Multiplexed mobilization and expression of biosynthetic gene clusters.

Bacterial genomes contain large reservoirs of biosynthetic gene clusters (BGCs) that are predicted to encode unexplored natural products. Heterologous expression of previously unstudied BGCs should facilitate the discovery of additional therapeutically relevant bioactive molecules from bacterial culture collections, but the large-scale manipulation of BGCs remains cumbersome. Here, we describe a method to parallelize the identification, mobilization and heterologous expression of BGCs. Our solution simultaneously captures large numbers of BGCs by cloning the genomes of a strain collection in a large-insert library and uses the CONKAT-seq (co-occurrence network analysis of targeted sequences) sequencing pipeline to efficiently localize clones carrying intact BGCs which represent candidates for heterologous expression. Our discovery of several natural products, including an antibiotic that is active against multi-drug resistant Staphylococcus aureus, demonstrates the potential of leveraging economies of scale with this approach to systematically interrogate cryptic BGCs contained in strain collections.

Functional reconstitution of a bacterial CO2 concentrating mechanism in Escherichia coli.

Many photosynthetic organisms employ a CO2 concentrating mechanism (CCM) to increase the rate of CO2 fixation via the Calvin cycle. CCMs catalyze ≈50% of global photosynthesis, yet it remains unclear which genes and proteins are required to produce this complex adaptation. We describe the construction of a functional CCM in a non-native host, achieved by expressing genes from an autotrophic bacterium in an Escherichia coli strain engineered to depend on rubisco carboxylation for growth. Expression of 20 CCM genes enabled E. coli to grow by fixing CO2 from ambient air into biomass, with growth in ambient air depending on the components of the CCM. Bacterial CCMs are therefore genetically compact and readily transplanted, rationalizing their presence in diverse bacteria. Reconstitution enabled genetic experiments refining our understanding of the CCM, thereby laying the groundwork for deeper study and engineering of the cell biology supporting CO2 assimilation in diverse organisms.

Highly active rubiscos discovered by systematic interrogation of natural sequence diversity.

CO2 is converted into biomass almost solely by the enzyme rubisco. The poor carboxylation properties of plant rubiscos have led to efforts that made it the most kinetically characterized enzyme, yet these studies focused on < 5% of its natural diversity. Here, we searched for fast-carboxylating variants by systematically mining genomic and metagenomic data. Approximately 33,000 unique rubisco sequences were identified and clustered into ≈ 1,000 similarity groups. We then synthesized, purified, and biochemically tested the carboxylation rates of 143 representatives, spanning all clusters of form-II and form-II/III rubiscos. Most variants (> 100) were active in vitro, with the fastest having a turnover number of 22 ± 1 s-1 -sixfold faster than the median plant rubisco and nearly twofold faster than the fastest measured rubisco to date. Unlike rubiscos from plants and cyanobacteria, the fastest variants discovered here are homodimers and exhibit a much simpler folding and activation kinetics. Our pipeline can be utilized to explore the kinetic space of other enzymes of interest, allowing us to get a better view of the biosynthetic potential of the biosphere.

Point mutations in topoisomerase I alter the mutation spectrum in E. coli and impact the emergence of drug resistance genotypes.

Identifying the molecular mechanisms that give rise to genetic variation is essential for the understanding of evolutionary processes. Previously, we have used adaptive laboratory evolution to enable biomass synthesis from CO2 in Escherichia coli. Genetic analysis of adapted clones from two independently evolving populations revealed distinct enrichment for insertion and deletion mutational events. Here, we follow these observations to show that mutations in the gene encoding for DNA topoisomerase I (topA) give rise to mutator phenotypes with characteristic mutational spectra. Using genetic assays and mutation accumulation lines, we find that point mutations in topA increase the rate of sequence deletion and duplication events. Interestingly, we observe that a single residue substitution (R168C) results in a high rate of head-to-tail (tandem) short sequence duplications, which are independent of existing sequence repeats. Finally, we show that the unique mutation spectrum of topA mutants enhances the emergence of antibiotic resistance in comparison to mismatch-repair (mutS) mutators, and leads to new resistance genotypes. Our findings highlight a potential link between the catalytic activity of topoisomerases and the fundamental question regarding the emergence of de novo tandem repeats, which are known modulators of bacterial evolution.

Conversion of Escherichia coli to Generate All Biomass Carbon from CO2.

The living world is largely divided into autotrophs that convert CO2 into biomass and heterotrophs that consume organic compounds. In spite of widespread interest in renewable energy storage and more sustainable food production, the engineering of industrially relevant heterotrophic model organisms to use CO2 as their sole carbon source has so far remained an outstanding challenge. Here, we report the achievement of this transformation on laboratory timescales. We constructed and evolved Escherichia coli to produce all its biomass carbon from CO2. Reducing power and energy, but not carbon, are supplied via the one-carbon molecule formate, which can be produced electrochemically. Rubisco and phosphoribulokinase were co-expressed with formate dehydrogenase to enable CO2 fixation and reduction via the Calvin-Benson-Bassham cycle. Autotrophic growth was achieved following several months of continuous laboratory evolution in a chemostat under intensifying organic carbon limitation and confirmed via isotopic labeling.

Uncovering the biosynthetic potential of rare metagenomic DNA using co-occurrence network analysis of targeted sequences.

Sequencing of DNA extracted from environmental samples can provide key insights into the biosynthetic potential of uncultured bacteria. However, the high complexity of soil metagenomes, which can contain thousands of bacterial species per gram of soil, imposes significant challenges to explore secondary metabolites potentially produced by rare members of the soil microbiome. Here, we develop a targeted sequencing workflow termed CONKAT-seq (co-occurrence network analysis of targeted sequences) that detects physically clustered biosynthetic domains, a hallmark of bacterial secondary metabolism. Following targeted amplification of conserved biosynthetic domains in a highly partitioned metagenomic library, CONKAT-seq evaluates amplicon co-occurrence patterns across library subpools to identify chromosomally clustered domains. We show that a single soil sample can contain more than a thousand uncharacterized biosynthetic gene clusters, most of which originate from low frequency genomes which are practically inaccessible through untargeted sequencing. CONKAT-seq allows scalable exploration of largely untapped biosynthetic diversity across multiple soils, and can guide the discovery of novel secondary metabolites from rare members of the soil microbiome.

The genetic basis for the adaptation of E. coli to sugar synthesis from CO2.

Understanding the evolution of a new metabolic capability in full mechanistic detail is challenging, as causative mutations may be masked by non-essential "hitchhiking" mutations accumulated during the evolutionary trajectory. We have previously used adaptive laboratory evolution of a rationally engineered ancestor to generate an Escherichia coli strain able to utilize CO2 fixation for sugar synthesis. Here, we reveal the genetic basis underlying this metabolic transition. Five mutations are sufficient to enable robust growth when a non-native Calvin-Benson-Bassham cycle provides all the sugar-derived metabolic building blocks. These mutations are found either in enzymes that affect the efflux of intermediates from the autocatalytic CO2 fixation cycle toward biomass (prs, serA, and pgi), or in key regulators of carbon metabolism (crp and ppsR). Using suppressor analysis, we show that a decrease in catalytic capacity is a common feature of all mutations found in enzymes. These findings highlight the enzymatic constraints that are essential to the metabolic stability of autocatalytic cycles and are relevant to future efforts in constructing non-native carbon fixation pathways.

Engineering carbon fixation in E. coli: from heterologous RuBisCO expression to the Calvin-Benson-Bassham cycle.

Carbon fixation is the gateway of inorganic carbon into the biosphere. Our ability to engineer carbon fixation pathways in living organisms is expected to play a crucial role in the quest towards agricultural and energetic sustainability. Recent successes to introduce non-native carbon fixation pathways into heterotrophic hosts offer novel platforms for manipulating these pathways in genetically malleable organisms. Here, we focus on past efforts and future directions for engineering the dominant carbon fixation pathway in the biosphere, the Calvin-Benson cycle, into the well-known model organism Escherichia coli. We describe how central carbon metabolism of this heterotrophic bacterium can be manipulated to allow directed evolution of carbon fixing enzymes. Finally, we highlight future directions towards synthetic autotrophy.

Design principles of autocatalytic cycles constrain enzyme kinetics and force low substrate saturation at flux branch points.

A set of chemical reactions that require a metabolite to synthesize more of that metabolite is an autocatalytic cycle. Here, we show that most of the reactions in the core of central carbon metabolism are part of compact autocatalytic cycles. Such metabolic designs must meet specific conditions to support stable fluxes, hence avoiding depletion of intermediate metabolites. As such, they are subjected to constraints that may seem counter-intuitive: the enzymes of branch reactions out of the cycle must be overexpressed and the affinity of these enzymes to their substrates must be relatively weak. We use recent quantitative proteomics and fluxomics measurements to show that the above conditions hold for functioning cycles in central carbon metabolism of E. coli. This work demonstrates that the topology of a metabolic network can shape kinetic parameters of enzymes and lead to seemingly wasteful enzyme usage.

Sugar Synthesis from CO2 in Escherichia coli.

Can a heterotrophic organism be evolved to synthesize biomass from CO2 directly? So far, non-native carbon fixation in which biomass precursors are synthesized solely from CO2 has remained an elusive grand challenge. Here, we demonstrate how a combination of rational metabolic rewiring, recombinant expression, and laboratory evolution has led to the biosynthesis of sugars and other major biomass constituents by a fully functional Calvin-Benson-Bassham (CBB) cycle in E. coli. In the evolved bacteria, carbon fixation is performed via a non-native CBB cycle, while reducing power and energy are obtained by oxidizing a supplied organic compound (e.g., pyruvate). Genome sequencing reveals that mutations in flux branchpoints, connecting the non-native CBB cycle to biosynthetic pathways, are essential for this phenotype. The successful evolution of a non-native carbon fixation pathway, though not yet resulting in net carbon gain, strikingly demonstrates the capacity for rapid trophic-mode evolution of metabolism applicable to biotechnology. PAPERCLIP.

Pyruvate Formate-Lyase Enables Efficient Growth of Escherichia coli on Acetate and Formate.

Pyruvate formate-lyase (PFL) is a ubiquitous enzyme that supports increased ATP yield during sugar fermentation. While the PFL reaction is known to be reversible in vitro, the ability of PFL to support microbial growth by condensing acetyl-CoA and formate in vivo has never been directly tested. Here, we employ Escherichia coli mutant strains that cannot assimilate acetate via the glyoxylate shunt and use carbon labeling experiments to unequivocally demonstrate PFL-dependent co-assimilation of acetate and formate. Moreover, PFL-dependent growth is faster than growth on acetate using the glyoxylate shunt. Hence, growth via the reverse activity of PFL could have substantial ecological and biotechnological significance.

An in vivo metabolic approach for deciphering the product specificity of glycerate kinase proves that both E. coli's glycerate kinases generate 2-phosphoglycerate.

Apart from addressing humanity's growing demand for fuels, pharmaceuticals, plastics and other value added chemicals, metabolic engineering of microbes can serve as a powerful tool to address questions concerning the characteristics of cellular metabolism. Along these lines, we developed an in vivo metabolic strategy that conclusively identifies the product specificity of glycerate kinase. By deleting E. coli's phosphoglycerate mutases, we divide its central metabolism into an 'upper' and 'lower' metabolism, each requiring its own carbon source for the bacterium to grow. Glycerate can serve to replace the upper or lower carbon source depending on the product of glycerate kinase. Using this strategy we show that while glycerate kinase from Arabidopsis thaliana produces 3-phosphoglycerate, both E. coli's enzymes generate 2-phosphoglycerate. This strategy represents a general approach to decipher enzyme specificity under physiological conditions.

ADAM metalloproteases promote a developmental switch in responsiveness to the axonal repellant Sema3A.

During embryonic development, axons can gain and lose sensitivity to guidance cues, and this flexibility is essential for the correct wiring of the nervous system. Yet, the underlying molecular mechanisms are largely unknown. Here we show that receptor cleavage by ADAM (A Disintegrin And Metalloprotease) metalloproteases promotes murine sensory axons loss of responsiveness to the chemorepellant Sema3A. Genetic ablation of ADAM10 and ADAM17 disrupts the developmental downregulation of Neuropilin-1 (Nrp1), the receptor for Sema3A, in sensory axons. Moreover, this is correlated with gain of repulsive response to Sema3A. Overexpression of Nrp1 in neurons reverses axonal desensitization to Sema3A, but this is hampered in a mutant Nrp1 with high susceptibility to cleavage. Lastly, we detect guidance errors of proprioceptive axons in ADAM knockouts that are consistent with enhanced response to Sema3A. Our results provide the first evidence for involvement of ADAMs in regulating developmental switch in responsiveness to axonal guidance cues.

Assembly of the TLR2/6 transmembrane domains is essential for activation and is a target for prevention of sepsis.

TLR2, together with TLR1 and TLR6, is essential for detecting lipopeptides and bacterial cell wall components such as lipoteichoic acid from Gram-positive bacteria. In this study, we report that transmembrane domain (TMD)-derived peptides from TLR2 and TLR6 specifically inhibit TLR2 activation. Secretion of the cytokines TNF-α and IL-6 by cultured macrophages (RAW264.7 cell line) was inhibited by these peptides in response to TLR2 activation by lipoteichoic acid (TLR2/6 activator) or palmitoyl (3)-Cys-Ser-Lys(4)-OH (TLR2/1 activator) but not by LPS (TLR4 activator). Extensive biophysical and biochemical assays, combined with GALLEX experiments, show that these peptides heterodimerize with their complementary TMDs on their reciprocal protein. These results suggest that TLR2/6/1 TMD assembly is essential for activating this complex. Importantly, when administered to mice inflicted by TLR2, but not TLR4-driven lethal inflammation, a selected peptide rescued 60% of these septic mice, showing potent in vivo inhibition of TNF-α and IL-6 secretion. Furthermore, this peptide also showed high protection in a whole bacteria model. Owing to the importance of TLR2 regulation under a variety of pathological conditions, compounds that can fine-tune this activity are of great importance.

Quantifying translational coupling in E. coli synthetic operons using RBS modulation and fluorescent reporters.

Translational coupling is the interdependence of translation efficiency of neighboring genes encoded within an operon. The degree of coupling may be quantified by measuring how the translation rate of a gene is modulated by the translation rate of its upstream gene. Translational coupling was observed in prokaryotic operons several decades ago, but the quantitative range of modulation translational coupling leads to and the factors governing this modulation were only partially characterized. In this study, we systematically quantify and characterize translational coupling in E. coli synthetic operons using a library of plasmids carrying fluorescent reporter genes that are controlled by a set of different ribosome binding site (RBS) sequences. The downstream gene expression level is found to be enhanced by the upstream gene expression via translational coupling with the enhancement level varying from almost no coupling to over 10-fold depending on the upstream gene's sequence. Additionally, we find that the level of translational coupling in our system is similar between the second and third locations in the operon. The coupling depends on the distance between the stop codon of the upstream gene and the start codon of the downstream gene. This study is the first to systematically and quantitatively characterize translational coupling in a synthetic E. coli operon. Our analysis will be useful in accurate manipulation of gene expression in synthetic biology and serves as a step toward understanding the mechanisms involved in translational expression modulation.

Spanning high-dimensional expression space using ribosome-binding site combinatorics.

Protein levels are a dominant factor shaping natural and synthetic biological systems. Although proper functioning of metabolic pathways relies on precise control of enzyme levels, the experimental ability to balance the levels of many genes in parallel is a major outstanding challenge. Here, we introduce a rapid and modular method to span the expression space of several proteins in parallel. By combinatorially pairing genes with a compact set of ribosome-binding sites, we modulate protein abundance by several orders of magnitude. We demonstrate our strategy by using a synthetic operon containing fluorescent proteins to span a 3D color space. Using the same approach, we modulate a recombinant carotenoid biosynthesis pathway in Escherichia coli to reveal a diversity of phenotypes, each characterized by a distinct carotenoid accumulation profile. In a single combinatorial assembly, we achieve a yield of the industrially valuable compound astaxanthin 4-fold higher than previously reported. The methodology presented here provides an efficient tool for exploring a high-dimensional expression space to locate desirable phenotypes.

HIV-1 gp41 and TCRalpha trans-membrane domains share a motif exploited by the HIV virus to modulate T-cell proliferation.

Viruses have evolved several strategies to modify cellular processes and evade the immune response in order to successfully infect, replicate, and persist in the host. By utilizing in-silico testing of a transmembrane sequence library derived from virus protein sequences, we have pin-pointed a nine amino-acid motif shared by a group of different viruses; this motif resembles the transmembrane domain of the alpha-subunit of the T-cell receptor (TCRalpha). The most striking similarity was found within the immunodeficiency virus (SIV and HIV) glycoprotein 41 TMD (gp41 TMD). Previous studies have shown that stable interactions between TCRalpha and CD3 are localized to this nine amino acid motif within TCRalpha, and a peptide derived from it (TCRalpha TMD, GLRILLLKV) interfered and intervened in the TCR function when added exogenously. We now report that the gp41 TMD peptide co-localizes with CD3 within the TCR complex and inhibits T cell proliferation in vitro. However, the inhibitory mechanism of gp41 TMD differs from that of the TCRalpha TMD and also from the other two known immunosuppressive regions within gp41.