Laboratory evolution reveals general and specific tolerance mechanisms for commodity chemicals.

Although strain tolerance to high product concentrations is a barrier to the economically viable biomanufacturing of industrial chemicals, chemical tolerance mechanisms are often unknown. To reveal tolerance mechanisms, an automated platform was utilized to evolve Escherichia coli to grow optimally in the presence of 11 industrial chemicals (1,2-propanediol, 2,3-butanediol, glutarate, adipate, putrescine, hexamethylenediamine, butanol, isobutyrate, coumarate, octanoate, hexanoate), reaching tolerance at concentrations 60%-400% higher than initial toxic levels. Sequencing genomes of 223 isolates from 89 populations, reverse engineering, and cross-compound tolerance profiling were employed to uncover tolerance mechanisms. We show that: 1) cells are tolerized via frequent mutation of membrane transporters or cell wall-associated proteins (e.g., ProV, KgtP, SapB, NagA, NagC, MreB), transcription and translation machineries (e.g., RpoA, RpoB, RpoC, RpsA, RpsG, NusA, Rho), stress signaling proteins (e.g., RelA, SspA, SpoT, YobF), and for certain chemicals, regulators and enzymes in metabolism (e.g., MetJ, NadR, GudD, PurT); 2) osmotic stress plays a significant role in tolerance when chemical concentrations exceed a general threshold and mutated genes frequently overlap with those enabling chemical tolerance in membrane transporters and cell wall-associated proteins; 3) tolerization to a specific chemical generally improves tolerance to structurally similar compounds whereas a tradeoff can occur on dissimilar chemicals, and 4) using pre-tolerized starting isolates can hugely enhance the subsequent production of chemicals when a production pathway is inserted in many, but not all, evolved tolerized host strains, underpinning the need for evolving multiple parallel populations. Taken as a whole, this study provides a comprehensive genotype-phenotype map based on identified mutations and growth phenotypes for 223 chemical tolerant isolates.

Ethanol-lactate transition of Lachancea thermotolerans is linked to nitrogen metabolism.

Climate change increases sugar content in grapes, resulting in unwanted increase in ethanol content of wine. Lachancea thermotolerans ferments glucose and fructose into both ethanol and lactate, decreasing final ethanol content and positively affecting wine acidity. Reported Lachancea thermotolerans strains show big variation in lactate production during fermentation. However, a mechanistic understanding of this lactate producing phenotype is currently lacking. Through a combination of metabolomics, transcriptomics, genomics and computational methods we show that the lactate production is induced by amino acid limitation in a high lactate producing strain. We found in fermentations in synthetic grape juice media that lactate production starts in the last stages of growth, marked by decreased growth rate and increased expression levels of stress related genes. This onset of lactate production is specific for the high lactate producing strain and independent of oxygen availability. The onset of lactate production was changed by increased amino acid content of the media, and it is shown by both computational methods and amino acid measurements that at the onset of lactate production amino acids become limiting for growth. This study shows that lactate production of Lachancea thermotolerans is directly linked to nitrogen availability in the media, an insight that can further aid in the improvement of wine quality.

Rational and evolutionary engineering of Saccharomyces cerevisiae for production of dicarboxylic acids from lignocellulosic biomass and exploring genetic mechanisms of the yeast tolerance to the biomass hydrolysate.

BACKGROUND: Lignosulfonates are significant wood chemicals with a $700 million market, produced by sulfite pulping of wood. During the pulping process, spent sulfite liquor (SSL) is generated, which in addition to lignosulfonates contains hemicellulose-derived sugars-in case of hardwoods primarily the pentose sugar xylose. The pentoses are currently underutilized. If they could be converted into value-added chemicals, overall economic profitability of the process would increase. SSLs are typically very inhibitory to microorganisms, which presents a challenge for a biotechnological process. The aim of the present work was to develop a robust yeast strain able to convert xylose in SSL to carboxylic acids. RESULTS: The industrial strain Ethanol Red of the yeast Saccharomyces cerevisiae was engineered for efficient utilization of xylose in a Eucalyptus globulus lignosulfonate stream at low pH using CRISPR/Cas genome editing and adaptive laboratory evolution. The engineered strain grew in synthetic medium with xylose as sole carbon source with maximum specific growth rate (µmax) of 0.28 1/h. Selected evolved strains utilized all carbon sources in the SSL at pH 3.5 and grew with µmax between 0.05 and 0.1 1/h depending on a nitrogen source supplement. Putative genetic determinants of the increased tolerance to the SSL were revealed by whole genome sequencing of the evolved strains. In particular, four top-candidate genes (SNG1, FIT3, FZF1 and CBP3) were identified along with other gene candidates with predicted important roles, based on the type and distribution of the mutations across different strains and especially the best performing ones. The developed strains were further engineered for production of dicarboxylic acids (succinic and malic acid) via overexpression of the reductive branch of the tricarboxylic acid cycle (TCA). The production strain produced 0.2 mol and 0.12 mol of malic acid and succinic acid, respectively, per mol of xylose present in the SSL. CONCLUSIONS: The combined metabolic engineering and adaptive evolution approach provided a robust SSL-tolerant industrial strain that converts fermentable carbon content of the SSL feedstock into malic and succinic acids at low pH.in production yields reaching 0.1 mol and 0.065 mol per mol of total consumed carbon sources.. Moreover, our work suggests potential genetic background of the tolerance to the SSL stream pointing out potential gene targets for improving the tolerance to inhibitory industrial feedstocks.

A dual-reporter system for investigating and optimizing protein translation and folding in E. coli.

Strategies for investigating and optimizing the expression and folding of proteins for biotechnological and pharmaceutical purposes are in high demand. Here, we describe a dual-reporter biosensor system that simultaneously assesses in vivo protein translation and protein folding, thereby enabling rapid screening of mutant libraries. We have validated the dual-reporter system on five different proteins and find an excellent correlation between reporter signals and the levels of protein expression and solubility of the proteins. We further demonstrate the applicability of the dual-reporter system as a screening assay for deep mutational scanning experiments. The system enables high throughput selection of protein variants with high expression levels and altered protein stability. Next generation sequencing analysis of the resulting libraries of protein variants show a good correlation between computationally predicted and experimentally determined protein stabilities. We furthermore show that the mutational experimental data obtained using this system may be useful for protein structure calculations.

Updated Genome Sequence for the Probiotic Bacterium Bifidobacterium animalis subsp. lactis BB-12.

The genome of Bifidobacterium animalis subsp. lactis BB-12 was sequenced using Oxford Nanopore Technologies long-read and Illumina short-read sequencing platforms. A hybrid genome assembly approach was used to construct an updated complete genome sequence for BB-12 containing 1,944,152 bp, with a G+C content of 60.5% and 1,615 genes.

Screening for texturing Leuconostoc and genomics behind polysaccharide production.

Synthesis of polysaccharides by Leuconostoc can result in improved texture of fermented products. A total of 249 Leuconostoc strains were screened for homo-polysaccharide production and for texturing capabilities in milk. A total of six Ln. mesenteroides strains with superior texturing properties had the genetic blueprint for both homo- (HoPS) and hetero-polysaccharide (HePS) synthesis. Only one strain produced texture in milk without added sucrose, suggesting HePS synthesis via the Wzy dependent pathway. In milk acidification experiments with added sucrose, all six strains depleted the sucrose and released fructose. Thus, they can be used for both texture and possibly also for sweetness enhancement.

Three biofilm types produced by a model pseudomonad are differentiated by structural characteristics and fitness advantage.

Model bacterial biofilm systems suggest that bacteria produce one type of biofilm, which is then modified by environmental and physiological factors, although the diversification of developing populations might result in the appearance of adaptive mutants producing altered structures with improved fitness advantage. Here we compare the air-liquid (A-L) interface viscous mass (VM) biofilm produced by Pseudomonas fluorescens SBW25 and the wrinkly spreader (WS) and complementary biofilm-forming strain (CBFS) biofilm types produced by adaptive SBW25 mutants in order to better understand the link between these physical structures and the fitness advantage they provide in experimental microcosms. WS, CBFS and VM biofilms can be differentiated by strength, attachment levels and rheology, as well as by strain characteristics associated with biofilm formation. Competitive fitness assays demonstrate that they provide similar advantages under static growth conditions but respond differently to increasing levels of physical disturbance. Pairwise competitions between biofilms suggest that these strains must be competing for at least two growth-limiting resources at the A-L interface, most probably O2 and nutrients, although VM and CBFS cells located lower down in the liquid column might provide an additional fitness advantage through the colonization of a less competitive zone below the biofilm. Our comparison of different SBW25 biofilm types illustrates more generally how varied biofilm characteristics and fitness advantage could become among adaptive mutants arising from an ancestral biofilm-forming strain and raises the question of how significant these changes might be in a range of medical, biotechnological and industrial contexts where diversification and change may be problematic.

Genome-wide identification of tolerance mechanisms toward p-coumaric acid in Pseudomonas putida.

The soil bacterium Pseudomonas putida KT2440 has gained increasing biotechnological interest due to its ability to tolerate different types of stress. Here, the tolerance of P. putida KT2440 toward eleven toxic chemical compounds was investigated. P. putida was found to be significantly more tolerant toward three of the eleven compounds when compared to Escherichia coli. Increased tolerance was for example found toward p-coumaric acid, an interesting precursor for polymerization with a significant industrial relevance. The tolerance mechanism was therefore investigated using the genome-wide approach, Tn-seq. Libraries containing a large number of miniTn5-Km transposon insertion mutants were grown in the presence and absence of p-coumaric acid, and the enrichment or depletion of mutants was quantified by high-throughput sequencing. Several genes, including the ABC transporter Ttg2ABC and the cytochrome c maturation system (ccm), were identified to play an important role in the tolerance toward p-coumaric acid of this bacterium. Most of the identified genes were involved in membrane stability, suggesting that tolerance toward p-coumaric acid is related to transport and membrane integrity.

Adaptive radiation of Pseudomonas fluorescens SBW25 in experimental microcosms provides an understanding of the evolutionary ecology and molecular biology of A-L interface biofilm formation.

Combined experimental evolutionary and molecular biology approaches have been used to investigate the adaptive radiation of Pseudomonas fluorescens SBW25 in static microcosms leading to the colonisation of the air-liquid interface by biofilm-forming mutants such as the Wrinkly Spreader (WS). In these microcosms, the ecosystem engineering of the early wild-type colonists establishes the niche space for subsequent WS evolution and colonisation. Random WS mutations occurring in the developing population that deregulate diguanylate cyclases and c-di-GMP homeostasis result in cellulose-based biofilms at the air-liquid interface. These structures allow Wrinkly Spreaders to intercept O2 diffusing into the liquid column and limit the growth of competitors lower down. As the biofilm matures, competition increasingly occurs between WS lineages, and niche divergence within the biofilm may support further diversification before system failure when the structure finally sinks. A combination of pleiotropic and epistasis effects, as well as secondary mutations, may explain variations in WS phenotype and fitness. Understanding how mutations subvert regulatory networks to express intrinsic genome potential and key innovations providing a selective advantage in novel environments is key to understanding the versatility of bacteria, and how selection and ecological opportunity can rapidly lead to substantive changes in phenotype and in community structure and function.

Biofilm formation and cellulose expression by Bordetella avium 197N, the causative agent of bordetellosis in birds and an opportunistic respiratory pathogen in humans.

Although bacterial cellulose synthase (bcs) operons are widespread within the Proteobacteria phylum, subunits required for the partial-acetylation of the polymer appear to be restricted to a few γ-group soil, plant-associated and phytopathogenic pseudomonads, including Pseudomonas fluorescens SBW25 and several Pseudomonas syringae pathovars. However, a bcs operon with acetylation subunits has also been annotated in the unrelated ß-group respiratory pathogen, Bordetella avium 197N. Our comparison of subunit protein sequences and GC content analyses confirms the close similarity between the B. avium 197N and pseudomonad operons and suggests that, in both cases, the cellulose synthase and acetylation subunits were acquired as a single unit. Using static liquid microcosms, we can confirm that B. avium 197N expresses low levels of cellulose in air-liquid interface biofilms and that biofilm strength and attachment levels could be increased by elevating c-di-GMP levels like the pseudomonads, but cellulose was not required for biofilm formation itself. The finding that B. avium 197N is capable of producing cellulose from a highly-conserved, but relatively uncommon bcs operon raises the question of what functional role this modified polymer plays during the infection of the upper respiratory tract or survival between hosts, and what environmental signals control its production.

Rapid resistome mapping using nanopore sequencing.

The emergence of antibiotic resistance in human pathogens has become a major threat to modern medicine. The outcome of antibiotic treatment can be affected by the composition of the gut. Accordingly, knowledge of the gut resistome composition could enable more effective and individualized treatment of bacterial infections. Yet, rapid workflows for resistome characterization are lacking. To address this challenge we developed the poreFUME workflow that deploys functional metagenomic selections and nanopore sequencing to resistome mapping. We demonstrate the approach by functionally characterizing the gut resistome of an ICU (intensive care unit) patient. The accuracy of the poreFUME pipeline is with >97% sufficient for the annotation of antibiotic resistance genes. The poreFUME pipeline provides a promising approach for efficient resistome profiling that could inform antibiotic treatment decisions in the future.

Increased production of L-serine in Escherichia coli through Adaptive Laboratory Evolution.

L-serine is a promising building block biochemical with a high theoretical production yield from glucose. Toxicity of L-serine is however prohibitive for high-titer production in E. coli. Here, E. coli lacking L-serine degradation pathways was evolved for improved tolerance by gradually increasing L-serine concentration from 3 to 100g/L using adaptive laboratory evolution (ALE). Genome sequencing of isolated clones revealed multiplication of genetic regions, as well as mutations in thrA, thereby showing a potential mechanism of serine inhibition. Additional mutations were evaluated by MAGE combined with amplicon sequencing, revealing role of rho, lrp, pykF, eno, and rpoB on tolerance and fitness in minimal medium. Production using the tolerant strains resulted in 37g/L of L-serine with a 24% mass yield. The resulting titer is similar to the highest production reported for any organism thereby highlighting the potential of ALE for industrial biotechnology.

Multi-omics Quantification of Species Variation of Escherichia coli Links Molecular Features with Strain Phenotypes.

Escherichia coli strains are widely used in academic research and biotechnology. New technologies for quantifying strain-specific differences and their underlying contributing factors promise greater understanding of how these differences significantly impact physiology, synthetic biology, metabolic engineering, and process design. Here, we quantified strain-specific differences in seven widely used strains of E. coli (BL21, C, Crooks, DH5a, K-12 MG1655, K-12 W3110, and W) using genomics, phenomics, transcriptomics, and genome-scale modeling. Metabolic physiology and gene expression varied widely with downstream implications for productivity, product yield, and titer. These differences could be linked to differential regulatory structure. Analyzing high-flux reactions and expression of encoding genes resulted in a correlated and quantitative link between these sets, with strain-specific caveats. Integrated modeling revealed that certain strains are better suited to produce given compounds or express desired constructs considering native expression states of pathways that enable high-production phenotypes. This study yields a framework for quantitatively comparing strains in a species with implications for strain selection.

Is genotyping of single isolates sufficient for population structure analysis of Pseudomonas aeruginosa in cystic fibrosis airways?

BACKGROUND: The primary cause of morbidity and mortality in cystic fibrosis (CF) patients is lung infection by Pseudomonas aeruginosa. Therefore much work has been done to understand the adaptation and evolution of P. aeruginosa in the CF lung. However, many of these studies have focused on longitudinally collected single isolates, and only few have included cross-sectional analyses of entire P. aeruginosa populations in sputum samples. To date only few studies have used the approach of metagenomic analysis for the purpose of investigating P. aeruginosa populations in CF airways. RESULTS: We analysed five metagenomes together with longitudinally collected single isolates from four recently chronically infected CF patients. With this approach we were able to link the clone type and the majority of SNP profiles of the single isolates to that of the metagenome(s) for each individual patient. CONCLUSION: Based on our analysis we find that when having access to comprehensive collections of longitudinal single isolates it is possible to rediscover the genotypes of the single isolates in the metagenomic samples. This suggests that information gained from genome sequencing of comprehensive collections of single isolates is satisfactory for many investigations of adaptation and evolution of P. aeruginosa to the CF airways.

EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is widely used in the biotechnology industry for production of ethanol, recombinant proteins, food ingredients and other chemicals. In order to generate highly producing and stable strains, genome integration of genes encoding metabolic pathway enzymes is the preferred option. However, integration of pathway genes in single or few copies, especially those encoding rate-controlling steps, is often not sufficient to sustain high metabolic fluxes. By exploiting the sequence diversity in the long terminal repeats (LTR) of Ty retrotransposons, we developed a new set of integrative vectors, EasyCloneMulti, that enables multiple and simultaneous integration of genes in S. cerevisiae. By creating vector backbones that combine consensus sequences that aim at targeting subsets of Ty sequences and a quickly degrading selective marker, integrations at multiple genomic loci and a range of expression levels were obtained, as assessed with the green fluorescent protein (GFP) reporter system. The EasyCloneMulti vector set was applied to balance the expression of the rate-controlling step in the ß-alanine pathway for biosynthesis of 3-hydroxypropionic acid (3HP). The best 3HP producing clone, with 5.45 g.L(-1) of 3HP, produced 11 times more 3HP than the lowest producing clone, which demonstrates the capability of EasyCloneMulti vectors to impact metabolic pathway enzyme activity.

Adaptation of the autotrophic acetogen Sporomusa ovata to methanol accelerates the conversion of CO2 to organic products.

Acetogens are efficient microbial catalysts for bioprocesses converting C1 compounds into organic products. Here, an adaptive laboratory evolution approach was implemented to adapt Sporomusa ovata for faster autotrophic metabolism and CO2 conversion to organic chemicals. S. ovata was first adapted to grow quicker autotrophically with methanol, a toxic C1 compound, as the sole substrate. Better growth on different concentrations of methanol and with H2-CO2 indicated the adapted strain had a more efficient autotrophic metabolism and a higher tolerance to solvent. The growth rate on methanol was increased 5-fold. Furthermore, acetate production rate from CO2 with an electrode serving as the electron donor was increased 6.5-fold confirming that the acceleration of the autotrophic metabolism of the adapted strain is independent of the electron donor provided. Whole-genome sequencing, transcriptomic, and biochemical studies revealed that the molecular mechanisms responsible for the novel characteristics of the adapted strain were associated with the methanol oxidation pathway and the Wood-Ljungdahl pathway of acetogens along with biosynthetic pathways, cell wall components, and protein chaperones. The results demonstrate that an efficient strategy to increase rates of CO2 conversion in bioprocesses like microbial electrosynthesis is to evolve the microbial catalyst by adaptive laboratory evolution to optimize its autotrophic metabolism.

The presence of anti-LPS antibodies and human serum activity against Proteus mirabilis S/R forms in correlation with TLR4 (Thr399Ile) gene polymorphism in rheumatoid arthritis.

OBJECTIVES: Proteus mirabilis strains are human pathogens responsible for urinary tract infections, which may also be involved in rheumatoid arthritis (RA). DESIGN AND METHODS: We determined whether the binding site of anti-LPS antibodies on the O-polysaccharide part of P. mirabilis LPS correlates with the level of TLR4 (Thr399Ile) gene polymorphism in the sera of RA patients. We investigated the deposition of C3d and C5b complement components on the P. mirabilis LPS. The ELISA method used in this study was optimized with LAL test and laser interferometry. RESULTS: Depending on LPS P. mirabilis used in these studies, the amount of antibodies in RA patients sera varied. We did not observe a correlation between anti-LPS antibodies binding and the level of TLR4 (Thr399Ile) gene polymorphism. We found that the lower complement components deposition by O49 in contrast to O9 LPS correlates with its reduced sensitivities to human complement-mediated killing. CONCLUSION: The immunological response against P. mirabilis LPS might play a role in rheumatoid arthritis.

Surfactants expressed by soil pseudomonads alter local soil-water distribution, suggesting a hydrological role for these compounds.

Several biological roles have been demonstrated for surfactants expressed by soil and rhizosphere Pseudomonas spp., but the impact of these powerful surface-active agents on the local soil-water distribution within the partially saturated soil pore network has not been examined. To investigate this potential hydrological role, the liquid surface tension (γ)-reducing activities (LSTRA) of 72 pseudomonads isolated from a sandy loam soil by tensiometry of culture supernatants were characterized. Of these, 67% exhibited LSTRA, reducing γ to a minimum (γ(Min)) of 24 mN m(-1) established by individual distribution identification analysis. Soil microcosms were then used to examine the impact of surfactant expression on the local soil-water distribution. The volumetric water content (θ) of soil microcosms was significantly lowered (0.78 ×) by Pseudomonas fluorescens SBW25 expressing the surfactant viscosin compared with a surfactant-deficient mutant (P<0.002). Six of 15 soil pseudomonad isolates examined were found to have a similar impact on θ when compared with sterile microcosms (P<0.05). These findings indicate that surfactant-expressing pseudomonads could modify the local soil-water distributions and that surfactants may therefore play a significant hydrological role in soils, in addition to their recognized biological activities.

Environmental modification and niche construction: developing O2 gradients drive the evolution of the Wrinkly Spreader.

The evolutionary success of the novel Wrinkly Spreader (WS) genotypes in diversifying Pseudomonas fluorescens SBW25 populations in static liquid microcosms has been attributed to the greater availability of O(2) at the air-liquid (A-L) interface where the WS produces a physically cohesive-class biofilm. However, the importance of O(2) gradients in SBW25 adaptation has never been examined. We have explicitly tested the role of O(2) in evolving populations using microsensor profiling and experiments conducted under high and low O(2) conditions. Initial colonists of static microcosms were found to establish O(2) gradients before significant population growth had occurred, converting a previously homogenous environment into one containing a resource continuum with high and low O(2) regions. These gradients were found to persist for long periods by which time significant numbers of WS had appeared colonising the high O(2) niches. Growth was O(2) limited in static microcosms, but high O(2) conditions like those found near the A-L interface supported greater growth and favoured the emergence of WS-like genotypes. A fitness advantage to biofilm formation was seen under high but not low O(2) conditions, suggesting that the cost of biofilm production could only be offset when O(2) levels above the A-L interface were high. Profiling of mature WS biofilms showed that they also contained high and low O(2) regions. Niches within these may support further diversification and succession of the developing biofilm population. O(2) availability has been found to be a major factor underlying the evolutionary success of the WS genotype in static microcosms and illustrates the importance of this resource continuum in microbial diversification and adaptation.

Characterization of a novel air-liquid interface biofilm of Pseudomonas fluorescens SBW25.

Pseudomonads are able to form a variety of biofilms that colonize the air-liquid (A-L) interface of static liquid microcosms, and differ in matrix composition, strength, resilience and degrees of attachment to the microcosm walls. From Pseudomonas fluorescens SBW25, mutants have evolved during prolonged adaptation-evolution experiments which produce robust biofilms of the physically cohesive class at the A-L interface, and which have been well characterized. In this study we describe a novel A-L interface biofilm produced by SBW25 that is categorized as a viscous mass (VM)-class biofilm. Several metals were found to induce this biofilm in static King's B microcosms, including copper, iron, lead and manganese, and we have used iron to allow further examination of this structure. Iron was demonstrated to induce SBW25 to express cellulose, which provided the matrix of the biofilm, a weak structure that was readily destroyed by physical disturbance. This was confirmed in situ by a low (0.023-0.047 g) maximum deformation mass and relatively poor attachment as measured by crystal violet staining. Biofilm strength increased with increasing iron concentration, in contrast to attachment levels, which decreased with increasing iron. Furthermore, iron added to mature biofilms significantly increased strength, suggesting that iron also promotes interactions between cellulose fibres that increase matrix interconnectivity. Whilst weak attachment is important in maintaining the biofilm at the A-L interface, surface-interaction effects involving cellulose, which reduced surface tension by approximately 3.8 mN m(-1), may also contribute towards this localization. The fragility and viscoelastic nature of the biofilm were confirmed by controlled-stress amplitude sweep tests to characterize critical rheological parameters, which included a shear modulus of 0.75 Pa, a zero shear viscosity of 0.24 Pa s(-1) and a flow point of 0.028 Pa. Growth and morphological data thus far support a non-specific metal-associated physiological, rather than mutational, origin for production of the SBW25 VM biofilm, which is an example of the versatility of bacteria to inhabit optimal niches within their environment.