Background Klebsiella pneumoniae contains an endogenous isobutanol synthesis pathway. The ipdC gene annotated as an indole-3-pyruvate decarboxylase (Kp-IpdC), was identified to catalyze the formation of isobutyraldehyde from 2-ketoisovalerate. Results Compared with 2-ketoisovalerate decarboxylase from Lactococcus lactis (KivD), a decarboxylase commonly used in artificial isobutanol synthesis pathways, Kp-IpdC has an 2.8-fold lower K m for 2-ketoisovalerate, leading to higher isobutanol production without induction. However, expression of ipdC by IPTG induction resulted in a low isobutanol titer. In vitro enzymatic reactions showed that Kp-IpdC exhibits promiscuous pyruvate decarboxylase activity, which adversely consume the available pyruvate precursor for isobutanol synthesis. To address this, we have engineered Kp-IpdC to reduce pyruvate decarboxylase activity. From computational modeling, we identified 10 amino acid residues surrounding the active site for mutagenesis. Ten designs consisting of eight single-point mutants and two double-point mutants were selected for exploration. Mutants L546W and T290L that showed only 5.1% and 22.1% of catalytic efficiency on pyruvate compared to Kp-IpdC, were then expressed in K. pneumoniae for in vivo testing. Isobutanol production by K. pneumoniae T290L was 25% higher than that of the control strain, and a final titer of 5.5 g/L isobutanol was obtained with a substrate conversion ratio of 0.16 mol/mol glucose. Conclusions This research provides a new way to improve the efficiency of the biological route of isobutanol production.

We report a dramatic effect on product outcomes of the lithium ion enabled amino-Cope-like anionic asymmetric cascade when different γ-dienolate heteroatom substituents are employed. For dienolates with azide, thiomethyl, and trifluoromethylthiol substituents, a Mannich/amino-Cope/cyclization cascade ensues to form chiral cyclohexenone products with two new stereocenters in an anti-relationship. For fluoride-substituted nucleophiles, a Mannich/amino-Cope cascade proceeds to afford chiral acyclic products with two new stereocenters in a syn-relationship. Bromide- and chloride-substituted nucleophiles appear to proceed via the same pathway as the fluoride albeit with the added twist of a 3-exo-trig cyclization to yield chiral cyclopropane products with three stereocenters. When this same class of nucleophiles is substituted with a γ-nitro group, the Mannich-initiated cascade is now diverted to a β-lactam product instead of the amino-Cope pathway. These anionic asymmetric cascades are solvent- and counterion-dependent, with a lithium counterion being essential in combination with etheral solvents such as MTBE and CPME. By altering the geometry of the imine double bond from E to Z, the configurations at the R1 and X stereocenters are flipped. Mechanistic, computational, substituent, and counterion studies suggest that these cascades proceed via a common Mannich-product intermediate, which then proceeds via either a chair (X = N3, SMe, or SCF3) or boat-like (X = F, Cl, or Br) transition state to afford amino-Cope-like products or β-lactam in the case of X = NO2.

SUMMARYSpecialized diterpenoid metabolites are important mediators of plant–environment interactions in monocot crops. To understand metabolite functions in plant environmental adaptation that ultimately can enable crop improvement strategies, a deeper knowledge of the underlying species‐specific biosynthetic pathways is required. Here, we report the genomics‐enabled discovery of five cytochrome P450 monooxygenases (CYP71Z25–CYP71Z29) that form previously unknown furanoditerpenoids in the monocot bioenergy crop Panicum virgatum (switchgrass). Combinatorial pathway reconstruction showed that CYP71Z25–CYP71Z29 catalyze furan ring addition directly to primary diterpene alcohol intermediates derived from distinct class II diterpene synthase products. Transcriptional co‐expression patterns and the presence of select diterpenoids in switchgrass roots support the occurrence of P450‐derived furanoditerpenoids in planta. Integrating molecular dynamics, structural analysis and targeted mutagenesis identified active site determinants that contribute to the distinct catalytic specificities underlying the broad substrate promiscuity of CYP71Z25–CYP71Z29 for native and non‐native diterpenoids.

Background Klebsiella pneumoniae contains an endogenous isobutanol synthesis pathway. ipdC , annotated as an indole-3-pyruvate decarboxylase (Kp-IpdC), was identified to catalyze the formation of isobutyraldehyde from 2-ketoisovalerate. Results Compared with 2-ketoisovalerate decarboxylase from Lactococcus lactis (KivD), a decarboxylase commonly used in artificial isobutanol synthesis, Kp-IpdC has an 2.8-fold lower K m for 2-ketoisovalerate, leading to higher isobutanol production without induction. However, high level expression of ipdC by induction resulted in a low isobutanol titer. In vitro enzymatic reactions showed that Kp-IpdC exhibits promiscuous pyruvate decarboxylase activity, which adversely consume the available pyruvate precursor for isobutanol synthesis. To address this we have engineered Kp-IpdC to reduce pyruvate decarboxylase activity. From computational modeling we identified 10 residues surrounding the active site for mutagenesis. Ten designs consisting of eight single-point mutants and two double-mutants were selected for exploration. Mutants L546W and T290L showed 5.1% and 22.1% of catalytic efficiency on pyruvate, which were then expressed in K. pneumoniae for in vivo test. Isobutanol production by K. pneumoniae T290L was 25% higher than the control strain, and a final titer of 5.5 g/L isobutanol was obtained with a substrate conversion ratio of 0.16 mol/mol glucose. Conclusions This research provides a new way to improve the efficiency of the biological route of isobutanol production.

Specialized diterpenoid metabolites are important mediators of stress resilience in monocot crops. A deeper understanding of how species-specific diterpenoid-metabolic pathways and functions contribute to plant chemical defenses can enable crop improvement strategies. Here, we report the genomics-enabled discovery of five cytochrome P450 monooxygenases (CYP71Z25-29) that form previously unknown furanoditerpenoids in the monocot bioenergy crop switchgrass ( Panicum virgatum ). Combinatorial pathway reconstruction showed that CYP71Z25-29 catalyze furan ring addition to diterpene alcohol intermediates derived from distinct class II diterpene synthases, thus bypassing the canonical role of class I diterpene synthases in plant diterpenoid metabolism. Transcriptional co-expression patterns and presence of select diterpenoids in droughted switchgrass roots support possible roles of CYP71Z25-29 in abiotic stress responses. Integrating molecular dynamics, structural analysis, and targeted mutagenesis, identified active site determinants controlling distinct CYP71Z25-29 catalytic specificities and, combined with broad substrate promiscuity for native and non-native diterpenoids, highlights the potential of these P450s for natural product engineering. Significance Statement Diterpenoids play important roles in stress resilience and chemically mediated interactions in many plant species, including major food and bioenergy crops. Enzymes of the cytochrome P450 monooxygenase family catalyze the various functional decorations of core diterpene scaffolds that determine the large diversity of biologically active diterpenoids. This study describes the identification and mechanistic analysis of an unusual group of cytochrome P450 monooxygenases, CYP71Z25-29, from the bioenergy crop switchgrass ( Panicum virgatum ). These enzymes catalyze the furan ring addition directly to class II diterpene synthase products, thus bypassing the conserved pairwise reaction of class II and class I diterpene synthases in labdane diterpenoid metabolism. Insight into the distinct substrate-specificity of CYP71Z25-29 offers opportunity for engineering of furanoditerpenoid bioproducts.

Each year vast international resources are wasted on irreproducible research. The scientific community has been slow to adopt standard software engineering practices, despite the increases in high-dimensional data, complexities of workflows, and computational environments. Here we show how scientific software applications can be created in a reproducible manner when simple design goals for reproducibility are met. We describe the implementation of a test server framework and 40 scientific benchmarks, covering numerous applications in Rosetta bio-macromolecular modeling. High performance computing cluster integration allows these benchmarks to run continuously and automatically. Detailed protocol captures are useful for developers and users of Rosetta and other macromolecular modeling tools. The framework and design concepts presented here are valuable for developers and users of any type of scientific software and for the scientific community to create reproducible methods. Specific examples highlight the utility of this framework and the comprehensive documentation illustrates the ease of adding new tests in a matter of hours.

Interest in animal cell-based meat (ACBM) or laboratory grown meat has been increasing, however the economic viability of these potential products has not been thoroughly vetted. Recent studies suggest monoclonal antibody production technology can be adapted for the industrialization of ACBM production. This study provides a scenario-based assessment of the projected cost per kilogram of ACBM based on cellular metabolic requirements and process/chemical engineering conventions. A sensitivity analysis of the model identified the nine most influential cost factors for ACBM production out of 67 initial parameters. The results indicate that technological performance will need to approach technical limits for ACBM to achieve profitably as a commodity. However, the model also suggests that low-volume high-value specialty products could be viable based on current technology. One Sentence Summary A model based upon cellular metabolism and engineering conventions was created to examine the economic viability of animal cell-based meat. Significance statement Animal cell-based meat (ACBM) has received a significant amount of media attention (as well as corporate investment) in recent years based on its perceived potential to displace traditional meat production, whether beef, poultry, or fish. However, a robust techno-economic assessment (TEA) of these potential products is not publicly available. Our study examined the capital and operating expenditures for potential ACBM products based upon fundamental cellular attributes, the use of proposed near-term/existing technology, and process engineering conventions. Our findings suggest that the current production pathways are far from producing cost-competitive ACBM products, as well as highlight the technical metrics that must be achieved for an ACBM product to become economically viable. ### Competing Interest Statement The authors have declared no competing interest.

Interest in animal cell-based meat (ACBM) or laboratory-grown meat has been increasing; however, the economic viability of these potential products has not been thoroughly vetted. Recent studies suggest monoclonal antibody production technology can be adapted for the industrialization of ACBM production. This study provides a scenario-based assessment of the projected cost per kilogram of ACBM produced in the United States based on cellular metabolic requirements and process/chemical engineering conventions. A sensitivity analysis of the model identified the nine most influential cost factors for ACBM production out of 67 initial parameters. The results indicate that technological performance will need to approach technical limits for ACBM to achieve profitably as a commodity. However, the model also suggests that low-volume high-value specialty products could be viable based on current technology.

ABSTRACTBCR-ABLtyrosine kinase inhibitors (TKI) are used to treat the chronic myeloid leukemia (CML). Many TKI have been developed as the primary treatment to the CML.Imatinib, a first generation TKI, directly targetsBCR-ABLwith effective results. As the disease becomes more advanced, patients start to develop resistance toimatinib. Due to this effect it is necessary to generate novel treatments for advanced stage CML. Computational tools can predict new drug candidates to targetBCR-ABL. We have designed two new drug candidates with different levels of modification, based on the predicted structure activity relationships withBCR-ABL. These new drug candidates are predicted to have better binding affinities withBCR-ABLthanimatinib, which can be more potent treatments of the disease.

A key goal of protein engineering is to accurately model the stability and catalytic activity of enzymes. However, the limitations of functional predictive abilities pose a major challenge for modeling algorithm design, and can be attributed to the lack of large data sets quantifying the functional properties of enzymes. Here, the thermal stability (TM) and Michaelis-Menten constants ( k cat, KM, and k cat/KM) of six new variants of the β-glucosidase B (BglB) protein are quantitatively characterized. Molecular stability of the enzyme variants were hypothesized using the Foldit software and BglB was synthesized in E. coli cells. Testing was done through a colorimetric kinetic assay and thermal stability fluorescence-based protein unfolding assay. Results from the assays suggest that all mutations, with the exception of variant Y169M, all experienced reduced catalytic efficiency compared to the wildtype. Assay results indicate that variant W123R is more thermally stable compared to the wildtype, while the differences in thermal stability between the other variants, and the wildtype are negligible. The collected thermal stability and catalytic efficiency data has been added to a data set with the aim of improving Rosetta algorithms for modeling and predicting the functional interactions between biomolecules through a machine learning algorithm and facilitate the precise engineering of protein catalysts.

The mechanism and origins of syn and anti selectivity of cross-benzoin reactions between furfural and α-amino aldehydes, catalyzed by a triazolium-based NHC, were investigated using density functional theory calculations. N-Boc-α-amino aldehydes were found to react with anti selectivity, while N-Bn-N-Boc-α-amino aldehydes react with syn selectivity. We find that the anti product is more thermodynamically favored than the syn product for reactions with N-Boc-α-amino aldehydes, and that the formation of the syn product for reactions involving N-Bn-N-Boc-α-amino aldehydes is kinetically favored. The switch in selectivity is a result of an intramolecular hydrogen bond in the N-Boc-α-amino aldehyde, whereas switching to N-Bn-N-Boc-α-amine removes the hydrogen bond. The steric and electronic interactions in the transition state are rationalized by a Felkin-Anh model.

Diastereoselective Lewis acid-mediated additions of nucleophilic alkenes to N-sulfonyl imines are reported. The canonical polar Felkin-Anh model describing additions to carbonyls does not adequately describe analogous additions to N-sulfonyl imines. Herein, we describe the development of conditions to produce both syn and anti products with high diastereoselectivity and good yields. A stereoelectronic model consistent with experimental outcomes is also proposed.

Author(s): Fell, Jason Scott | Advisor(s): Houk, Kendall N | Abstract: This dissertation describes research that delves into exploring puzzling chemical phenomena utilizing modern computational chemistry methods. Theoretical chemical models that are coupled with quantum mechanical (QM) calculations can dissect complex chemical reactions into many components that influence chemical reactivity and selectivity. Each chapter of this dissertation demonstrates that QM calculations can help predict and explain the complexities of chemical phenomena involving reaction mechanisms. The first section (hapters 1 thru 4) details computational explorations of reactivities and selectivities of pericyclic reactions. Pericyclic reactions are an important class of chemical reactions wherein the reacting species form a cyclic transition state with aromatic delocalization. Often these reactions occur with high degrees of regio- and stereoselectivity. Chapter 1 explores the large differences in reactivity between cyclic 1- and 2-azadienes in Diels-Alder reactions. Chapter 2 investigates how thiol addition to substituted oxanorbornadienes promotes a retro-Diels-Alder reaction, as well as how the substitution pattern on the norbornadiene affects the rates of fragmentation. Chapter 3 probes how an anion-accelerated Cope rearrangement is inherently stereoselective. Chapter 4 explores if the enzyme iridoid synthase is a natural Diels-Alderase by modeling the uncatalyzed Diels-Alder reaction and determining if the background reaction is achievable and inherently stereoselective. The second section, Chapter 5, delves into utilizing QM calculations to predict the potential reactivity of a proposed catalyst to selectivity perform anti-Markovnikov hydrations of olefins. Our calculations predict that the proposed di-manganese catalyst would produce the anti-Markovnikov product preferentially to the Markovnikov product at a ratio as low as 12:1 and as high as 100:1 depending on the catalyst ligands. The third section (Chapters 6 and 7) tackle the stereoselectivity of chemical reactions involving nucleophilic additions to aldehydes and imines. Chapter 6 explores the mechanism and origins of the diastereoselectivity of cross-benzoin reactions of furfural and α-amino aldehydes catalyzed by a triazolium-based NHC. Chapter 7 the polar-Felkin-Anh stereoselectivity of nucleophilic addition to α-chiral imines in the presence of Lewis acid catalysts.

We report useful new lithium-assisted asymmetric anion-accelerated amino-Cope rearrangement cascades. A strategic nitrogen atom chiral auxiliary serves three critical roles, by (1) enabling in situ assembly of the chiral 3-amino-1,5-diene precursor, (2) facilitating the rearrangement via a lithium enolate chelate, and (3) imparting its influence on consecutive inter- or intramolecular C-C or C-X bond-forming events via resulting chiral enamide intermediates or imine products. The mechanism of the amino-Cope rearrangement was explored with density functional theory. A stepwise dissociation-recombination mechanism was found to be favored. The stereochemistry of the chiral auxiliary determines the stereochemistry of the Cope product by influencing the orientation of the lithium dienolate and sulfinylimine fragments in the recombination step. These robust asymmetric anion-accelerated amino-Cope enabled cascades open the door for rapid and predictable assembly of complex chiral acyclic and cyclic nitrogen-containing motifs in one pot.

The reactivities of butadiene, cyclopentadiene, furan, thiophene, pyrrole, and their 1-aza- and 2-aza-derivatives in Diels-Alder reactions with ethylene and fumaronitrile were investigated with density functional theory (M06-2X/6-311G(d,p)). The activation free energies for the Diels-Alder reactions of cyclic 1-azadienes are 10-14 kcal mol-1 higher than those of cyclic 2-azadienes, and the reaction free energies are 17-20 kcal mol-1 more endergonic. The distortion/interaction model shows that the increased activation energies of cyclic 1-azadienes originate from increased transition state distortion energies and unfavorable interaction energies, arising from addition to the nitrogen terminus of the C═N bond.

Additions of amines or thiols to 7-oxanorbornadienes promote retro-[4 + 2] reactions to yield furans. Substitution at the bridgehead position also greatly influences the stability of the oxanorbornene adducts. Activation and reaction energies were computed with the M06-2X density functional, the origins of amine and thiol promoted fragmentation, and how substituent effects control fragmentation rates and reaction energetics are reported.

The metalloenzyme peptide deformylase (PDF) plays a crucial role in the biosynthesis of proteins by eubacteria, making the enzyme a promising target for antibacterial agents. In a reaction catalyzed by an Fe(II) coordination complex in the enzyme active site, PDF cleaves a formyl group from the N-terminus of nascent eubacterial proteins. Computational chemistry methods are combined with the use of in silico models of the enzyme chemistry to examine specific effects on the enzymatic catalysis. In particular, density functional theory calculations have been carried out on a biomimetic model system based on a heteroscorpionate N2Sthiolate biomimetic ligand system bearing a range of electron-donating and electron-withdrawing substituents. In this way, the effects of electronic changes to the metal coordination environment on the thermodynamics and kinetics of the deformylation reaction are determined. The reaction was found to be more thermodynamically favorable as the added substituents shifted from electron-withdrawing to electron-donating [or, equivalently, with decreasing Hammett parameters (σ p) for the substituents]. As the substituents change from electron-withdrawing to electron-donating, the hydroxide ligand responsible for initiating nucleophilic attack on the carbonyl group of the formyl-terminated peptide substrate is shown to become decreasingly strongly bound to the metal, making the reaction less endergonic. The rate of reaction was found to be fastest for substituents with σ p ≈ 0, and progressively slower as the substituents became increasingly electron-donating or electron-withdrawing. At σ p ≈ 0, a balance is found between the reactivity of the hydroxide ligand involved in the nucleophilic attack and activation of the carbonyl group by the iron center toward that nucleophilic attack.

The use of chiral Ni(II)-salen derivatives was examined in mediated electrohydrocyclization reactions. Cyclic voltammetry established the existence of a catalytic current. Bulk electrolysis revealed a slight change in the diastereoselectivity of the cyclizations. Computational studies were conducted that showed that Ni(II) and Zn(II) were the best metals for electron transfer, while the analogous Co(II) and Cu(II) compounds would likely not result in effective electron transfer.