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.

Escherichia coli Data-Driven Strain Design Using Aggregated Adaptive Laboratory Evolution Mutational Data.

Microbes are being engineered for an increasingly large and diverse set of applications. However, the designing of microbial genomes remains challenging due to the general complexity of biological systems. Adaptive Laboratory Evolution (ALE) leverages nature's problem-solving processes to generate optimized genotypes currently inaccessible to rational methods. The large amount of public ALE data now represents a new opportunity for data-driven strain design. This study describes how novel strain designs, or genome sequences not yet observed in ALE experiments or published designs, can be extracted from aggregated ALE data and demonstrates this by designing, building, and testing three novel Escherichia coli strains with fitnesses comparable to ALE mutants. These designs were achieved through a meta-analysis of aggregated ALE mutations data (63 Escherichia coli K-12 MG1655 based ALE experiments, described by 93 unique environmental conditions, 357 independent evolutions, and 13 957 observed mutations), which additionally revealed global ALE mutation trends that inform on ALE-derived strain design principles. Such informative trends anticipate ALE-derived strain designs as largely gene-centric, as opposed to noncoding, and composed of a relatively small number of beneficial variants (approximately 6). These results demonstrate how strain design efforts can be enhanced by the meta-analysis of aggregated ALE data.

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.

Generation of a platform strain for ionic liquid tolerance using adaptive laboratory evolution.

BACKGROUND: There is a need to replace petroleum-derived with sustainable feedstocks for chemical production. Certain biomass feedstocks can meet this need as abundant, diverse, and renewable resources. Specific ionic liquids (ILs) can play a role in this process as promising candidates for chemical pretreatment and deconstruction of plant-based biomass feedstocks as they efficiently release carbohydrates which can be fermented. However, the most efficient pretreatment ILs are highly toxic to biological systems, such as microbial fermentations, and hinder subsequent bioprocessing of fermentative sugars obtained from IL-treated biomass. METHODS: To generate strains capable of tolerating residual ILs present in treated feedstocks, a tolerance adaptive laboratory evolution (TALE) approach was developed and utilized to improve growth of two different Escherichia coli strains, DH1 and K-12 MG1655, in the presence of two different ionic liquids, 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) and 1-butyl-3-methylimidazolium chloride ([C4C1Im]Cl). For multiple parallel replicate populations of E. coli, cells were repeatedly passed to select for improved fitness over the course of approximately 40 days. Clonal isolates were screened and the best performing isolates were subjected to whole genome sequencing. RESULTS: The most prevalent mutations in tolerant clones occurred in transport processes related to the functions of mdtJI, a multidrug efflux pump, and yhdP, an uncharacterized transporter. Additional mutations were enriched in processes such as transcriptional regulation and nucleotide biosynthesis. Finally, the best-performing strains were compared to previously characterized tolerant strains and showed superior performance in tolerance of different IL and media combinations (i.e., cross tolerance) with robust growth at 8.5% (w/v) and detectable growth up to 11.9% (w/v) [C2C1Im][OAc]. CONCLUSION: The generated strains thus represent the best performing platform strains available for bioproduction utilizing IL-treated renewable substrates, and the TALE method was highly successful in overcoming the general issue of substrate toxicity and has great promise for use in tolerance engineering.

Systems biology solutions for biochemical production challenges.

There is an urgent need to significantly accelerate the development of microbial cell factories to produce fuels and chemicals from renewable feedstocks in order to facilitate the transition to a biobased society. Methods commonly used within the field of systems biology including omics characterization, genome-scale metabolic modeling, and adaptive laboratory evolution can be readily deployed in metabolic engineering projects. However, high performance strains usually carry tens of genetic modifications and need to operate in challenging environmental conditions. This additional complexity compared to basic science research requires pushing systems biology strategies to their limits and often spurs innovative developments that benefit fields outside metabolic engineering. Here we survey recent advanced applications of systems biology methods in engineering microbial production strains for biofuels and -chemicals.

Genome-wide Escherichia coli stress response and improved tolerance towards industrially relevant chemicals.

BACKGROUND: Economically viable biobased production of bulk chemicals and biofuels typically requires high product titers. During microbial bioconversion this often leads to product toxicity, and tolerance is therefore a critical element in the engineering of production strains. RESULTS: Here, a systems biology approach was employed to understand the chemical stress response of Escherichia coli, including a genome-wide screen for mutants with increased fitness during chemical stress. Twelve chemicals with significant production potential were selected, consisting of organic solvent-like chemicals (butanol, hydroxy-γ-butyrolactone, 1,4-butanediol, furfural), organic acids (acetate, itaconic acid, levulinic acid, succinic acid), amino acids (serine, threonine) and membrane-intercalating chemicals (decanoic acid, geraniol). The transcriptional response towards these chemicals revealed large overlaps of transcription changes within and between chemical groups, with functions such as energy metabolism, stress response, membrane modification, transporters and iron metabolism being affected. Regulon enrichment analysis identified key regulators likely mediating the transcriptional response, including CRP, RpoS, OmpR, ArcA, Fur and GadX. These regulators, the genes within their regulons and the above mentioned cellular functions therefore constitute potential targets for increasing E. coli chemical tolerance. Fitness determination of genome-wide transposon mutants (Tn-seq) subjected to the same chemical stress identified 294 enriched and 336 depleted mutants and experimental validation revealed up to 60 % increase in mutant growth rates. Mutants enriched in several conditions contained, among others, insertions in genes of the Mar-Sox-Rob regulon as well as transcription and translation related gene functions. CONCLUSIONS: The combination of the transcriptional response and mutant screening provides general targets that can increase tolerance towards not only single, but multiple chemicals.

Transient overexpression of DNA adenine methylase enables efficient and mobile genome engineering with reduced off-target effects.

Homologous recombination of single-stranded oligonucleotides is a highly efficient process for introducing precise mutations into the genome of E. coli and other organisms when mismatch repair (MMR) is disabled. This can result in the rapid accumulation of off-target mutations that can mask desired phenotypes, especially when selections need to be employed following the generation of combinatorial libraries. While the use of inducible mutator phenotypes or other MMR evasion tactics have proven useful, reported methods either require non-mobile genetic modifications or costly oligonucleotides that also result in reduced efficiencies of replacement. Therefore a new system was developed, Transient Mutator Multiplex Automated Genome Engineering (TM-MAGE), that solves problems encountered in other methods for oligonucleotide-mediated recombination. TM-MAGE enables nearly equivalent efficiencies of allelic replacement to the use of strains with fully disabled MMR and with an approximately 12- to 33-fold lower off-target mutation rate. Furthermore, growth temperatures are not restricted and a version of the plasmid can be readily removed by sucrose counterselection. TM-MAGE was used to combinatorially reconstruct mutations found in evolved salt-tolerant strains, enabling the identification of causative mutations and isolation of strains with up to 75% increases in growth rate and greatly reduced lag times in 0.6 M NaCl.

Seven gene deletions in seven days: Fast generation of Escherichia coli strains tolerant to acetate and osmotic stress.

Generation of multiple genomic alterations is currently a time consuming process. Here, a method was established that enables highly efficient and simultaneous deletion of multiple genes in Escherichia coli. A temperature sensitive plasmid containing arabinose inducible lambda Red recombineering genes and a rhamnose inducible flippase recombinase was constructed to facilitate fast marker-free deletions. To further speed up the procedure, we integrated the arabinose inducible lambda Red recombineering genes and the rhamnose inducible FLP into the genome of E. coli K-12 MG1655. This system enables growth at 37 °C, thereby facilitating removal of integrated antibiotic cassettes and deletion of additional genes in the same day. Phosphorothioated primers were demonstrated to enable simultaneous deletions during one round of electroporation. Utilizing these methods, we constructed strains in which four to seven genes were deleted in E. coli W and E. coli K-12. The growth rate of an E. coli K-12 quintuple deletion strain was significantly improved in the presence of high concentrations of acetate and NaCl. In conclusion, we have generated a method that enables efficient and simultaneous deletion of multiple genes in several E. coli variants. The method enables deletion of up to seven genes in as little as seven days.

Combinatorial strategies for improving multiple-stress resistance in industrially relevant Escherichia coli strains.

High-cell-density fermentation for industrial production of chemicals can impose numerous stresses on cells due to high substrate, product, and by-product concentrations; high osmolarity; reactive oxygen species; and elevated temperatures. There is a need to develop platform strains of industrial microorganisms that are more tolerant toward these typical processing conditions. In this study, the growth of six industrially relevant strains of Escherichia coli was characterized under eight stress conditions representative of fed-batch fermentation, and strains W and BL21(DE3) were selected as platforms for transposon (Tn) mutagenesis due to favorable resistance characteristics. Selection experiments, followed by either targeted or genome-wide next-generation-sequencing-based Tn insertion site determination, were performed to identify mutants with improved growth properties under a subset of three stress conditions and two combinations of individual stresses. A subset of the identified loss-of-function mutants were selected for a combinatorial approach, where strains with combinations of two and three gene deletions were systematically constructed and tested for single and multistress resistance. These approaches allowed identification of (i) strain-background-specific stress resistance phenotypes, (ii) novel gene deletion mutants in E. coli that confer single and multistress resistance in a strain-background-dependent manner, and (iii) synergistic effects of multiple gene deletions that confer improved resistance over single deletions. The results of this study underscore the suboptimality and strain-specific variability of the genetic network regulating growth under stressful conditions and suggest that further exploration of the combinatorial gene deletion space in multiple strain backgrounds is needed for optimizing strains for microbial bioprocessing applications.

Microbial production of fatty acid-derived fuels and chemicals.

Fatty acid metabolism is an attractive route to produce liquid transportation fuels and commodity oleochemicals from renewable feedstocks. Recently, genes and enzymes, which comprise metabolic pathways for producing fatty acid-derived compounds (e.g. esters, alkanes, olefins, ketones, alcohols, polyesters) have been elucidated and used in engineered microbial hosts. The resulting strains often generate products at low percentages of maximum theoretical yields, leaving significant room for metabolic engineering. Economically viable processes will require strains to approach theoretical yields, particularly for replacement of petroleum-derived fuels. This review will describe recent progress toward this goal, highlighting the scientific discoveries of each pathway, ongoing biochemical studies to understand each enzyme, and metabolic engineering strategies that are being used to improve strain performance.

Modulating membrane composition alters free fatty acid tolerance in Escherichia coli.

Microbial synthesis of free fatty acids (FFA) is a promising strategy for converting renewable sugars to advanced biofuels and oleochemicals. Unfortunately, FFA production negatively impacts membrane integrity and cell viability in Escherichia coli, the dominant host in which FFA production has been studied. These negative effects provide a selective pressure against FFA production that could lead to genetic instability at industrial scale. In prior work, an engineered E. coli strain harboring an expression plasmid for the Umbellularia californica acyl-acyl carrier protein (ACP) thioesterase was shown to have highly elevated levels of unsaturated fatty acids in the cell membrane. The change in membrane content was hypothesized to be one underlying cause of the negative physiological effects associated with FFA production. In this work, a connection between the regulator of unsaturated fatty acid biosynthesis in E. coli, FabR, thioesterase expression, and unsaturated membrane content was established. A strategy for restoring normal membrane saturation levels and increasing tolerance towards endogenous production of FFAs was implemented by modulating acyl-ACP pools with a second thioesterase (from Geobacillus sp. Y412MC10) that primarily targets medium chain length, unsaturated acyl-ACPs. The strategy succeeded in restoring membrane content and improving viability in FFA producing E. coli while maintaining FFA titers. However, the restored fitness did not increase FFA productivity, indicating the existence of additional metabolic or regulatory barriers.

Identification of transport proteins involved in free fatty acid efflux in Escherichia coli.

Escherichia coli has been used as a platform host for studying the production of free fatty acids (FFA) and other energy-dense compounds useful in biofuel applications. Most of the FFA produced by E. coli are found extracellularly. This finding suggests that a mechanism for transport across the cell envelope exists, yet knowledge of proteins that may be responsible for export remains incomplete. Production of FFA has been shown to cause cell lysis, induce stress responses, and impair basic physiological processes. These phenotypes could potentially be diminished if efflux rates were increased. Here, a total of 15 genes and operons were deleted and screened for their impact on cell viability and titer in FFA-producing E. coli. Deletions of acrAB and rob and, to a lower degree of statistical confidence, emrAB, mdtEF, and mdtABCD reduced multiple measures of viability, while deletion of tolC nearly abolished FFA production. An acrAB emrAB deletion strain exhibited greatly reduced FFA titers approaching the tolC deletion phenotype. Expression of efflux pumps on multicopy plasmids did not improve endogenous FFA production in an acrAB(+) strain, but plasmid-based expression of acrAB, mdtEF, and an mdtEF-tolC artificial operon improved the MIC of exogenously added decanoate for an acrAB mutant strain. The findings suggest that AcrAB-TolC is responsible for most of the FFA efflux in E. coli, with residual activity provided by other resistance-nodulation-cell division superfamily-type efflux pumps, including EmrAB-TolC and MdtEF-TolC. While the expression of these proteins on multicopy plasmids did not improve production over the basal level, their identification enables future engineering efforts.

Engineering Escherichia coli to synthesize free fatty acids.

Fatty acid metabolism has received significant attention as a route for producing high-energy density, liquid transportation fuels and high-value oleochemicals from renewable feedstocks. If microbes can be engineered to produce these compounds at yields that approach the theoretical limits of 0.3-0.4 g/g glucose, then processes can be developed to replace current petrochemical technologies. Here, we review recent metabolic engineering efforts to maximize production of free fatty acids (FFA) in Escherichia coli, the first step towards production of downstream products. To date, metabolic engineers have succeeded in achieving higher yields of FFA than any downstream products. Regulation of fatty acid metabolism and the physiological effects of fatty acid production will also be reviewed from the perspective of identifying future engineering targets.

Kinetic modeling of free fatty acid production in Escherichia coli based on continuous cultivation of a plasmid free strain.

The microbial production of free fatty acids (FFAs) and reduced derivatives is an attractive process for the renewable production of diesel fuels. Toward this goal, a plasmid-free strain of Escherichia coli was engineered to produce FFAs by integrating three copies of a thioesterase gene from Umbellularia californica (BTE) under the control of an inducible promoter onto the chromosome. In batch culture, the resulting strain produced identical titers to a previously reported strain that expressed the thioesterase from a plasmid. The growth rate, glucose consumption rate, and FFA production rate of this strain were studied in continuous cultivation under carbon limitation. The highest yield of FFA on glucose was observed at a dilution rate of 0.05 h(-1) with the highest specific productivity observed at a dilution rate of 0.2 h(-1). The observed yields under the lowest dilution rate were 15% higher than that observed in batch cultures. An increase in both productivity and yield (≈ 40%) was observed when the composition of the nutrients was altered to shift the culture toward non-carbon limitation. A deterministic model of the production strain has been proposed and indicates that maintenance requirements for this strain are significantly higher than wild-type E. coli.

Isolation of improved free fatty acid overproducing strains of Escherichia coli via Nile red based high-throughput screening.

Biological production of hydrocarbons is an attractive strategy to produce drop-in replacement transportation fuels. Several methods for converting microbially-produced fatty acids into reduced compounds compatible with petrodiesel have been reported. For these processes to become economically viable, microorganisms must be engineered to approach the theoretical yield of fatty acid products from renewable feedstocks such as glucose. Strains with increased titers can be obtained through both rational and random approaches. While powerful, random approaches require a genetic selection or facile screen that is amenable to high throughput platforms. Here, we present the use of a high throughput screen for fatty acids based on the hydrophobic dye Nile red. The method was applied to screening a transposon library of a free fatty acid overproducing strain of Escherichia coli in search of high producing mutants. Ten gene targets were identified via primary and secondary screening. A strain comprising a clean knockout of one of the identified genes led to a 20% increase in titer over the baseline strain. A selection strategy that combines these findings and can act in an iterative fashion has been developed and can be used for future strain optimization in hydrocarbon producing strains.

Membrane stresses induced by overproduction of free fatty acids in Escherichia coli.

Microbially produced fatty acids are potential precursors to high-energy-density biofuels, including alkanes and alkyl ethyl esters, by either catalytic conversion of free fatty acids (FFAs) or enzymatic conversion of acyl-acyl carrier protein or acyl-coenzyme A intermediates. Metabolic engineering efforts aimed at overproducing FFAs in Escherichia coli have achieved less than 30% of the maximum theoretical yield on the supplied carbon source. In this work, the viability, morphology, transcript levels, and protein levels of a strain of E. coli that overproduces medium-chain-length FFAs was compared to an engineered control strain. By early stationary phase, an 85% reduction in viable cell counts and exacerbated loss of inner membrane integrity were observed in the FFA-overproducing strain. These effects were enhanced in strains endogenously producing FFAs compared to strains exposed to exogenously fed FFAs. Under two sets of cultivation conditions, long-chain unsaturated fatty acid content greatly increased, and the expression of genes and proteins required for unsaturated fatty acid biosynthesis were significantly decreased. Membrane stresses were further implicated by increased expression of genes and proteins of the phage shock response, the MarA/Rob/SoxS regulon, and the nuo and cyo operons of aerobic respiration. Gene deletion studies confirmed the importance of the phage shock proteins and Rob for maintaining cell viability; however, little to no change in FFA titer was observed after 24 h of cultivation. The results of this study serve as a baseline for future targeted attempts to improve FFA yields and titers in E. coli.

Bacterial production of free fatty acids from freshwater macroalgal cellulose.

The predominant strategy for using algae to produce biofuels relies on the overproduction of lipids in microalgae with subsequent conversion to biodiesel (methyl-esters) or green diesel (alkanes). Conditions that both optimize algal growth and lipid accumulation rarely overlap, and differences in growth rates can lead to wild species outcompeting the desired lipid-rich strains. Here, we demonstrate an alternative strategy in which cellulose contained in the cell walls of multicellular algae is used as a feedstock for cultivating biofuel-producing microorganisms. Cellulose was extracted from an environmental sample of Cladophora glomerata-dominated periphyton that was collected from Lake Mendota, WI, USA. The resulting cellulose cake was hydrolyzed by commercial enzymes to release fermentable glucose. The hydrolysis mixture was used to formulate an undefined medium that was able to support the growth, without supplementation, of a free fatty acid (FFA)-overproducing strain of Escherichia coli (Lennen et. al 2010). To maximize free fatty acid production from glucose, an isopropyl ß-D-1-thiogalactopyranoside (IPTG)-inducible vector was constructed to express the Umbellularia californica acyl-acyl carrier protein (ACP) thioesterase. Thioesterase expression was optimized by inducing cultures with 50 µM IPTG. Cell density and FFA titers from cultures grown on algae-based media reached 50% of those (∼90 µg/mL FFA) cultures grown on rich Luria-Bertani broth supplemented with 0.2% glucose. In comparison, cultures grown in two media based on AFEX-pretreated corn stover generated tenfold less FFA than cultures grown in algae-based media. This study demonstrates that macroalgal cellulose is a potential carbon source for the production of biofuels or other microbially synthesized compounds.

A process for microbial hydrocarbon synthesis: Overproduction of fatty acids in Escherichia coli and catalytic conversion to alkanes.

The development of renewable alternatives to diesel and jet fuels is highly desirable for the heavy transportation sector, and would offer benefits over the production and use of short-chain alcohols for personal transportation. Here, we report the development of a metabolically engineered strain of Escherichia coli that overproduces medium-chain length fatty acids via three basic modifications: elimination of beta-oxidation, overexpression of the four subunits of acetyl-CoA carboxylase, and expression of a plant acyl-acyl carrier protein (ACP) thioesterase from Umbellularia californica (BTE). The expression level of BTE was optimized by comparing fatty acid production from strains harboring BTE on plasmids with four different copy numbers. Expression of BTE from low copy number plasmids resulted in the highest fatty acid production. Up to a seven-fold increase in total fatty acid production was observed in engineered strains over a negative control strain (lacking beta-oxidation), with a composition dominated by C(12) and C(14) saturated and unsaturated fatty acids. Next, a strategy for producing undecane via a combination of biotechnology and heterogeneous catalysis is demonstrated. Fatty acids were extracted from a culture of an overproducing strain into an alkane phase and fed to a Pd/C plug flow reactor, where the extracted fatty acids were decarboxylated into saturated alkanes. The result is an enriched alkane stream that can be recycled for continuous extractions. Complete conversion of C(12) fatty acids extracted from culture to alkanes has been demonstrated yielding a concentration of 0.44 g L(-1) (culture volume) undecane.

Non-chemical biofouling control in heat exchangers and seawater piping systems using acoustic pulses generated by an electrical discharge.

Acoustic pulses generated by an electrical discharge (pulsed acoustics) were investigated as a means for biofouling control in two test formats, viz. a 5/8" outside diameter titanium tube and a mockup heat exchanger. The pulsed acoustic device, when operated at 17 kV, demonstrated 95% inhibition of microfouling over a 20 ft length of titanium tube over a 4-week period, comparable to chlorination in combination with a high-velocity flush. The pulsed acoustic device inhibited microfouling over a downstream distance of 15 ft, therefore, a single pulsed acoustic device is theoretically capable of protecting at least 30 ft of tube from microfouling (15 ft on either side of the device). A correlation between acoustic intensity in the frequency range 0.01-1 MHz and the level of biofouling inhibition was observed. The threshold acoustic intensity for microfouling inhibition was determined for this frequency range. It was also observed that the orientation of the device is critical to obtaining microfouling inhibition.