Ryan T. Gill

Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides

A fundamental goal in biotechnology and biology is the development of approaches to better understand the genetic basis of traits. Here we report a versatile method, trackable multiplex recombineering (TRMR), whereby thousands of specific genetic modifications are created and evaluated simultaneously. To demonstrate TRMR, in a single day we modified the expression of >95% of the genes in Escherichia coli by inserting synthetic DNA cassettes and molecular barcodes upstream of each gene. Barcode sequences and microarrays were then used to quantify population dynamics. Within a week we mapped thousands of genes that affect E. coli growth in various media (rich, minimal and cellulosic hydrolysate) and in the presence of several growth inhibitors (β-glucoside, D-fucose, valine and methylglyoxal). This approach can be applied to a broad range of traits to identify targets for future genome-engineering endeavors.

Cellulosic hydrolysate toxicity and tolerance mechanisms in Escherichia coli.

The sustainable production of biofuels will require the efficient utilization of lignocellulosic biomass. A key barrier involves the creation of growth-inhibitory compounds by chemical pretreatment steps, which ultimately reduce the efficiency of fermentative microbial biocatalysts. The primary toxins include organic acids, furan derivatives, and phenolic compounds. Weak acids enter the cell and dissociate, resulting in a drop in intracellular pH as well as various anion-specific effects on metabolism. Furan derivatives, dehydration products of hexose and pentose sugars, have been shown to hinder fermentative enzyme function. Phenolic compounds, formed from lignin, can disrupt membranes and are hypothesized to interfere with the function of intracellular hydrophobic targets. This review covers mechanisms of toxicity and tolerance for these compounds with a specific focus on the important industrial organism Escherichia coli. Recent efforts to engineer E. coli for improved tolerance to these toxins are also discussed.

Organic acid toxicity, tolerance, and production in Escherichia coli biorefining applications

Organic acids are valuable platform chemicals for future biorefining applications. Such applications involve the conversion of low-cost renewable resources to platform sugars, which are then converted to platform chemicals by fermentation and further derivatized to large-volume chemicals through conventional catalytic routes. Organic acids are toxic to many of the microorganisms, such as Escherichia coli, proposed to serve as biorefining platform hosts at concentrations well below what is required for economical production. The toxicity is two-fold including not only pH based growth inhibition but also anion-specific effects on metabolism that also affect growth. E. coli maintain viability at very low pH through several different tolerance mechanisms including but not limited to the use of decarboxylation reactions that consume protons, ion transporters that remove protons, increased expression of known stress genes, and changing membrane composition. The focus of this mini-review is on organic acid toxicity and associated tolerance mechanisms as well as several examples of successful organic acid production processes for E. coli.

SCALEs: multiscale analysis of library enrichment

We report a genome-wide, multiscale approach to simultaneously measure the effect that the increased copy of each gene and/or operon has on a desired trait or phenotype. The method involves (i) growth selections on a mixture of several different plasmid-based genomic libraries of defined insert sizes or SCALEs, (ii) microarray studies of enriched plasmid DNA, and a (iii) mathematical multiscale analysis that precisely identifies the relevant genetic elements. This approach allows for identification of all single open reading frames and larger multigene fragments within a genomic library that alter the expression of a given phenotype. We have demonstrated this method in Escherichia coli by monitoring, in parallel, a population of >106 genomic library clones of different insert sizes, throughout continuous selections over a period of 100 generations.

Application and engineering of fatty acid biosynthesis in Escherichia coli for advanced fuels and chemicals.

Research towards the commercialization of fungible biofuels has received a great deal of recent interest and investment. To this end the microbial production of fatty acid-derived fuels from sustainable feedstocks is emerging as a viable option with rapid advances from both industry and academia. The manipulation of the fatty acid biosynthesis pathway, especially in Escherichia coli, has been widely studied and several approaches that increase fatty acid production have been identified. However, further advances will be required for the economic large-scale production of fatty acid-derived biofuels. Here we present an overview of fatty acid biosynthesis and its regulation in E. coli from a metabolic engineering viewpoint and offer potential approaches and considerations for the microbial overproduction of custom designed fatty acids for use as biofuels or in the manufacture of oleochemicals.

Genome-Wide Dynamic Transcriptional Profiling of the Light-to-Dark Transition in Synechocystis sp. Strain PCC 6803

We report the results of whole-genome transcriptional profiling of the light-to-dark transition with the model photosynthetic prokaryote Synechocystis sp. strain PCC 6803 (Synechocystis). Experiments were conducted by growing Synechocystis cultures to mid-exponential phase and then exposing them to two cycles of light/dark conditions, during which RNA samples were obtained. These samples were probed with a full-genome DNA microarray (3,169 genes, 20 samples) as well as a partial-genome microarray (88 genes, 29 samples). We concluded that (i) 30-min sampling intervals accurately captured transcriptional dynamics throughout the light/dark transition, (ii) 25% of the Synechocystis genes (783 genes) responded positively to the presence of light, and (iii) the response dynamics varied greatly for individual genes, with a delay of up to 120 to 150 min for some genes. Four classes of genes were identified on the basis of their dynamic gene expression profiles: class I (108 genes, 30-min response time), class II (279 genes, 60 to 90 min), class III (258 genes, 120 to 150 min), and class IV (138 genes, 180 min). The dynamics of several transcripts from genes involved in photosynthesis and primary energy generation are discussed. Finally, we applied Fisher discriminant analysis to better visualize the progression of the overall transcriptional program throughout the light/dark transition and to determine those genes most indicative of the lighting conditions during growth.

Genome-wide screening for trait conferring genes using DNA microarrays

We report a DNA microarray-based method for genome-wide monitoring of competitively grown transformants to identify genes whose overexpression confers a specific cellular phenotype. Whereas transcriptional profiling identifies differentially expressed genes that are correlated with particular aspects of the cellular phenotype, this functional genomics approach determines genes that result in a specific physiology. This parallel gene-trait mapping method consists of transforming a strain with a genomic library, enriching the cell population in transformants containing the trait conferring gene(s), and finally using DNA microarrays to simultaneously isolate and identify the enriched gene inserts. Various methods of enrichment can be used; here, genes conferring low-level antibiotic resistance were identified by growth in selective media. We demonstrated the method by transforming Escherichia coli cells with a genomic E. coli library and selecting for transformants exhibiting a growth advantage in the presence of the anti-microbial agent Pine-Sol. Genes conferring Pine-Sol tolerance (19 genes) or sensitivity (27 genes) were identified by hybridizing, on DNA microarrays containing 1,160 E. coli gene probes, extra-chromosomal DNA isolated from transformed cells grown in the presence of various levels of Pine-Sol. Results were further validated by plating and sequencing of individual colonies, and also by assessing the Pine-Sol resistance of cells transformed with enriched plasmid library or individual resistance genes identified by the microarrays. Applications of this method beyond antibiotic resistance include identification of genes resulting in resistance to chemotherapeutic agents, genes yielding resistance to toxic products (recombinant proteins, chemical feedstocks) in industrial fermentations, genes providing enhanced growth in cell culture or high cell density fermentations, genes facilitating growth on unconventional substrates, and others.

Manipulating redox and ATP balancing for improved production of succinate in E. coli

Redox and energy balance plays a key role in determining microbial fitness. Efforts to redirect bacterial metabolism often involve overexpression and deletion of genes surrounding key central metabolites, such as pyruvate and acetyl-coA. In the case of metabolic engineering of Escherichia coli for succinate production, efforts have mainly focused on the manipulation of key pyruvate metabolizing enzymes. E. coli AFP111 strain lacking ldhA, pflB and ptsG encoded activities accumulates acetate and ethanol as well as shows poor anaerobic growth on rich and minimal media. To address these issues, we first deleted genes (adhE, ackA-pta) involved in byproduct formation downstream of acetyl-CoA followed by the deletion of iclR and pdhR to activate the glyoxylate pathway. Based on data from these studies, we hypothesized that the succinate productivity was limited by the insufficient ATP generation. Genome-scale thermodynamics-based flux balance analysis indicated that overexpression of ATP-forming PEPCK from Actinobacillus succinogenes in an ldhA, pflB and ptsG triple mutant strain could result in an increase in biomass and succinate flux. Testing of this prediction confirmed that PEPCK overexpression resulted in a 60% increase in biomass and succinate formation in the ldhA, pflB, ptsG mutant strain.

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.

Genes restoring redox balance in fermentation-deficient E. coli NZN111

The objectives of this study were to improve understanding of the biochemical mechanisms underlying the growth defects resulting from deletion of pflB and ldhA in E. coli (strain NZN111) and identify genes for which overexpression would relieve this growth defect. Our approach involved the application of a mixed library selection method [Lynch et al., 2007. SCALEs: multiscale analysis of library enrichment. Nature Methods 4, 87-93.] to identify genes for which increased copy number improved growth of E. coli NZN111 under microaerobic conditions. This method employs libraries that cover the genome at a higher resolution relative to the conventional library methods. Our results indicate that NZN111 is growth impaired primarily due to unusually high intracellular NADH/NAD(+) ratios as opposed to limitations in intracellular acetyl-coA pools or pyruvate accumulation. We report the effect of several genetic and biochemical methods for decreasing the intracellular redox ratio on NZN111 growth and succinate production, including strategies that result in a 5-fold increase in growth in M9 media under microaerobic conditions and up to a 20% increase in succinate production. This work provides new insights into the growth defects resulting from ldhA and pflB growth defects in E. coli as well as a list of genes that can be used to improve growth and production of succinate and other metabolic products where redox imbalances are a key limitation.