Enhancing transcription in Escherichia coli and Pseudomonas putida using bacteriophage lambda anti-terminator protein Q.

Functional characterization of metagenomic DNA often involves expressing heterologous DNA in genetically tractable microorganisms such as Escherichia coli. Functional expression of heterologous genes can suffer from limitations due to the lack of recognition of foreign promoters or presence of intrinsic terminators on foreign DNA between a vector-based promoter and the transcription start site. Anti-terminator proteins are a possible solution to overcome this limitation. When bacteriophage lambda infects E. coli, it relies on the host transcription machinery to transcribe and express phage DNA. Lambda anti-terminator protein Q (λQ) regulates the expression of late-genes of phage lambda. E. coli RNA polymerase recognizes the PR' promoter on the lambda genome and forms a complex with λQ, to overcome the terminator tR'. Here we show the use of λQ to efficiently transcribe a capsular polysaccharide cluster, cps3, from Lactobacillus plantarum containing intrinsic terminators in Escherichia coli. In addition, we expand the use of anti-terminator λQ in Pseudomonas putida. The results show ~ fivefold higher expression of a fluorescent reporter located ~ 12.5kbp downstream from the promoter, when the transcription is driven by PR' promoter in presence of λQ compared to a lac promoter. These results suggest that λQ could be used in metabolic engineering to enhance expression of heterologous DNA.

Microbe-Assisted Nanocomposite Anodes for Aqueous Li-Ion Batteries.

With the rapid increase in the use of lithium-ion batteries (LIBs), the development of safe LIBs has become an important social issue. Replacing flammable organic liquid electrolytes in current LIBs with water can be an alternative route to resolve this safety concern. The water-in-salt (WIS) electrolytes received great attention as next-generation electrolytes due to their large electrochemical stability window. However, their high cathodic limit remains as a challenge, impeding the use of low-potential anodes. Here, we report the first biodirected synthesis of carbonaceous layers on anodes to use them as interlayers that prevent a direct contact of water molecules to anode particles. High-aspect ratio microbes are utilized as precursors of carbonaceous layers on TiO2 nanoparticles (m-TiO2) to enhance the conductivity and to reduce the electrolysis of WIS electrolytes. We selected the cylindrical shape of microbes that offers geometric diversity, providing us a toolkit to investigate the effect of microbe length in forming the network in binary composites and their impacts on the battery performance with WIS electrolytes. Using microbes with varying aspect ratios, the optimal microbe size to maximize the battery performance is determined. The effects of storage time on microbe size are also studied. Compared to uncoated TiO2 anodes, m-TiO2 exhibited 49% higher capacity at the 40th cycle and enhanced the cycle life close to anodes made with a conventional carbon precursor while using an 11% less amount of carbon. We performed density functional theory calculations to unravel the underlying mechanism of the performance improvement using microbe-derived carbon layers. Computational results show that high amounts of pyridinic nitrogen present in the peptide bonds in microbes are expected to slow down the water diffusion. Our findings provide key insights into the design of an interlayer for WIS anodes and open an avenue to fabricate energy storage materials using biomaterials.

Adaptive laboratory evolution of ß-caryophyllene producing Saccharomyces cerevisiae.

BACKGROUND: ß-Caryophyllene is a plant terpenoid with therapeutic and biofuel properties. Production of terpenoids through microbial cells is a potentially sustainable alternative for production. Adaptive laboratory evolution is a complementary technique to metabolic engineering for strain improvement, if the product-of-interest is coupled with growth. Here we use a combination of pathway engineering and adaptive laboratory evolution to improve the production of ß-caryophyllene, an extracellular product, by leveraging the antioxidant potential of the compound. RESULTS: Using oxidative stress as selective pressure, we developed an adaptive laboratory evolution that worked to evolve an engineered ß-caryophyllene producing yeast strain for improved production within a few generations. This strategy resulted in fourfold increase in production in isolated mutants. Further increasing the flux to ß-caryophyllene in the best evolved mutant achieved a titer of 104.7 ± 6.2 mg/L product. Genomic analysis revealed a gain-of-function mutation in the a-factor exporter STE6 was identified to be involved in significantly increased production, likely as a result of increased product export. CONCLUSION: An optimized selection strategy based on oxidative stress was developed to improve the production of the extracellular product ß-caryophyllene in an engineered yeast strain. Application of the selection strategy in adaptive laboratory evolution resulted in mutants with significantly increased production and identification of novel responsible mutations.

Adaptive laboratory evolution for growth coupled microbial production.

Adaptive laboratory evolution (ALE) is a powerful tool to select for strains with growth-coupled phenotypes. When coupled with next-generation sequencing and omic technologies, genotype-to-phenotype relationships and the molecular mechanisms underlying desired complex phenotypes can now be uncovered using ALE. However, in order for ALE to be effective in generating strains with increased productivity, the product-of-interest needs to be coupled with cellular growth or survival. Advances in computational metabolic modeling can now identify metabolic engineering strategies to force the coupling of desired product formation with growth for a wide range of different compounds. Such strategies can potentially be coupled with ALE to further enhance productivity of microbial hosts. In addition to metabolic strategies, if the compound of interest is known to impart beneficial traits to the host, such as stress tolerance, then an environment can be designed to allow product formation to be coupled with growth or survival. This mini-review will cover recent advances in both the metabolic and environmental engineering and synthetic biology strategies to couple production with microbial fitness, successful cases for the use of these strategies with ALE to improve product formation, discuss limitations, and future perspectives.

CgSTE11 mediates cross tolerance to multiple environmental stressors in Candida glabrata.

Candida glabrata is a human commensal and an opportunistic human fungal pathogen. It is more closely related to the model yeast Saccharomyces cerevisiae than other Candida spp. Compared with S. cerevisiae, C. glabrata exhibits higher innate tolerance to various environmental stressors, including hyperthermal stress. Here we investigate the molecular mechanisms of C. glabrata adaptation to heat stress via adaptive laboratory evolution. We show that all parallel evolved populations readily adapt to hyperthermal challenge (from 47 °C to 50 °C) and exhibit convergence in evolved phenotypes with extensive cross-tolerance to various other environmental stressors such as oxidants, acids, and alcohols. Genome resequencing identified fixation of mutations in CgSTE11 in all parallel evolved populations. The CgSTE11 homolog in S. cerevisiae plays crucial roles in various mitogen-activated protein kinase (MAPK) signaling pathways, but its role is less understood in C. glabrata. Subsequent verification confirmed that CgSTE11 is important in hyperthermal tolerance and the observed extensive cross-tolerance to other environmental stressors. These results support the hypothesis that CgSTE11 mediates cross-talks between MAPK signaling pathways in C. glabrata in response to environmental challenges.

Beneficial mutations for carotenoid production identified from laboratory-evolved Saccharomyces cerevisiae.

Adaptive laboratory evolution (ALE) is a powerful tool used to increase strain fitness in the presence of environmental stressors. If production and strain fitness can be coupled, ALE can be used to increase product formation. In earlier work, carotenoids hyperproducing mutants were obtained using an ALE strategy. Here, de novo mutations were identified in hyperproducers, and reconstructed mutants were explored to determine the exact impact of each mutation on production and tolerance. A single mutation in YMRCTy1-3 conferred increased carotenoid production, and when combined with other beneficial mutations led to further increased ß-carotene production. Findings also suggest that the ALE strategy selected for mutations that confer increased carotenoid production as primary phenotype. Raman spectroscopy analysis and total lipid quantification revealed positive correlation between increased lipid content and increased ß-carotene production. Finally, we demonstrated that the best combinations of mutations identified for ß-carotene production were also beneficial for production of lycopene.

Identifying novel genetic determinants for oxidative stress tolerance in Candida glabrata via adaptive laboratory evolution.

Candida glabrata (C glabrata) is an important yeast of industrial and medical significance. Resistance to oxidative stress is an important trait affecting its robustness as a production host or virulence as a pathogenic agent, but current understanding of resistance mechanisms is still limited in this fungus. In this study, we rapidly evolved C glabrata population to adapt to oxidative challenge (from 80mM to 350mM of H2 O2 ) through short-term adaptive laboratory evolution. Adaptive mutants were isolated from evolved populations and subjected to phenotypic and omics analyses to identify potential mechanisms of tolerance to H2 O2 . Phenotypic characterizations revealed faster detoxification of H2 O2 and ability to initiate growth at a higher concentration of the oxidant in the isolated adaptive mutants compared with the wild type. Genome resequencing and genome-wide transcriptome analysis revealed multiple genetic determinants (eg, CAGL0E01243g, CAGL0F06831g, and CAGL0C00385g) that potentially contribute to enhanced H2 O2 resistance. Subsequent experimental verification confirmed that CgCth2 (CAGL0E01243g) and CgMga2 (CAGL0F06831g) are important in C glabrata tolerance to oxidative stress. Transcriptome profiling of adaptive mutants and bioinformatic analysis suggest that NADPH regeneration, modulation of membrane composition, cell wall remodeling, and/or global regulatory changes are involved in C glabrata tolerance to H2 O2 .

Relative Abundances of Candida albicans and Candida glabrata in In Vitro Coculture Biofilms Impact Biofilm Structure and Formation.

Candida is a member of the normal human microbiota and often resides on mucosal surfaces such as the oral cavity or the gastrointestinal tract. In addition to their commensality, Candida species can opportunistically become pathogenic if the host microbiota is disrupted or if the host immune system becomes compromised. An important factor for Candida pathogenesis is its ability to form biofilm communities. The two most medically important species-Candida albicans and Candida glabrata-are often coisolated from infection sites, suggesting the importance of Candida coculture biofilms. In this work, we report that biofilm formation of the coculture population depends on the relative ratio of starting cell concentrations of C. albicans and C. glabrata When using a starting ratio of C. albicans to C. glabrata of 1:3, ∼6.5- and ∼2.5-fold increases in biofilm biomass were observed relative to those of a C. albicans monoculture and a C. albicans/C. glabrata ratio of 1:1, respectively. Confocal microscopy analysis revealed the heterogeneity and complex structures composed of long C. albicans hyphae and C. glabrata cell clusters in the coculture biofilms, and reverse transcription-quantitative PCR (qRT-PCR) studies showed increases in the relative expression of the HWP1 and ALS3 adhesion genes in the C. albicans/C. glabrata 1:3 biofilm compared to that in the C. albicans monoculture biofilm. Additionally, only the 1:3 C. albicans/C. glabrata biofilm demonstrated an increased resistance to the antifungal drug caspofungin. Overall, the results suggest that interspecific interactions between these two fungal pathogens increase biofilm formation and virulence-related gene expression in a coculture composition-dependent manner.IMPORTANCECandida albicans and Candida glabrata are often coisolated during infection, and the occurrence of coisolation increases with increasing inflammation, suggesting possible synergistic interactions between the two Candida species in pathogenesis. During the course of an infection, the prevalence of each Candida species may change over time due to differences in metabolism and in the resistance of each species to antifungal therapies. Therefore, it is necessary to understand the dynamics between C. albicans and C. glabrata in coculture to develop better therapeutic strategies against Candida infections. Existing in vitro work has focused on understanding how an equal-part culture of C. albicans and C. glabrata impacts biofilm formation and pathogenesis. What is not understood, and what is investigated in this work, is how the composition of Candida species in coculture impacts overall biofilm formation, virulence gene expression, and the therapeutic treatment of biofilms.

Growth-Coupled Carotenoids Production Using Adaptive Laboratory Evolution.

Adaptive laboratory evolution is a powerful technique for strain development. However, the target phenotypes using this strategy have been limited by the required coupling of the phenotype-of-interest with fitness or survival, and thus adaptive evolution is generally not used to improve product formation. If the desired product confers a benefit to the host, then adaptive evolution can be an effective approach to improve host productivity. In this book chapter, we describe an effective adaptive laboratory evolution strategy for improving product formation of carotenoids, a class of compounds with antioxidant potential, in the yeast Saccharomyces cerevisiae.

Sexual recombination and increased mutation rate expedite evolution of Escherichia coli in varied fitness landscapes.

Sexual recombination and mutation rate are theorized to play different roles in adaptive evolution depending on the fitness landscape; however, direct experimental support is limited. Here we examine how these factors affect the rate of adaptation utilizing a "genderless" strain of Escherichia coli capable of continuous in situ sexual recombination. The results show that the populations with increased mutation rate, and capable of sexual recombination, outperform all the other populations. We further characterize two sexual and two asexual populations with increased mutation rate and observe maintenance of beneficial mutations in the sexual populations through mutational sweeps. Furthermore, we experimentally identify the molecular signature of a mating event within the sexual population that combines two beneficial mutations to generate a fitter progeny; this evidence suggests that the recombination event partially alleviates clonal interference. We present additional data suggesting that stochasticity plays an important role in the combinations of mutations observed.

Insights on Osmotic Tolerance Mechanisms in Escherichia coli Gained from an rpoC Mutation.

An 84 bp in-frame duplication (K370_A396dup) within the rpoC subunit of RNA polymerase was found in two independent mutants selected during an adaptive laboratory evolution experiment under osmotic stress in Escherichia coli, suggesting that this mutation confers improved osmotic tolerance. To determine the role this mutation in rpoC plays in osmotic tolerance, we reconstructed the mutation in BW25113, and found it to confer improved tolerance to hyperosmotic stress. Metabolite analysis, exogenous supplementation assays, and cell membrane damage analysis suggest that the mechanism of improved osmotic tolerance by this rpoC mutation may be related to the higher production of acetic acid and amino acids such as proline, and increased membrane integrity in the presence of NaCl stress in exponential phase cells. Transcriptional analysis led to the findings that the overexpression of methionine related genes metK and mmuP improves osmotic tolerance in BW25113. Furthermore, deletion of a stress related gene bolA was found to confer enhanced osmotic tolerance in BW25113 and MG1655. These findings expand our current understanding of osmotic tolerance in E. coli, and have the potential to expand the utilization of high saline feedstocks and water sources in microbial fermentation.

Benefits of a Recombination-Proficient Escherichia coli System for Adaptive Laboratory Evolution.

Adaptive laboratory evolution typically involves the propagation of organisms asexually to select for mutants with the desired phenotypes. However, asexual evolution is prone to competition among beneficial mutations (clonal interference) and the accumulation of hitchhiking and neutral mutations. The benefits of horizontal gene transfer toward overcoming these known disadvantages of asexual evolution were characterized in a strain of Escherichia coli engineered for superior sexual recombination (genderless). Specifically, we experimentally validated the capacity of the genderless strain to reduce the mutational load and recombine beneficial mutations. We also confirmed that inclusion of multiple origins of transfer influences both the frequency of genetic exchange throughout the chromosome and the linkage of donor DNA. We built a simple kinetic model to estimate recombination frequency as a function of transfer size and relative genotype enrichment in batch transfers; the model output correlated well with the experimental data. Our results provide strong support for the advantages of utilizing the genderless strain over its asexual counterpart during adaptive laboratory evolution for generating beneficial mutants with reduced mutational load. IMPORTANCE: Over 80 years ago Fisher and Muller began a debate on the origins of sexual recombination. Although many aspects of sexual recombination have been examined at length, experimental evidence behind the behaviors of recombination in many systems and the means to harness it remain elusive. In this study, we sought to experimentally validate some advantages of recombination in typically asexual Escherichia coli and determine if a sexual strain of E. coli can become an effective tool for strain development.

Characterization of an evolved carotenoids hyper-producer of Saccharomyces cerevisiae through bioreactor parameter optimization and Raman spectroscopy.

An evolutionary engineering approach for enhancing heterologous carotenoids production in an engineered Saccharomyces cerevisiae strain was used previously to isolate several carotenoids hyper-producers from the evolved populations. ß-Carotene production was characterized in the parental and one of the evolved carotenoids hyper-producers (SM14) using bench-top bioreactors to assess the impact of pH, aeration, and media composition on ß-carotene production levels. The results show that with maintaining a low pH and increasing the carbon-to-nitrogen ratio (C:N) from 8.8 to 50 in standard YNB medium, a higher ß-carotene production level at 25.52 ± 2.15 mg ß-carotene g(-1) (dry cell weight) in the carotenoids hyper-producer was obtained. The increase in C:N ratio also significantly increased carotenoids production in the parental strain by 298 % [from 5.68 ± 1.24 to 22.58 ± 0.11 mg ß-carotene g(-1) (dcw)]. In this study, it was shown that Raman spectroscopy is capable of monitoring ß-carotene production in these cultures. Raman spectroscopy is adaptable to large-scale fermentations and can give results in near real-time. Furthermore, we found that Raman spectroscopy was also able to measure the relative lipid compositions and protein content of the parental and SM14 strains at two different C:N ratios in the bioreactor. The Raman analysis showed a higher total fatty acid content in the SM14 compared with the parental strain and that an increased C:N ratio resulted in significant increase in total fatty acid content of both strains. The data suggest a positive correlation between the yield of ß-carotene per biomass and total fatty acid content of the cell.

Recent progress in biobutanol tolerance in microbial systems with an emphasis on Clostridium.

Biobased production of butanol promises a more sustainable route for industrial production. However, butanol toxicity remains a barrier for achieving high product titers. Investigation into butanol stress has shed some light on its modes of toxicity. Unfortunately, there still remain significant shortfalls in our understanding of the complex interactions of butanol with cells. To address this knowledge gap, a diverse range of tools have been employed to gain a better understanding of the adverse effects of butanol on the cell. These findings have lead to the identification of possible molecular mechanisms associated with butanol tolerance, which can be harnessed for future strain development efforts. This review focuses on recent efforts to address the toxicity of butanol in microbial producers and offers some perspectives on the future direction of this research sector.

Genome shuffling to generate recombinant yeasts for tolerance to inhibitors present in lignocellulosic hydrolysates.

OBJECTIVES: To investigate the use of genome shuffling to generate recombinants from previously generated hydrolysates-tolerant strains to improve tolerance of Saccharomyces cerevisiae to one or more inhibitory by-products present in lignocellulosic hydrolysates. RESULTS: Recombinants of previously evolved strains of S. cerevisiae were generated and analyzed for their relative performance in the individual inhibitors furfural, acetic acid, 5-(hydroxymethyl)-furfural (HMF) and in synthetic hydrolysates. One recombinant exhibited a 100 % fitness increase in the presence of HMF as compared to the wild-type diploid, while another stain exhibited a 13 % fitness increase in the presence of furfural. Furthermore, for one of these recombinants, these increases in fitness were specific to the inhibitor HMF and to synthetic hydrolysates rather than being due to a general increase in fitness. Mutations present in the evolved hydrolysates-tolerant mutants were identified via whole-genome resequencing. CONCLUSION: Recombinants of S. cerevisiae were produced with increased tolerance to inhibitory by-products present in hydrolysates of lignocellulosic biomass and identified potential genetic determinants associated with this phenotype.

Recent advances in the evolutionary engineering of industrial biocatalysts.

Evolutionary engineering has been used to improve key industrial strain traits, such as carbon source utilization, tolerance to adverse environmental conditions, and resistance to chemical inhibitors, for many decades due to its technical simplicity and effectiveness. The lack of need for prior genetic knowledge underlying the phenotypes of interest makes this a powerful approach for strain development for even species with minimal genotypic information. While the basic experimental procedure for laboratory adaptive evolution has remained broadly similar for many years, a range of recent advances show promise for improving the experimental workflows for evolutionary engineering by accelerating the pace of evolution, simplifying the analysis of evolved mutants, and providing new ways of linking desirable phenotypes to selectable characteristics. This review aims to highlight some of these recent advances and discuss how they may be used to improve industrially relevant microbial phenotypes.

Evolved osmotolerant Escherichia coli mutants frequently exhibit defective N-acetylglucosamine catabolism and point mutations in cell shape-regulating protein MreB.

Biocatalyst robustness toward stresses imposed during fermentation is important for efficient bio-based production. Osmotic stress, imposed by high osmolyte concentrations or dense populations, can significantly impact growth and productivity. In order to better understand the osmotic stress tolerance phenotype, we evolved sexual (capable of in situ DNA exchange) and asexual Escherichia coli strains under sodium chloride (NaCl) stress. All isolates had significantly improved growth under selection and could grow in up to 0.80 M (47 g/liter) NaCl, a concentration that completely inhibits the growth of the unevolved parental strains. Whole genome resequencing revealed frequent mutations in genes controlling N-acetylglucosamine catabolism (nagC, nagA), cell shape (mrdA, mreB), osmoprotectant uptake (proV), and motility (fimA). Possible epistatic interactions between nagC, nagA, fimA, and proV deletions were also detected when reconstructed as defined mutations. Biofilm formation under osmotic stress was found to be decreased in most mutant isolates, coupled with perturbations in indole secretion. Transcriptional analysis also revealed significant changes in ompACGL porin expression and increased transcription of sulfonate uptake systems in the evolved mutants. These findings expand our current knowledge of the osmotic stress phenotype and will be useful for the rational engineering of osmotic tolerance into industrial strains in the future.

Improving carotenoids production in yeast via adaptive laboratory evolution.

Adaptive laboratory evolution is an important tool for the engineering of strains for industrially relevant phenotypes. Traditionally, adaptive laboratory evolution has been implemented to improve robustness of industrial strains under diverse operational conditions; however due to the required coupling between growth and survival, its application for increased production of secondary metabolites generally results in decreased production due to the metabolic burden imposed by, or toxicity of, the produced compound. In this study, adaptive laboratory evolution was successfully applied to improve carotenoids production in an engineered Saccharomyces cerevisiae producer strain by exploiting the antioxidant properties of carotenoids. Short-term evolution experiment using periodic hydrogen peroxide shocking schemes resulted in a 3-fold increase in carotenoids production (from 6 mg/g dry cell weight to up to 18 mg/g dry cell weight). Subsequent transcriptome analysis was used to elucidate the molecular mechanisms for increased carotenoids production. Upregulation of genes related with lipid biosynthesis and mevalonate biosynthesis pathways were commonly observed in the carotenoids hyper-producers analyzed.