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At least 325 records · Page 18
All sourcesNASA NTRSDOE OSTI
DOE OSTI2010

Interconversion of the Specificities of Human Lysosomal Enzymes Associated with Fabry and Schindler Diseases

The human lysosomal enzymes {alpha}-galactosidase ({alpha}-GAL, EC 3.2.1.22) and {alpha}-N-acetylgalactosaminidase ({alpha}-NAGAL, EC 3.2.1.49) share 46% amino acid sequence identity and have similar folds. The active sites of the two enzymes share 11 of 13 amino acids, differing only where they interact with the 2-position of the substrates. Using a rational protein engineering approach, we interconverted the enzymatic specificity of {alpha}-GAL and {alpha}-NAGAL. The engineered {alpha}-GAL (which we call {alpha}-GALSA) retains the antigenicity of {alpha}-GAL but has acquired the enzymatic specificity of {alpha}-NAGAL. Conversely, the engineered {alpha}-NAGAL (which we call {alpha}-NAGAL{sup EL}) retains the antigenicity of {alpha}-NAGAL but has acquired the enzymatic specificity of the {alpha}-GAL enzyme. Comparison of the crystal structures of the designed enzyme {alpha}-GAL{sup SA} to the wild-type enzymes shows that active sites of {alpha}-GAL{sup SA} and {alpha}-NAGAL superimpose well, indicating success of the rational design. The designed enzymes might be useful as non-immunogenic alternatives in enzyme replacement therapy for treatment of lysosomal storage disorders such as Fabry disease.

36 MATERIALS SCIENCE↗
DOE OSTI2018

Engineering Anaerobic Gut Fungi for Lignocellulose Breakdown

Biofuels derived from plant biomass (lignocellulose) are attractive alternatives to petroleum-based products, as they avoid many of the economic and environmental problems that plague traditional energy sources. Breakdown of plant waste into simple sugars would translate into the production of renewable fuels via microbial fermentation. However, existing technologies are insufficient to allow for industrial-scale production of these products due to difficulties associated with the recalcitrance of crude lignin-rich biomass, and the high cost/poor performance of known cellulolytic enzymes (cellulases). Therefore, there is a critical need to develop new technologies to break down lignocellulosic biomass into fermentable sugars for downstream fuel development. Towards this goal, much can be learned by studying how anaerobic microbes depolymerize lignocellulose in biomass-rich environments, such as the digestive tract of large herbivores. Anaerobic gut fungi are native to the gut and rumen of these animals, where they have evolved unique abilities to break down lignocellulosic biomass through invasive growth and the secretion of powerful enzymes. The goal of this project was to engineer anaerobic gut fungi as novel platform organisms for biofuel production from plant material. To accomplish this goal, a panel of anaerobic fungi was isolated form different herbivores and screened for their ability to degrade lignocellulose. Four novel strains were isolated and genetically sequenced, including Piromyces finnis, Neocallimastix californiae, Anaeromyces robustus, and Caecomyces churrovis. Transcriptomic and genomic characterization of these anaerobic fungi revealed that they harbor the largest collection of biomass-degrading enzymes of any sequenced fungus, and delineated the basic metabolic networks that govern lignocellulose hydrolysis within anaerobic fungi, RNA sequencing enabled the dynamics and mechanism associated with carbon catabolite repression to be delineated, which revealed how important enzyme groups are coordinated during breakdown. Through cluster analysis, a set of putative novel enzymes associated with lignocellulose breakdown was discovered, which also revealed a set of proteins critical to the formation of fungal cellulosomes (multi-enzyme complexes). Using this information, genetic transformation strategies to manipulate gut fungi were developed, which would endow them with enhanced functionality against a range of industrially relevant substrates. Collectively, this information will establish the molecular framework for anaerobic fungal hydrolysis, and will guide in the development of lignocellulosic biofuels.

09 BIOMASS FUELS↗
DOE OSTI2018

Comparative studies of glycolytic pathways and channeling under in vitro and in vivo modes

This study constructed cell‐free glycolytic enzyme systems and compared them to their in vivo functions in Escherichia coli . Under in vitro conditions, flux regulation followed enzyme concentrations and kinetics. In E. coli, only one of the isozymes of phosphofructokinase (PfkA) and fructose‐bisphosphate aldolase (FbaA) facilitate Embden‐Meyerhof‐Parnas (EMP) flux, but under in vitro assays, these isozymes were interchangeable. Additionally, in vitro introduction of the Entner–Doudoroff (ED) pathway improved glycolysis rates, while in vivo overexpression of the ED pathway could not capture significant flux unless its phosphotransferase system (PTS) was knocked out. Lastly, in vivo dynamic 13 C‐experiments revealed that the labeling order of EMP pathway intermediates was not strictly cascade, indicating intracellular metabolites were not well mixed. These enigmatic observations cannot be fully explained by thermodynamics or substrate level regulations. This article supports the long‐time conjecture that EMP enzymes are channeled, and the PTS may be an anchor point to initiate enzyme assemblies. © 2018 American Institute of Chemical Engineers AIChE J , 65: 483–490, 2019

Abernathy, Mary H.↗
DOE OSTI2014

Search, engineering, and applications of new oxidative biocatalysts

Abstract Most industrial enzymes are hydrolases, such as glycosidases and esterases. However, oxidoreductases have an unexploited potential for substituting harsh (and scarcely selective) chemical processes. A group of basidiomycetes are the only organisms degrading the aromatic lignin polymer, enabling the subsequent use of plant polysaccharides. Therefore, these fungi and their ligninolytic peroxidases are the biocatalysts of choice for industrial delignification and oxidative biotransformations of aromatic and other organic compounds. The latter also include oxygenation reactions, which are catalyzed with high regio/stereo selectivity by fungal peroxygenases. In search for novel and more robust peroxidases/peroxygenases, basidiomycetes from unexplored habitats were screened, and hundreds of genes identified in basidiomycete genomes (in collaboration with the DOE JGI). The most interesting genes were heterologously expressed, and the corresponding enzymes structurally‐functionally characterized. The information obtained enabled us to improve the enzyme operational and catalytic properties by directed mutagenesis. However, the structural‐functional relationships explaining some desirable properties are not established yet and, therefore, their introduction was addressed by ‘non‐rational’ directed evolution. Then, over 100 oxidative biotransformations were analyzed. Among them, it is noteworthy to mention the regio/stereo selective hydroxylation of long/short‐chain alkanes (a chemically challenging reaction), epoxidation of alkenes, and production of hydroxy‐fatty acids. Concerning aromatic oxygenations, the regioselective hydroxylation of flavonoids, and stereoselective hydroxylation/epoxidation of alkyl/alkenyl‐benzenes were among the most remarkable reactions, together with enzymatic hydroxylation of benzene (as an alternative for harsh chemical process). Finally, peroxidases and peroxygenases also showed a potential as delignification biocatalysts and in the decolorization of contaminant dyes from textile industries. © 2014 The Authors. Biofuels, Bioproducts and Biorefi ning published by Society of Chemical Industry and John Wiley & Sons, Ltd.

Martínez, Angel T.↗
DOE OSTI2018

Biosynthesis of monoethylene glycol in Saccharomyces cerevisiae utilizing native glycolytic enzymes

Monoethylene glycol (MEG) is an important commodity chemical with applications in numerous industrial processes, primarily in the manufacture of polyethylene terephthalate (PET) polyester used in packaging applications. In the drive towards a sustainable chemical industry, bio-based production of MEG from renewable biomass has attracted growing interest. Recent attempts for bio-based MEG production have investigated metabolic network modifications in Escherichia coli, specifically rewiring the xylose assimilation pathways for the synthesis of MEG. In the present study, we examined the suitability of Saccharomyces cerevisiae, a preferred organism for industrial applications, as platform for MEG biosynthesis. Here, based on combined genetic, biochemical and fermentation studies, we report evidence for the existence of an endogenous biosynthetic route for MEG production from D-xylose in S. cerevisiae which consists of phosphofructokinase and fructose-bisphosphate aldolase, the two key enzymes in the glycolytic pathway. Further metabolic engineering and process optimization yielded a strain capable of producing up to 4.0 g/L MEG, which is the highest titer reported in yeast to-date.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450 BM3 Variants

Cytochrome P450 BM3 is a heme-containing enzyme from Bacillus megaterium that displays high monooxygenase activity and has a self-sufficient electron transfer system in the full-length enzyme. Its potential synthetic applications drive protein engineering efforts to produce variants capable of oxidizing nonnative substrates such as pharmaceuticals and aromatic pollutants. However, promiscuous P450 BM3 mutants often exhibit lower stability, thereby hindering their industrial application. This study demonstrated that the heme domain R47L/F87V/L188Q/E267V/F81I pentuple mutant (PM) is destabilized because of the disruption of hydrophobic contacts and salt bridge interactions. This was directly observed from crystal structures of PM in the presence and absence of ligands (palmitic acid and metyrapone). The instability of the tertiary structure and heme environment of substrate-free PM was confirmed by pulse proteolysis and circular dichroism, respectively. Binding of the inhibitor, metyrapone, significantly stabilized PM, but the presence of the native substrate, palmitic acid, had no effect. On the basis of high-temperature molecular dynamics simulations, the lid domain, β-sheet 1, and Cys ligand loop (a β-bulge segment connected to the heme) are the most labile regions and, thus, potential sites for stabilizing mutations. Possible approaches to stabilization include improvement of hydrophobic packing interactions in the lid domain and introduction of new salt bridges into β-sheet 1 and the heme region. An understanding of the molecular factors behind the loss of stability of P450 BM3 variants therefore expedites site-directed mutagenesis studies aimed at developing thermostability.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2014

Elements Required for an Efficient NADP-Malic Enzyme Type C4 Photosynthesis

Abstract C4 photosynthesis has higher light, nitrogen, and water use efficiencies than C3 photosynthesis. Although the basic anatomical, cellular, and biochemical features of C4 photosynthesis are well understood, the quantitative significance of each element of C4 photosynthesis to the high photosynthetic efficiency are not well defined. Here, we addressed this question by developing and using a systems model of C4 photosynthesis, which includes not only the Calvin-Benson cycle, starch synthesis, sucrose synthesis, C4 shuttle, and CO2 leakage, but also photorespiration and metabolite transport between the bundle sheath cells and mesophyll cells. The model effectively simulated the CO2 uptake rates, and the changes of metabolite concentrations under varied CO2 and light levels. Analyses show that triose phosphate transport and CO2 leakage can help maintain a high photosynthetic rate by balancing ATP and NADPH amounts in bundle sheath cells and mesophyll cells. Finally, we used the model to define the optimal enzyme properties and a blueprint for C4 engineering. As such, this model provides a theoretical framework for guiding C4 engineering and studying C4 photosynthesis in general.

09 BIOMASS FUELS↗
DOE OSTI2026

Multi-omic characterization of a soil microbial consortium reveals critical role of succinate and glutamate metabolism during calcium carbonate precipitation

Microbially induced calcium carbonate precipitation (MICP) holds potential for use in soil stabilization and carbon sequestration, with the overall efficiency of the process being a major determinant for use in many environmental and civil engineering applications. While the biogeochemical pathways and enzymes driving MICP are known, the microbial metabolic networks and community dynamics underlying such precipitation remain poorly characterized. To address this gap, we developed a four-member consortium of soil bacteria (Curtobacterium flaccumfaciens, Rhodococcus qingshengii, Microbacterium sp., and Bacillus toyonensis), termed carbon storing consortium - A (CSC-A), that is capable of MICP. Prior work shows that MICP production is higher in CSC-A compared to the sum of carbonate produced by each member, suggesting carbonate production is driven by consortium dynamics. To that end we used a multi-omic integration approach of genomics, transcriptomics, and metabolomics to investigate potential inter-species interactions that may influence the MICP phenotype. Genomic life history characterizations identified evidence of niche specialization by B. toyonensis and Microbacterium, while metatranscriptomic analysis suggests R. qingshengii is a keystone species during growth in urea. By comparing individual species’ metabolomes to the metabolic profile of a shared well of precipitated metabolites, we identified over 200 metabolites predicted to be produced or consumed by CSC-A members. Integrating both data types to search the KEGG reactome highlighted a network centered around glutamine metabolism and branched chain amino acid biosynthesis under regulation during CSC-A growth in urea. Succinate metabolism was also a major node in this network and laboratory assays confirmed that increasing the amount of succinate in the growth medium leads to increased carbonate precipitation by CSC-A, a critical confirmation of our modeling approach. By isolating and identifying the interconnected metabolic components underlying MICP in CSC-A, we identified keystone taxa, metabolites, and pathways important for future optimization of the application of this consortia to carbonate precipitation.

carbon storing consortium - A (CSC-A)↗
DOE OSTI2022

Understanding of bacterial lignin extracellular degradation mechanisms by Pseudomonas putida KT2440 via secretomic analysis

Abstract Background Bacterial lignin degradation is believed to be primarily achieved by a secreted enzyme system. Effects of such extracellular enzyme systems on lignin structural changes and degradation pathways are still not clearly understood, which remains as a bottleneck in the bacterial lignin bioconversion process. Results This study investigated lignin degradation using an isolated secretome secreted by Pseudomonas putida KT2440 that grew on glucose as the only carbon source. Enzyme assays revealed that the secretome harbored oxidase and peroxidase/Mn 2+ -peroxidase capacity and reached the highest activity at 120 h of the fermentation time. The degradation rate of alkali lignin was found to be only 8.1% by oxidases, but increased to 14.5% with the activation of peroxidase/Mn 2+ -peroxidase. Gas chromatography–mass spectrometry (GC–MS) and two-dimensional 1 H– 13 C heteronuclear single-quantum coherence (HSQC) NMR analysis revealed that the oxidases exhibited strong C–C bond ( β-β , β -5, and β -1) cleavage. The activation of peroxidases enhanced lignin degradation by stimulating C–O bond ( β -O-4) cleavage, resulting in increased yields of aromatic monomers and dimers. Further mass spectrometry-based quantitative proteomics measurements comprehensively identified different groups of enzymes particularly oxidoreductases in P. putida secretome, including reductases, peroxidases, monooxygenases, dioxygenases, oxidases, and dehydrogenases, potentially contributed to the lignin degradation process. Conclusions Overall, we discovered that bacterial extracellular degradation of alkali lignin to vanillin, vanillic acid, and other lignin-derived aromatics involved a series of oxidative cleavage, catalyzed by active DyP-type peroxidase, multicopper oxidase, and other accessory enzymes. These results will guide further metabolic engineering design to improve the efficiency of lignin bioconversion. Graphical Abstract

09 BIOMASS FUELS↗
DOE OSTI2021

Identification and structural analysis of a thermophilic β-1,3-glucanase from compost

β-1,3-glucanase can specifically hydrolyze glucans to oligosaccharides and has potential applications in biotechnology. We used the metatranscriptomic technology to discover a thermophilic β-1,3-glucanase from compost. The phylogenetic study shows that it belongs to the family 16 glycoside hydrolase (GH16) and is most homologous with an enzyme from Streptomyces sioyaensis, an actinobacterium. It has the activity of 146.9 U/mg in the optimal reaction condition (75 °C and pH 5.5). Its catalytic domain was crystallized and diffracted to 1.14 Å resolution. The crystal structure shows a sandwich-like β-jelly-roll fold with two disulfide bonds. After analyzing the occurring frequencies of these cysteine residues, we designed two mutants (C160G and C180I) to study the role of these disulfide bonds. Both mutants have decreased their optimal temperature from 75 to 70 °C, which indicate that the disulfide bonds are important to maintain thermostability. Interestingly, the activity of C160G has increased ~ 17% to reach 171.4 U/mg. We speculate that the increased activity of C160G mutant is due to increased dynamics near the active site. Our studies give a good example of balancing the rigidity and flexibility for enzyme activity, which is helpful for protein engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Robust Expression and Secretion of Xylanase1 in Chlamydomonas reinhardtii by Fusion to a Selection Gene and Processing with the FMDV 2A Peptide

Microalgae have recently received attention as a potential low-cost host for the production of recombinant proteins and novel metabolites. However, a major obstacle to the development of algae as an industrial platform has been the poor expression of heterologous genes from the nuclear genome. Here we describe a nuclear expression strategy using the foot-and-mouth-disease-virus 2A self-cleavage peptide to transcriptionally fuse heterologous gene expression to antibiotic resistance in Chlamydomonas reinhardtii. We demonstrate that strains transformed with ble-2A-GFP are zeocin-resistant and accumulate high levels of GFP that is properly ‘cleaved’ at the FMDV 2A peptide resulting in monomeric, cytosolic GFP that is easily detectable by in-gel fluorescence analysis or fluorescent microscopy. Furthermore, we used our ble2A nuclear expression vector to engineer the heterologous expression of the industrial enzyme, xylanase. We demonstrate that linking xyn1 expression to ble2A expression on the same open reading frame led to a dramatic (~100-fold) increase in xylanase activity in cells lysates compared to the unlinked construct. Finally, by inserting an endogenous secretion signal between the ble2A and xyn1 coding regions, we were able to target monomeric xylanase for secretion. The novel microalgae nuclear expression strategy described here enables the selection of transgenic lines that are efficiently expressing the heterologous gene-of-interest and should prove valuable for basic research as well as algal biotechnology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae

Despite the extensive use of Saccharomyces cerevisiae as a platform for synthetic biology, strain engineering remains slow and laborious. Here, we employ CRISPR/Cas9 technology to build a cloning-free toolkit that addresses commonly encountered obstacles in metabolic engineering, including chromosomal integration locus and promoter selection, as well as protein localization and solubility. The toolkit includes 23 Cas9-sgRNA plasmids, 37 promoters of various strengths and temporal expression profiles, and 10 protein-localization, degradation and solubility tags. We facilitated the use of these parts via a web-based tool, that automates the generation of DNA fragments for integration. Our system builds upon existing gene editing methods in the thoroughness with which the parts are standardized and characterized, the types and number of parts available and the ease with which our methodology can be used to perform genetic edits in yeast. We demonstrated the applicability of this toolkit by optimizing the expression of a challenging but industrially important enzyme, taxadiene synthase (TXS). This approach enabled us to diagnose an issue with TXS solubility, the resolution of which yielded a 25-fold improvement in taxadiene production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The Moderately (D)efficient Enzyme: Catalysis-Related Damage In Vivo and Its Repair

Enzymes have in vivo life spans. Analysis of life spans, i.e., lifetime totals of catalytic turnovers, suggests that nonsurvivable collateral chemical damage from the very reactions that enzymes catalyze is a common but underdiagnosed cause of enzyme death. Analysis also implies that many enzymes are moderately deficient in that their active-site regions are not naturally as hardened against such collateral damage as they could be, leaving room for improvement by rational design or directed evolution. Enzyme life span might also be improved by engineering systems that repair otherwise fatal active-site damage, of which a handful are known and more are inferred to exist. Unfortunately, the data needed to design and execute such improvements are lacking: there are too few measurements of in vivo life span, and existing information about the extent, nature, and mechanisms of active-site damage and repair during normal enzyme operation is too scarce, anecdotal, and speculative to act on. Fortunately, advances in proteomics, metabolomics, cheminformatics, comparative genomics, and structural biochemistry now empower a systematic, data-driven approach for identifying, predicting, and validating instances of active-site damage and its repair. These capabilities would be practically useful in enzyme redesign and improvement of in-use stability and could change our thinking about which enzymes die young in vivo, and why.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2015

Deletion of nfnAB in Thermoanaerobacterium saccharolyticum and Its Effect on Metabolism

NfnAB catalyzes the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP + . The NfnAB complex has been hypothesized to be the main enzyme for ferredoxin oxidization in strains of Thermoanaerobacterium saccharolyticum engineered for increased ethanol production. NfnAB complex activity was detectable in crude cell extracts of T. saccharolyticum. In this paper, activity was also detected using activity staining of native PAGE gels. The nfnAB gene was deleted in different strains of T. saccharolyticum to determine its effect on end product formation. In wild-type T. saccharolyticum, deletion of nfnAB resulted in a 46% increase in H 2 formation but otherwise little change in other fermentation products. In two engineered strains with 80% theoretical ethanol yield, loss of nfnAB caused two different responses: in one strain, ethanol yield decreased to about 30% of the theoretical value, while another strain had no change in ethanol yield. Biochemical analysis of cell extracts showed that the ΔnfnAB strain with decreased ethanol yield had NADPH-linked alcohol dehydrogenase (ADH) activity, while the ΔnfnAB strain with unchanged ethanol yield had NADH-linked ADH activity. Deletion of nfnAB caused loss of NADPH-linked ferredoxin oxidoreductase activity in all cell extracts. Significant NADH-linked ferredoxin oxidoreductase activity was seen in all cell extracts, including those that had lost nfnAB. This suggests that there is an unidentified NADH:ferredoxin oxidoreductase (distinct from nfnAB) playing a role in ethanol formation. The NfnAB complex plays a key role in generating NADPH in a strain that had become reliant on NADPH-ADH activity. Importance: Thermophilic anaerobes that can convert biomass-derived sugars into ethanol have been investigated as candidates for biofuel formation. Many anaerobes have been genetically engineered to increase biofuel formation; however, key aspects of metabolism remain unknown and poorly understood. One example is the mechanism for ferredoxin oxidation and transfer of electrons to NAD(P) + . The electron-bifurcating enzyme complex NfnAB is known to catalyze the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP + and is thought to play key roles linking NAD(P)(H) metabolism with ferredoxin metabolism. Finally, we report the first deletion of nfnAB and demonstrate a role for NfnAB in metabolism and ethanol formation in Thermoanaerobacterium saccharolyticum and show that this may be an important feature among other thermophilic ethanologenic anaerobes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Complete replacement of Arabidopsis oil-producing enzymes with heterologous diacylglycerol acyltransferases

Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1) share responsibility for triacylglycerol (TAG) biosynthesis, and their selectivities control TAG fatty acid (FA) compositions. For rational metabolic engineering of seed oils, replacing endogenous TAG biosynthesis with exogenous enzymes containing different substrate FA selectivities is desirable; however, the dgat1-1/pdat1-2 double mutant is pollen lethal. Here, we evaluated the ability of 3 DGAT1s, from phylogenetically diverse plants with distinct TAG assembly processes, to completely replace endogenous TAG biosynthesis in Arabidopsis ( Arabidopsis thaliana ). We transformed dgat1-1 mutant plants with expression constructs for DGAT1 s from Camelina sativa , Physaria fendleri , and castor ( Ricinus communis ). Transgene expression was properly “contextualized” by using a previously determined minimum necessary expression unit containing the promoter/5′ UTR and first intron of native AtDGAT1 ; both of these DNA elements are essential for pollen expression. Next, we crossed homozygous lines with a DGAT1/DGAT1/PDAT1/pdat1-2 parent. C. sativa and P. fendleri DGAT1s restored the FA compositions and transcriptional differences of dgat1-1 to near wild-type and rescued the dgat1-1/pdat1-2 pollen lethality. R. communis DGAT1 was active in dgat1-1 seeds but produced unique oil profiles and alterations in the expression of lipid metabolic genes; it also failed to rescue dgat1-1/pdat1-2 lethality. This study confirms that the promoter and first intron of AtDGAT1 can modulate the expression of foreign DGAT1 genes to fit the correct spatiotemporal profile necessary for completely replacing endogenous TAG biosynthesis. Furthermore, it demonstrates an additional layer of unexpected enzyme incompatibility between oilseed lineages, which may complicate bioengineering approaches that seek to replace essential genes with orthologs.

McGuire, Sean T. [Washington State Univ., Pullman,↗
DOE OSTI2019

Sensor-Enabled Alleviation of Product Inhibition in Chorismate Pyruvate-Lyase

Product inhibition is a frequent bottleneck in industrial enzymes, and testing mutations to alleviate product inhibition via traditional methods remains challenging as many variants need to be tested against multiple substrate and product concentrations. Further, traditional screening methods are conducted in vitro, and resulting enzyme variants may perform differently in vivo in the context of whole-cell metabolism and regulation. In this study, we address these two problems by establishing a high-throughput screening method to alleviate product inhibition in an industrially relevant enzyme, chorismate pyruvate-lyase (UbiC). First, we engineered a highly specific, genetically encoded biosensor for 4-hydroxybenzoate (4HB) in an industrially relevant host, Pseudomonas putida KT2440. We subsequently applied the biosensor to detect the activity of a heterologously expressed UbiC that converts chorismate into 4HB and pyruvate. By using benzoate as a product surrogate that inhibits UbiC without activating the biosensor, we were able to efficiently create and screen a diversified library for UbiC variants with reduced product inhibition. Introduction of the improved UbiC enzyme variant into an experimental production strain for the industrial precursor cis,cis-muconic acid (muconate), enabled a >2-fold yield improvement for glucose to muconate conversion when the new UbiC variant was expressed from a plasmid and a 60% yield increase when the same UbiC variant was genomically integrated into the strain. Overall, this work demonstrates that by coupling a library of enzyme variants to whole-cell catalysis and biosensing, variants with reduced product inhibition can be identified, and that this improved enzyme can result in increased titers of a downstream molecule of interest.

09 BIOMASS FUELS↗
DOE OSTI2022

Debottlenecking 4-hydroxybenzoate hydroxylation in Pseudomonas putida KT2440 improves muconate productivity from p -coumarate

The transformation of 4-hydroxybenzoate (4-HBA) to protocatechuate (PCA) is catalyzed by flavoprotein oxygenases known as para-hydroxybenzoate-3-hydroxylases (PHBHs). In Pseudomonas putida KT2440 (P. putida) strains engineered to convert lignin-related aromatic compounds to muconic acid (MA), PHBH activity is rate-limiting, as indicated by the accumulation of 4-HBA, which ultimately limits MA productivity. Here, we hypothesized that replacement of PobA, the native P. putida PHBH, with PraI, a PHBH from Paenibacillus sp. JJ-1b with a broader nicotinamide cofactor preference, could alleviate this bottleneck. Biochemical assays confirmed the strict preference of NADPH for PobA, while PraI can utilize either NADH or NADPH. Kinetic assays demonstrated that both PobA and PraI can utilize NADPH with comparable catalytic efficiency and that PraI also efficiently utilizes NADH at roughly half the catalytic efficiency. The X-ray crystal structure of PraI was solved and revealed absolute conservation of the active site architecture to other PHBH structures despite their differing cofactor preferences. To understand the effect in vivo, we compared three P. putida strains engineered to produce MA from p-coumarate (pCA), showing that expression of praI leads to lower 4-HBA accumulation and decreased NADP+/NADPH ratios relative to strains harboring pobA, indicative of a relieved 4-HBA bottleneck due to increased NADPH availability. In bioreactor cultivations, a strain exclusively expressing praI achieved a titer of 40 g/L MA at 100% molar yield and a productivity of 0.5 g/L/h. Altogether, this study demonstrates the benefit of sampling readily available natural enzyme diversity for debottlenecking metabolic flux in an engineered strain for microbial conversion of lignin-derived compounds to value-added products.

09 BIOMASS FUELS↗
DOE OSTI2014

Protein design for pathway engineering

Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. (C) 2013 Elsevier Inc. All rights reserved.

Eriksen, DT↗