Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening
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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
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Results for “Enzyme Engineering”
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Conformational Dynamics and Catalytic Backups in a Hyper-thermostable Engineered Archaeal Protein Tyrosine Phosphatase
Protein tyrosine phosphatases (PTPs) are a family of enzymes that play important roles in regulating cellular signaling pathways. The activity of these enzymes is regulated by the motion of a catalytic loop that places a critical conserved aspartic acid side chain into the active site for acid–base catalysis upon loop closure. These enzymes also have a conserved phosphate-binding loop that is typically highly rigid and forms a well-defined anion-binding nest. The intimate links between loop dynamics and chemistry in these enzymes make PTPs an excellent model system for understanding the role of loop dynamics in protein function and evolution. In this context, archaeal PTPs, which have often evolved in extremophilic organisms, are highly understudied, despite their unusual biophysical properties. We present here an engineered chimeric PTP (ShufPTP) generated by shuffling the amino acid sequence of five extant hyperthermophilic archaeal PTPs. Despite ShufPTP’s high sequence similarity to its natural counterparts, it presents a suite of unique properties, including high flexibility of the phosphate binding P-loop, facile oxidation of the active-site cysteine, mechanistic promiscuity, and, most notably, hyperthermostability, with a denaturation temperature likely >130 °C (>8 °C higher than the highest recorded growth temperature of any archaeal strain). Our combined structural, biochemical, biophysical, and computational analysis provides insight both into how small steps in evolutionary space can radically modulate the biophysical properties of an enzyme and showcases the tremendous potential of archaeal enzymes for biotechnology, to generate novel enzymes capable of operating under extreme conditions.
archaea↗
High-Throughput Large-Scale Targeted Proteomics Assays for Quantifying Pathway Proteins in Pseudomonas putida KT2440
Targeted proteomics is a mass spectrometry-based protein quantification technique with high sensitivity, accuracy, and reproducibility. As a key component in the multi-omics toolbox of systems biology, targeted liquid chromatography-selected reaction monitoring (LC-SRM) measurements are critical for enzyme and pathway identification and design in metabolic engineering. To fulfill the increasing need for analyzing large sample sets with faster turnaround time in systems biology, high-throughput LC-SRM is greatly needed. Even though nanoflow LC-SRM has better sensitivity, it lacks the speed offered by microflow LC-SRM. Recent advancements in mass spectrometry instrumentation significantly enhance the scan speed and sensitivity of LC-SRM, thereby creating opportunities for applying the high speed of microflow LC-SRM without losing peptide multiplexing power or sacrificing sensitivity. Here, we studied the performance of microflow LC-SRM relative to nanoflow LC-SRM by monitoring 339 peptides representing 132 enzymes in Pseudomonas putida KT2440 grown on various carbon sources. The results from the two LC-SRM platforms are highly correlated. In addition, the response curve study of 248 peptides demonstrates that microflow LC-SRM has comparable sensitivity for the majority of detected peptides and better mass spectrometry signal and chromatography stability than nanoflow LC-SRM.
59 BASIC BIOLOGICAL SCIENCES↗
Growth-uncoupled propanediol production in a Thermoanaerobacterium thermosaccharolyticum strain engineered for high ethanol yield
Abstract Cocultures of engineered thermophilic bacteria can ferment lignocellulose without costly pretreatment or added enzymes, an ability that can be exploited for low cost biofuel production from renewable feedstocks. The hemicellulose-fermenting species Thermoanaerobacterium thermosaccharolyticum was engineered for high ethanol yield, but we found that the strains switched from growth-coupled production of ethanol to growth uncoupled production of acetate and 1,2-propanediol upon growth cessation, producing up to 6.7 g/L 1,2-propanediol from 60 g/L cellobiose. The unique capability of this species to make 1,2-propanediol from sugars was described decades ago, but the genes responsible were not identified. Here we deleted genes encoding methylglyoxal reductase, methylglyoxal synthase and glycerol dehydrogenase. Deletion of the latter two genes eliminated propanediol production. To understand how carbon flux is redirected in this species, we hypothesized that high ATP levels during growth cessation downregulate the activity of alcohol and aldehyde dehydrogenase activities. Measurements with cell free extracts show approximately twofold and tenfold inhibition of these activities by 10 mM ATP, supporting the hypothesized mechanism of metabolic redirection. This result may have implications for efforts to direct and maximize flux through alcohol dehydrogenase in other species.
09 BIOMASS FUELS↗
Engineering an Extremely Hybrid PKS for Adipic Acid Production
Polyketide synthases (PKSs) are modular enzymes with exceptional potential as biocatalysts for producing non-native compounds. Here, we report the first PKS-based pathway for adipic acid (AA), an industrial monomer for nylon production, by engineering one of the most extensively hybridized PKS systems to date. Using a retrobiosynthetic approach, we identified EtnB, a succinyl-CoA-loading module that uniquely retains the terminal carboxyl group, enabling access to dicarboxylic polyketide products, rarely produced by canonical PKSs. EtnB was coupled to a fully reducing extension module through an engineered communication linker, which improved ACP–KS interactions, enhanced titers, and demonstrated selective succinyl-CoA loading in vivo . This construct integrates genes from five organisms─with domains from seven PKS modules joined across six non-natural junctions─and functions in both Escherichia coli and Pseudomonas putida . Additional engineering that included AT domain exchanges, optimization of extender unit supply, and host strain metabolic rewiring further increased AA production. Together, this work demonstrates that highly chimeric PKSs can be rendered functional through rational design, expands the PKS toolkit with a carboxyl-retaining loading module, and establishes a versatile platform for engineering diacids and other noncanonical products through PKS pathways.
biomanufacturing↗
Metabolic engineering of Escherichia coli for quinolinic acid production by assembling L-aspartate oxidase and quinolinate synthase as an enzyme complex
Not provided.
Biotechnology & Applied Microbiology↗
Experimental and theoretical insights into the effects of pH on catalysis of bond-cleavage by the lignin peroxidase isozyme H8 from Phanerochaete chrysosporium
Abstract Background Lignin peroxidases catalyze a variety of reactions, resulting in cleavage of both β-O-4′ ether bonds and C–C bonds in lignin, both of which are essential for depolymerizing lignin into fragments amendable to biological or chemical upgrading to valuable products. Studies of the specificity of lignin peroxidases to catalyze these various reactions and the role reaction conditions such as pH play have been limited by the lack of assays that allow quantification of specific bond-breaking events. The subsequent theoretical understanding of the underlying mechanisms by which pH modulates the activity of lignin peroxidases remains nascent. Here, we report on combined experimental and theoretical studies of the effect of pH on the enzyme-catalyzed cleavage of β-O-4′ ether bonds and of C–C bonds by a lignin peroxidase isozyme H8 from Phanerochaete chrysosporium and an acid stabilized variant of the same enzyme. Results Using a nanostructure initiator mass spectrometry assay that provides quantification of bond breaking in a phenolic model lignin dimer we found that catalysis of degradation of the dimer to products by an acid-stabilized variant of lignin peroxidase isozyme H8 increased from 38.4% at pH 5 to 92.5% at pH 2.6. At pH 2.6, the observed product distribution resulted from 65.5% β-O-4′ ether bond cleavage, 27.0% C α -C 1 carbon bond cleavage, and 3.6% C α -oxidation as by-product. Using ab initio molecular dynamic simulations and climbing-image Nudge Elastic Band based transition state searches, we suggest the effect of lower pH is via protonation of aliphatic hydroxyl groups under which extremely acidic conditions resulted in lower energetic barriers for bond-cleavages, particularly β-O-4′ bonds. Conclusion These coupled experimental results and theoretical explanations suggest pH is a key driving force for selective and efficient lignin peroxidase isozyme H8 catalyzed depolymerization of the phenolic lignin dimer and further suggest that engineering of lignin peroxidase isozyme H8 and other enzymes involved in lignin depolymerization should include targeting stability at low pH.
09 BIOMASS FUELS↗
From the bench to the reactor: engineered filamentous fungi for biochemical and biomaterial production
Filamentous fungi can convert a wide variety of naturally occurring chemical compounds, including organic biomass and waste streams, into a range of products. They have long been used for industrial organic acid production and food preparation. In this review, we will discuss production of products such as organic acids, lipids, small molecules, enzymes, materials, and foods, and highlight advances in metabolic and protein engineering, including CRISPR-Cas9-mediated strain improvements. We discuss to what extent these products are already being made on a commercial scale, as well as what is still required to make certain promising concepts industrially and commercially relevant. Despite significant progress, the systematic application of synthetic biology to filamentous fungi remains in its infancy, with many opportunities for discovery and innovation as new strains and genetic tools are developed. The integration of fungal biotechnology into circular and bio-based economies promises to address critical challenges in waste management, resource sustainability, and the development of new materials for terrestrial and extraterrestrial applications, but requires further developments in genetic engineering and process design.
09 BIOMASS FUELS↗
Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae
The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. Here, we applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L –1 nerolidol. Harnessing Oxford Nanopore’s long-read genomic sequencing, we reveal a potential genetic cause—the chromosome integration of a 2μ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L –1 nerolidol and ~0.96 g L –1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2μ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.
59 BASIC BIOLOGICAL SCIENCES↗
Accelerating polyketide synthase engineering for high TRY production of biofuels and bioproducts (Final Technical Report)
Polyketide synthase (PKS) enzymes have a modular, deterministic logic that holds the potential to act as a flexible chemical factory for the biological production of a huge diversity of valuable small molecule compounds. However, engineering a custom PKS to produce a specific desired product currently requires years of trial and error, for reasons that remain poorly understood. In this project, we have developed a rapid, high throughput, Design-Build-Test-Learn (DBTL) cycle for polyketide synthases (PKSs) and demonstrate its utility for production of materials precursors. The objectives are 1) to develop a rapid, high-throughput (HT) DBTL cycle for PKSs that will enable production of a large number of unnatural, organic molecules on demand at high titer, rate, and yield (TRY); 2) to demonstrate the utility of the PKS DBTL cycle to produce three molecules: one commodity chemical (caprolactam or valerolactam) and two novel materials precursors (caprolactam or valerolactam derivatives); and 3) to demonstrate the utility of the PKS DBTL cycle to increase the TRY of one molecule (caprolactam or valerolactam). In this project, we have successfully demonstrated our high throughput PKS DBTL pipeline, and have biologically produced valerolactam and several other novel nylon monomers.
60 APPLIED LIFE SCIENCES↗
Accelerating polyketide synthase engineering for high TRY production of biofuels and bioproducts (CRADA Final Report)
Polyketide synthase (PKS) enzymes have a modular, deterministic logic that holds the potential to act as a flexible chemical factory for the biological production of a huge diversity of valuable small molecule compounds. However, engineering a custom PKS to produce a specific desired product currently requires years of trial and error, for reasons that remain poorly understood. In this project, we have developed a rapid, high throughput, Design-Build-Test-Learn (DBTL) cycle for polyketide synthases (PKSs) and demonstrate its utility for production of materials precursors. The objectives are 1) to develop a rapid, high-throughput (HT) DBTL cycle for PKSs that will enable production of a large number of unnatural, organic molecules on demand at high titer, rate, and yield (TRY); 2) to demonstrate the utility of the PKS DBTL cycle to produce three molecules: one commodity chemical (caprolactam or valerolactam) and two novel materials precursors (caprolactam or valerolactam derivatives); and 3) to demonstrate the utility of the PKS DBTL cycle to increase the TRY of one molecule (caprolactam or valerolactam). In this project, we have successfully demonstrated our high throughput PKS DBTL pipeline, and have biologically produced valerolactam and several other novel nylon monomers.
09 BIOMASS FUELS↗
Toward a Circular Bioeconomy: Designing Microbes and Polymers for Biodegradation
Polymer production is rapidly increasing, but there are no large-scale technologies available to effectively mitigate the massive accumulation of these recalcitrant materials. One potential solution is the development of a carbon-neutral polymer life cycle, where microorganisms convert plant biomass to chemicals, which are used to synthesize biodegradable materials that ultimately contribute to the growth of new plants. Realizing a circular carbon life cycle requires the integration of knowledge across microbiology, bioengineering, materials science, and organic chemistry, which itself has hindered large-scale industrial advances. This review addresses the biodegradation status of common synthetic polymers, identifying novel microbes and enzymes capable of metabolizing these recalcitrant materials and engineering approaches to enhance their biodegradation pathways. Design considerations for the next generation of biodegradable polymers are also reviewed, and finally, opportunities to apply findings from lignocellulosic biodegradation to the design and biodegradation of similarly recalcitrant synthetic polymers are discussed.
59 BASIC BIOLOGICAL SCIENCES↗
Acetate as a Platform for Carbon-Negative Production of Renewable Fuels and Chemicals (Final Technical Report)
This project was an industrial-academic collaboration between experts at the University of Wisconsin-Madison, the University of Kentucky, and LanzaTech, a world leader in the use of gas fermentation to sustainably produce fuels and chemicals. The project developed technologies to create an integrated process for converting carbon dioxide and renewable hydrogen into molecules that can be blended with liquid transportation fuels or used in an array of chemical applications. The project was motivated by the Program Objectives of eliminating carbon dioxide release in the production of chemicals by integrating the unique and efficient capabilities of two microorganisms into a single process. The first microbe, an acetogen, produces acetate from carbon dioxide and hydrogen while the second microbe upgrades acetate from acetogen fermentation permeates to higher-value chemical products. The carbon dioxide released in the upgrading process is recycled internally to produce more acetate. As such, the process can be designed to operate with zero carbon dioxide release and net positive carbon dioxide capture. The process has the potential to provide an alternative paradigm to the current bioeconomy – one in which acetate is the primary energy carrier instead of sugars. Our process by-passes photosynthesis and the barriers created by biomass as primary chemical feedstock. As such, the process can be scaled to meet existing sources of carbon dioxide emissions and located anywhere renewable hydrogen can be provided. Our work developed microorganisms with optimized metabolism for producing acetate and other microorganisms with improved conversion of acetate to dodecanol and dodecyl-acetate. We developed synthetic biology tools for a promising non-model bacterium that could enhance metabolic engineering efforts to convert acetate to chemical products. We conducted protein engineering studies to improve the activity of key enzymes involved in our metabolic pathways. We conducted a full technoeconomic analysis that set technical targets for each strain to meet economic goals. We identified key technical barriers in our process and proposed strategies to overcome them.
09 BIOMASS FUELS↗
Continuous multimodal technologies in industrial microbiology: potential for achieving high process performance and agility
The field of industrial microbiology has great potential to tackle the most pressing challenges facing society, but today, this entails much more than classical fermentation technology followed by product upgrading. Methods are needed to confer unit-operation-level functionality and whole-system-level robustness, as well as flexibility. As examples of this concept, we focus here on methodologies often deployed at industrial scale as discrete processes—such as biological, chemical, and electrochemical unit operations—that are also capable of substantial complementarity when combined sequentially and operated continuously. A key and relatively new type of conversion step is made possible by catalyst immobilization methods at both the enzymatic and cellular level. Cases drawn from electro-fermentation for acetoin production and syngas-mediated microbial electrosynthesis of ethanol exemplify the synergy of combined operational approaches. Because modular processes can be treated as a series of reactor elements, considerable flexibility is possible. As discussed, however, not all processes are amenable to this intensification.
09 BIOMASS FUELS↗
Structural Characterization and Dynamics of AdhE Ultrastructures from Clostridium thermocellum Show a Containment Strategy for Toxic Intermediates
Clostridium thermocellum, a cellulolytic thermophilic anaerobe, is considered by many to be a prime candidate for the realization of consolidated bioprocessing (CBP) and is known as an industry standard for biofuel production. C. thermocellum is among the best biomass degraders identified to date in nature and produces ethanol as one of its main products. Many studies have helped increase ethanol titers in this microbe; however, ethanol production using C. thermocellum is still not economically viable. Therefore, a better understanding of its ethanol synthesis pathway is required. The main pathway for ethanol production in C. thermocellum involves the bifunctional aldehyde-alcohol dehydrogenase (AdhE). To better understand the function of the C. thermocellum AdhE, we used cryo-electron microscopy (cryo-EM) to obtain a 3.28 A structure of the AdhE complex. This high-resolution structure, in combination with molecular dynamics simulations, provides insight into the substrate channeling of the toxic intermediate acetaldehyde, indicates the potential role of C. thermocellum AdhE to regulate activity and cofactor pools, and establishes a basis for future engineering studies. The containment strategy found in this enzyme offers a template that could be replicated in other systems where toxic intermediates need to be sequestered to increase the production of valuable biochemicals.
09 BIOMASS FUELS↗
Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9
Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.
59 BASIC BIOLOGICAL SCIENCES↗
Engineered enzymatic cascade converts diols to amino alcohols
Aliphatic amino alcohols such as 6-amino-1-hexanol are potential platform chemicals for a variety of advanced materials, but applications are currently limited by reagent costs. Aliphatic amino alcohols can currently be synthesized from biomass-derived diols at elevated temperatures and pressures using Ru-based catalysts that produce a mixture of amino-alcohol, diamine, and cyclic amine products. Replacing chemical amination with an enzymatic cascade would reduce resource needs and enable reactions under milder conditions. In this work, we characterized a two-enzyme cascade that selectively converts C4–C7 diols to the corresponding amino alcohols under aqueous conditions at room temperature and pressure. By engineering the rate-limiting enzyme and optimizing reaction conditions, we increased amino alcohol production nearly 30-fold, achieving a selectivity of 99%. The same enzyme cascade could also be used to convert amino alcohols into cyclic amines through reduction of the corresponding cyclic imine. This engineered cascade provides a green opportunity to sustainably synthesize asymmetric bifunctional platform chemicals.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Verazine biosynthesis from simple sugars in engineered Saccharomyces cerevisiae
Steroidal alkaloids are FDA-approved drugs (e.g., Zytiga) and promising drug candidates/leads (e.g., cyclopamine); yet many of the ≥697 known steroidal alkaloid natural products remain underutilized as drugs because it can be challenging to scale their biosynthesis in their producing organisms. Cyclopamine is a steroidal alkaloid produced by corn lily (Veratrum spp.) plants, and it is an inhibitor of the Hedgehog (Hh) signaling pathway. Therefore, cyclopamine is an important drug candidate/lead to treat human diseases that are associated with dysregulated Hh signaling, such as basal cell carcinoma and acute myeloid leukemia. Cyclopamine and its semi-synthetic derivatives have been studied in (pre)clinical trials as Hh inhibitor-based drugs. However, challenges in scaling the production of cyclopamine have slowed efforts to improve its efficacy and safety profile through (bio)synthetic derivatization, often limiting drug development to synthetic analogs of cyclopamine such as the FDA-approved drugs Odomzo, Daurismo, and Erivedge. If a platform for the scalable and sustainable production of cyclopamine were established, then its (bio)synthetic derivatization, clinical development, and, ultimately, widespread distribution could be accelerated. Ongoing efforts to achieve this goal include the biosynthesis of cyclopamine in Veratrum plant cell culture and the semi-/total chemical synthesis of cyclopamine. Herein, this work advances efforts towards a promising future approach: the biosynthesis of cyclopamine in engineered microorganisms. We completed the heterologous microbial production of verazine (biosynthetic precursor to cyclopamine) from simple sugars (i.e., glucose and galactose) in engineered Saccharomyces cerevisiae (S. cerevisiae) through the inducible upregulation of the native yeast mevalonate and lanosterol biosynthetic pathways, diversion of biosynthetic flux from ergosterol (i.e., native sterol in S. cerevisiae) to cholesterol (i.e., biosynthetic precursor to verazine), and expression of a refactored five-step verazine biosynthetic pathway. The engineered S. cerevisiae strain that produced verazine contains eight heterologous enzymes sourced from seven different species. Importantly, S. cerevisiae-produced verazine was indistinguishable via liquid chromatography-mass spectrometry from both a commercial standard (Veratrum spp. plant-produced) and Nicotiana benthamiana-produced verazine. To the best of our knowledge, this is the first report describing the heterologous production of a steroidal alkaloid in an engineered yeast. Verazine production was ultimately increased through design-build-test-learn cycles to a final titer of 83 ± 3 μg/L (4.1 ± 0.1 μg/g DCW). Finally, this research lays the groundwork for future microbial biosynthesis of cyclopamine, (bio)synthetic derivatives of cyclopamine, and other steroidal alkaloid natural products.
59 BASIC BIOLOGICAL SCIENCES↗