Pharmacy residency training program increases residents' confidence in inpatient medical emergency response.

DISCLAIMER: In an effort to expedite the publication of articles, AJHP is posting manuscripts online as soon as possible after acceptance. Accepted manuscripts have been peer-reviewed and copyedited, but are posted online before technical formatting and author proofing. These manuscripts are not the final version of record and will be replaced with the final article (formatted per AJHP style and proofed by the authors) at a later time. PURPOSE: The benefits of pharmacists' involvement in medical emergencies are well established, but optimal methods of training pharmacists for emergency response are unknown. The primary objective of this report is to describe the design and evaluation of a pharmacy resident medical emergency response training (PR-MERT) program for preparing trainees to respond to hospital medical emergencies, including cardiac arrest and rapid sequence intubation. SUMMARY: The PR-MERT program was a year-long longitudinal experience designed to prepare postgraduate year 1 pharmacy residents for medical emergency response. During the first month, the residents completed an orientation session that encompassed several lectures, certification by the American Heart Association in basic life support and advanced cardiovascular life support, standardized simulation scenarios, and mock medical emergencies. The trainees continued to utilize these skills and clinical knowledge through a longitudinal didactic lecture series, resident case conferences, and practice-based application by responding to real-life medical emergencies. Residents were assessed and coached throughout the program by clinical pharmacy preceptors and a "code coach" with extensive medical emergency response experience. After the year-long training, residents completed an anonymous survey assessing self-confidence and the structure of the program. The results showed improved confidence in medication selection and dosing, as well as anticipating the needs of the team and speaking up in cardiac arrest and RSI situations. Residents were satisfied with the training offered and structure of the program. CONCLUSION: The development of a PR-MERT program at an academic medical center was successful in achieving longitudinal learning objectives and improving residents' confidence in responding to medical emergencies. The implementation of a similar medical emergency training curriculum in inpatient pharmacy residency programs may be beneficial.

Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield.

Phage therapy to treat life-threatening drug-resistant infections has been hampered by technical challenges in phage production. Cell-free bacteriophage synthesis (CFBS) can overcome the limitations of standard phage production methods by manufacturing phage virions in vitro. CFBS mimics intracellular phage assembly using transcription/translation machinery (TXTL) harvested from bacterial lysates and combined with reagents to synthesize proteins encoded by a phage genomic DNA template. These systems may enable rapid phage production and engineering to accelerate phages from bench-to-bedside. TXTL harvested from wild type or commonly used bacterial strains was not optimized for bacteriophage production. Here, we demonstrate that TXTL from genetically modified E. coli BL21 can be used to enhance phage T7 yields in vitro by CFBS. Expression of 18 E. coli BL21 genes was manipulated by inducible CRISPR interference (CRISPRi) mediated by nuclease deficient Cas12a from F. novicida (dFnCas12a) to identify genes implicated in T7 propagation as positive or negative effectors. Genes shown to have a significant effect were overexpressed (positive effectors) or repressed (negative effectors) to modify the genetic background of TXTL harvested for CFBS. Phage T7 CFBS yields were improved by up to 10-fold in vitro through overexpression of translation initiation factor IF-3 (infC) and small RNAs OxyS and CyaR and by repression of RecC subunit exonuclease RecBCD. Continued improvement of CFBS will mitigate phage manufacturing bottlenecks and lower hurdles to widespread adoption of phage therapy.

Single and multiplexed gene repression in solventogenic Clostridium via Cas12a-based CRISPR interference.

The Gram-positive, spore-forming, obligate anaerobic firmicute species that make up the Clostridium genus have broad feedstock consumption capabilities and produce value-added metabolic products, but genetic manipulation is difficult, limiting their broad appeal. CRISPR-Cas systems have recently been applied to Clostridium species, primarily using Cas9 as a counterselection marker in conjunction with plasmid-based homologous recombination. CRISPR interference is a method that reduces gene expression of specific genes via precision targeting of a nuclease deficient Cas effector protein. Here, we develop a dCas12a-based CRISPR interference system for transcriptional gene repression in multiple mesophilic Clostridium species. We show the Francisella novicida Cas12a-based system has a broader applicability due to the low GC content in Clostridium species compared to CRISPR Cas systems derived from other bacteria. We demonstrate >99% reduction in transcript levels of targeted genes in Clostridium acetobutylicum and >75% reduction in Clostridium pasteurianum. We also demonstrate multiplexed repression via use of a single synthetic CRISPR array, achieving 99% reduction in targeted gene expression and elucidating a unique metabolic profile for their reduced expression. Overall, this work builds a foundation for high throughput genetic screens without genetic editing, a key limitation in current screening methods used in the Clostridium community.

Elucidation of Sequence-Function Relationships for an Improved Biobutanol In Vivo Biosensor in E. coli.

Transcription factor (TF)-promoter pairs have been repurposed from native hosts to provide tools to measure intracellular biochemical production titer and dynamically control gene expression. Most often, native TF-promoter systems require rigorous screening to obtain desirable characteristics optimized for biotechnological applications. High-throughput techniques may provide a rational and less labor-intensive strategy to engineer user-defined TF-promoter pairs using fluorescence-activated cell sorting and deep sequencing methods (sort-seq). Based on the designed promoter library's distribution characteristics, we elucidate sequence-function interactions between the TF and DNA. In this work, we use the sort-seq method to study the sequence-function relationship of a σ54-dependent, butanol-responsive TF-promoter pair, BmoR-PBMO derived from Thauera butanivorans, at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range. Activities of promoters from a mutagenized PBMO library were sorted based on gfp expression and subsequently deep sequenced to correlate site-specific sequences with changes in dynamic range. We identified site-specific mutations that increase the sensor output. Double mutant and a single mutant, CA(129,130)TC and G(205)A, in PBMO promoter increased dynamic ranges of 4-fold and 1.65-fold compared with the native system, respectively. In addition, sort-seq identified essential sites required for the proper function of the σ54-dependent promoter biosensor in the context of the host. This work can enable high-throughput screening methods for strain development.

Transient ammonia stress on Chinese hamster ovary (CHO) cells yield alterations to alanine metabolism and IgG glycosylation profiles.

BACKGROUND: Ammonia concentrations typically increase during mammalian cell cultures, mainly due to glutamine and other amino acid consumption. An early ammonia stress indicator is a metabolic shift with respect to alanine. To determine the underlying mechanisms of this metabolic shift, a Chinese hamster ovary (CHO) cell line with two distinct ages (standard and young) was cultured in parallel fed-batch bioreactors with 0 mM or 10 mM ammonia added at 12 h. Reduced viable cell densities were observed for the stressed cells, while viability was not significantly affected. The stressed cultures had higher alanine, lactate, and glutamate accumulation. Interestingly, the ammonia concentrations were similar by Day 8.5 for all cultures. We hypothesized the ammonia was converted to alanine as a coping mechanism. Interestingly, no significant differences were observed for metabolite profiles due to cell age. Glycosylation analysis showed the ammonia stress reduced galactosylation, sialylation, and fucosylation. Transcriptome analysis of the standard-aged cultures indicated the ammonia stress had a limited impact on the transcriptome, where few of the significant changes were directly related metabolite or glycosylation reactions. These results indicate that mechanisms used to alleviate ammonia stress are most likely controlled post-transcriptionally, and this is where future research should focus.

Transcription factor-based biosensors and inducible systems in non-model bacteria: current progress and future directions.

Genetic diversity within the geobiosphere encompasses enormous sensing capabilities and many non-model bacteria are of biotechnological interest. Biosensing, or more generally inducible, systems are a vital component of metabolic engineering, as they allow tight control of gene expression as well as the basis for high-throughput screens on non-growth-related phenotypes. While these inducible systems, primarily transcription factor/promoter pairs, have been utilized extensively in Escherichia coli, progress in other bacteria is limited because of differences in transcription machinery, physiological compatibility of parts and proteins, and other nuances. Here, we provide an overview of the available genetic biosensing elements in non-model organisms and state-of-the-art efforts to engineer them, and then discuss challenges preventing these methods from common use in non-model bacteria.

Clusters of Nanoscale Liposomes Modulate the Release of Encapsulated Species and Mimic the Compartmentalization Intrinsic in Cell Structures.

We report the ability to place a high concentration of liposomes in a confined volume as a multicompartment cluster that mimics biological cells and allows for the modulation of release of encapsulated species. The formation of these coated multicompartmental structures is achieved by first binding liposomes into clusters before encapsulating them within a two-dimensional metal-organic framework composed of tannic acid coordinated with a metal ion. The essential feature is a molecularly thin skin over a ssystem of clustered liposomes in a pouch. The structural features of these pouches are revealed by small-angle scattering and electron microscopy. Through cryogenic electron microscopy, clusters with intact liposomes are observed that appear to be encapsulated within a pouch. Small-angle X-ray scattering shows the emergence of a relatively weak Bragg peak at q = 0.125 Å-1, possibly indicating the attachment of the bilayers of adjacent liposomes. The metal-phenolic network (MPN) forms a nanosized conformal coating around liposome clusters, resulting in the reduced release rate of the encapsulated rhodamine B dye. We further show the possibility of communication between the adjacent nanocompartments in the cluster by demonstrating enhanced energy transfer using fluorescence resonance energy transfer (FRET) experiments where the lipophilic donor dye 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) incorporated within one liposomal compartment transfers energy upon excitation to the lipophilic acceptor dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) in a neighboring liposomal compartment due to their close proximity within the multicompartmental cluster. These observations have significance in adapting these multicompartmental structures that mimic biological cells for cascade reactions and as new depot drug delivery systems.

Recent Developments of the Synthetic Biology Toolkit for Clostridium.

The Clostridium genus is a large, diverse group consisting of Gram-positive, spore-forming, obligate anaerobic firmicutes. Among this group are historically notorious pathogens as well as several industrially relevant species with the ability to produce chemical commodities, particularly biofuels, from renewable biomass. Additionally, other species are studied for their potential use as therapeutics. Although metabolic engineering and synthetic biology have been instrumental in improving product tolerance, titer, yields, and feed stock consumption capabilities in several organisms, low transformation efficiencies and lack of synthetic biology tools and genetic parts make metabolic engineering within the Clostridium genus difficult. Progress has recently been made to overcome challenges associated with engineering various Clostridium spp. For example, developments in CRISPR tools in multiple species and strains allow greater capability to produce edits with greater precision, faster, and with higher efficiencies. In this mini-review, we will highlight these recent advances and compare them to established methods for genetic engineering in Clostridium. In addition, we discuss the current state and development of Clostridium-based promoters (constitutive and inducible) and reporters. Future progress in this area will enable more rapid development of strain engineering, which would allow for the industrial exploitation of Clostridium for several applications including bioproduction of several commodity products.

Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol.

Tight and tunable control of gene expression is a highly desirable goal in synthetic biology for constructing predictable gene circuits and achieving preferred phenotypes. Elucidating the sequence-function relationship of promoters is crucial for manipulating gene expression at the transcriptional level, particularly for inducible systems dependent on transcriptional regulators. Sort-seq methods employing fluorescence-activated cell sorting (FACS) and high-throughput sequencing allow for the quantitative analysis of sequence-function relationships in a robust and rapid way. Here we utilized a massively parallel sort-seq approach to analyze the formaldehyde-inducible Escherichia coli promoter (Pfrm) with single-nucleotide resolution. A library of mutated formaldehyde-inducible promoters was cloned upstream of gfp on a plasmid. The library was partitioned into bins via FACS on the basis of green fluorescent protein (GFP) expression level, and mutated promoters falling into each expression bin were identified with high-throughput sequencing. The resulting analysis identified two 19 base pair repressor binding sites, one upstream of the -35 RNA polymerase (RNAP) binding site and one overlapping with the -10 site, and assessed the relative importance of each position and base therein. Key mutations were identified for tuning expression levels and were used to engineer formaldehyde-inducible promoters with predictable activities. Engineered variants demonstrated up to 14-fold lower basal expression, 13-fold higher induced expression, and a 3.6-fold stronger response as indicated by relative dynamic range. Finally, an engineered formaldehyde-inducible promoter was employed to drive the expression of heterologous methanol assimilation genes and achieved increased biomass levels on methanol, a non-native substrate of E. coli.

Enzyme I facilitates reverse flux from pyruvate to phosphoenolpyruvate in Escherichia coli.

The bacterial phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) consists of cascading phosphotransferases that couple the simultaneous import and phosphorylation of a variety of sugars to the glycolytic conversion of phosphoenolpyruvate (PEP) to pyruvate. As the primary route of glucose uptake in E. coli, the PTS plays a key role in regulating central carbon metabolism and carbon catabolite repression, and is a frequent target of metabolic engineering interventions. Here we show that Enzyme I, the terminal phosphotransferase responsible for the conversion of PEP to pyruvate, is responsible for a significant in vivo flux in the reverse direction (pyruvate to PEP) during both gluconeogenic and glycolytic growth. We use 13C alanine tracers to quantify this back-flux in single and double knockouts of genes relating to PEP synthetase and PTS components. Our findings are relevant to metabolic engineering design and add to our understanding of gene-reaction connectivity in E. coli.

Engineering membrane and cell-wall programs for tolerance to toxic chemicals: Beyond solo genes.

Metabolite toxicity in microbes, particularly at the membrane, remains a bottleneck in the production of fuels and chemicals. Under chemical stress, native adaptation mechanisms combat hyper-fluidization by modifying the phospholipids in the membrane. Recent work in fluxomics reveals the mechanism of how membrane damage negatively affects energy metabolism while lipidomic and transcriptomic analyses show that strains evolved to be tolerant maintain membrane fluidity under stress through a variety of mechanisms such as incorporation of cyclopropanated fatty acids, trans-unsaturated fatty acids, and upregulation of cell wall biosynthesis genes. Engineered strains with modifications made in the biosynthesis of fatty acids, peptidoglycan, and lipopolysaccharide have shown increased tolerance to exogenous stress as well as increased production of desired metabolites of industrial importance. We review recent advances in elucidation of mechanisms or toxicity and tolerance as well as efforts to engineer the bacterial membrane and cell wall.

Co-utilization of glucose and xylose by evolved Thermus thermophilus LC113 strain elucidated by (13)C metabolic flux analysis and whole genome sequencing.

We evolved Thermus thermophilus to efficiently co-utilize glucose and xylose, the two most abundant sugars in lignocellulosic biomass, at high temperatures without carbon catabolite repression. To generate the strain, T. thermophilus HB8 was first evolved on glucose to improve its growth characteristics, followed by evolution on xylose. The resulting strain, T. thermophilus LC113, was characterized in growth studies, by whole genome sequencing, and (13)C-metabolic flux analysis ((13)C-MFA) with [1,6-(13)C]glucose, [5-(13)C]xylose, and [1,6-(13)C]glucose+[5-(13)C]xylose as isotopic tracers. Compared to the starting strain, the evolved strain had an increased growth rate (~2-fold), increased biomass yield, increased tolerance to high temperatures up to 90°C, and gained the ability to grow on xylose in minimal medium. At the optimal growth temperature of 81°C, the maximum growth rate on glucose and xylose was 0.44 and 0.46h(-1), respectively. In medium containing glucose and xylose the strain efficiently co-utilized the two sugars. (13)C-MFA results provided insights into the metabolism of T. thermophilus LC113 that allows efficient co-utilization of glucose and xylose. Specifically, (13)C-MFA revealed that metabolic fluxes in the upper part of metabolism adjust flexibly to sugar availability, while fluxes in the lower part of metabolism remain relatively constant. Whole genome sequence analysis revealed two large structural changes that can help explain the physiology of the evolved strain: a duplication of a chromosome region that contains many sugar transporters, and a 5x multiplication of a region on the pVV8 plasmid that contains xylose isomerase and xylulokinase genes, the first two enzymes of xylose catabolism. Taken together, (13)C-MFA and genome sequence analysis provided complementary insights into the physiology of the evolved strain.

Characterization of physiological responses to 22 gene knockouts in Escherichia coli central carbon metabolism.

Understanding the impact of gene knockouts on cellular physiology, and metabolism in particular, is centrally important to quantitative systems biology and metabolic engineering. Here, we present a comprehensive physiological characterization of wild-type Escherichia coli and 22 knockouts of enzymes in the upper part of central carbon metabolism, including the PTS system, glycolysis, pentose phosphate pathway and Entner-Doudoroff pathway. Our results reveal significant metabolic changes that are affected by specific gene knockouts. Analysis of collective trends and correlations in the data using principal component analysis (PCA) provide new, and sometimes surprising, insights into E. coli physiology. Additionally, by comparing the data-to-model predictions from constraint-based approaches such as FBA, MOMA and RELATCH we demonstrate the important role of less well-understood kinetic and regulatory effects in central carbon metabolism.

Whole-genome sequence of an evolved Clostridium pasteurianum strain reveals Spo0A deficiency responsible for increased butanol production and superior growth.

BACKGROUND: Biodiesel production results in crude glycerol waste from the transesterification of fatty acids (10 % w/w). The solventogenic Clostridium pasteurianum, an anaerobic Firmicute, can produce butanol from glycerol as the sole carbon source. Coupling butanol fermentation with biodiesel production can improve the overall economic viability of biofuels. However, crude glycerol contains growth-inhibiting byproducts which reduce feedstock consumption and solvent production. RESULTS: To obtain a strain with improved characteristics, a random mutagenesis and directed evolution selection technique was used. A wild-type C. pasteurianum (ATCC 6013) culture was chemically mutagenized, and the resulting population underwent 10 days of selection in increasing concentrations of crude glycerol (80-150 g/L). The best-performing mutant (M150B) showed a 91 % increase in butanol production in 100 g/L crude glycerol compared to the wild-type strain, as well as increased growth rate, a higher final optical density, and less production of the side product PDO (1,3-propanediol). Wild-type and M150B strains were sequenced via Single Molecule Real-Time (SMRT) sequencing. Mutations introduced to the M150B genome were identified by sequence comparison to the wild-type and published closed sequences. A major mutation (a deletion) in the gene of the master transcriptional regulator of sporulation, Spo0A, was identified. A spo0A single gene knockout strain was constructed using a double--crossover genome-editing method. The Spo0A-deficient strain showed similar tolerance to crude glycerol as the evolved mutant strain M150B. Methylation patterns on genomic DNA identified by SMRT sequencing were used to transform plasmid DNA to overcome the native C. pasteurianum restriction endonuclease. CONCLUSIONS: Solvent production in the absence of Spo0A shows C. pasteurianum differs in solvent-production regulation compared to other solventogenic Clostridium. Growth-associated butanol production shows C. pasteurianum to be an attractive option for further engineering as it may prove a better candidate for butanol production through continuous fermentation.

Expression of heterologous sigma factors enables functional screening of metagenomic and heterologous genomic libraries.

A key limitation in using heterologous genomic or metagenomic libraries in functional genomics and genome engineering is the low expression of heterologous genes in screening hosts, such as Escherichia coli. To overcome this limitation, here we generate E. coli strains capable of recognizing heterologous promoters by expressing heterologous sigma factors. Among seven sigma factors tested, RpoD from Lactobacillus plantarum (Lpl) appears to be able of initiating transcription from all sources of DNA. Using the promoter GFP-trap concept, we successfully screen several heterologous and metagenomic DNA libraries, thus enlarging the genomic space that can be functionally sampled in E. coli. For an application, we show that screening fosmid-based Lpl genomic libraries in an E. coli strain with a chromosomally integrated Lpl rpoD enables the identification of Lpl genetic determinants imparting strong ethanol tolerance in E. coli. Transcriptome analysis confirms increased expression of heterologous genes in the engineered strain.

Synthetic methylotrophy: engineering the production of biofuels and chemicals based on the biology of aerobic methanol utilization.

Synthetic methylotrophy is the development of non-native methylotrophs that can utilize methane and methanol as sole carbon and energy sources or as co-substrates with carbohydrates to produce metabolites as biofuels and chemicals. The availability of methane (from natural gas) and its oxidation product, methanol, has been increasing, while prices have been decreasing, thus rendering them as attractive fermentation substrates. As they are more reduced than most carbohydrates, methane and methanol, as co-substrates, can enhance the yields of biologically produced metabolites. Here we discuss synthetic biology and metabolic engineering strategies based on the native biology of aerobic methylotrophs for developing synthetic strains grown on methanol, with Escherichia coli as the prototype.

Comparison of genome-wide selection strategies to identify furfural tolerance genes in Escherichia coli.

Engineering both feedstock and product tolerance is important for transitioning towards next-generation biofuels derived from renewable sources. Tolerance to chemical inhibitors typically results in complex phenotypes, for which multiple genetic changes must often be made to confer tolerance. Here, we performed a genome-wide search for furfural-tolerant alleles using the TRackable Multiplex Recombineering (TRMR) method (Warner et al. (2010), Nature Biotechnology), which uses chromosomally integrated mutations directed towards increased or decreased expression of virtually every gene in Escherichia coli. We employed various growth selection strategies to assess the role of selection design towards growth enrichments. We also compared genes with increased fitness from our TRMR selection to those from a previously reported genome-wide identification study of furfural tolerance genes using a plasmid-based genomic library approach (Glebes et al. (2014) PLOS ONE). In several cases, growth improvements were observed for the chromosomally integrated promoter/RBS mutations but not for the plasmid-based overexpression constructs. Through this assessment, four novel tolerance genes, ahpC, yhjH, rna, and dicA, were identified and confirmed for their effect on improving growth in the presence of furfural.

Genome-wide mapping of furfural tolerance genes in Escherichia coli.

Advances in genomics have improved the ability to map complex genotype-to-phenotype relationships, like those required for engineering chemical tolerance. Here, we have applied the multiSCale Analysis of Library Enrichments (SCALEs; Lynch et al. (2007) Nat. Method.) approach to map, in parallel, the effect of increased dosage for >10(5) different fragments of the Escherichia coli genome onto furfural tolerance (furfural is a key toxin of lignocellulosic hydrolysate). Only 268 of >4,000 E. coli genes (∼ 6%) were enriched after growth selections in the presence of furfural. Several of the enriched genes were cloned and tested individually for their effect on furfural tolerance. Overexpression of thyA, lpcA, or groESL individually increased growth in the presence of furfural. Overexpression of lpcA, but not groESL or thyA, resulted in increased furfural reduction rate, a previously identified mechanism underlying furfural tolerance. We additionally show that plasmid-based expression of functional LpcA or GroESL is required to confer furfural tolerance. This study identifies new furfural tolerant genes, which can be applied in future strain design efforts focused on the production of fuels and chemicals from lignocellulosic hydrolysate.

Strategy for directing combinatorial genome engineering in Escherichia coli.

We describe a directed genome-engineering approach that combines genome-wide methods for mapping genes to traits [Warner JR, Reeder PJ, Karimpour-Fard A, Woodruff LBA, Gill RT (2010) Nat Biotechnol 28:856-862] with strategies for rapidly creating combinatorial ribosomal binding site (RBS) mutation libraries containing billions of targeted modifications [Wang HH, et al. (2009) Nature 460:894-898]. This approach should prove broadly applicable to various efforts focused on improving production of fuels, chemicals, and pharmaceuticals, among other products. We used barcoded promoter mutation libraries to map the effect of increased or decreased expression of nearly every gene in Escherichia coli onto growth in several model environments (cellulosic hydrolysate, low pH, and high acetate). Based on these data, we created and evaluated RBS mutant libraries (containing greater than 100,000,000 targeted mutations), targeting the genes identified to most affect growth. On laboratory timescales, we successfully identified a broad range of mutations (>25 growth-enhancing mutations confirmed), which improved growth rate 10-200% for several different conditions. Although successful, our efforts to identify superior combinations of growth-enhancing genes emphasized the importance of epistatic interactions among the targeted genes (synergistic, antagonistic) for taking full advantage of this approach to directed genome engineering.

Elucidating acetate tolerance in E. coli using a genome-wide approach.

Engineering organisms for improved performance using lignocellulose feedstocks is an important step towards a sustainable fuel and chemical industry. Cellulosic feedstocks contain carbon and energy in the form of cellulosic and hemicellulosic sugars that are not metabolized by most industrial microorganisms. Pretreatment processes that hydrolyze these polysaccharides often also result in the accumulation of growth inhibitory compounds, such as acetate and furfural among others. Here, we have applied a recently reported strategy for engineering tolerance towards the goal of increasing Escherichia coli growth in the presence of elevated acetate concentrations (Lynch et al., 2007). We performed growth selections upon an E. coli genome library developed using a moderate selection pressure to identify genomic regions implicated in acetate toxicity and tolerance. These studies identified a range of high-fitness genes that are normally involved in membrane and extracellular processes, are key regulated steps in pathways, and are involved in pathways that yield specific amino acids and nucleotides. Supplementation of the products and metabolically related metabolites of these pathways significantly increased growth rate (a 130% increase in specific growth) at inhibitory acetate concentrations. Our results suggest that acetate tolerance will not involve engineering of a single pathway; rather we observe a range of potential mechanisms for overcoming acetate based inhibition.