https://www.chemistryworld.com/news/what-is-directed-evolution-and-why-did-it-win-the-chemistry-nobel-prize/3009584.article

https://youtu.be/FWEN2pxoXoQ

https://cen.acs.org/people/nobel-prize/directed-evolution-changing-world/96/i44

Refers to the process of separating desired products from biosynthetic pathways

Functional group protection and deprotection increases the number of steps and auxiliary agents during synthesis and is not considered green. The methodology described in this research obeys one of the principles of green chemistry: "Unnecessary derivatization (use of blocking groups, protection/deprotection, temporary modification of physical/chemical processes) should be minimized or avoided if possible, because such steps require additional reagents and can generate waste."

Free alcohols and primary amines are very reactive functional groups that are inert during this carbene transfer reaction.

Via site saturation mutagenesis, V75 and M103 positions along the protein sequence were identified as likely beneficial mutations and were randomized, i.e., the amino acids at these positions are replaced by random ones. A large number of random variants, which together constitute a library, are produced and then screened in an attempt to discover a highly active variant among them. The evolved triple mutant fits the bill.

Recombinant Protein is a protein encoded by a gene that has been cloned in a system that supports expression of the gene (in this case, it is M100). Modification of the gene by recombinant DNA technology can lead to expression of a mutant protein. In this study, M100D mutation is more highly activating than the wild-type protein (as it occurs in nature).

M100 is the specific amino acid residue within the protein sequence that has been identified to be critical for the protein’s function. it is very important to determine the ideal amino acid residue for this position. Site saturation mutagenesis is a form of random mutagenesis, allowing the substitution of specific sites against all 20 possible amino acids at once. In this study, this technique is employed to generate a series of enzymes with enhanced activity and enantiospecificity.

Certain iridium catalytic systems show 97% ee while copper catalysts show 35% ee and rhodium has been shown to exhibit 77% ee.

Any reagent that exhibits chirality (or asymmetry) in its molecular structure

carbene is a neutral reactive intermediate; a carbene insertion reaction is the insertion of carbene in a carbon-hydrogen bond.

Proposed theory for the binding mode for the iron-carbene complex is one where the carbene complex forms such that it takes the place of the axial methionine. The silane may approach from the more exposed side in the wild-type protein. This further explains the observed stereochemistry of the organosilicon product. The V75T, M100D, and M103E mutations may improve reactivity by providing better access of the substrate to the iron center.

A complete carbene transfer to the protein may be the reason for the catalyst to be inactivated. The activity and lifetime of Rma cyt c may be improved with further mutagenesis.

Turnover frequency for each variant relative to wild type protein: WT: 1 M100D 2.8 +/- 0.2 V75T M100D 4.6 +/- 0.3 V75T M100D M103E 7.1 +/- 0.4

From this experimental data, it is clear that directed evolution has resulted from changing the enzyme from unselective wild type into a highly enantioselective variant.

Is a method of engineering proteins towards a defined goal or purpose. Directed evolution mimics 'real' evolution and is accelerated in the laboratory by focusing on individual genes expressed in fast‐growing microorganisms such as E.Coli. Enzyme chosen (known as wild type) must show at least a minimal desired reactivity. Mutations are introduced at strategic locations in the wild type protein. Then, the library of mutant proteins is screened for the mutated enzymes with enhanced reactivity. The improved enzymes are used as parents for the next round of mutation and screening. Additional beneficial mutations are introduced if needed. This can continue for several cycles until a desired and beneficial evolution of the enzyme is attained.

conditions that occur in nature for an organism in contrast to laboratory conditions

Additional diazo compounds that were successful were are R3 = -CH3, -CH2CH3, -Ph.

1. Rma cyt c V75T M100D M103E shows excellent enantioselectivity and turnover over a wide range of substrates. Silicon substrates with weakly electron donating or activating methyl substituents (4), strongly electron donating -OMe (5), weakly deactivating -Cl (6), strongly deactivating -CF3 (7), and moderately deactivating COOMe and CONMe (9 and 10 respectively) show moderate to excellent turnover and high selectivity. No direct relationship exists between turnover number and substituents effects from this study. Enantioselectivity is excellent in all substrates. All products were identified using GC-MS and no traditional organic chemistry techniques were used.

The wildtype protein chosen must exhibit some degree of activity for the desired reaction. When compared to hemin, hemin with bovine serum, a range of protein engineered cytochrome P450 and myoglobin variants, it was found that Rmacyt c showed 97% ee. This was chosen as the enzyme that would form the basis for directed evolution.

In this study, Professor Arnold's group used protein engineered variants of cytochrome P450BM3 to bring about highly diastereoselective and enantioselective cyclopropanation reaction of styrenes from diazoester. Variant BM3-CIS was identified as a competent cyclopropanation catalyst. It exhibits a strong preference for the cis product and forms both diasteromers over 90% ee and is as stable as the wild type enzyme. P450-BM3 works on a wide range of substrates with both electron donating and electron withdrawing substituents in styrene.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5901037/

https://www.nobelprize.org/uploads/2018/10/advanced-chemistryprize-2018.pdf

With each mutation, the chemoselectivity of the enzyme is greatly enhanced. V75T M100D M103E favors the carbon-silicon bond 29 times more than the wild type. This is attributed to the improved binding and orientation of the silicon donor in the enzyme's active site.

A generally applicable protocol, as a result of directed evolution, over a variety of substrates illustrates the synthetic utility of the mutant.

Heme proteins that were readily available were screened to identify the one that gave the most enantioselectivity. This served as starting point for directed evolution. Purified heme protein, silane, diazoester, thiosulfate, methyl cyanide and M9-N buffer as the medium for microbial growth were stirred at room temperature in anaerobic conditions. Reactions were performed in triplicate. Unreacted starting materials was obtained in all cases and no further purification was carried out.

A new approach of conducting chemical reactions where reactants must react rapidly and selectively with each other under physiological conditions. Two key and relevant features of bioorthogonal reactions is high selectivity and compatibility with naturally occurring functional groups.

Metabolic engineering is the production of specific target chemicals in high yield and stereoselectivity by altering the metabolic pathways. Metabolic pathway is changed via recombinant DNA technology.

aromatic compounds where one or more ring carbon atoms are replaced by a heteroatom such as nitrogen, sulfur or oxygen.

compounds that contain two fused benzene rings; also referred to as polycyclic aromatic hydrocarbon

organic compounds that contain -CONH2 structural feature

organic compounds that contain -COOR functional group

organic compounds that contain a halogen connected to an alkyl group such as methyl ethyl etc.

organic compounds that contain a halogen connected to a benzene ring

organic compounds that contains C-O-C structural feature

oxygen free conditions

At the theoretical level, the steps needed for obtaining a recombinant protein are straightforward. Take your gene of interest, clone it, transform it into the host of choice (here it is E.Coli), induce and then, the protein is ready for purification and characterization.

an active site created by chance rather than by design

The amnio acid, methionine

located at a further distance

The amino acid, histadine

located at a closer distance

repels or has no affinity towards water

cells with membrane bound organelles

relatively unchanged when one goes back in genealogical time

An organism that thrives in extreme high temperature and high salt concentrations

Enantioinduction is also popularly known as asymmetric induction. This process is the preferential formation of one enantiomer over the other as a result of the influence of a chiral feature present in reactants or the catalyst.

branch of biology that deals with structure and function of nucleic acids and proteins

Is the order of nucleotides that make up the genetic codes that is translated into proteins

elements that have the same number of electrons in the outermost shell (also known as valence shell) and have similar electronic properties

not harmful to living cells

Compounds that contain carbon-silicon bonds

A compound available in one enantiomeric form

The number of moles of substrata that a catalyst can convert into the desired product before becoming inactive

Chemoselectivity is the preferential reaction of a reagent with a specific functional group over others. Enantioselectivity is the extent to which one enantiomer is formed over the other in a chemical reaction.

is a type of metalloprotein and contains a heme group which is required for the functionality of the protein

speed up a reaction with the use of external agent, typically a chemical compound

Although transition metal catalysis offer several advantages, the metals typically used are toxic. Removal of these metals after the synthetic process is time consuming and expensive. The process described in the research ensures sustainability and reduces cost.