Towards synthetic PETtrophy: Engineering Pseudomonas putida for concurrent polyethylene terephthalate (PET) monomer metabolism and PET hydrolase expression.

BACKGROUND: Biocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts. RESULTS: P. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive. CONCLUSION: Our results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.

Directed Evolution of a Cytochrome P450 Carbene Transferase for Selective Functionalization of Cyclic Compounds.

Transfers of carbene moieties to heterocycles or cyclic alkenes to obtain C(sp2)-H alkylation or cyclopropane products are valuable transformations for synthesis of pharmacophores and chemical building blocks. Through their readily tunable active-site geometries, hemoprotein "carbene transferases" could provide an alternative to traditional transition metal catalysts by enabling heterocycle functionalizations with high chemo-, regio-, and stereocontrol. However, carbene transferases accepting heterocyclic substrates are scarce; the few enzymes capable of heterocycle or cyclic internal alkene functionalization described to date are characterized by low turnovers or depend on artificially introduced, costly iridium-porphyrin cofactors. We addressed this challenge by evolving a cytochrome P450 for highly efficient carbene transfer to indoles, pyrroles, and cyclic alkenes. We first developed a spectrophotometric high-throughput screening assay based on 1-methylindole C3-alkylation that enabled rapid analysis of thousands of P450 variants and comprehensive directed evolution via random and targeted mutagenesis. This effort yielded a P450 variant with 11 amino acid substitutions and a large deletion of the non-catalytic P450 reductase domain, which chemoselectively C3-alkylates indoles with up to 470 turnovers per minute and 18 000 total turnovers. We subsequently used this optimized alkylation variant for parallel evolution toward more challenging heterocycle carbene functionalizations, including C2/ C3 regioselective pyrrole alkylation, enantioselective indole alkylation with ethyl 2-diazopropanoate, and cyclic internal alkene cyclopropanation. The resulting set of efficient biocatalysts showcases the tunability of hemoproteins for highly selective functionalization of cyclic targets and the power of directed evolution to enhance the scope of new-to-nature enzyme catalysts.

Predicting HIV-1 transmission and antibody neutralization efficacy in vivo from stoichiometric parameters.

The potential of broadly neutralizing antibodies targeting the HIV-1 envelope trimer to prevent HIV-1 transmission has opened new avenues for therapies and vaccines. However, their implementation remains challenging and would profit from a deepened mechanistic understanding of HIV-antibody interactions and the mucosal transmission process. In this study we experimentally determined stoichiometric parameters of the HIV-1 trimer-antibody interaction, confirming that binding of one antibody is sufficient for trimer neutralization. This defines numerical requirements for HIV-1 virion neutralization and thereby enables mathematical modelling of in vitro and in vivo antibody neutralization efficacy. The model we developed accurately predicts antibody efficacy in animal passive immunization studies and provides estimates for protective mucosal antibody concentrations. Furthermore, we derive estimates of the probability for a single virion to start host infection and the risks of male-to-female HIV-1 transmission per sexual intercourse. Our work thereby delivers comprehensive quantitative insights into both the molecular principles governing HIV-antibody interactions and the initial steps of mucosal HIV-1 transmission. These insights, alongside the underlying, adaptable modelling framework presented here, will be valuable for supporting in silico pre-trial planning and post-hoc evaluation of HIV-1 vaccination or antibody treatment trials.

Alternate Heme Ligation Steers Activity and Selectivity in Engineered Cytochrome P450-Catalyzed Carbene-Transfer Reactions.

We report a biocatalytic platform of engineered cytochrome P450 enzymes to carry out carbene-transfer reactions using a lactone-based carbene precursor. By simply altering the heme-ligating residue, we obtained two enzymes that catalyze olefin cyclopropanation (Ser) or S-H bond insertion (Cys). Both enzymes exhibit high catalytic efficiency and stereoselectivity, thus enabling facile access to structurally diverse spiro[2.4]lactones and α-thio-γ-lactones. Computational studies revealed the mechanism of carbene S-H insertion and explain how the axial ligand controls reactivity and selectivity. This work expands the catalytic repertoire of hemeproteins and offers insights into how these enzymes can be tuned for new chemistry.

Different infectivity of HIV-1 strains is linked to number of envelope trimers required for entry.

HIV-1 enters target cells by virtue of envelope glycoprotein trimers that are incorporated at low density in the viral membrane. How many trimers are required to interact with target cell receptors to mediate virus entry, the HIV entry stoichiometry, still awaits clarification. Here, we provide estimates of the HIV entry stoichiometry utilizing a combined approach of experimental analyses and mathematical modeling. We demonstrate that divergent HIV strains differ in their stoichiometry of entry and require between 1 to 7 trimers, with most strains depending on 2 to 3 trimers to complete infection. Envelope modifications that perturb trimer structure lead to an increase in the entry stoichiometry, as did naturally occurring antibody or entry inhibitor escape mutations. Highlighting the physiological relevance of our findings, a high entry stoichiometry correlated with low virus infectivity and slow virus entry kinetics. The entry stoichiometry therefore directly influences HIV transmission, as trimer number requirements will dictate the infectivity of virus populations and efficacy of neutralizing antibodies. Thereby our results render consideration of stoichiometric concepts relevant for developing antibody-based vaccines and therapeutics against HIV.

Partial rescue of V1V2 mutant infectivity by HIV-1 cell-cell transmission supports the domain's exceptional capacity for sequence variation.

BACKGROUND: Variable loops 1 and 2 (V1V2) of the HIV-1 envelope glycoprotein gp120 perform two key functions: ensuring envelope trimer entry competence and shielding against neutralizing antibodies. While preserving entry functionality would suggest a high need for V1V2 sequence optimization and conservation, shielding efficacy is known to depend on a high flexibility of V1V2 giving rise to its substantial sequence variability. How entry competence of the trimer is maintained despite the continuous emergence of antibody escape mutations within V1V2 has not been resolved. Since HIV cell-cell transmission is considered a highly effective means of virus dissemination, we investigated whether cell-cell transmission may serve to enhance infectivity of V1V2 variants with debilitated free virus entry. RESULTS: In a detailed comparison of wt and V1V2 mutant envelopes, V1V2 proved to be a key factor in ascertaining free virus infectivity, with V1V2 mutants displaying significantly reduced trimer integrity. Despite these defects, cell-cell transmission was able to partially rescue infectivity of V1V2 mutant viruses. We identified two regions, encompassing amino acids 156 to 160 (targeted by broadly neutralizing antibodies) and 175 to 180 (encompassing the α4ß7 binding site) which were particularly prone to free virus infectivity loss upon mutation but maintained infectivity in cell-cell transmission. Of note, V1V2 antibody shielding proved important during both free virus infection and cell-cell transmission. CONCLUSIONS: Based on our data we propose a model for V1V2 evolution that centers on cell-cell transmission as a salvage pathway for virus replication. Escape from antibody neutralization may frequently result in V1V2 mutations that reduce free virus infectivity. Cell-cell transmission could provide these escape viruses with sufficiently high replication levels that enable selection of compensatory mutations, thereby restoring free virus infectivity while ensuring antibody escape. Thus, our study highlights the need to factor in cell-cell transmission when considering neutralization escape pathways of HIV-1.

Swab-Seq: A high-throughput platform for massively scaled up SARS-CoV-2 testing.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is due to the high rates of transmission by individuals who are asymptomatic at the time of transmission1,2. Frequent, widespread testing of the asymptomatic population for SARS-CoV-2 is essential to suppress viral transmission. Despite increases in testing capacity, multiple challenges remain in deploying traditional reverse transcription and quantitative PCR (RT-qPCR) tests at the scale required for population screening of asymptomatic individuals. We have developed SwabSeq, a high-throughput testing platform for SARS-CoV-2 that uses next-generation sequencing as a readout. SwabSeq employs sample-specific molecular barcodes to enable thousands of samples to be combined and simultaneously analyzed for the presence or absence of SARS-CoV-2 in a single run. Importantly, SwabSeq incorporates an in vitro RNA standard that mimics the viral amplicon, but can be distinguished by sequencing. This standard allows for end-point rather than quantitative PCR, improves quantitation, reduces requirements for automation and sample-to-sample normalization, enables purification-free detection, and gives better ability to call true negatives. After setting up SwabSeq in a high-complexity CLIA laboratory, we performed more than 80,000 tests for COVID-19 in less than two months, confirming in a real world setting that SwabSeq inexpensively delivers highly sensitive and specific results at scale, with a turn-around of less than 24 hours. Our clinical laboratory uses SwabSeq to test both nasal and saliva samples without RNA extraction, while maintaining analytical sensitivity comparable to or better than traditional RT-qPCR tests. Moving forward, SwabSeq can rapidly scale up testing to mitigate devastating spread of novel pathogens.

Massively scaled-up testing for SARS-CoV-2 RNA via next-generation sequencing of pooled and barcoded nasal and saliva samples.

Frequent and widespread testing of members of the population who are asymptomatic for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for the mitigation of the transmission of the virus. Despite the recent increases in testing capacity, tests based on quantitative polymerase chain reaction (qPCR) assays cannot be easily deployed at the scale required for population-wide screening. Here, we show that next-generation sequencing of pooled samples tagged with sample-specific molecular barcodes enables the testing of thousands of nasal or saliva samples for SARS-CoV-2 RNA in a single run without the need for RNA extraction. The assay, which we named SwabSeq, incorporates a synthetic RNA standard that facilitates end-point quantification and the calling of true negatives, and that reduces the requirements for automation, purification and sample-to-sample normalization. We used SwabSeq to perform 80,000 tests, with an analytical sensitivity and specificity comparable to or better than traditional qPCR tests, in less than two months with turnaround times of less than 24 h. SwabSeq could be rapidly adapted for the detection of other pathogens.

Engineering Chemoselectivity in Hemoprotein-Catalyzed Indole Amidation.

Here we report a cytochrome P450 variant that catalyzes C2-amidation of 1-methylindoles with tosyl azide via nitrene transfer. Before evolutionary optimization the enzyme exhibited two undesired side reactivities resulting in reduction of the putative iron-nitrenoid intermediate or cycloaddition between the two substrates to form triazole products. We speculated that triazole formation was a promiscuous cycloaddition activity of the P450 heme domain, while sulfonamide formation likely arose from surplus electron transfer from the reductase domain. Directed evolution involving mutagenesis of both the heme and reductase domains delivered an enzyme providing the desired indole amidation products with up to 8400 turnovers, 90% yield, and a shift in chemoselectivity from 2:19:1 to 110:12:1 in favor of nitrene transfer over reduction or triazole formation. This work expands the substrate scope of hemoprotein nitrene transferases to heterocycles and highlights the adaptability of the P450 scaffold to solve challenging chemoselectivity problems in non-natural enzymatic catalysis.

Diverse Engineered Heme Proteins Enable Stereodivergent Cyclopropanation of Unactivated Alkenes.

Developing catalysts that produce each stereoisomer of a desired product selectively is a longstanding synthetic challenge. Biochemists have addressed this challenge by screening nature's diversity to discover enzymes that catalyze the formation of complementary stereoisomers. We show here that the same approach can be applied to a new-to-nature enzymatic reaction, alkene cyclopropanation via carbene transfer. By screening diverse native and engineered heme proteins, we identified globins and serine-ligated "P411" variants of cytochromes P450 with promiscuous activity for cyclopropanation of unactivated alkene substrates. We then enhanced their activities and stereoselectivities by directed evolution: just 1-3 rounds of site-saturation mutagenesis and screening generated enzymes that transform unactivated alkenes and electron-deficient alkenes into each of the four stereoisomeric cyclopropanes with up to 5,400 total turnovers and 98% enantiomeric excess. These fully genetically encoded biocatalysts function in whole Escherichia coli cells in mild, aqueous conditions and provide the first example of enantioselective, intermolecular iron-catalyzed cyclopropanation of unactivated alkenes.

Exploiting and engineering hemoproteins for abiological carbene and nitrene transfer reactions.

The surge in reports of heme-dependent proteins as catalysts for abiotic, synthetically valuable carbene and nitrene transfer reactions dramatically illustrates the evolvability of the protein world and our nascent ability to exploit that for new enzyme chemistry. We highlight the latest additions to the hemoprotein-catalyzed reaction repertoire (including carbene Si-H and C-H insertions, Doyle-Kirmse reactions, aldehyde olefinations, azide-to-aldehyde conversions, and intermolecular nitrene C-H insertion) and show how different hemoprotein scaffolds offer varied reactivity and selectivity. Preparative-scale syntheses of pharmaceutically relevant compounds accomplished with these new catalysts are beginning to demonstrate their biotechnological relevance. Insights into the determinants of enzyme lifetime and product yield are providing generalizable cues for engineering heme-dependent proteins to further broaden the scope and utility of these non-natural activities.

The HIV-1 Entry Process: A Stoichiometric View.

HIV-1 infection starts with fusion of the viral and the host cell membranes, a process mediated by the HIV-1 envelope glycoprotein trimer. The number of trimers required to complete membrane fusion, referred to as HIV-1 entry stoichiometry, remains under debate. A precise definition of HIV-1 entry stoichiometry is important as it reflects the efficacy of the viral entry process and steers the infectivity of HIV-1 virion populations. Initial estimates suggested a unanimous entry stoichiometry across HIV-1 strains while recent findings showed that HIV-1 strains can differ in entry stoichiometry. Here, we review current analyses of HIV-1 entry stoichiometry and point out future research directions to further define the interplay between entry stoichiometry, virus entry fitness, transmission, and susceptibility to antibody neutralization.

Mathematical models: a key to understanding HIV envelope interactions?

The spikes of the human immunodeficiency virus (HIV) mediate viral entry and are the most important targets for neutralizing antibodies. Each spike consists of three identical subunits. The role of the spike's subunits in antibody binding is not fully understood. One experimental approach to analyze trimer function uses assays with mixed envelope trimer expressing cells or viruses. As these experiments do not allow direct observation of subunit functions, mathematical models are required to interpret them. Here we describe a modeling framework to study (i) the interaction of the V1V2 loop with epitopes on the V3 loop and (ii) the composition of quaternary epitopes. In a first step we identify which trimers can form in these assays and how they function under antibody binding. We then derive the behavior of an average trimer. We contrast two experimental reporting systems and list their advantages and disadvantages. In these experiments trimer formation might not be perfectly random and we show how these effects can be tested. As we still lack a potent vaccine against HIV, and this vaccine surely has to stimulate the production of neutralizing antibodies, mixed trimer approaches in combination with mathematical models will help to identify vulnerable sites of the HIV spike.

Interaction of the gp120 V1V2 loop with a neighboring gp120 unit shields the HIV envelope trimer against cross-neutralizing antibodies.

The HIV-1 envelope trimer adopts a quaternary conformation that effectively shields neutralization-sensitive domains and thus represents a major obstacle for natural and vaccine-elicited antibody responses. By using a structure-function analysis based on a specifically devised mathematical model, we demonstrate in this study that protection from neutralization is enforced by intersubunit contact between the variable loops 1 and 2 (V1V2) and domains of neighboring gp120 subunits in the trimer encompassing the V3 loop. Our data are consistent with an interaction of the V1V2 and V3 loop at the spike apex as proposed by cryoelectron tomography experiments. By defining the orientation of the V1V2 loop within the trimer toward the neighboring gp120 subunit's V3 loop, our data close an important gap in the understanding of the architecture of the trimeric spike. Knowledge on how the V1V2 barrier functions in the context of the trimer to mask conserved epitopes on gp120 may aid future vaccine design.

MPER-specific antibodies induce gp120 shedding and irreversibly neutralize HIV-1.

Interference with virus entry is known to be the principle mechanism of HIV neutralization by antibodies, including 2F5 and 4E10, which bind to the membrane-proximal external region (MPER) of the gp41 envelope protein. However, to date, the precise molecular events underlying neutralization by MPER-specific antibodies remain incompletely understood. In this study, we investigated the capacity of these antibodies to irrevocably sterilize HIV virions. Long-term effects of antibodies on virions can differ, rendering neutralization either reversible or irreversible. MPER-specific antibodies irreversibly neutralize virions, and this capacity is associated with induction of gp120 shedding. Both processes have similar thermodynamic properties and slow kinetics requiring several hours. Antibodies directed to the CD4 binding site, V3 loop, and the MPER can induce gp120 shedding, and shedding activity is detected with high frequency in plasma from patients infected with divergent genetic HIV-1 subtypes. Importantly, as we show in this study, induction of gp120 shedding is closely associated with MPER antibody inhibition, constituting either a primary event leading to virion neutralization or representing an immediate consequence thereof, and thus needs to be factored into the mechanistic processes underlying their activity.