. Furthermore, TMW 2.2256 shows an increase in pathways required for balancing out the intracellular NADH/NADH+ ratios. Conclusions Our study reveals for the first time, that both versions of tagatose-6P pathways encoded in both strains are simultaneously active together with the Leloir pathway and contribute to the degradation of galactose. These findings will help to understand the strain dependent features that might be required for a starter strain in lupine moromi.

This thesis describes a progression of experimental work from proof of concept (ie can laboratory generated interspecific yeast strains be developed for industrial application) through to realisation of the potential of novel interspecific wine yeast for improved outputs in the winery. A competitive market requires winemakers to not only be aware of production costs, but also to find market niches by differentiating their wine styles. Developing new yeast with improved fermentation traits and/or potential to produce diverse wine flavours and aromas can provide tools to the winemaker that are readily and easily utilised in the winery without any extra (or costly) processing intervention. With consumer reluctance to the acceptance of genetically modified organisms, yeast breeding remains an important technique for yeast strain development. Traditionally, yeast breeding programs have centred around mating between different Saccharomyces cerevisiae yeast strains. Incorporating a higher level of genomic diversity into a Saccharomyces cerevisiae wine yeast by hybridisation with other Saccharomyces species has the potential to deliver novel flavour and aroma profiles through the production of a wider range of yeast-derived, flavour-active metabolites. This research reports on the development of laboratory-generated yeast interspecific hybrids created by natural breeding techniques. Initially, interspecific hybrids between species most closely related to S. cerevisiae were assessed. Grape juice fermentation by hybrids from crosses between a commercial S. cerevisiae wine yeast and either Saccharomyces paradoxus or Saccharomyces kudriavzevii showed that the hybrids had robust fermentation properties and produced wines with different concentrations of aromatic products relative to the commercial wine yeast parent. Progeny from crosses utilising a more divergent species (Saccharomyces mikatae) were fermentation competent and could deliver wines with novel flavours and aromas, including flavour compounds more commonly associated with non- Saccharomyces species. Next, a targeted approach to determine whether hybrids could be generated with a predictable phenotype that could address an explicit problematic fermentation trait was used. Elevated volatile acidity levels when producing dessert wines from highsugar juices pose a challenge to winemakers. Hybrids from a mating with a S. cerevisiae wine yeast and Saccharomyces uvarum (a species previously reported to produce wines with low concentrations of acetic acid) displayed the desired targeted phenotypes; strong fermentation properties in high-sugar juice and wines with low volatile acidity. Subsequent experiments indicated that the hybrids were less robust in grape juice than their S. cerevisiae wine yeast parent. With this in mind, it was decided to attempt to increase fitness of one S. cerevisiae x S. uvarum hybrid by an adaptive evolution approach in grape juice. To avoid the problem of selecting end-point collateral mutations that shape phenotypes in addition to that which is targeted, isolates were progressively screened from the evolving population. An evolved isolate with loss of S. uvarum Chromosome 14 (the overriding chromosomal alteration) but no other detectable changes in karyotype demonstrated that loss of S. uvarum Chromosome 14 alone conferred increased fitness. Fermentation kinetics showed that the evolved strain had an increased fermentation performance relative to the original hybrid and retained the desirable fermentation trait of the parent: wines with low volatile acidity. This research establishes that Saccharomyces interspecific hybridisation can deliver tools to the winemaking industry in the realm of wine style differentiation through the formation of novel yeast volatile fermentation metabolite profiles, and improved yeast fermentation properties. In addition, adopting an evolutionary approach in a fermentative context can deliver increased fitness to a wine yeast interspecific hybrid.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2018