Engineered Biosynthesis of β‐Alkyl Tryptophan Analogues

Boville, Christina E.; Scheele, Remkes A.; Koch, Philipp; Brinkmann‐Chen, Sabine; Buller, Andrew R.; Arnold, Frances H.

doi:10.1002/ange.201807998

“…In previous work where TrpB was engineered for the synthesis of tryptophan derivatives, enzymes from the thermophilic organisms Pyrococcus furiosus (PfTrpB) and Thermotoga maritima (TmTrpB) had sufficient activity with the indole derivatives to enable screening for beneficial mutations. [2][3][5][6][7] When we tested the wild-type enzymes and engineered variants from our collection via mass spectrometry-based screening with 3-methyloxindole 1 as a nucleophile and serine 2 as the electrophile, several variants showed activity, with up to 28% HPLC yield for the expected mass (SI, Figure 1). However, these enzymes formed the N-alkylated oxindole 3a (Figure 3a) rather than the desired C-alkylated product 3b.…”

Section: Screening For Activity With 3-methyloxindolementioning

confidence: 99%

“…2 We evolved this enzyme to accept a variety of indole analogues and nitroalkanes with activated sp³-carbon atoms as nucleophiles (Figure 1b). [3][4][5][6][7][8] However, these studies revealed limitations including chemo-and stereoselective control we would need to address in order to realize TrpB's broad potential as a biocatalyst for C-C bond formation. Previous biocatalytic reactions catalyzed by variants of TrpB [5][6]8 and proposed stereo-and chemo-selective reaction using engineered TrpB.…”

Section: Introductionmentioning

confidence: 99%

“…2,10 TrpB is highly stereoselective and can retain the absolute configuration of the Cα of L-serine after product formation (Figure 1b). [3][4][5][6][7] However, the stereoselectivity at potential new stereogenic centers is unknown. Substrates with nucleophilic sp 3 -hybridized carbons have the ability to form a new quaternary carbon at the γ-position.…”

Section: Introductionmentioning

confidence: 99%

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Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation

Dick¹,

Sarai²,

Martynowycz³

et al. 2019

Preprint

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<div><div><div><p>We previously engineered the tryptophan synthase beta-subunit (TrpB), which catalyzes the condensation reaction between L-serine and indole to form L-tryptophan, to synthesize a range of modified tryptophans from serine and indole derivatives. In this study, we used directed evolution to engineer TrpB to accept 3-substituted oxindoles and form C–C bonds leading to new quaternary stereocenters. At first, the TrpBs that could use 3-substituted oxindoles preferentially formed N–C bonds by attacking the oxindole N<sub>1 </sub>atom. We found, however, that protecting the nitrogen encouraged evolution towards C-alkylation, which persisted even when this protection was removed. After seven rounds of evolution leading to a 400-fold improvement in activity, variant <i>Pf</i><sub>quat</sub> efficiently alkylates 3-substituted oxindoles to selectively form new stereocenters at the γ-position of the amino acid products. The configuration of the new γ-stereocenter of one of the products was determined from the crystal structure obtained by microcrystal electron diffraction (MicroED). Substrates structurally related to 3-methyloxindole such as lactones and ketones can also be used by the enzyme for quaternary carbon bond formation, where the biocatalyst exhibits excellent regioselectivity for the tertiary carbon atom. Highly thermostable and expressed at > 500 mg/L <i>E. coli</i> culture, TrpB <i>Pf</i><sub>quat </sub>provides an efficient and environmentally-friendly platform for the preparation of noncanonical amino acids bearing quaternary carbons.</p></div></div></div>

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“…2,10 TrpB is highly stereoselective and can retain the absolute configuration of the Cα of L-serine after product formation (Figure 1b). [3][4][5][6][7] However, the stereoselectivity at potential new stereogenic centers is unknown. Substrates with nucleophilic sp 3 -hybridized carbons have the ability to form a new quaternary carbon at the γ-position.…”

Section: Introductionmentioning

confidence: 99%

Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation

Dick¹,

Sarai²,

Martynowycz³

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

<div><div><div><p>We previously engineered the tryptophan synthase beta-subunit (TrpB), which catalyzes the condensation reaction between L-serine and indole to form L-tryptophan, to synthesize a range of modified tryptophans from serine and indole derivatives. In this study, we used directed evolution to engineer TrpB to accept 3-substituted oxindoles and form C–C bonds leading to new quaternary stereocenters. At first, the TrpBs that could use 3-substituted oxindoles preferentially formed N–C bonds by attacking the oxindole N<sub>1 </sub>atom. We found, however, that protecting the nitrogen encouraged evolution towards C-alkylation, which persisted even when this protection was removed. After seven rounds of evolution leading to a 400-fold improvement in activity, variant <i>Pf</i><sub>quat</sub> efficiently alkylates 3-substituted oxindoles to selectively form new stereocenters at the γ-position of the amino acid products. The configuration of the new γ-stereocenter of one of the products was determined from the crystal structure obtained by microcrystal electron diffraction (MicroED). Substrates structurally related to 3-methyloxindole such as lactones and ketones can also be used by the enzyme for quaternary carbon bond formation, where the biocatalyst exhibits excellent regioselectivity for the tertiary carbon atom. Highly thermostable and expressed at > 500 mg/L <i>E. coli</i> culture, TrpB <i>Pf</i><sub>quat </sub>provides an efficient and environmentally-friendly platform for the preparation of noncanonical amino acids bearing quaternary carbons.</p></div></div></div>

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“…Also of note, Frances and her team have addressed limitations to producing certain biofuels by using directed evolution to engineer key enzymes within metabolic pathways, and to improve cellulase properties . They have also targeted biosynthesis of a wide range of unnatural tryptophan analogs . Finally, collaborations with Ron Weiss beginning circa 2002 combined directed evolution with a still burgeoning field of synthetic biology, resulting in seminal papers …”

mentioning

confidence: 99%

Commemorating Frances Arnold

Collins

¹

,

Cirino

²

2020

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of Sciences (2008) (the first woman to be elected to all three), the 2011 NAE Draper Prize (the first woman to receive it), and the 2016 Millennium Technology Prize (first woman to win). At the time of this writing, Google Scholar reports her co-authored publications receiving over 50,000 citations, with an h-index of 122. She is co-founder of Gevo, Inc (producing carbon-neutral biofuels) 3 and Provivi (producing environmentally safe biopesticides), 4 and was recently appointed to the board of directors for Alphabet, Inc (the parent company of Google). 5Many edited texts and countless review articles describe directed evolution techniques and applications (e.g., References 6-9 ). While many key players have emerged in this field throughout the years (most with research ties to Frances), the Arnold lab has always been at the forefront, melding molecular biology, chemistry, and biomolecular engineering to "one-up" nature and create novel and often extraordinary biocatalysts. At the same time, Arnold and coworkers continuously push the envelope with regard to method development and mechanistic understanding, thus offering the protein engineering community proven strategies for, and new insights into, molecular evolution. In the following paragraphs, we summarize the "evolution" of research in the Arnold lab and discuss her legacy as a protein pioneer and a devoted mentor.The concept of a directed evolution experiment is rather simple-create a library of mutant genes (as compared to a parent gene), and screen or select for those mutants (offspring) which confer an improved property of interest. Then, repeat (refer to journal cover image). This was not a novel idea in 1991 when the Arnold lab first reported examples of evolving an enzyme (subtilisin E) for enhanced activity and stability in organic solvent. 10-13 At the time, however, the practice of directed evolution was not easily implemented, and its potential was not yet appreciated by the protein engineering community. Implementing this simple concept was not simple. How could, or should, mutant libraries be constructed? How large or diverse should they be? How does one search through such libraries for the rare improvements, in a reasonable timeframe? Are there limits to evolving protein properties or reaction chemistries? Frances, her students, and her post docs sought to answer such questions, riding waves of advances in molecular and cell biology, synthetic biology, and computational methods (among others). The early years of research following her initial subtilisin studies furnished numerous, now prevalent directed evolution techniques, both for library creation and for library screening. The formalization of such methods led to the publication in 2003 of two volumes of directed evolution protocols, edited by Frances Arnold and GeorgeGeorgiou. 14 Through the years, these volumes have served as "directed evolution 101" for many labs. A wide variety of enzymes have served as targets of directed evolution in the Arnold lab (e.g., proteases, esterases, oxygena...

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Engineered Biosynthesis of β‐Alkyl Tryptophan Analogues

Cited by 21 publications

References 30 publications

Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation

Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation

Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation

Commemorating Frances Arnold

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