Deep mutational scanning reveals the molecular determinants of RNA polymerase-mediated adaptation and tradeoffs.

RNA polymerase (RNAP) is emblematic of complex biological systems that control multiple traits involving trade-offs such as growth versus maintenance. Laboratory evolution has revealed that mutations in RNAP subunits, including RpoB, are frequently selected. However, we lack a systems view of how mutations alter the RNAP molecular functions to promote adaptation. We, therefore, measured the fitness of thousands of mutations within a region of rpoB under multiple conditions and genetic backgrounds, to find that adaptive mutations cluster in two modules. Mutations in one module favor growth over maintenance through a partial loss of an interaction associated with faster elongation. Mutations in the other favor maintenance over growth through a destabilized RNAP-DNA complex. The two molecular handles capture the versatile RNAP-mediated adaptations. Combining both interaction losses simultaneously improved maintenance and growth, challenging the idea that growth-maintenance tradeoff resorts only from limited resources, and revealing how compensatory evolution operates within RNAP.

Cross-resistance to cefiderocol and ceftazidime-avibactam in KPC ß-lactamase mutants and the inoculum effect.

OBJECTIVES: Ceftazidime-avibactam (CZA) and cefiderocol are recently commercialized molecules active against highly drug-resistant bacteria, including carbapenem-resistant members of the Enterobacteriaceae. Mutants resistant to CZA have been described, notably in Klebsiella pneumoniae carbapenemase (KPC) producers. Considering the structural similarities between ceftazidime and cefiderocol, we hypothesized that resistance to CZA in KPC-producing members of the Enterobacterales may lead to cross-resistance to cefiderocol. METHODS: CZA-resistant mutants from three clinical isolates of the Enterobacterales carrying either blaKPC-2 or blaKPC-3 were selected in vitro. Mutants with increased MIC to CZA compared to the ancestral allele were cloned in a pBR322 plasmid and expressed in Escherichia coli TOP10. We evaluated the impact of these mutations on cefiderocol MICs and minimal bactericidal concentrations (MBCs), and we assessed the impact of bacterial inoculum size on cefiderocol MICs. RESULTS: We used 37 KPC mutants with increased CZA MICs. Of these, six have been described previously in clinical isolates. Compared to the wild-type alleles, increases in the cefiderocol MICs of 4- to 32-fold were observed for 75.6% of tested mutants (28/37), MICs reaching up to 4 mg/L in E. coli TOP10 for KPC-31 (D179Y-H274Y mutations). MBCs and MICs of cefiderocol were similar, confirming the bactericidal activity of this drug. Finally, when using higher inocula (107 CFU/mL), a large increase in cefiderocol MIC was observed, and all isolates were categorized as resistant. CONCLUSION: We observed that most of the CZA-resistant KPC variants have a possible impact on cefiderocol by increasing the cefiderocol MICs. In addition, cefiderocol is greatly impacted by the inoculum effect, suggesting that precautions should be taken when treating infections with a suspected high inoculum.

KPC Beta-Lactamases Are Permissive to Insertions and Deletions Conferring Substrate Spectrum Modifications and Resistance to Ceftazidime-Avibactam.

To explore the mutational possibilities of insertions and deletions (indels) in the Klebsiella pneumoniae carbapenemase (KPC) beta-lactamase, we selected for ceftazidime-avibactam-resistant mutants. Of 96 screened mutants, we obtained 19 indels (2 to 15 amino acids), all located in the loops surrounding the active site. Three antibiotic susceptibility phenotypes emerged: an extended-spectrum-beta-lactamase-like phenotype, an activity restricted to ceftazidime, and a carbapenem-susceptible KPC-like phenotype. Tolerance for indels reflects the evolvability of KPC beta-lactamase, which could challenge the therapeutic management of patients.

Engineering regulatory networks for complex phenotypes in E. coli.

Regulatory networks describe the hierarchical relationship between transcription factors, associated proteins, and their target genes. Regulatory networks respond to environmental and genetic perturbations by reprogramming cellular metabolism. Here we design, construct, and map a comprehensive regulatory network library containing 110,120 specific mutations in 82 regulators expected to perturb metabolism. We screen the library for different targeted phenotypes, and identify mutants that confer strong resistance to various inhibitors, and/or enhanced production of target compounds. These improvements are identified in a single round of selection, showing that the regulatory network library is universally applicable and is convenient and effective for engineering targeted phenotypes. The facile construction and mapping of the regulatory network library provides a path for developing a more detailed understanding of global regulation in E. coli, with potential for adaptation and use in less-understood organisms, expanding toolkits for future strain engineering, synthetic biology, and broader efforts.

Integrating CRISPR-Enabled Trackable Genome Engineering and Transcriptomic Analysis of Global Regulators for Antibiotic Resistance Selection and Identification in Escherichia coli.

It is important to expedite our understanding of antibiotic resistance to address the increasing numbers of fatalities and environmental pollution due to the emergence of antibiotic resistance and multidrug-resistant strains. Here, we combined the CRISPR-enabled trackable genome engineering (CREATE) technology and transcriptomic analysis to investigate antibiotic tolerance in Escherichia coli We developed rationally designed site saturation mutagenesis libraries targeting 23 global regulators to identify fitness-conferring mutations in response to diverse antibiotic stresses. We identified seven novel mutations that confer resistance to the ribosome-targeting antibiotics doxycycline, thiamphenicol, and gentamicin in E. coli To the best of our knowledge, these mutations that we identified have not been reported previously during treatment with the indicated antibiotics. Transcriptome sequencing-based transcriptome analysis was further employed to evaluate the genome-wide changes in gene expression in E. coli for SoxR G121P and cAMP receptor protein (CRP) V140W reconstructions, and improved fitness in response to doxycycline and gentamicin was seen. In the case of doxycycline, we speculated that SoxR G121P significantly increased the expression of genes involved in carbohydrate metabolism and energy metabolism to promote cell growth for improved adaptation. In the CRP V140W mutant with improved gentamicin tolerance, the expression of several amino acid biosynthesis genes and fatty acid degradation genes was significantly changed, and these changes probably altered the cellular energy state to improve adaptation. These findings have important significance for understanding such nonspecific mechanisms of antibiotic resistance and developing new antibacterial drugs.IMPORTANCE The growing threat of antimicrobial resistance poses a serious threat to public health care and motivates efforts to understand the means by which resistance acquisition occurs and how this can be combatted. To address these challenges, we expedited the identification of novel mutations that enable complex phenotypic changes that result in improved tolerance to antibiotics by integrating CREATE and transcriptomic analysis of global regulators. The results give us a better understanding of the mechanisms of resistance to tetracycline antibiotics and aminoglycoside antibiotics and also indicate that the method may be used for quickly identifying resistance-related mutations.

Determinants for Efficient Editing with Cas9-Mediated Recombineering in Escherichia coli.

In E. coli, editing efficiency with Cas9-mediated recombineering varies across targets due to differences in the level of Cas9:gRNA-mediated DNA double-strand break (DSB)-induced cell death. We found that editing efficiency with the same gRNA and repair template can also change with target position, cas9 promoter strength, and growth conditions. Incomplete editing, off-target activity, nontargeted mutations, and failure to cleave target DNA even if Cas9 is bound also compromise editing efficiency. These effects on editing efficiency were gRNA-specific. We propose that differences in the efficiency of Cas9:gRNA-mediated DNA DSBs, as well as possible differences in binding of Cas9:gRNA complexes to their target sites, account for the observed variations in editing efficiency between gRNAs. We show that editing behavior using the same gRNA can be modified by mutating the gRNA spacer, which changes the DNA DSB activity. Finally, we discuss how variable editing with different gRNAs could limit high-throughput applications and provide strategies to overcome these limitations.

Small molecule regulated sgRNAs enable control of genome editing in E. coli by Cas9.

CRISPR-Cas9 has led to great advances in gene editing for a broad spectrum of applications. To further the utility of Cas9 there have been efforts to achieve temporal control over its nuclease activity. While different approaches have focused on regulation of CRISPR interference or editing in mammalian cells, none of the reported methods enable control of the nuclease activity in bacteria. Here, we develop RNA linkers to combine theophylline- and 3-methylxanthine (3MX)-binding aptamers with the sgRNA, enabling small molecule-dependent editing in Escherichia coli. These activatable guide RNAs enable temporal and post-transcriptional control of in vivo gene editing. Further, they reduce the death of host cells caused by cuts in the genome, a major limitation of CRISPR-mediated bacterial recombineering.

CRISPR/Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli.

Deep mutational scanning can provide significant insights into the function of essential genes in bacteria. Here, we developed a high-throughput method for mutating essential genes of Escherichia coli in their native genetic context. We used Cas9-mediated recombineering to introduce a library of mutations, created by error-prone PCR, within a gene fragment on the genome using a single gRNA pre-validated for high efficiency. Tracking mutation frequency through deep sequencing revealed biases in the position and the number of the introduced mutations. We overcame these biases by increasing the homology arm length and blocking mismatch repair to achieve a mutation efficiency of 85% for non-essential genes and 55% for essential genes. These experiments also improved our understanding of poorly characterized recombineering process using dsDNA donors with single nucleotide changes. Finally, we applied our technology to target rpoB, the beta subunit of RNA polymerase, to study resistance against rifampicin. In a single experiment, we validate multiple biochemical and clinical observations made in the previous decades and provide insights into resistance compensation with the study of double mutants.

Multiplex navigation of global regulatory networks (MINR) in yeast for improved ethanol tolerance and production.

Multiplex navigation of global regulatory networks (MINR) is an approach for combinatorially reprogramming gene expression to manipulate complex phenotypes. We designed, constructed, and mapped MINR libraries containing 43,020 specific mutations in 25 regulatory genes expected to perturb the yeast regulatory network. We selected growth competition experiments for library mutants conferring increased ethanol and/or glucose tolerance. We identified specific mutants that not only possessed improved ethanol and/or glucose tolerance but also produced ethanol at concentrations up to 2-fold higher than those produced by the wild-type strain. We further determined that mutations increasing ethanol tolerance were transferable to a diploid industrial yeast strain. The facile construction and mapping of 43,020 designer regulatory mutations provide a roadmap for how to access and engineer complex phenotypes in future synthetic biology and broader efforts.

Deep scanning lysine metabolism in Escherichia coli.

Our limited ability to predict genotype-phenotype relationships has called for strategies that allow testing of thousands of hypotheses in parallel. Deep scanning mutagenesis has been successfully implemented to map genotype-phenotype relationships at a single-protein scale, allowing scientists to elucidate properties that are difficult to predict. However, most phenotypes are dictated by several proteins that are interconnected through complex and robust regulatory and metabolic networks. These sophisticated networks hinder our understanding of the phenotype of interest and limit our capabilities to rewire cellular functions. Here, we leveraged CRISPR-EnAbled Trackable genome Engineering to attempt a parallel and high-resolution interrogation of complex networks, deep scanning multiple proteins associated with lysine metabolism in Escherichia coli We designed over 16,000 mutations to perturb this pathway and mapped their contribution toward resistance to an amino acid analog. By doing so, we identified different routes that can alter pathway function and flux, uncovering mechanisms that would be difficult to rationally design. This approach sets a framework for forward investigation of complex multigenic phenotypes.

Genomic Deoxyxylulose Phosphate Reductoisomerase (DXR) Mutations Conferring Resistance to the Antimalarial Drug Fosmidomycin in E. coli.

Sequence to activity mapping technologies are rapidly developing, enabling the generation and isolation of mutations conferring novel phenotypes. Here we used the CRISPR enabled trackable genome engineering (CREATE) technology to investigate the inhibition of the essential ispC gene in its native genomic context in Escherichia coli. We created a full saturation library of 33 sites proximal to the ligand binding pocket and challenged this library with the antimalarial drug fosmidomycin, which targets the ispC gene product, DXR. This selection is especially challenging since it is relatively weak in E. coli, with multiple naturally occurring pathways for resistance. We identified several previously unreported mutations that confer fosmidomycin resistance, in highly conserved sites that also exist in pathogens including the malaria-inducing Plasmodium falciparum. This approach may have implications for the isolation of resistance-conferring mutations and may affect the design of future generations of fosmidomycin-based drugs.

Iterative genome editing of Escherichia coli for 3-hydroxypropionic acid production.

Synthetic biology requires strategies for the targeted, efficient, and combinatorial engineering of biological sub-systems at the molecular level. Here, we report the use of the iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method for the rapid construction of combinatorially modified genomes. We coupled this genome engineering strategy with high-throughput phenotypic screening and selections to recursively engineer multiple traits in Escherichia coli for improved production of the platform chemical 3-hydroxypropionic acid (3HP). Specifically, we engineered i) central carbon metabolism, ii) 3HP synthesis, and (iii) 3HP tolerance through design, construction and testing of ~ 162,000 mutations across 115 genes spanning global regulators, transcription factors, and enzymes involved in 3HP synthesis and tolerance. The iCREATE process required ~ 1 month to perform 13 rounds of combinatorial genome modifications with targeted gene knockouts, expression modification by ribosomal binding site (RBS) engineering, and genome-level site-saturation mutagenesis. Specific mutants conferring increased 3HP titer, yield, and productivity were identified and then combined to produce 3HP at a yield and concentration ~ 60-fold higher than the wild-type strain.

Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering.

Strain engineering for industrial production requires a targeted improvement of multiple complex traits, which range from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method to engineer rapid glucose and xylose co-consumption and tolerance to hydrolysate inhibitors in E. coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target ~40,000 mutations across 30 genes. These libraries included global and high-level regulators that regulate global gene expression, transcription factors that play important roles in genome-level transcription, enzymes that function in the sugar transport system, NAD(P)H metabolism, and the aldehyde reduction system. Specific mutants that conferred increased growth in mixed sugars and hydrolysate tolerance conditions were isolated, confirmed, and evaluated for changes in genome-wide expression levels. We tested the strain with positive combinatorial mutations for 3-hydroxypropionic acid (3HP) production under high furfural and high acetate hydrolysate fermentation, which demonstrated a 7- and 8-fold increase in 3HP productivity relative to the parent strain, respectively.

The Resistome: A Comprehensive Database of Escherichia coli Resistance Phenotypes.

The microbial ability to resist stressful environmental conditions and chemical inhibitors is of great industrial and medical interest. Much of the data related to mutation-based stress resistance, however, is scattered through the academic literature, making it difficult to apply systematic analyses to this wealth of information. To address this issue, we introduce the Resistome database: a literature-curated collection of Escherichia coli genotypes-phenotypes containing over 5,000 mutants that resist hundreds of compounds and environmental conditions. We use the Resistome to understand our current state of knowledge regarding resistance and to detect potential synergy or antagonism between resistance phenotypes. Our data set represents one of the most comprehensive collections of genomic data related to resistance currently available. Future development will focus on the construction of a combined genomic-transcriptomic-proteomic framework for understanding E. coli's resistance biology. The Resistome can be downloaded at https://bitbucket.org/jdwinkler/resistome_release/overview .

Lytic Polysaccharide Monooxygenases ScLPMO10B and ScLPMO10C Are Stable in Ionic Liquids As Determined by Molecular Simulations.

Lytic polysaccharide monooxygenases (LPMOs) are a newly discovered family of enzymes proposed to work synergistically with cellulases and aid in the decomposition of cellulose for the creation of environmentally friendly fuels and chemicals. To our knowledge, evaluation of the stability of LPMOs in ionic liquid (IL) solvents at relevant biomass processing conditions has not been explored. Herein, molecular dynamics simulations of ScLPMO10B and ScLPMO10C in three ILs at 10 and 20 wt% in water and in pure water have been performed. Enzyme stability was predicted to be high on the basis of structural and dynamic analyses we performed. We used the simulations to identify key areas that deviate from the crystal structures as a starting place for surface charge modifications to increase stability in ILs. Results show that, in general, both enzymes have a high degree of stability across the range of IL solutions tested. For each enzyme, two regions were identified that showed notable deviations from the crystal structure. In addition to providing a basis for future rational design efforts, this work represents a first step toward engineering LPMOs to function efficiently in enzyme cocktails for use in industrial biomass processing applications with ILs.

Yeast knockout library allows for efficient testing of genomic mutations for cell-free protein synthesis.

Cell-free protein synthesis (CFPS) systems from crude lysates have benefitted from modifications to their enzyme composition. For example, functionally deleting enzymes in the source strain that are deleterious to CFPS can improve protein synthesis yields. However, making such modifications can take substantial time. As a proof-of-concept to accelerate prototyping capabilities, we assessed the feasibility of using the yeast knockout collection to identify negative effectors in a Saccharomyces cerevisiae CFPS platform. We analyzed extracts made from six deletion strains that targeted the single deletion of potentially negative effectors (e.g., nucleases). We found a statistically significant increase in luciferase yields upon loss of function of GCN3, PEP4, PPT1, NGL3, and XRN1 with a maximum increase of over 6-fold as compared to the wild type. Our work has implications for yeast CFPS and for rapidly prototyping strains to enable cell-free synthetic biology applications.

A cell-free expression and purification process for rapid production of protein biologics.

Cell-free protein synthesis has emerged as a powerful technology for rapid and efficient protein production. Cell-free methods are also amenable to automation and such systems have been extensively used for high-throughput protein production and screening; however, current fluidic systems are not adequate for manufacturing protein biopharmaceuticals. In this work, we report on the initial development of a fluidic process for rapid end-to-end production of recombinant protein biologics. This process incorporates a bioreactor module that can be used with eukaryotic or prokaryotic lysates that are programmed for combined transcription/translation of an engineered DNA template encoding for specific protein targets. Purification of the cell-free expressed product occurs through a series of protein separation modules that are configurable for process-specific isolation of different proteins. Using this approach, we demonstrate production of two bioactive human protein therapeutics, erythropoietin and granulocyte-macrophage colony-stimulating factor, in yeast and bacterial extracts, respectively, each within 24 hours. This process is flexible, scalable and amenable to automation for rapid production at the point-of-need of proteins with significant pharmaceutical, medical, or biotechnological value.

Complex systems in metabolic engineering.

Metabolic engineers manipulate intricate biological networks to build efficient biological machines. The inherent complexity of this task, derived from the extensive and often unknown interconnectivity between and within these networks, often prevents researchers from achieving desired performance. Other fields have developed methods to tackle the issue of complexity for their unique subset of engineering problems, but to date, there has not been extensive and comprehensive examination of how metabolic engineers use existing tools to ameliorate this effect on their own research projects. In this review, we examine how complexity affects engineering at the protein, pathway, and genome levels within an organism, and the tools for handling these issues to achieve high-performing strain designs. Quantitative complexity metrics and their applications to metabolic engineering versus traditional engineering fields are also discussed. We conclude by predicting how metabolic engineering practices may advance in light of an explicit consideration of design complexity.