Aromatic aldehydes are useful in numerous applications, especially as flavors, fragrances, and pharmaceutical precursors. However, microbial synthesis of aldehydes is hindered by rapid, endogenous, and redundant conversion of aldehydes to their corresponding alcohols. We report the construction of an Escherichia coli K-12 MG1655 strain with reduced aromatic aldehyde reduction (RARE) that serves as a platform for aromatic aldehyde biosynthesis. Six genes with reported activity on the model substrate benzaldehyde were rationally targeted for deletion: three genes that encode aldo-keto reductases and three genes that encode alcohol dehydrogenases. Upon expression of a recombinant carboxylic acid reductase in the RARE strain and addition of benzoate during growth, benzaldehyde remained in the culture after 24 h, with less than 12% conversion of benzaldehyde to benzyl alcohol. Although individual overexpression results demonstrated that all six genes could contribute to benzaldehyde reduction in vivo, additional experiments featuring subset deletion strains revealed that two of the gene deletions were dispensable under the conditions tested. The engineered strain was next investigated for the production of vanillin from vanillate and succeeded in preventing formation of the byproduct vanillyl alcohol. A pathway for the biosynthesis of vanillin directly from glucose was introduced and resulted in a 55-fold improvement in vanillin titer when using the RARE strain versus the wild-type strain. Finally, synthesis of the chiral pharmaceutical intermediate L-phenylacetylcarbinol (L-PAC) was demonstrated from benzaldehyde and glucose upon expression of a recombinant mutant pyruvate decarboxylase in the RARE strain. Beyond allowing accumulation of aromatic aldehydes as end products in E. coli, the RARE strain expands the classes of chemicals that can be produced microbially via aldehyde intermediates.

Bacteria within biofilms secrete and surround themselves with an extracellular matrix, which serves as a first line of defense against antibiotic attack. Polysaccharides constitute major elements of the biofilm matrix and are implied in surface adhesion and biofilm organization, but their contributions to the resistance properties of biofilms remain largely elusive. Using a combination of static and continuous-flow biofilm experiments we show that Psl, one major polysaccharide in the Pseudomonas aeruginosa biofilm matrix, provides a generic first line of defense toward antibiotics with diverse biochemical properties during the initial stages of biofilm development. Furthermore, we show with mixed-strain experiments that antibiotic-sensitive “non-producing” cells lacking Psl can gain tolerance by integrating into Psl-containing biofilms. However, non-producers dilute the protective capacity of the matrix and hence, excessive incorporation can result in the collapse of resistance of the entire community. Our data also reveal that Psl mediated protection is extendible to E. coli and S. aureus in co-culture biofilms. Together, our study shows that Psl represents a critical first bottleneck to the antibiotic attack of a biofilm community early in biofilm development.

The depletion of disruptive variation caused by purifying natural selection (constraint) has been widely used to investigate protein-coding genes underlying human disorders, but attempts to assess constraint for non-protein-coding regions have proven more difficult. Here we aggregate, process, and release a dataset of 76,156 human genomes from the Genome Aggregation Database (gnomAD), the largest public open-access human genome reference dataset, and use this dataset to build a mutational constraint map for the whole genome. We present a refined mutational model that incorporates local sequence context and regional genomic features to detect depletions of variation across the genome. As expected, protein-coding sequences overall are under stronger constraint than non-coding regions. Within the non-coding genome, constrained regions are enriched for known regulatory elements and variants implicated in complex human diseases and traits, facilitating the triangulation of biological annotation, disease association, and natural selection to non-coding DNA analysis. More constrained regulatory elements tend to regulate more constrained protein-coding genes, while non-coding constraint captures additional functional information underrecognized by gene constraint metrics. We demonstrate that this genome-wide constraint map provides an effective approach for characterizing the non-coding genome and improving the identification and interpretation of functional human genetic variation.

The constitutive activation of the anoxic redox control transcriptional regulator (ArcA) in Escherichia coli during aerobic growth, with the consequent production of a strain that exhibits anaerobic physiology even in the presence of air, is reported in this work. Removal of three terminal cytochrome oxidase genes (cydAB, cyoABCD, and cbdAB) and a quinol monooxygenase gene (ygiN) from the E. coli K-12 MG1655 genome resulted in the activation of ArcA aerobically. These mutations resulted in reduction of the oxygen uptake rate by nearly 98% and production of D-lactate as a sole by-product under oxic and anoxic conditions. The knockout strain exhibited nearly identical physiological behaviors under both conditions, suggesting that the mutations resulted in significant metabolic and regulatory perturbations. In order to fully understand the physiology of this mutant and to identify underlying metabolic and regulatory reasons that prevent the transition from an aerobic to an anaerobic phenotype, we utilized whole-genome transcriptome analysis, 13 C tracing experiments, and physiological characterization. Our analysis showed that the deletions resulted in the activation of anaerobic respiration under oxic conditions and a consequential shift in the content of the quinone pool from ubiquinones to menaquinones. An increase in menaquinone concentration resulted in the activation of ArcA. The activation of the ArcB/ArcA regulatory system led to a major shift in the metabolic flux distribution through the central metabolism of the mutant strain. Flux analysis indicated that the mutant strain had undetectable fluxes around the tricarboxylic acid (TCA) cycle and elevated flux through glycolysis and anaplerotic input to oxaloacetate. Flux and transcriptomics data were highly correlated and showed similar patterns.Escherichia coli has been studied extensively with respect to its physiology, genetics, and metabolism. One of the unique features of its metabolism is the ability to support robust growth under both oxic and anoxic conditions (38). During aerobic growth, when oxygen is used as a terminal electron acceptor, E. coli divides rapidly and produces carbon dioxide and acetate as major growth by-products (38), representing an efficient form of energy metabolism. In the absence of oxygen, E. coli and other microorganisms rely on anaerobic respiration and fermentation in order to oxidize substrates, recycle electron carriers, and produce ATP (38). This metabolic versatility of E. coli allows it to survive and thrive over a wide range of conditions.Since the ability to produce a number of reduced by-products, such as organic acids and ethanol, is of importance in the field of metabolic engineering, the majority of metabolic engineering designs rely on anaerobic conditions (15,30,31). It has also been shown that E. coli strains developed for overproduction of commodity chemicals can further be improved using adaptive evolution strategies (15). Adaptive evolution is often performed through a series of dilutions allowing cells to...

Hyperthermophilic bacteria from the Thermotogales lineage can produce hydrogen by fermenting a wide range of carbohydrates. Previous experimental studies identified a large fraction of genes committed to carbohydrate degradation and utilization in the model bacterium Thermotoga maritima. Knowledge of these genes enabled comprehensive reconstruction of biochemical pathways comprising the carbohydrate utilization network. However, transcriptional factors (TFs) and regulatory mechanisms driving this network remained largely unknown. Here, we used an integrated approach based on comparative analysis of genomic and transcriptomic data for the reconstruction of the carbohydrate utilization regulatory networks in 11 Thermotogales genomes. We identified DNA-binding motifs and regulons for 19 orthologous TFs in the Thermotogales. The inferred regulatory network in T. maritima contains 181 genes encoding TFs, sugar catabolic enzymes and ABC-family transporters. In contrast to many previously described bacteria, a transcriptional regulation strategy of Thermotoga does not employ global regulatory factors. The reconstructed regulatory network in T. maritima was validated by gene expression profiling on a panel of mono- and disaccharides and by in vitro DNA-binding assays. The observed upregulation of genes involved in catabolism of pectin, trehalose, cellobiose, arabinose, rhamnose, xylose, glucose, galactose, and ribose showed a strong correlation with the UxaR, TreR, BglR, CelR, AraR, RhaR, XylR, GluR, GalR, and RbsR regulons. Ultimately, this study elucidated the transcriptional regulatory network and mechanisms controlling expression of carbohydrate utilization genes in T. maritima. In addition to improving the functional annotations of associated transporters and catabolic enzymes, this research provides novel insights into the evolution of regulatory networks in Thermotogales.

The generation of genome-scale data is becoming more routine, yet the subsequent analysis of omics data remains a significant challenge. Here, an approach that integrates multiple omics datasets with bioinformatics tools was developed that produces a detailed annotation of several microbial genomic features. This methodology was used to characterize the genome of Thermotoga maritima—a phylogenetically deep-branching, hyperthermophilic bacterium. Experimental data were generated for whole-genome resequencing, transcription start site (TSS) determination, transcriptome profiling, and proteome profiling. These datasets, analyzed in combination with bioinformatics tools, served as a basis for the improvement of gene annotation, the elucidation of transcription units (TUs), the identification of putative non-coding RNAs (ncRNAs), and the determination of promoters and ribosome binding sites. This revealed many distinctive properties of the T. maritima genome organization relative to other bacteria. This genome has a high number of genes per TU (3.3), a paucity of putative ncRNAs (12), and few TUs with multiple TSSs (3.7%). Quantitative analysis of promoters and ribosome binding sites showed increased sequence conservation relative to other bacteria. The 5′UTRs follow an atypical bimodal length distribution comprised of “Short” 5′UTRs (11–17 nt) and “Common” 5′UTRs (26–32 nt). Transcriptional regulation is limited by a lack of intergenic space for the majority of TUs. Lastly, a high fraction of annotated genes are expressed independent of growth state and a linear correlation of mRNA/protein is observed (Pearson r = 0.63, p<2.2×10−16 t-test). These distinctive properties are hypothesized to be a reflection of this organism's hyperthermophilic lifestyle and could yield novel insights into the evolutionary trajectory of microbial life on earth.

3-hydroxy-γ-butyrolactone (3HBL) is a versatile chiral synthon, deemed a top value-added chemical from biomass by the DOE. We recently reported the first biosynthetic pathway towards 3HBL and its hydrolyzed form, 3,4-dihydroxybutyric acid (3,4-DHBA) in recombinant Escherichia coli using glucose and glycolic acid as feedstocks and briefly described their synthesis solely from glucose. Synthesis from glucose requires integration of the endogenous glyoxylate shunt with the 3,4-DHBA/3HBL pathway and co-overexpression of seven genes, posing challenges with respect to expression, repression of the glyoxylate shunt and optimal carbon distribution between the two pathways. Here we discuss engineering this integration. While appropriate media and over-expression of glyoxylate shunt enzymes helped overcome repression, two orthogonal expression systems were employed to address the expression and carbon distribution challenge. Synthesis of up to 0.3g/L of 3HBL and 0.7g/L of 3,4-DHBA solely from glucose was demonstrated, amounting to 24% of the theoretical maximum.

Metabolic engineering efforts require enzymes that are both highly active and specific toward the synthesis of a desired output product to be commercially feasible. The 3-hydroxyacid (3HA) pathway, also known as the reverse β-oxidation or coenzyme-A-dependent chain-elongation pathway, can allow for the synthesis of dozens of useful compounds of various chain lengths and functionalities. However, this pathway suffers from byproduct formation, which lowers the yields of the desired longer chain products, as well as increases downstream separation costs. The thiolase enzyme catalyzes the first reaction in this pathway, and its substrate specificity at each of its two catalytic steps sets the chain length and composition of the chemical scaffold upon which the other downstream enzymes act. However, there have been few attempts reported in the literature to rationally engineer thiolase substrate specificity. In this study, we present a model-guided, rational design study of ordered substrate binding applied to two biosynthetic thiolases, with the goal of increasing the ratio of C6/C4 products formed by the 3HA pathway, 3-hydroxy-hexanoic acid and 3-hydroxybutyric acid. We identify thiolase mutants that result in nearly 10-fold increases in C6/C4 selectivity. Our findings can extend to other pathways that employ the thiolase for chain elongation, as well as expand our knowledge of sequence-structure-function relationship for this important class of enzymes.