Biosynthesis of diverse phenylalanine derivatives from aldehydes, carboxylic acids or alcohols

Abstract

The present invention relates to a recombinant cell. The recombinant cell may express a first enzyme, a second enzyme and a third enzyme and produce a nonstandard amino acid (nsAA). The first enzyme may be a L-threonine transaldolase (L-TTA) or a threonine aldolase (TAs). The second enzyme may be a phenylserine dehydratase or a threonine deaminase (TD). The third enzyme may be an aminotransferase (AT), a L-amino acid dehydrogenase (L-AADH) or a D-amino acid aminotransferase. At least one of the first enzyme, the second enzyme and the third enzyme may be heterologous to the recombinant cell. Also provided are a method for producing a nonstandard amino acid (nsAA) by the recombinant cell and a method for producing a nsAA in a container in the presence of the first enzyme, the second enzyme and the third enzyme.

Description

[0001] BIOSYNTHESIS OF DIVERSE PHENYLALANINE DERIVATIVES FROM ALDEHYDES, CARBOXYLIC ACIDS OR ALCOHOLS

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to United States Provisional Application No. 63 / 663,759, filed June 25, 2024, and United States Provisional Application No. 63 / 730,012, filed December 10, 2024, the contents of each of which are incorporated herein by reference in their entireties for all purposes.

[0004] REFERENCE TO U.S. GOVERNMENT SUPPORT

[0005] This invention was made with government support under grant number N00014- 22-1-2536 awarded by Office of Naval Research. The United States has certain rights in the invention.

[0006] REFERENCE TO SEQUENCE LISTING

[0007] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 2101715- 001309_SequenceListing.xml, created June 24, 2025, which is 21 KB in size. The information in XML file format of the Sequence Listing is incorporated herein by reference in its entirety.

[0008] FIELD OF THE INVENTION

[0009] The invention relates to production of non-standard amino acids (nsAAs) from aldehydes, carboxylic acids or alcohol, optionally without supplying external aldehyde, and incorporation of the nsAAs into target proteins.

[0010] BACKGROUND OF THE INVENTION

[0011] Non-standard amino acids (nsAAs) are valuable building blocks for chemical diversification of products that are prepared by synthetic or biological means. Diverse commercially relevant applications have been enabled by the introduction of sidechain chemistries that do not belong to the standard twenty ribosomally translated amino acids. An illustrative though not exhaustive list includes the formation or augmentation of bioactive natural products, synthesis of stimuli-responsive materials, improved protein stability, increased enzyme catalytic efficiency, synthesis of artificial metalloenzymes and (photo)catalytic active sites, design of metal-responsive protein switches, immunochemical termination of protein self-tolerance, bio-orthogonal conjugation of therapeutics, and intrinsic biological containment of genetically modified organisms. Notably, each of the applications described above has prominently featured derivatives of L-phenylalanine, which are the most common class of nsAAs used in genetic code expansion approaches within live cells. However, efficient and cost- effective production of enantioenriched phenylalanine derivatives has been a longstanding challenge. Biocatalysis presents a promising solution by utilizing enantioselective enzymes under mild reaction conditions, eliminating the need for protecting groups, and offering an increasingly scalable, sustainable, and economical approach compared to traditional catalytic routes.

[0012] Two classes of pyridoxal 5'-phosphate (PLP)-dependent biocatalysts that exhibit wide substrate scope during synthesis of amino acids and that harness inexpensive precursors are L-threonine aldolases (L-TAs) and L-threonine transaldolases (L-TTAs). These enzymes catalyze aldol-like condensations of aldehydes and either L-glycine or L-threonine (L-Thr), respectively, to generate p-hydroxy a-amino acids (P-OH AAs). They exhibit activity on a broad range of aldehydes with aryl side chains containing diverse ring substitutions. L-TTAs are especially promising compared to L-TAs given their ability to form a glycyl quinonoid intermediate whose protonation is kinetically disfavored, enabling efficient desired reactivity with aryl aldehydes. While p- hydroxylated nsAAs could find substantial use in drug discovery, the formation of the p- hydroxy group results in chemical and steric differences from standard phenylalanine analogs and are currently less well-explored industrially.

[0013] Additionally, whether nsAA synthesis occurs chemically or biologically, the current synthesis paradigms for genetic code expansion applications in live cells necessitate downstream processing steps of nsAA isolation and supplementation to cell culture media. In addition to the costs and labor of the nsAA synthesis and isolation steps, the requirement of nsAA supplementation also imposes constraints that limit the performance or scalability of genetic code expansion in diverse application contexts.

[0014] Non-standard amino acids (nsAAs) that are L-phenylalanine derivatives with aryl ring functionalization have long been harnessed in natural product synthesis, therapeutic peptide synthesis, and diverse applications of genetic code expansion. Yet, to date these chiral molecules have often been the products of poorly enantioselective and environmentally harsh organic synthesis routes.

[0015] There remains a need for inexpensive and polyspecific processes to generate desirable nsAAs in order to expand and scale the technology of genetic code expansion.

[0016] SUMMARY OF THE INVENTION

[0017] The present invention relates to recombinant cells expressing enzymes for producing non-standard amino acids (nsAAs) and related production methods. The present invention is based on the inventors' surprising discovery of enzymes to catalyze sequential removal of a p-hydroxy group from p-hydroxylated nsAAs.

[0018] The present invention provides a recombinant cell. The recombinant cell expresses a first enzyme, a second enzyme and a third enzyme and produces a nonstandard amino acid (nsAA). The first enzyme is a L-threonine transaldolase (L- TTA) or a threonine aldolase (TAs). The second enzyme is a phenylserine dehydratase or a threonine deaminase (TD). The third enzyme is an aminotransferase (AT), a L- amino acid dehydrogenase (L-AADH) or a D-amino acid aminotransferase. At least one of the first enzyme, the second enzyme and the third enzyme is heterologous to the recombinant cell.

[0019] The L-TTA may be from Pseudomonas fluorescens (ObiH) (SEQ ID NO: 1) or from Parachlamydiales bacterium (PbTTA) (SEQ ID NO: 2).

[0020] The phenylserine dehydratase may be from Ralstonia pickettii PS22 (RpPSDH) (SEQ ID NO: 3).

[0021] The AT may be a tyrosine aminotransferase from E. coli (TyrB) (SEQ ID NO: 4).

[0022] The recombinant cell may further express a carboxylic acid reductase (CAR) and a phosphopantetheinyl transferase. The CAR may be from SegniHparus rotundus (SrCAR) (SEQ ID NO: 5) or from Mycobacterium avium (MavCAR) (SEQ ID NO: 6). The phosphopantetheinyl transferase may be from Bacillus subtilis (sfp) (SEQ ID NO: 7).

[0023] The recombinant cell may further express an alcohol oxidase (AO) or alcohol dehydrogenase (ADH).

[0024] The recombinant cell may further express a target protein and comprise an orthogonal translation system (OTS). The target protein may comprise a site for amber stop codon suppression. The OTS may comprise an aminoacyl-tRNA synthetase and a tRNA pair decoding the amber stop codon. The nsAA may be incorporated into the target protein at the site.

[0025] The recombinant cell may be E. coli.

[0026] The present invention also provides a method for producing a nonstandard amino acid (nsAA) by a recombinant cell. This in vivo production method comprises (a) growing a recombinant cell in a culture medium, wherein the culture medium comprises an aldehyde, a L-amino acid and an amine donor, wherein the L-amino acid is L- threonine (L-Thr) or L-glycine (L-Gly); (b) expressing a first enzyme, a second enzyme and a third enzyme by the recombinant cell, wherein the first enzyme is a L-threonine transaldolase (L-TTA) when the L-amino acid is L-Thr or a threonine aldolase (TA) when the L-amino acid is L-Gly, the second enzyme is a phenylserine dehydratase or a threonine deaminase (TD), and the third enzyme is an aminotransferase (AT), a L- amino acid dehydrogenase (L-AADH) or a D-amino acid aminotransferase, wherein at least one of the first enzyme, the second enzyme and the third enzyme is heterologous to the recombinant cell; (c) reacting the aldehyde with the L-Thr or L-Gly, whereby a 0- hydroxy a-amino acid (0-OH AA) is generated; (d) converting the 0-OH AA to a keto acid; and (e) reacting the keto acid with the amine donor, whereby a nonstandard amino acid (nsAA) is produced by the recombinant cell. The method may exclude supplying the -OH AA and / or the keto acid into the culture medium.

[0027] Where the culture medium further comprises a carboxylic acid, the in vivo production method may further comprise expressing a carboxylic acid reductase (CAR) and a phosphopantetheinyl transferase by the recombinant cell, activating the CAR and converting the carboxylic acid to the aldehyde. The in vivo production method may exclude supplying the aldehyde into the culture medium.

[0028] Where the culture medium further comprises an alcohol, the in vivo production method may further comprise expressing an alcohol oxidase (AO) or an alcohol dehydrogenase (ADH), and converting the alcohol to the aldehyde. The in vivo production method may exclude supplying the aldehyde into the culture medium.

[0029] Where the recombinant cell further expresses a target protein and comprises an orthogonal translation system (OTS), the target protein comprises a site for amber stop codon suppression, and the OTS comprises an aminoacyl-tRNA synthetase and a tRNA pair decoding the amber stop codon, the in vivo production method may further comprise incorporating the nsAA into the target protein at the site such that the target protein comprising the nsAA is produced by the recombinant cell.

[0030] According to the in vivo production method, the aldehyde may be an aryl aldehyde, the nsAA may be a L-phenylalanine derivative, and the AT may be an aromatic amino acid aminotransferase.

[0031] The present invention further provides a method for producing a nonstandard amino acid (nsAA) in a container. The container comprises an aldehyde, an L-amino acid, an amine donor, a first enzyme, a second enzyme and a third enzyme, wherein the L-amino acid is L-threonine (L-Thr) or L-glycine (L-Gly), the first enzyme is a L- threonine transaldolase (L-TTA) when the L-amino acid is L-Thr or a threonine aldolase (TAs) when the L-amino acid is L-Gly, the second enzyme is a phenylserine dehydratase or threonine deaminase (TD), and the third enzyme is an aminotransferase (AT), a L-amino acid dehydrogenase (L-AADH), or a D-amino acid aminotransferase. This in vitro production method comprises: (a) reacting the aldehyde with the L-Thr or the L-Gly in the container, whereby a 0-hydroxy a-amino acid (fJ-OH AA) is generated in the container; (b) converting the 0-OH AA to a keto acid in the container; and (c) reacting the keto acid with the amine donor, whereby a nonstandard amino acid (nsAA) is produced in the container. The in vitro production method may exclude supplying the 0-OH AA and / or the keto acid into the container.

[0032] Where the container further comprises a carboxylic acid and a carboxylic acid reductase (CAR), a phosphopantetheinyl transferase, ATP, and a NADPH and / or cofactor regeneration system, and the NADPH and / or cofactor regeneration system comprises a type 2-III polyphosphate kinase from Erysipelotrichaceae organism (PPK12) (SEQ ID NO: 8), a Bacillus megaterium glucose dehydrogenase (bmGDH) (SEQ ID NO: 9), and an E. coll inorganic pyrophosphatase (ecPPase) (SEQ ID NO: 10), the in vitro production method may further comprise activating the CAR, and converting the carboxylic acid to the aldehyde in the container. The in vitro production method may exclude supplying the aldehyde into container.

[0033] Where the container further comprises an alcohol and either an alcohol oxidase (AO) or an alcohol dehydrogenase (ADH), the in vitro production method may further comprise converting the alcohol to the aldehyde in the container. The in vitro production method may exclude supplying the aldehyde into the container.

[0034] Where the container further comprises a recombinant cell, the recombinant cell comprises a target protein and an orthogonal translation system (OTS), the target protein comprises a site for amber stop codon suppression, and the OTS comprises an aminoacyl-tRNA synthetase and a tRNA pair decoding the amber stop codon, the in vitro production method may further comprise incorporating the nsAA into the target protein at the site, whereby a target protein comprising the nsAA is produced by the recombinant cell.

[0035] According to the in vitro production method, the aldehyde may be an aryl aldehyde, the nsAA may be a L-phenylalanine derivative, and the AT may be an aromatic amino acid aminotransferase.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1 shows a one-pot reaction prepared by adding equimolar enzyme concentration (2 pM) of each purified enzyme (s-ObiH (0.13 mg / mL), RpPSDH (0.07 mg / mL), and TyrB (0.09 mg / mL)) to a reaction mixture containing 0.4 mM PLP, 15 mM MgCI2, 100 mM L-Thr, and 25 mM L-Glu. The concentration of phenylalanine, the desired final product, is measured by HPLC 30 min after reaction initiation upon supplementing 2 mM of one of the respective substrates (benzaldehyde, phenylserine, or phenylpyruvate).

[0038] FIGS. 2A-2B demonstrate a broad specificity one-pot biocatalytic cascade to prepare L-phenylalanine derivatives from aryl aldehydes. (A) Reaction diagram and conditions. Three enzymes are purified and supplied at 2 pM protein concentrations: s- ObiH, RpPSDH, and TyrB. Aldehydes are supplied at 2 mM (2% (v / v) DMSO). Other components in the reaction buffer are indicated in the top horizontal pink bar, unless otherwise specified. (B) The chemical structures and average HPLC analytical yields of triplicate reactions are shown for 18 biosynthesized phenylalanine derivatives. We observed >99% e.e. for all products analyzed through Marfey's analysis. The enantiomeric excess for product lie was not determined (N.D.). tReaction performed with 1 mM aldehyde, 5 pM s-ObiH, 2 pM RpPSDH, 2 pM TyrB, pH 7.5 with 0.4 mM PLP, 15 mM MgCI2, 100 mM L-Thr, 10 mM L-Glu, and 1% (v / v) DMSO at 30 °C with an endpoint of 24 h.

[0039] FIGS. 3A-3B show extension of the one-pot biocatalytic cascade to include carboxylic acids as the precursor. (A) Reaction diagram that includes the carboxylic acid reductase SrCAR (0.26 mg / mL) in addition to the enzymes previously supplied. Full reaction conditions are in the top horizontal pink bar. (B) Table of HPLC yields of target nsAA obtained when supplying one of 18 distinct carboxylic acids to the reaction cascade after 24 h. Reactions were performed in triplicate. tProduction of 9e confirmed via MS without quantification.

[0040] FIG. 4 shows preparative-scale nsAA synthesis using clarified lysate from the engineered E. coli strain RARE.A16. Reaction chemistry performed at 40 mL scale to convert 25 mM terephthalaldehyde (3b) to 4-formyl-L-phenylalanine (3e) under conditions shown below the reaction arrow. Final HPLC analytical yield at 18 h was 64% at greater than 99% e.e. and time-course data is shown.

[0041] FIGS. 5A-5C show reactions catalyzed by engineered live E. coli strains that were transformed for heterologous expression of L-threonine transaldolases (L-TTAs), which catalyze the first step of the pathway starting from aldehyde substrates. (A) Reaction catalyzed by L-TTA. (B) Illustration of the reaction format with a live strain engineered both for aldehyde retention and for expression of SUMO-tagged L-TTAs. (C) HPLC traces obtained when supplying either just the aldehyde substrate (2 mM), an endpoint reaction catalyzed by the purified s-ObiH after provision of 2 mM aldehyde substrate, or an endpoint reaction catalyzed by cells that express s-ObiH and were supplied 1 mM aldehyde substrate. Four different aldehyde substrates were tested (#b), each corresponding to a commonly used nsAA for genetic code expansion.

[0042] FIGS. 6A-6C demonstrate biosynthesis of encodable phenylalanine derivatives from aldehydes supplemented to live cells. (A) Designed biochemical pathway featuring reactions catalyzed by an L-TTA, PSDH, and AT. (B) Concentration of nsAA formed by the supplementation of 1 mM of each of the model aldehydes 4b, 2b, 5b, and 15b to cells that are transformed to express a SUMO-tagged L-TTA (either s-ObiH or s-PbTTA) and s-RpPSDH, with a reliance on native expression levels of endogenous aminotransferases to form the desired nsAA products. (C) Concentration of nsAA formed by the supplementation of 1 mM of each of the aldehydes to four different strains (wildtype MG1655, RARE, RARE.A16, and ROAR) transformed to express s- PbTTA and s-RpPSDH.

[0043] FIG. 7 shows biosynthesis of phenylalanine derivatives from carboxylic acids supplemented to live cells. Concentration of nsAA formed by the supplementation of -1- each of the model carboxylic acids to cells that are transformed to express MavCAR, s- ObiH, and s-RpPSDH, with a reliance on native expression levels of endogenous aminotransferases to form the desired nsAA products 4e, 2e, 5e, and 15e from 1, 2 and 4 mM of the carboxylic acid precursors.

[0044] FIGS. 8A-8D show site-specific incorporation of biosynthesized nsAAs from aldehyde or carboxylic acid precursors using a single engineered strain of E. coli. (A) Genetic construct design and digested peptide mass spectrometry result indicating high-fidelity site-specific incorporation of biphenylalanine (15e) within GFP reporter protein. (B) Genetic construct design and digested peptide mass spectrometry indicating high-fidelity site-specific incorporation of 4-azido-phenylalanine (4e) within GFP reporter protein. (C) Solid-media growth and escape from biocontainment assay indicating the successful extension of synthetic auxotrophy to include reliance on the aldehyde 15b. This was done by transforming a previously engineered and evolved strain that requires 4e (DEP.e5) for heterologous expression of our core biosynthetic pathway. The strain already contains a BipA-dependent BipARS and essential genes that are modified to contain in-frame UAG codons.

[0045] FIG. 9 shows incorporation of nsAAs biosynthesized from diverse aryl aldehyde precursors via a lysate-based reaction platform. Reaction mixtures containing the bioproduced nsAAs were directly supplemented to live cells containing an aaRS and tRNA pair for amber stop codon suppression as well as a GFP reporter protein containing a site for nsAA incorporation (TAG). Incorporation of the biosynthesized nsAAs was analyzed as an increase in the fold change in FL / OD600 relative to the "no substrate" negative control. Screening was performed on 14 different aldehyde precursors (ald-1 - ald-14) with five aaRSs (aaRS-1 - aaRS-5).

[0046] FIG. 10 shows incorporation of nsAAs biosynthesized from diverse carboxylic acid precursors via a resting whole cell-based reaction platform. Reaction mixtures containing the bio-produced nsAAs were directly supplemented to live cells containing an OTS and reporter protein. Incorporation of the biosynthesized nsAAs was analyzed as an increase in the fold change in FL / OD600 relative to the "no substrate" negative control. Four different carboxylic acid precursors (4-Azidobenzoic acid, 4- Benzoylbenzoic acid, MHET, and 4-(Phenylazo)benzoic acid) were supplemented to reaction mixtures containing CAR, L-TTA, and PSDH resting whole cells, and subsequently supplied to live cells for incorporation.

[0047] FIGS. 11A-11B show (A) Conversion of 4-methoxylbenzyl alcohol to o- methyltyrosine (OMeTyr) using resting cells containing either BuAO or TTA and PSDH; and (B) One-pot production of OMeTyr using either ObiH or PbTTA coupled with BuAO. Reactions were performed with 5 mM loading of 4-methoxylbenzyl alcohol (AO and AO + TTA-PSDH coupled reactions) or 4-methoxylbenzaldehdye (ObiH and PbTTA only cases). Reaction buffer consisted of 50 mg cell wet weight / mL (100 mg cell wet weight / mL for coupled assay), 200 mM HEPES, 1 mM PLP, 10 mM MgCI2, 200 mM L-Thr and 50 mM L-Glu at a pH of 7.5.

[0048] DETAILED DESCRIPTION OF THE INVENTION

[0049] The present invention relates to production of a nonstandard amino acid (nsAA) from a L-amino acid such as L-threonine (L-Thr) or L-glycine (L-Gly) via a cascade of reactions catalyzed by a plurality of enzymes. The present invention is based on the inventors' surprising discovery of remarkably broad substrate tolerance of two natural pyridoxal 5'-phosphate (PLP)-dependent enzymes, a phenylserine dehydratase and an aromatic amino acid aminotransferase, when coupled with a natural L-threonine transaldolases (L-TTAs) to design a high-yield cascade, for example, at a yield of at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95% or 99%, capable of converting numerous non-native substrates as, for example, functionalized aryl aldehyde precursors, to unnatural products of considerable demonstrated value, for example, with use an a bio-orthogonal handle (4-acetyl-L-phenalanine, 4-azido-L- phenylalanine, 4-formyl-L-phenylalanine, in the creation of designer enzymes (4- borono-L-phenylalanine, 4-benzoyl-L-phenylalanine), and more. Upon investigation of the phenylserine dehydratase from Ralstonia picketin' PS22 (RpPSDH) coupled with an E. coli aromatic amino acid aminotransferase, TyrB, the inventors have developed a one-pot cascade of purified enzymes for generating phenylalanine derivatives with high yield and diversity from readily available and inexpensive aryl aldehyde precursors by coupling the purified enzymes to an L-TTA from Pseudomonas fluorescens (ObiH). The inventors have expanded the versatility of the cascade through the addition of a promiscuous carboxylic acid reductase from Segniliparus rotundus (SrCAR) for generation of aryl aldehydes from carboxylic acids, which are often less expensive and more stable than aryl aldehydes. The inventors have further investigated an alternative module consisting of an alcohol oxidase (AO) instead of a carboxylic acid reductase to initialize the cascade from aromatic alcohols, which decreases the co-factor demands of the pathway.

[0050] One way to address the issue of nsAA isolation from living cells is to remove the downstream processing steps of small molecule separation through the development of a methodology to directly supplement reaction mixtures containing biocata lytica lly produced nsAAs to live cells for subsequent incorporation into proteins. The inventors have demonstrated the application of a polyspecific cascade in clarified lysate-based or resting whole cell-based reactions to further improve the economic feasibility of the platform by obviating the need for costly and time-intensive enzyme purification. The inventors have further discovered that incorporation of the bio-produced nsAAs may then be rapidly screened for incorporation into a protein of interest, facilitating the discovery and implementation of novel or otherwise cost-prohibitive chemistries in genetic code expansion technologies.

[0051] The inventors have developed a further option, the combined nsAA biosynthesis and site-specific incorporation within proteins using a single engineered organism, creating opportunities to harness principles from synthetic biology, such as on-demand nsAA synthesis, nsAA sensing, and intercellular communication. Pioneering efforts during the last two decades have begun to demonstrate coupling of nsAA biosynthesis and site-specific incorporation for certain nsAAs. However, past efforts featured biosynthetic pathways that exhibit highly restricted substrate specificity such that each desired nsAA requires a different pathway. In contrast, biocatalysis-oriented efforts have leveraged polyspecific enzymes as platforms for synthesis of diverse phenylalanine derivatives in cell-free contexts. However, these biocatalytic strategies have not been demonstrated to function in growing cells that are most actively translating new proteins, which is necessary when combining biosynthesis and incorporation.

[0052] Given the extensive application of >80 phenylalanine derivatives in genetic code expansion technologies and the lack of a promiscuous biosynthetic pathway to access these encodable nsAAs, the inventors have aimed to design a platform to produce these chemistries from inexpensive and readily accessible precursors. Using strains of E. coli that are engineered to stabilize aryl aldehydes, the inventors have shown successful biosynthesis of multiple highly relevant nsAAs that vary in size and polarity from either aryl aldehyde or carboxylic acid precursors in metabolically active cells. The inventors have demonstrated coupling of biosynthesis and high-fidelity encoding of nsAAs starting from either an aldehyde or carboxylic acid precursor. Finally, the inventors have illustrated how this platform for bioconversion from readily accessible precursors to nsAAs offers new synthetic biology applications based on control of protein translation, such as extending the permissive chemical range of the biological containment technique of synthetic auxotrophy to synthetic aryl aldehydes.

[0053] With respect to non-standard amino acids (nsAAs) such as L-phenylalanine derivatives with aryl ring functionalization, the inventors have revealed the broad specificity of multiple natural pyridoxal 5'-phosphate (PLP)-dependent enzymes, specifically an L-threonine transaldolase, a phenylserine dehydratase, and an aminotransferase, towards substrates that contain aryl side chains with diverse substitutions. The inventors have exploited this tolerance to construct a one-pot biocatalytic cascade that achieves high-yield synthesis of diverse enantiopure L- phenylalanine derivatives from aldehydes under mild aqueous reaction conditions. The inventors have demonstrated addition of a carboxylic acid reductase module to this cascade to enable the biosynthesis of L-phenylalanine derivatives from carboxylic acids that may be less expensive or less reactive than the corresponding aldehydes. The inventors have further shown an alternative module that allows biosynthesis of L- phenylalanine derivatives from alcohols by applying an alcohol oxidase to produce aryl aldehydes from alcohols for direct coupling with the downstream biocatalytic cascade. In principle, an alcohol dehydrogenase may also perform this reaction, though with different co-factor requirements. The economic feasibility and scalability of the polyspecific nsAA production platform is improved through the demonstration of both lysate-based and resting whole-cell based reactions, which remove the need for costly and time-intensive enzyme purification. Implementation of a co-factor regeneration system for the carboxylic acid reductase module significantly lowers the cost associated with equimolar supplementation of costly ATP and NADPH. The present invention offers an efficient, versatile, and scalable route with the potential to lower manufacturing cost and democratize synthesis for many valuable nsAAs.

[0054] To further improve translation of the platform for applications of genetic code expansion in live cells, the inventors have sought to address a key limitation - the reliance on the external supplementation of non-standard amino acids (nsAAs) to cell culturing media. In particular, the inventors have demonstrated two alternatives to eliminate the need for the isolation of a synthesized nsAA: 1) the direct supplementation of reaction mixtures containing biocatalytically produced nsAAs to live bacterial cells for subsequent incorporation into a target protein without nsAA separation and 2) the combination of phenylalanine derivative semi-synthesis and sitespecific incorporation using a single engineered bacterial host. The system of the present invention serves as a platform that exhibits broad substrate specificity towards commercially ubiquitous, achiral building blocks of aryl aldehydes, carboxylic acids, or alcohols, producing the family of nsAAs that are most frequently used for genetic code expansion. These platforms allow for the rapid production and screening of a broad range of industrially relevant and novel nsAAs for compatible orthogonal translation machinery. The inventors have additionally shown that the combination of nsAA biosynthesis and incorporation steps may extend the chemical reach of the intrinsic biological containment strategy of synthetic auxotrophy from reliance on nsAAs to instead reliance on low-cost and achiral building blocks. The present system is expected to aid industrial-scale manufacturing of peptides and proteins that contain nsAAs and facilitate access to expensive or commercially unavailable chemistries for labs that lack separations or traditional synthesis expertise. The modularity of the designed reaction pathway of the present invention may allow one to introduce alternative enzymes or expand to additional chemical classes. For example, alcohol dehydrogenases (ADHs), threonine aldolases (TAs), threonine deaminases (TDs), and L-amino acid dehydrogenases (L-AADHs) are alternative classes of enzymes that may readily be swapped into the reaction cascade as needed to provide additional screening protentional, and such substitutions should be governed by the current filing and deemed obvious for subsequent cases. For the production of D- amino acids, the final module in the biocatalytic cascade may simply be replaced with an enzyme to re-install the amine group with the desired configuration, such as a D- amino acid aminotransferase. Initialization from alkyl precursors, (alcohols, carboxylic acids, aldehydes) may allow for the extension of this platform towards aliphatic nsAA biosynthesis. Finally, additional modules may be added up or downstream of the biocatalytic pathway to initialize from alternative precursors or add further functionality.

[0055] The term "recombinant cell" as used herein refers to a cell that is not naturally occurring. A recombinant cell is made by engineering from a naturally occurring cell. The recombinant cell may overexpress a protein native to the corresponding naturally occurring cell or express a protein heterologous to the corresponding naturally occurring cell.

[0056] The term "nonstandard amino acid (nsAA)", also known as a noncanonical amino acid or unnatural amino acid, as used herein refers to a nonproteinogenic amino acid. As shown in Table 3, examples of nsAAs include 4-nitro-L-phenylalanine (le), 4- acetyl-L-phenylalanine (2e), 4-formyl-L-phenylalanine (3e), 4-azido-L-phenylalanine (4e), 4-cyano-L-phenylalanine (5e), 4-bromo-L-phenylalanine (6e), 2-fluoro-L- phenylalanine (7e), 3-fluoro-L-phenylalanine (8e), 4-fluoro-L-phenylalanine (9e), (S)- 2-Amino-3-(3-(trifluoromethyl)phenyl)propanoic acid (lOe), O-methyl-L-tyrosine (lie), 4-borono-L-phenylalanine (12e), 2-nitro-L-phenylalanine (13e), 3-nitro-L-phenylalanine (14e), Biphenylalanine (15e), 3-(l-Naphthyl)-L-alanine (16e), 3-(2-Naphthyl)-L-alanine (17e), and 4-benzoyl-L-phenylalanine (18e).

[0057] The term "L-threonine transaldolase (L-TTA)" as used herein refers to a protein capable of catalyzing the production of -hydroxy nsAAs from aldehydes and L-Thr. Examples of the L-TTAs include ObiH, PbTTA, PiTTA, CsTTA, BuTTA, and KaTTA.

[0058] The nsAA may be produced from an aldehyde. As shown in Table 3, examples of aldehydes include 4-nitrobenzaldehyde (lb), 4-acetylbenzaldehyde (2b), terephthalaldehyde (3b), 4-azidobenzaldehyde (4b), 4-formylbenzonitrile (5b), 4- bromobenzaldehyde (6b), 2-fluorobenzaldehyde (7b), 3-fluorobenzaldehyde (8b), 4- fluorobenzaldehyde (9b), 3-(trifluoromethyl)benzaldehyde (10b), 4-anisaldehyde (lib), 4-formylphenylboronoic acid (12b), 2-nitrobenzaldehyde (13b), 3- nitrobenzaldehyde (14b), biphenyl-4-carboxaldehyde (15b), 1-naphthaldehyde (16b), 2-naphthaldehyde (17b), and 4-benzoylbenzaldehyde (18b).

[0059] The nsAA may be produced from a carboxylic acid. As shown in Table 3, examples of carboxylic acids include 4-nitrobenzoic acid (la), 4-acetylbenzoic acid (2a), 4-Formylbenzoic acid (3a), 4-azidobenzoic acid (4a), 4-cyanobenzoic acid (5a), 4- bromobenzoic acid (6a), 2-fluorobenzoic acid (7a), 3-fluorobenzoic acid (8a), 4- fluorobenzoic acid (9a), 3-(trifluoromethyl)benzoic acid (10a), 4-methoxybenzoic acid (11a), 4-boronobenzoic acid (12a), 2-nitrobenzoic acid (13a), 3-nitrobenzoic acid (14a), biphenyl-4-carboxylic acid (15a), 1-naphthoic acid (16a), 2-naphthoic acid (17a), and 4-benzoylbenzoic acid (18a).

[0060] The term "threonine aldolase (TA)" as used herein refers to a protein capable of catalyzing production of a p-hydroxy nsAAs from an aldehyde and glycine. Examples of the TAs include TmTA from Thermotoga maritima, EcTA from E. coli, AjTA from Aeromonas jandaei, PpTA from Pseudomonas putida, and ScTA from Streptomyces coelicolor.

[0061] The term "phenylserine dehydratase" as used herein refers to a protein capable of catalyzing deamination of a p-hydroxy nsAA to yield a keto acid and ammonia. Examples of the phenylserine dehydratases include RpPSDH, and PxPSDH from Paraburkholderia xenovorans.

[0062] The term "threonine deaminase (TD)" as used herein refers a protein capable of catalyzing deamination of a p-hydroxy nsAA to yield a keto acid and ammonia. Examples of the TDs include CgTD from Corynebacterium glutamicum, and EcTD from E. coli.

[0063] The term "aminotransferase (AT)" as used herein refers to a protein capable of catalyzing production of an amino acid from a keto acid in the presence of an amine donor. Examples of the ATs include TyrB, AspC from E. coli, and GlnAT from Thermus thermophilus HB8.

[0064] The term "L-amino acid dehydrogenase (L-AADH)" as used herein refers to a protein capable of catalyzing production of an amino acid from a keto acid in the presence of NADH. Examples of the L-AADHs include Bb-L-AADH from Bacillus badius.

[0065] The term "D-amino acid aminotransferase" as used herein refers to a protein capable of catalyzing production of a D-amino acid from a keto acid in the presence of an amine donor. Examples of the D-amino acid aminotransferases include Bs-D-AT from Bacillus subtilis.

[0066] The term "carboxylic acid reductase (CAR)" as used herein refers to a protein capable of catalyzing production of an aldehyde from a carboxylic acid in the presence of NADPH and ATP. Examples of the CARs include SrCAR, MavCAR, MmCAR, MabCAR, NiCAR, and AfCAR.

[0067] The term "phosphopantetheinyl transferase" as used herein refers to protein capable of transferring a phosphopantetheinyl moiety to a serine residue for activation. Examples of the phosphopantetheinyl transferases include BsSfp.

[0068] The term "alcohol oxidase (AO)" as used herein refers to protein capable of catalyzing production of an aldehyde from an alcohol with hydrogen peroxide as a byproduct. Examples of the Aos include BuAO.

[0069] The term "alcohol dehydrogenase (ADH)" as used herein refers to protein capable of catalyzing the production of aldehydes from alcohols with NAD+ . Examples of the ADHs include TkADH from Thermococcus kodakarensis.

[0070] The term "orthogonal translation system (OTS)" as used herein refers to aminoacyl-tRNA synthetase and a tRNA pair decoding a site for nsAA incorporation (e.g. the amber stop codon). The OTS may further comprise of engineering variants of MjTyRS / tRNA, and / or PylRS / tRNA.

[0071] The term "nicotinamide adenine dinucleotide phosphate (NADPH) and / or cofactor regeneration system" as used herein refers to a system to produce NADPH from NADP+ or ATP from AMP. The NADPH and / or cofactor regeneration system may comprise a type 2-III polyphosphate kinase (PPK12) from an unclassified Erysipelotrichaceae organism, BmGDH, and EcPPase.

[0072] The term "container" as used herein refers to an enclosed space outside a cell, a tissue or a biological organism. The container may be a device, for example, a bioreactor.

[0073] The present invention provides a recombinant cell. The recombinant cell expresses a first enzyme, a second enzyme and a third enzyme. The recombinant cell produces a nonstandard amino acid (nsAA). The first enzyme may be a L-threonine transaldolase (L-TTA) or a threonine aldolase (TAs). The second enzyme may be a phenylserine dehydratase or a threonine deaminase (TD). The third enzyme may be an aminotransferase (AT), a L-amino acid dehydrogenase (L-AADH) or a D-amino acid aminotransferase. At least one of the first enzyme, the second enzyme and the third enzyme may be heterologous to the recombinant cell. The recombinant cell may produce a nonstandard amino acid (nsAA) in an amount of about 5-800 pM from about 1 mM of a precursor, for example, an aldehyde, a carboxylic acid, or an alcohol. The yield may be about 0.1-99.9%, 10-99.9%, 15%-99.9%, 20-99.9%, 30-99.9%, 40- 99.9%, 50-99.9%, 60-99.9%, 70-99.9%, 80-99.9%, 90-99.9%, 95-99.9%, or at least about 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95% or 99%.

[0074] The L-TTA may be from Pseudomonas fluorescens (ObiH). The L-TTA may comprise an amino acid sequence at least about 80%, 85%, 90%, 95%, 99% or 100% homologous to the amino acid sequence of SEQ ID NO: 1. The L-TTA may consist of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99% or 100% homologous to the amino acid sequence of SEQ ID NO: 1. The L-TTA may comprise the amino acid sequence of SEQ ID NO: 1.

[0075] The L-TTA may be from Parachlamydiales bacterium (PbTTA). The PbTTA may comprise an amino acid sequence at least about 80%, 85%, 90%, 95%, 99% or 100% homologous to the amino acid sequence of SEQ ID NO: 2. The PbTTA may consist of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99% or 100% homologous to the amino acid sequence of SEQ ID NO: 2. The PbTTA may consist of the amino acid sequence of SEQ ID NO: 2.

[0076] The phenylserine dehydratase may be from Ralstonia pickettii PS22 (RpPSDH). The RpPSDH may comprise an amino acid sequence at least about 80%, 85%, 90%, 95%, 99% or 100% homologous to the amino acid sequence of SEQ ID NO: 3. The RpPSDH may consist of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99% or 100% homologous to the amino acid sequence of SEQ ID NO: 3. The RpPSDH may consist of the amino acid sequence of SEQ ID NO: 3.

[0077] The AT may be a tyrosine aminotransferase from E. coli (TyrB). The TyrB may comprise an amino acid sequence at least about 80%, 85%, 90%, 95%, 99% or 100% homologous to the amino acid sequence of SEQ ID NO: 4. The TyrB may consist of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99% or 100% homologous to the amino acid sequence of SEQ ID NO: 4. The TyrB may consist of the amino acid sequence of SEQ ID NO: 4.

[0078] The recombinant cell may further express a carboxylic acid reductase (CAR), a phosphopantetheinyl transferase, or both. The phosphopantetheinyl transferase may be required for CAR activation.

[0079] The CAR may be from Segniliparus rotundus (SrCAR). The SrCAR may comprise an amino acid sequence at least about 80%, 85%, 90%, 95%, 99% or 100% homologous to the amino acid sequence of SEQ ID NO: 5. The SrCAR may consist of the amino acid sequence of SEQ ID NO: 5. The SrCAR may consist of the amino acid sequence of SEQ ID NO: 5. The CAR may be from Mycobacterium avium (MavCAR). The MavCAR may comprise an amino acid sequence at least about 80%, 85%, 90%, 95%, 99% or 100% homologous to the amino acid sequence of SEQ ID NO: 6. The MavCAR may consist of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99% or 100% homologous to the amino acid sequence of SEQ ID NO: 6. The MavCAR may consist of the amino acid sequence of SEQ ID NO: 6.

[0080] The phosphopantetheinyl transferase may be from Bacillus subtilis (sfp). The sfp may comprise an amino acid sequence at least about 80%, 85%, 90%, 95%, 99% or 100% homologous to the amino acid sequence of SEQ ID NO: 7. The sfp may consist of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99% or 100% homologous to the amino acid sequence of SEQ ID NO: 7. The sfp may consist of the amino acid sequence of SEQ ID NO: 7.

[0081] The recombinant cell may further express an alcohol oxidase (AO) or an alcohol dehydrogenase (ADH).

[0082] The AO may be from Burkholderia (BuAO). The BuAO may comprise an amino acid sequence at least about 80%, 85%, 90%, 95%, 99% or 100% homologous to the amino acid sequence of SEQ ID NO: 11. The BuAO may consist of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99% or 100% homologous to the amino acid sequence of SEQ ID NO: 11. The BuAO may consist of the amino acid sequence of SEQ ID NO: 11.

[0083] The ADH may be from Thermococcus kodakarensis KOD1 (TkADH). The TkADH may comprise an amino acid sequence at least about 80%, 85%, 90%, 95%, 99% or 100% homologous to the amino acid sequence of SEQ ID NO: 12. The TkADH may consist of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99% or 100% homologous to the amino acid sequence of SEQ ID NO: 12. The TkADH may consist of the amino acid sequence of SEQ ID NO: 12.

[0084] The recombinant cell may further express a target protein and comprise an orthogonal translation system (OTS). The target protein may comprise a site for amber stop codon suppression. The OTS may comprise an aminoacyl-tRNA synthetase and a tRNA pair decoding the amber stop codon. The nsAA may be incorporated into the target protein at the site.

[0085] The recombinant cell may be E. coll.

[0086] For each recombinant cell of the present invention, a method for producing a nonstandard amino acid (nsAA) is provided. This in vivo production method may comprise growing a recombinant cell in a culture medium. The culture medium may comprise an aldehyde, a L-amino acid and an amine donor. The L-amino acid may be L-threonine (L-Thr) or L-glycine (L-Gly). The method may further comprise expressing a first enzyme, a second enzyme and a third enzyme by the recombinant cell. The first enzyme may be a L-threonine transaldolase (L-TTA) when the L-amino acid is L-Thr or a threonine aldolase (TA) when the L-amino acid is L-Gly. The second enzyme may be a phenylserine dehydratase or a threonine deaminase (TD). The third enzyme may be an aminotransferase (AT), a L-amino acid dehydrogenase (L-AADH) or a D-amino acid aminotransferase. At least one of the first enzyme, the second enzyme and the third enzyme may be heterologous to the recombinant cell. The method may further comprise reacting the aldehyde with the L-Thr or L-Gly so that a 0-hydroxy a-amino acid (fJ-OH AA) is generated. The method may further comprise converting the p-OH AA to a keto acid; and reacting the keto acid with the amine donor. As a result, a nonstandard amino acid (nsAA) is produced by the recombinant cell.

[0087] The in vivo production method may exclude supplying the p-OH AA, the keto acid or both into the culture medium. No external p-OH AA may be supplied into the culture medium. No external keto acid may be supplied into the culture medium. No external p-OH AA and no external keto acid may be supplied into the culture medium.

[0088] Where the culture medium further comprises a carboxylic acid, the in vivo production method may further comprise expressing a carboxylic acid reductase (CAR) and a phosphopantetheinyl transferase, for example, by the same recombinant cell or one or more additional recombinant cells, activating the CAR, and converting the carboxylic acid to the aldehyde. The in vivo production method may exclude supplying the aldehyde into the culture medium. No external aldehyde may be supplied into the culture medium.

[0089] Where the culture medium further comprises an alcohol, the in vivo production method may further comprise expressing an alcohol oxidase (AO) or an alcohol dehydrogenase (ADH), for example, by the same recombinant cell or one or more additional recombinant cells, and converting the alcohol to the aldehyde. The in vivo production method may exclude supplying the aldehyde into the culture medium. No external aldehyde may be supplied into the culture medium.

[0090] In one embodiment, the recombinant cell may further express a target protein and comprise an orthogonal translation system (OTS), the target protein may comprise a site for amber stop codon suppression, the OTS may comprise an aminoacyl-tRNA synthetase and a tRNA pair decoding the amber stop codon, and the in vivo production method may further comprise incorporating the nsAA into the target protein at the site such that a target protein comprising the nsAA may be produced by the recombinant cell.

[0091] According to the in vivo production method of the present invention, the aldehyde may be an aryl aldehyde, the nsAA may be a L-phenylalanine derivative, and the AT may be an aromatic amino acid aminotransferase.

[0092] According to the in vivo production method of the present invention, a nonstandard amino acid (nsAA) may be produced by the recombinant cell in an amount of about 5-800 pM from about 1 mM of precursor, for example, an aldehyde, a carboxylic acid, or an alcohol. The yield may be about 0.1-99.9%, 10-99.9%, 15%- 99.9%, 20-99.9%, 30-99.9%, 40-99.9%, 50-99.9%, 60-99.9%, 70-99.9%, 80-99.9%, 90-99.9%, 95-99.9%, or at least about 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95% or 99%.

[0093] The present invention further provides a method for producing a nonstandard amino acid (nsAA) in a container. The container may comprise an aldehyde, an L-amino acid, an amine donor, a first enzyme, a second enzyme and a third enzyme. The L- amino acid may be L-threonine (L-Thr) or L-glycine (L-Gly). The first enzyme may be a L-threonine transaldolase (L-TTA) when the L-amino acid is L-Thr or a threonine aldolase (TAs) when the L-amino acid is L-Gly. The second enzyme may be a phenylserine dehydratase or threonine deaminase (TD). The third enzyme may be an aminotransferase (AT), a L-amino acid dehydrogenase (L-AADH), or a D-amino acid aminotransferase. This in vitro production method may comprise reacting the aldehyde with the L-Thr or the L-Gly in the container, whereby a 0-hydroxy a-amino acid (0-OH AA) is generated in the container. The in vitro production method may further comprise converting the 0-OH AA to a keto acid in the container, and reacting the keto acid with the amine donor such that a nonstandard amino acid (nsAA) is produced in the container.

[0094] The in vitro production method may exclude supplying the 0-OH AA and / or the keto acid into the container. No external 0-OH AA may be supplied into the container. No external keto acid may be supplied into the container. No external 0-OH AA and no external keto acid may be supplied into the container.

[0095] In one embodiment, the container further comprises a carboxylic acid, a carboxylic acid reductase (CAR), a phosphopantetheinyl transferase, adenosine triphosphate (ATP), a nicotinamide adenine dinucleotide phosphate (NADPH) and / or cofactor regeneration system, and the in vitro production method further comprises activating the CAR, and converting the carboxylic acid to the aldehyde in the container. The NADPH and / or cofactor regeneration system may comprise a type 2-III polyphosphate kinase from Erysipelotrichaceae organism (PPK12) (SEQ ID NO: 8), a Bacillus megaterium glucose dehydrogenase (bmGDH) (SEQ ID NO: 9), and an E. coll inorganic pyrophosphatase (ecPPase) (SEQ ID NO: 10).

[0096] The in vitro production method may exclude supplying the aldehyde into container. No external aldehyde may be supplied into the container.

[0097] In one embodiment, the container may further comprise an alcohol and either an alcohol oxidase (AO) or an alcohol dehydrogenase (ADH), and the in vitro production method may further comprise converting the alcohol to the aldehyde in the container. The in vitro production method may exclude supplying the aldehyde into the container. No external aldehyde may be supplied into the container.

[0098] In another embodiment, the container may further comprise a recombinant cell, the recombinant cell may comprise a target protein and an orthogonal translation system (OTS), the target protein may comprise a site for amber stop codon suppression, the OTS may comprise an aminoacyl-tRNA synthetase and a tRNA pair decoding the amber stop codon, and the in vitro production method may further comprise incorporating the nonstandard amino acid into the target protein at the site such that a target protein comprising the nonstandard amino acid is produced by the recombinant cell.

[0099] According to the in vitro production method of the present invention, the aldehyde may be an aryl aldehyde, the nonstandard amino acid may be a L- phenylalanine derivative, and the AT may be an aromatic amino acid aminotransferase.

[0100] According to the in vitro production method of the present invention, a nonstandard amino acid (nsAA) may be produced in a container at a yield of about 0.1- 99.9%, 10-99.9%, 15%-99.9%, 20-99.9%, 30-99.9%, 40-99.9%, 50-99.9%, 60- 99.9%, 70-99.9%, 80-99.9%, 90-99.9%, 95-99.9%, or at least about 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95% or 99%. For example, the nsAA may be produced in a container in an amount of about 17 - 99% yield from 2 mM of aldehyde precursors.

[0101] The term "about" as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate.

[0102] Example 1. A One-Pot Biocatalytic Cascade to Access Diverse L-Phenylalanine

[0103] Derivatives from Aldehydes or Carboxylic Acids

[0104] 1. Materials & Methods 1.1 Strains and Plasmids

[0105] Escherichia coli strains and plasmids used are listed in Table 1. Expression of enzymes for purification was performed in E. coli BL21 (DE3). Expression of enzymes for lysate biotransformations was performed in E. coli RARE.A16.

[0106] 1.2 Materials and Chemicals

[0107] The following compounds were purchased from MilliporeSigma : kanamycin sulfate (Kan), chloramphenicol (Cm), streptomycin sulfate salt (Str), dimethyl sulfoxide (DMSO), potassium phosphate dibasic, potassium phosphate monobasic, magnesium sulfate, calcium chloride dihydrate, glycerol, Tris base, glycine, HEPES, ATP, and KOD XTREME Hot Start polymerase. The following were purchased from TCI America: D- glucose, phenylpyruvic acid, 4-nitrobenzoic acid (la), 4-acetylbenzoic acid (2a), 4- azidobenzoic acid (4a), 3-(trifluoromethyl)benzoic acid (10a), 2-nitrobenzoic acid (13a), 3-nitrobenzoic acid (14a), 2-naphthoic acid (17a), terephthalaldehyde (3b), 2- nitrobenzaldehyde (13b), 3-nitrobenzaldehyde (14b), biphenyl-4-carboxaldehyde (15b), 1-naphthaldehyde (16b), 2-fluoro-L-phenylalanine (7e), 3-fluoro-L- phenylalanine (8e), and O-methyl-L-tyrosine (16e). The following were purchased from Peptech: 4-nitro-L-phenylalanine (le), 4-acetyl-L-phenylalanine (2e), 4-cyano-L- phenylalanine (5e), 4-bromo-L-phenylalanine (6e), 4-fluoro-L-phenylalanine (9e), 2- nitro-L-phenylalanine (13e), 3-(l-Naphthyl)-L-alanine (16e), 3-(2-Naphthyl)-L-alanine (17e), and 4-benzoyl-L-phenylalanine (18e). The following were purchased from Thermo Scientific Chemicals: FDAA (Marfey's Reagent) (l-fluoro-2-4-dinitrophenyl-5-L- alanine amide, benzoic acid, 4-bromobenzoic acid (6a), 2-fluorobenzoic acid (7a), 3- fluorobenzoic acid (8a), 4-fluorobenzoic acid (9a), 1-naphthoic acid (16a), 4- benzoylbenzoic acid (18a), 4-bromobenzaldehyde (6b), 3-fluorobenzaldehyde (8b), 4- fluorobenzaldehyde (9b), and 3-nitro-L-phenylalanine (14e). The following were purchased from Sigma-Aldrich: benzaldehyde, terephthalic acid (3a), 4- methoxybenzoic acid (11a), biphenyl-4-carboxylic acid (15a), 4-nitrobenzaldehyde (lb), 4-formylbenzonitrile (5b), 4-anisaldehyde (11a), 4-formylphenylboronoic acid (12b), 2-naphthaldehyde (17b), and L-phenylalanine. Agarose, ethanol, 2- fluorobenzaldehyde (7b), L-glutamic acid monopotassium salt monohydrate, and L- threonine were purchased from Alfa Aesar. Acetonitrile, trifluoroacetic acid (TFA), sodium chloride, LB Broth powder (Lennox), and LB Agar powder (Lennox) were purchased from Fisher Chemical. Taq DNA ligase was purchased from GoldBio. L- glutamic acid, 3-(trifluoromethyl)benzaldehyde (10b), and 4-borono-L-phenylalanine (12e) were purchased from ACROS Organics. 4-boronobenzoic acid (12a), and (S)-2- Amino-3-(3-(trifluoromethyl)phenyl)propanoic acid (lOe) were purchased from Ambeed. 4-cyanobenzoic acid (5a), 4-azidobenzaldehyde (4b), and 4-carboxy-L- phenylalanine (19e) were purchased from ChemCruz. 4-azido-L-phenylalanine (4e) was purchased from Bachem. Biphenylalanine (15e) was purchased from Com bi- blocks. 4- nitrophenypyruvic acid was purchased from abcr GmbH. (2S)-2-amino-3-(4- formylphenyl)propanoic acid hydrochloride (3e) was custom chemically synthesized by ChiroBlock GmbH. Phusion DNA polymerase and T5 exonuclease were purchased from New England BioLabs (NEB). Sybr Safe DNA gel stain was purchased from Invitrogen. NADPH (tetrasodium salt) and 4-acetylbenzaldehyde (2b) were purchased from Santa Cruz Biotechnology. Anhydrotetracycline (aTc) was purchased from Cayman Chemical. KAPA2G FAST Multiplex Kit was purchased from Roche.

[0108] 1.3 Culture Conditions

[0109] Cultures were grown in LB-Lennox medium (LBL: 10 g / L bacto tryptone, 5 g / L sodium chloride, 5 g / L yeast extract) or 2xYT medium (16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride). Cultures were inoculated from an overnight culture and grown in appropriate antibiotic (30 pg / mL Kan) at 37 °C with shaking at 250 RPM in a 1 L baffled shake flask. At an ODeoo= 0.5-0.8, 0.2 pM anhydrotetracycline (aTc) added to induce enzyme expression. Cultures were incubated for an additional 5 h at 30 °C, followed by incubation at 18 °C for 16 h with shaking at 250 RPM unless otherwise specified.

[0110] 1.4 HPLC and LC-MS analysis

[0111] Metabolites of interest were quantified via high-performance liquid chromatography (HPLC) using an Agilent 1100 Infinity model equipped with a Zorbax Eclipse Plus-C18 column (part number: 959701-902, 5 pm, 95&, 2.1 x 150 mm). To quantify compounds of interest, an initial mobile phase of solvent A / B = 95 / 5 was used (solvent A, water, 0.1% TFA; solvent B, acetonitrile, 0.1% TFA) and maintained for 5 min. A gradient elution was performed (A / B) with: gradient from 95 / 5 to 50 / 50 for 5- 12 min, gradient from 50 / 50 to 0 / 100 for 12-13 min, gradient from 0 / 100 to 95 / 5 for 13-14, and equilibration at 95 / 5 for 14-15 min. A flow rate of 1 mL min1was maintained, and absorbance was monitored at 210, 250, 270, 280 and 300 nm. Confirmation of product production was performed using a Waters AQUITY Arc UPLC H- Class with a diode array coupled to a Waters AQUITY QDa Mass Detector. Product was analyzed using a Waters Cortecs UPLC C18 column with an initial mobile phase of solvent A / B = 95 / 5 (solvent A, water, 0.1% formic acid; solvent B, acetonitrile, 0.1% formic acid) and maintained for 5 min. A gradient elution was performed (A / B) with: gradient from 95 / 5 to 10 / 90 for 5-7 min, an isocratic flow at 10 / 90 for 7-10 min, then gradient from 10 / 90 to 95 / 5 for 10-10.5 min and a final isocratic step at 95 / 5 for 10-12 min at A flow rate of 1 mL min-1.

[0112] 1.5 Expression and Purification of Pathway Enzymes For expression, each plasmid encoding expression of each pathway enzyme was transformed into E. coli BL21(DE3) (NEB). 300 mL of LB in 1 L baffled flasks were supplemented with 30 pg / mL Kan and inoculated from an overnight culture at a 1 : 100 inoculation ratio and incubated at 37 °C with shaking at 250 RPM. Cultures were induced at mid-exponential phase (OD6oo=0.5-0.8) with 0.2 pM aTc, and then incubated at 30 °C for 5 h, followed by overnight expression at 18 °C. Cells were harvested by centrifugation (7000 x g at 4 °C for 6 min). The supernatant was removed, and the cell pellets stored at -80 °C prior to lysing.

[0113] For purification of s-ObiH and RpPSDH, cells E. coli Strains 1 and 2 in Table 1) were resuspended in Lysis buffer (25 mM HEPES pH 7.4, 250 mM NaCI, 0.4 mM PLP, 10 mM MgCh and 10 mM imidazole). The cells were lysed by sonication followed by centrifugation at 18213 x g for 1 h. The supernatant was sterile filtered through a 0.22 pm syringe filter and purified using Ni-Sepharose affinity chromatography (HisTrap HP, 5 mL) via an AKTA Pure fast protein liquid chromatography (FPLC) system using Lysis Buffer A (25 mM HEPES pH 7.4, 250 mM NaCI, 0.4 mM PLP, 10 mM MgCI2, and 10 mM imidazole) and Elution Buffer B (25 mM HEPES pH 7.4, 250 mM NaCI, 0.4 mM PLP, 10 mM MgCI2, and 250 mM imidazole). The column was equilibrated with 2.5% Buffer B and after performing an isocratic 12% Buffer B (40 mM imidazole) and 18% Buffer B (54 mM imidazole) wash, elution was performed at 100% Buffer B (250 mM imidazole). Purified fractions were pooled, concentrated, and dialyzed against dialysis buffer (100 mM HEPES pH 7.4, 300 mM NaCI, 0.4 mM PLP, and 10 mM MgCI2) with a 30 kDa molecular weight cutoff centrifugal filter (Amicon Ultra, Millipore). Samples were then flash frozen in microcentrifuge tubes in an ultracold ethanol bath and stored at -80 °C.

[0114] For TyrB and SrCAR purification, cells were resuspended in Lysis buffer (25 mM HEPES pH 7.4, 250 mM NaCI, and 10 mM imidazole). The cells were lysed by sonication followed by centrifugation at 18213 x g for 1 h. The cleared supernatant was sterile filtered through a 0.22 pm syringe filter and purified using Ni-Sepharose affinity chromatography (HisTrap HP, 5 mL) via an AKTA Pure fast protein liquid chromatography (FPLC) system using Lysis Buffer A (25 mM HEPES pH 7.4, 250 mM NaCI, and 10 mM imidazole) and Elution Buffer B (25 mM HEPES pH 7.4, 250 mM NaCI, and 250 mM imidazole). The column was equilibrated with 2.5% Buffer B and after performing an isocratic 12% Buffer B (40 mM imidazole) and 18% Buffer B (54 mM imidazole) wash, elution was performed at 100% Buffer B (250 mM imidazole). Purified fractions were pooled, concentrated, and dialyzed against dialysis buffer (100 mM HEPES pH 7.4, 300 mM NaCI, and 10 mM MgCI2) with a 30 kDa molecular weight cutoff centrifugal filter (Amicon Ultra, Millipore). Samples were then flash frozen in microcentrifuge tubes in an ultracold ethanol bath and stored at -80 °C. 1.6 In vitro PSDH Assay

[0115] Reactions were run at 100 pL scale in triplicate in 96-well plates, unless otherwise specified. The reaction was performed with 2 pM RpPSDH (0.07 mg / mL), 100 mM HEPES pH 7.5, 400 pM PLP, and 15 mM MgCh in both the absence and presence of 100 mM L-Thr. The reaction was initialized with addition of 1 mM phenylserine (100 mM stock in water) substrate and proceeded for 2 h at 30 °C with shaking in a Thermo Scientific plate shaker (catalog number 88882006) at 1000 RPM. Samples were prepared for analysis as follows: Samples were quenched with 1% TFA. Samples were chilled at -80 °C for at least 1 h prior to centrifugation (4000 x g at 4 °C for 12 min) to clear insoluble protein precipitate. Samples were then analyzed by HPLC-UV.

[0116] 1.7 In vitro AT Assay

[0117] The reaction was performed with 2 pM TyrB (0.09 mg / mL), 100 mM HEPES pH 7.5, 400 pM PLP, 15 mM MgCh, 10 mM L-Glu. The reaction was initialized with addition of 0.5 mM phenylpyruvate (100 mM stock in 70% ethanol) or 0.5 mM 4-nitro- phenylpyruvate (100 mM stock in 70% ethanol) and proceeded for 15 min at 30 °C at 1000 RPM. Samples were prepared for analysis as described in the "In vitro PSDH Assay" above and then analyzed by HPLC-UV.

[0118] 1.8 In vitro one-pot TTA, PSDH, AT Assay

[0119] The reaction was performed with 2 pM s-ObiH (0.13 mg / mL), 2 pM / ?pPSDH (0.07 mg / mL), 2 pM TyrB (0.09 mg / mL), 100 mM HEPES pH 7.5, 400 pM PLP, 15 mM MgCh, 100 mM L-Thr, and 25 mM L-Glu. For the one-pot production of phenylalanine, 2 mM of each substrate and possible intermediate (benzaldehyde, L-t / ireo-phenylserine, or phenylpyruvic acid) was added to the reaction mixture and quenched with 1% TFA after 0.5 h at 30 °C at 1000 RPM. For the production of diverse phenylalanine derivatives, 2 mM of each aldehyde tested (100 mM stock in DMSO) was added to the reaction mixture and subsequently incubated for 12 h at 30 °C at 1000 RPM. Samples were quenched at a 4x dilution in 3: 1 v / v ratio of methanol: IM HCL Samples were chilled at -80 °C for at least 1 h prior to centrifugation (4000 x g at 4 °C for 12 min) to clear insoluble protein precipitate. Samples were then analyzed by HPLC-UV.

[0120] 1.9 In vitro one-pot CAR, TTA, PSDH, AT Assay

[0121] The reaction was performed with 2 pM SrCAR (0.26 mg / mL), 2 pM s-ObiH (0.13 mg / mL), 2 pM RpPSDH (0.07 mg / mL), 2 pM TyrB (0.09 mg / mL), 100 mM HEPES pH 7.5, 2.5 mM ATP, 2.5 mM NADPH, 400 pM PLP, 15 mM MgCh, 100 mM L-Thr, 10 mM L- Glu, 1% (v / v) DMSO, and 1 mM of acid tested (100 mM stock in DMSO) for 24 h at 30 °C at 1000 RPM. Samples were prepared for analysis as described in the "In vitro PSDH Assay" above and then analyzed by HPLC-UV.

[0122] 1.10 Preparation of s-ObiH and s-RpPSDH Cell-free Extracts 5 mL starter cultures of RARE.A16 pZE-s-ObiH and RARE.A16 pZE-s-RpPSDH started from glycerol stocks were grown in LB media with 30 pg / mL Kan at 37 °C with shaking overnight until the cultures reached saturation. Then, 250 mL of 2xYT media with 30 pg / mL Kan in a 1 L baffled flask was inoculated from the overnight culture at a 1:100 inoculation ratio and incubated at 37 °C with shaking at 250 RPM. Cultures were induced at OD6oo=0.5-0.8 with 0.2 pM aTc and then incubated at 20 °C with shaking at 250 RPM for 24 h. Cells were harvested by centrifugation (7000 x g at 4 °C for 6 min).

[0123] Cells were washed once in 25 mL of 25 mM HEPES pH 8 and then resuspended in a cold lysis buffer (25 mM HEPES pH 8) at 5 mL lysis buffer per g cell pellet and sonicated using a QSonica Q125 sonicator with cycles of 5 s at 90% amplitude and 10 s off for 8 min on ice. Crude lysates were transferred to microcentrifuge tubes (ImL aliquots), centrifuged at 18213 x g for 30 min at 4 °C, and subsequently filtered using a 0.22 pM syringe filter.

[0124] 1.11 Clarified Lysate Assays for Synthesis of 4-acetylphenylalanine

[0125] The total protein concentrations of the s-ObiH and s- / ?pPSDH clarified lysates were measured by Bradford assay using bovine serum albumin as a reference. The reaction was performed with 3-4.5 mg / mL s-ObiH lysate and 3-4.5 mg / mL s- ?pPSDH lysate in reaction buffer containing 100 mM HEPES pH 8, 400 pM PLP, 15 mM MgCb, 100-200 mM L-Thr, 25-100 mM L-Glu, 0.1-2.5% (v / v) DMSO, and initialized with addition of 1-25 mM of 4-acetyl-benzaldehyde (2b) (1 M stock in DMSO). Reactions were run at 300 pL scale in triplicate in 96 deep-well plates for 24 h at 30 °C at 1000 RPM. Samples were prepared for analysis as described in the "one-pot TTA, PSDH, AT Assay" above and then analyzed by HPLC-UV.

[0126] 1.12 Preparative Scale Reaction for Synthesis of 4-formylphenylalanine

[0127] The soluble lysates were dialyzed using 3.5 kDa MWCO Spectra / Por 3 Dialysis Tubing against a dialysis buffer containing 50 mM HEPES pH 7.5, 0.2 mM pyridoxal-5'- phosphate monohydrate (PLP), 5 mM MgCb, and 150 mM NaCL Dialysis was performed at 4 °C with gentle stirring for 18 h. Lysate total protein concentrations were measured after dialysis by Bradford assay using bovine serum albumin as a reference.

[0128] To a 50 mL round bottom flask was added 26.7 mL DI water, 0.477 g HEPES (final concentration: 50 mM), 0.057 g MgCh (final concentration: 15 mM), 0.012 g PLP (final concentration: 1.2 mM), 0.953 g L-threonine (final concentration: 200 mM), and 0.813 g L-glutamic acid monopotassium salt monohydrate (final concentration: 100 mM). The reaction buffer was titrated to a pH of 8. s-ObiH lysate (final lysate concentration: 2.2 mg / mL) s-RpPSDH lysate (final lysate concentration 1.2 mg / mL) was then added to the reaction buffer. The reaction was gently stirred at 200 RPM using a stir bar and the temperature was maintained at 30 °C. 0.13 g of terephthalaldehyde (final concentration: 25 mM) dissolved in 1 mL of DMSO (final concentration 2.5% v / v) was added to initiate the reaction at 40 mL scale. Timepoints were taken at 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 14.5 h, and 18 h by quenching 4 pL of the reaction in 196 pL of 3:1 v / v ratio of methanol: lM HCI (50x dilution). The samples were vigorously mixed and centrifuged at 18213 x g for 1 minute. Samples were analyzed by HPLC-UV.

[0129] 1.13 Enantiomeric Excess by Marfey's Derivatization

[0130] To a 200 pL PCR tube was added 20 pL of 1 M sodium bicarbonate, 25 pL of 10 mM Marfey's reagent (prepared in acetonitrile), and 5 uL of quenched reaction (previous diluted 4-fold in methanol :1M HCI). The reaction was incubated at 40 °C for 20 h in a thermal heating block. The reactions were quenched by addition 50 pL of 4: 1 (v / v) mixture of acetonitrile:2 M HCI. Degassed samples were immediately loaded onto a Waters AQUITY Arc UPLC H-Class equipped with a diode array coupled to a Waters AQUITY QDa Mass Detector. Separation was achieved using a Waters Cortecs C18 column with solvent A, water, 0.1% formic acid; solvent B, acetonitrile, 0.1% formic acid. For method 1: a gradient elution was performed (A / B) with the following method : gradient from 90 / 10 to 40 / 60 for 5 min, a gradient from 40 / 60 to 10 / 90 for 5-14 min, an isocratic flow at 10 / 90 for 14-14.5 min, then gradient from 10 / 90 to 90 / 10 for 14.51-15 min. For method 2: a gradient from 90 / 10 to 20 / 80 for 14 min, then gradient from 20 / 80 to 90 / 10 for 14-14.5 min. For method 3: a gradient from 90 / 10 to 60 / 40 for 14 min, then gradient from 60 / 40 to 90 / 10 for 14-14.5 min. A flow rate of 1 mL min1was maintained, and absorbance was monitored at 340 nm.

[0131] 1.14 One-pot synthesis of 0-OH nsAAs from carboxylic acids with cofactor regeneration

[0132] 5 mL starter cultures of RARE.A16 pZE-s-ObiH and RARE.A16 pZE-srCAR, RARE.A16 pACYC-PPK12, RARE.A16 pACYC-bmGDH, and RARE.A16 pACYC-ecPPase were grown from fresh colony from a transformation plate at 37 °C with shaking at 250 rpm for 6 hours. The starter culture was used to inoculate a 250 mL of 2xYT or TB media with 50 pg / mL Kan or 34 pg / mL chloramphenicol in a 1 L baffled flask 1 :50 inoculation ratio and incubated at 37 °C with shaking at 250 RPM. Cultures were induced at ODGOO=0.8-1.0 with 0.1 pg / mL aTc or 1 mM ITPG and then incubated at 20 °C with shaking at 250 RPM for 20 h. Cells were harvested by centrifugation (3500 x g at 4 °C for 20 min). Lysates were produced by resuspending the cell pellets in 200 mM HEPES pH 8 at 5 mL / g-pellet. Resuspended cells were sonicated using a QSonica Q125 sonicator with cycles of 5 s at 90% amplitude and 10 s off for 8 min on ice. Crude lysates were transferred to 50 mL conical tubes and centrifuged at 28000 x g at 4 °C for 45 min. The soluble fraction was collected and frozen in 10 mL aliquots in liquid nitrogen or placed in a -80 °C freezer overnight. The frozen lysates were then lyophilized under vacuum for 48 h until samples were dry. Lyophilized lysate powder was then stored at -20 °C until use. The concentration of lysate used in biotransformation reactions refers to the total protein content of the lysate as measured by Bradford assay using bovine serum albumin (BSA) as a reference protein. P-OH nsAAs were produced from carboxylic acids with cofactor regeneration at 200 uL scale by adding 200 mM HEPES, 1 mM AMP, 0.5 mM NADP+, 30 mM carboxylic acid, 30 mM sodium hexametaphosphate, 0.4 mM PLP monohydrate, 100 mM glucose, 100 mM L-threonine, 22.5 mM MgCk hexahydrate, 5 mg / mL srCAR lysate, 2.2 mg / mL s-ObiH lysate, 1 mg / mL bmGDH lysate, 1.5 mg / mL PPK12 lysate, and 0.5 mg / mL ecPPase lysate to a 250 uL PCR tube. The PCR tube was then placed in an Eppendorf thermal mixer at 30 C for 24 h. Samples were quenched in a 19:1 solution of Methanol: IM HCI and centrifuged at 13000 x g. Supernatant was transferred to HPLC vials containing high recovery inserts prior to analysis on HPLC-MS (Waters, 12 min method).

[0133] Table 1. Strains and Plasmids used in Example 1

[0134] Table 2. Enzymes used in Example 1

[0135] Table 3. Compounds used in Example 1

[0136] 2. Results

[0137] 2.1 One-pot nsAA production from aldehyde precursors

[0138] We conducted a one-pot in vitro preparation of s-ObiH, RpPSDH, and TyrB, confirming endpoint phenylalanine production from benzaldehyde (FIG. 1). Building upon this foundation, we investigated the ability of this candidate cascade to produce diverse L-phenylalanine derivatives from their associated benzaldehyde derivatives. We observed generally high yields with our reaction cascade and high enantioselectivity as measured by HPLC and Marfey's analysis (FIGS. 2A-2B).

[0139] 2.2 One-pot nsAA production from carboxylic acid precursors

[0140] We performed a one-pot preparation of SrCAR, s-ObiH, RpPSDH, and TyrB, initializing from a carboxylic acid with the addition of the CAR module. We observed over 75% yield observed for 10 out of the 18 different carboxylic acid precursors screened, highlighting the promiscuity of the SrCAR (FIGS. 3A-3B). Additionally, utilizing a mutant CAR engineered for improved tolerance of ortho-ring substituents may improve yields for some chemistries. Use of co-factor regenerating enzymes or a resting whole cell catalyst platform could improve the economic viability of this cascade (examples described below).

[0141] 2.3 Preparative-scale synthesis of an nsAA using clarified lysate

[0142] To demonstrate an ability to utilize this cascade with higher substrate loadings and larger volumes, we investigated the potential to harness a clarified lysate system, using s-ObiH and s- / ?pPSDH individually expressed in the E. coli RARE.A16 strain. Additionally, we were able to harness the basal expression level of TyrB or other E. coli aminotransferases. We successfully produced the industrially relevant nsAA for bio- orthogonal conjugation, 4-acetyl-L-phenylalanine (2e), from aldehyde precursor (2b). Potential improvements towards the cascade may include the co-expression of an ornithine d-aminotransferase while maintaining the use of L-Glu as the donor, utilizing L-Asp as the amine donor with acetolactate synthase, or utilizing a "smart" amine donor such as lysine. Additionally, enzymatic oxidation or reduction of the acetaldehyde co-product of the L-TTA reaction offers a sink that can further shift equilibrium towards product formation. We further scaled-up our lysate-based reaction to produce 4-formyl-L- phenylalanine (3e), an nsAA that could be valuable for bio-orthogonal conjugation and that has limited commercial availability, achieving a final analytical yield of 64% and greater than 99% e.e. after 18 h as determined by HPLC and LC-MS (FIG. 4).

[0143] 2.4 One-pot synthesis of p-OH nsAAs from carboxylic acids with cofactor regeneration

[0144] To enable cost-effective and scalable biocatalytic synthesis of p-hydroxy nonstandard amino acids (P-OH nsAAs), a multienzyme cascade was developed using lyophilized lysates expressing individual recombinant enzymes. The biocatalytic system comprises a carboxylic acid reductase (srCAR), a type 2-III polyphosphate kinase (PPK12) from an unclassified Erysipelotrichaceae organism that catalyzes ATP regeneration from both AMP and ADP using polyphosphate as phosphate donor, a Bacillus megaterium glucose dehydrogenase (bmGDH) to regenerate NADPH, an E. coh inorganic pyrophosphatase (ecPPase) to hydrolyze inhibitory pyrophosphate, and an L- threonine transaldolase (s-ObiH) for C-C bond formation. To achieve one-pot synthesis of p-OH nsAAs, the aldehyde-producing cascade was directly coupled with lyophilized lysate containing s-ObiH. HPLC-MS analysis confirmed the formation of p-hydroxy- phenylalanine from benzoic acid, p-hydroxy-O-methyltyrosine from 4-methoxybenzoic acid, and p-hydroxy-4-acetylphenylalanine from 4-acetylbenzoic acid. This workflow eliminates the need for purified enzymes and exogenous cofactor supplementation, while maintaining the capacity to synthesize structurally diverse p-OH nsAAs from simple, inexpensive aryl carboxylic acid feedstocks.

[0145] 3. Conclusions

[0146] In summary, we have developed a one-pot biocatalytic cascade capable of synthesizing a wide range of high-value nsAAs from inexpensive and commercially available precursors by utilizing four polyspecific enzymes in a single pot. Our cascade presents a versatile method to generate industrially sought after phenylalanine derivatives, with valuable intermediates such as aldehydes (when starting from carboxylic acids; with uses in the fragrance industry), p-OH nsAAs (uses in pharmaceuticals), and aromatic a-keto acids (which may serve as key precursors in drug synthesis). Additionally, we have demonstrated initial scalability of this biosynthetic pathway using clarified lysate, which allows us to leverage endogenous E. coll aminotransferases for catalyzing the final step in the production of an nsAA that contains a handle for bio-orthogonal conjugation. Overall, this work offers a versatile and scalable approach to reducing nsAA manufacturing costs and broadening nsAA synthesis, including for building blocks that are not commercially available. Example 2. Combined biosynthesis and incorporation of nsAAs

[0147] 1. Materials & Methods

[0148] 1.1 Strains and plasmids

[0149] Escherichia coli strains and plasmids used are listed in Table 4. Overnight cultures of each E. coli strain to be tested were inoculated from a frozen glycerol stock (stored at -80 °C) and grown overnight in 3-5 mL of LBL media with appropriate antibiotic.

[0150] 1.2 Aldehyde Stability Assays

[0151] For aldehyde stability testing in metabolically active cells, overnight cultures were used to inoculate experimental cultures in 300 pL volumes in a 96-deep-well plate (Thermo Scientific™ 260251) at lOOx dilution. Cultures were supplemented with 5 mM of aldehyde substrates (prepared in 100 mM stocks in DMSO) at mid-exponential phase (ODsoo~0.5). Cultures were incubated at 37 °C with shaking at 1000 RPM and an orbital radius of 3 mm. Samples were taken by pipetting 200 pL from the cultures, centrifuging 4000 x g at 4 °C in a round bottom plate (SPL Life Sciences ISO 13485) and collecting the supernatant. Compounds were quantified after 20 h using HPLC.

[0152] 1.3 Biosynthesis Assays for CAR and L-TTA screening

[0153] For the in vivo L-TTA activity assay, overnight cultures of strains S1-S6 detailed in Table 4 were used to inoculate 300 pL volumes of MOPS EZ Rich media with 2% glucose and 100 mM L-Thr at a 1 : 100 inoculation ratio in a 96-deep-well plate with appropriate antibiotic (30 pg / mL Kan). Cultures were incubated at 37 °C, shaking 1000 RPM and ODeoo ~ 0.5 was reached. ODeoo was measured using a spectrophotometer. Then, 0.2 pM aTc was added for L-TTA expression and 1 mM of aldehyde precursor (lb-4b, prepared in 100 mM stocks in DMSO) was supplemented to the culture. Cultures were then incubated for 20 h at 30 °C, shaking 1000 RPM. Metabolite concentration in the supernatant was measured via analysis of the supernatant on HPLC.

[0154] For the CAR variant screening, we used strains S17-S22 (Table 4). In vivo activity assays were performed as described above, with metabolite concentration measured via HPLC after 2 h growth post induction and addition of 2 mM of the carboxylic acids la-3a or 1 mM of 4a.

[0155] 1.4 PSDH Expression and Activity Assays

[0156] To evaluate RpPSDH expression with and without the SUMO tag, overnight cultures of strains S7 and S8 in Table 4 were used to inoculate 3 mL LBL containing 30 pg / mL Kan and incubated at 37 °C in a shaking incubator at 250 RPM until an ODeoo of 0.5-0.8 was reached. PSDH expression was induced by the addition of 0.2 pM aTc and cultures were incubated at 30 °C for 18 h. Then, 1 mL of cells was transferred to a microcentrifuge tube and lysed with 0.05 mL of glass beads by vortexing using a Vortex Genie 2 for 15-30 min. Samples were centrifuged at 18213 x g at 4 °C for 15 min, lysate collected and subsequently denatured in Laemmli SDS reducing sample buffer for 10 min at 95 °C. Sample was then run on a sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel with a Thermo Scientific™ Spectra™ Multicolor Broad Range Protein ladder and then analyzed via western blot with an HRP-conjugated 6*His, His-Tag Mouse McAB primary antibody. An Amersham ECL Primer chemiluminescent detection reagent was used to visualize the blot.

[0157] 1.5 Coupled L-TTA and PSDH Biosynthesis Assays

[0158] Overnight cultures of strains S10 and Sil in Table 4 were used to inoculate 300 pL volumes of MOPS EZ Rich media with 2% glucose and 100 mM L-Thr at a 1 : 100 inoculation ratio in a 96-deep-well plate with appropriate antibiotic (50 pg / mL Carb). Cultures were incubated at 37 °C in a shaking incubator at 1000 RPM. At midexponential phase (ODGOO~0.5), cultures were induced with 1 mM IPTG and supplemented with 1 mM of aldehydes lb-4b (prepared in 100 mM stocks in DMSO). Product concentration was quantified after 20 h incubation at 30 °C using HPLC.

[0159] 1.6 Coupled CAR, L-TTA and PSDH Biosynthesis Assays

[0160] Overnight cultures of S23-S26 in Table 4 were used to inoculate 300 pL volumes of MOPS EZ Rich media with 2% glucose and 100 mM L-Thr at a 1 : 100 inoculation ratio in a 96-deep-well plate with appropriate antibiotic. Cultures were incubated at 37 °C in a shaking incubator at 250 RPM. At mid-exponential phase (ODeoo~0.5), cultures were induced with 1 mM IPTG and 1 mM of carboxylic acids la-4a (prepared in 100 mM stocks in DMSO) was added. Product concentration was quantified after 20 h incubation at 30 °C using HPLC.

[0161] 1.7 Coupled biosynthesis and incorporation

[0162] For the conversion of externally supplemented aldehyde 4b for subsequent incorporation of 4e, strain S32 in Table 4 was used. This strain was cultured at 37 °C in 150 mL of LBL media with 100 mM L-Thr in a 500 mL baffled shake flask in a shaking incubator at 250 RPM. At an ODeoo~0.4, the culture was induced with 1 mM IPTG and 0.2 pM aTc and 2 mM 4b precursor was added. Cultures were then grown at 30 °C for an additional 18 h. The cells were harvested by centrifugation (7000 x g at 4 °C for 5 min), supernatant was removed, and the pellet was stored at -80 °C pending purification.

[0163] For the conversion of externally supplemented aldehyde la for subsequent incorporation of le, strain S33 in Table 4 was used. This strain was cultured at 37 °C in 75 mL of LBL media with 100 mM L-Thr in a 250 mL baffled shake flask in duplicate in a shaking incubator at 250 RPM. At an ODeoo ~0.4, the culture was induced with 1 mM IPTG and 0.2 pM aTc and 2 mM la precursor was added. Cultures were then grown at 30 °C for an additional 18 h. The cells were harvested by centrifugation (7000 x g at 4 °C for 5 min), supernatant was removed, and the pellet was stored at -80 °C prior to purification.

[0164] We followed the protocol detailed in Expression and Purification of Pathway Enzymes in Example lfor purification of the his-tagged reporter protein (as well as TTA, PSDH, and CAR) and subsequent analysis by SDS-PAGE.

[0165] For LC-MS / MS measurements, protein bands corresponding to the molecular weight of the protein of interest were cut from the polyacrylamide gel, and the gel fragments were digested by in-gel tryptic digestion. Extracted peptides were desalted using Pierce pipette tips (Thermo Fisher Scientific), dried, and prepared for LC-MS / MS using a Thermo Fisher Scientific Orbitrap Eclipse Tribrid Mass Spectrometer (MS; Thermo Fisher Scientific) with an Ultimate 3000 nano-LC and a FAIMS Pro Interface (Thermo Fisher Scientific) following the protocol detailed in Butler, et al. (Platform for Distributed Production of Synthetic Nitrated Proteins in Live Bacteria. Nat. Chem. Biol. 2023, 19 (7), 911-920. https: / / doi.org / 10.1038 / s41589-023-01338-x) Proteomic analysis was performed in the MaxQuant-Andromeda software suite (v2.5.1.0). An E. coli reference proteome (taxonomyjd :83333) was used for the database search and the reporter protein with Tyr at the site for nsAA incorporation was used as the reference protein. Trypsin was specified at the digestion mode with 2 maximum missed cleavages. Variable modifications included methionine oxidation, Phe, Trp, 4e, le, or 4- amino-phenylalanine (pAF) substitutions for Tyr to account for potential misincorporation, reduction, or the target products. Other parameters were set to the default. Peptide intensity values from MaxQuant were used for quantification.

[0166] 1.8 Synthetic Auxotroph growth on aldehyde precursor

[0167] Cultures of strain S34 in Table 4 were inoculated from a frozen stock and grown overnight in 3 mL LBL media with 12.75 pg / mL Cm, 15 pg / mL Kan, 10 mM biphenylalanine (15e), 100 pM biphenylaldehyde (15b), 0.2% (wt / vol) L-arabinose, 0.2 pM aTc, 20 mM Tris-HCI pH 8, 0.005% SDS, and 100 mM L-Thr at 34 °C in a shaking incubator at 250 RPM. Cells were washed 4x in LBL media with 12.75 pg / mL Cm, 25 pg / mL Kan, 0.2% (wt / vol) L-arabinose, 0.2 pM aTc, 20 mM Tris-HCI pH 8, 0.005% SDS, and 100 mM L-Thr to remove the essential nutrient of biphenylalanine. Overnight cultures were then used to inoculate experimental cultures in 200 mL volumes a Greiner clear bottom 96 well plate at lOOx dilution in LBL media with 12.75 pg / mL Cm, 15 pg / mL Kan, 0.2% (wt / vol) L-arabinose, 0.2 pM aTc, 20 mM Tris-HCI pH 8, 0.005% SDS, and 100 mM L-Thr. Substrate was added at inoculation under the following experimental conditions performed in triplicate: no substrate, 10 pM nsAA, or 500 pM aldehyde precursor. Cultures were grown for 50 h in a Spectramax I3x plate reader with medium plate shaking at 30 °C and with absorbance readings at 600 nm.

[0168] After 50 h growth, 5 pL of each experimental triplicate was plated on both non- permissive and permissive LB-agar to the confirm growth observed in liquid culture was due to the presence of supplemented or biosynthesized essential nutrient, rather than escape of the synthetic auxotroph. Non-permissive agar contained 12.75 pg / mL Cm, 15 pg / mL Kan, 0.2% (wt / vol) L-arabinose, 20 mM Tris-HCI pH 8, 0.005% SDS. Permissive agar included the essential nutrient biphenylalanine at 10 pM. Plates were incubated at 30 °C for 18 h. Table 4. Strains and Plasmids used in Example 2

[0169] Table 5. Sequences of orthogonal SUMO-tags tested in Example 2

[0170] The start codon is underlined.

[0171] 2. Results

[0172] 2.1 Biosynthesis of model p-hydroxylated nsAAs in live cells

[0173] We examined the rate of formation of p-hyd roxylated nsAAs upon supplementing these aldehyde substrates to cultures of E. coli cells that were transformed for heterologous expression of s-ObiH . We observed the desired - hydroxylated nsAA products in the supernatant by HPLC at an end point of 20 h (FIGS. 5A-5C) . Next, we screened s-ObiH and five SUMO-tagged homologs of ObiH with the model aldehydes. All variants were active in live cell contexts under the conditions tested .

[0174] 2.2 Establishing a full biosynthetic pathway from aldehydes to nsAAs in live cells

[0175] We observed nearly 2-fold higher levels of phenylalanine formed when with cells that express a SUMO-tagged PSDH, s- / ?pPSDH, compared to RpPSDH when supplied L- t / ?reo-phenylserine. Next, coupled one of the two hig hest performing SUMO-tagged L- TTAs from our previous screen (either s-ObiH or s-P&TTA) with s- / ?pPSDH for heterologous expression and observed production of 4-acetylphenylalanine, 4- azidophenylalanine, 4-cyanophenylalanine, and biphenylalanine from the corresponding aldehyde precursors. We further investigated TyrB overexpression as well as supplementation of excess amine donor (L-Glu), althoug h we did not observe a benefit from either approach under the conditions tested . We also saw an great improvement in the titers of these nsAAs when the biosynthesis was performed in aldehyde stabilizing host strains (RARE, RARE.A16, and ROAR) compared to the wild-type MG1655 (FIGS. 6A-6C). These results highlighted the value of harnessing an aldehyde stabilizing chassis for this biochemical pathway in live cells.

[0176] 2.3 Demonstration of the extended biosynthetic pathway starting from carboxylic acids

[0177] We next investigated whether we could extend our pathway to demonstrate biosynthesis of our model nsAAs from their associated carboxylic acids 4a, 2a, 5a, and 15a. We tested a panel of of six CAR homologs from our previously published collection. The CARs were co-expressed with Sfp from Bacillus subtilis BsSfp), a phosphopantetheinyl transferase required for CAR activation. We detected aldehyde products in each condition tested. We proceeded with the MavC / R construct for subsequent experiments. Next, we transformed E. coli strains for heterologous expression of MavCAR, BsSfp, either s-RbTTA (for 1-3) or s-ObiH (for 4), and s- RpPSDH. Endpoint HPLC measurements of the culture media containing cells expressing this four-enzyme cascade indicated sucessful production of the desired nsAAs supplemented with carboxylic acid precursors (la-4a) (FIG. 7).

[0178] 2.4 Site-specific incorporation of biosynthesized nsAAs for control of protein translation using aldehydes or carboxylic acids

[0179] We prepared genetic constructs that enabled co-expression of the OTS, the reporter protein that contains the in-frame UAG codon, and the biosynthetic pathway, all in a single strain. We began with the core biosynthetic pathway starting from aldehydes for the incorporation of biosynthesized 15e from supplemented 2 mN 15b, ultimately observing high-fidelity incorporation of biosynthesized 15e (98%) (FIG. 8A).

[0180] To determine whether we could convert carboxylic acids to incorporated nsAAs, we selected 4e as a commercially relevant target given the significantly lower cost of 4a compared to 4e. Overall, with this biosynthesis and incorporation system, we observed high-fidelity incorporation of biosynthesized le (>99%), with minor azide reduction to 4-amino-phenylalanine (pAF) observed (0.3%) (FIG. 8B). This approach could offer substantial cost savings for commercial-scale manufacturing of therapeutic proteins that contain azides.

[0181] 2.5 Control of bacterial survival by provision of biphenylaldehyde

[0182] The ability to couple biosynthesis and incorporation of nsAAs through semi-synthesis strategies that harness low-cost synthetic precursors creates new opportunities in synthetic biology and biosensing where cellular function can now be governed by the concentration of certain aryl aldehydes or carboxylic acids. An exemplary cellular function is survival, where previous work has demonstrated the ability of nsAAs to control survival by mediating the full-length translation and proper folding of essential proteins. Pioneering efforts in this direction reported synthetic auxotrophs, which are intrinsically biologically contained due to their reliance on a chemical not found in nature. As chiral molecules, 15e and other nsAAs may be cost-prohibitive to supply at scales required for environmental applications of biocontained strains. Cells of the DEP.e5 strain (biphenylalanine-dependent) transformed with our pathway were only able to grow when 15e or 500 pM 15b were provided in liquid media, confirmed due to reliance on the supplemented synthetic molecules (FIGS. 8C-8D). These results indicate the ability to use our pathway to create synthetic auxotrophs that can grow in the presence of user-defined synthetic aldehydes.

[0183] 3. Conclusions

[0184] The current paradigm of nsAA synthesis, isolation, and supplementation to live cells cultures for site-specific incorporation within proteins is a major constraint on the application of genetic code expansion at industrially relevant scales and for emerging environmental uses. Here, we demonstrate the design of a synthetic biochemical pathway that functions in live bacterial cells and its coupling to orthogonal translation systems for combined nsAA biosynthesis and incorporation within a single strain. This approach reduces the problem of sourcing a phenylalanine derivative to a problem of sourcing an aryl aldehyde or aryl carboxylic acid, which are simple achiral building blocks. These synthetic compounds are widely available, and because carboxylic acids are also ubiquitous in metabolism, this creates opportunities to biosynthesize the precursors towards total biosynthesis from renewable feedstocks. This coupling of metabolic engineering to genetic code expansion could address other bottlenecks in genetic code expansion, such as transport, nsAA toxicity (by maintaining a lower steady state), or nsAA concentration (by increasing intracellular concentration). Engineering higher affinity OTSs should serve as a less burdensome means of improving target protein yield when coupling nsAA biosynthesis and incorporation. By concluding this study with the creation of a strain whose survival depends on the biosynthesis and incorporation of biphenylalanine from biphenylaldehyde, we now have a unique selection platform for directed evolution or adaptive laboratory evolution of any of the steps in this process.

[0185] Example 3. Incorporation of biocatalytically produced nsAAs

[0186] 1. Materials & Methods

[0187] 1.1 Strains and plasmids

[0188] Escherichia coli strains and plasmids used are listed in Table 7.

[0189] 1.2 Materials and Chemicals

[0190] The following were purchased from Sigma-Aldrich: piperonal, 4- ethoxybenzaldehyde. The following were purchased from Thermo Scientific Chemicals: 4-methylsulphonyl benzaldehyde. 4-isopropoxybenzaldehyde was purchased from Alfa Aesar. 2-chloro-4-nitrobenzaldehyde and MHET (2-Hydroxyethyl terephthalate) were purchased from Ambeed. 3-(Bromomethyl)benzaldehyde was purchased from Combi- blocks. 4-(Phenylazo)benzoic acid was purchased from AA blocks.

[0191] 1.3 Heterologous enzyme expression

[0192] 5 mL starter cultures of RARE.A16 pZE-MavCAR-sfp, RARE.A16 pZE-s-ObiH and RARE.A16 pZE-s-RpPSDH started from glycerol stocks were grown in LB media with 30 pg / mL Kan at 37 °C with shaking overnight. Then, 250 mL of 2xYT media with 30 pg / mL Kan in a 1 L baffled flask was inoculated from the overnight culture at a 1 :100 inoculation ratio and incubated at 37 °C with shaking at 250 RPM. Cultures were induced at OD6oo=0.5-0.8 with 0.2 pM aTc and then incubated at 20 °C with shaking at 250 RPM for 24 h. Cells were harvested by centrifugation (7000 x g at 4 °C for 6 min). Cell pellets were stored at -80 °C pending use as resting whole cell catalysts or for the preparation of clarified lysate.

[0193] 1.4 Preparation of clarified lysates

[0194] The protocol described in Preparation of s-ObiH and s-RpPSDH Cell-free Extracts in Example 1 was followed.

[0195] 1.5 Clarified Lysate Assays for nsAA synthesis

[0196] The total protein concentrations of the s-ObiH and s- / ?pPSDH clarified lysates were measured by Bradford assay using bovine serum albumin as a reference. The reaction was performed with 5 mg / mL s-ObiH lysate and 3 mg / mL s- / ?pPSDH lysate in reaction buffer containing 100 mM HEPES pH 7.5, 400 pM PLP, 15 mM MgCb, 100 mM L-Thr, 50 mM L-Glu, 5% (v / v) DMSO, and initialized with addition of 5 mM of an aldehyde precursor (prepared as 100 mM stocks in DMSO). Reactions were run at 300 pL scale in triplicate in 96 deep-well plates or at 1 mL scale in microcentrifuge tubes for 24 h at 30 °C at 1000 RPM. A small sample was quenched with a 3:1 v / v ratio of methanol:2M HCI for analysis by LC-MS.

[0197] 1.6 Resting Whole Cell Assays for nsAA synthesis

[0198] Cells pellets corresponding to the overexpression of CAR, TTA, and PSDH were resuspended at 50 mg / mL wet cell weight each in reaction buffer containing 100 mM HEPES pH 7.5, 15 mM MgCb, 25 mM glucose, 50 mM L-Glu, 100 mM L-Thr, and 5 mM of an aryl aldehyde (prepared as a 100 mM stock in DMSO). Reactions were run at 130 uL / well scale in triplicate in 96-well round bottom plates for 24 h at 30 °C at 1000 RPM. A small sample was quenched with a 3: 1 v / v ratio of methanol:2M HCI for analysis by LC-MS.

[0199] 1.7 Preparation of Culture Media for Incorporation Assays The reaction mixtures (either the lysate-based reactions or resting whole cellbased reactions) were then added to concentrated MOPS EZ rich media lacking the aromatic amino acids (Y, F, W) with 2% glucose for a final composition of 25% v / v reaction mixture. Antibiotics (15 pg / mL Kan and 17 pg / mL Cm) and inducers (0.2 pM aTc and 0.2% (wt / vol) L-arabinose) were added to culture media.

[0200] 1.8 Incorporation assay with culture media containing reaction mixtures with biocatalytically produced nsAAs

[0201] E. coli strains denoted "aaRS-1 - aaRS-5" in Table 7 were used to inoculate 300 pL volumes of the culture media in a 96-deep-well plate and were incubated at 37 °C in a shaking incubator at 1000 RPM for 20 h. Cells were then diluted 4x in PBS buffer, and then quantified for ODeoo and GFP fluorescence at excitation and emission wavelengths of 485 and 525 nm, respectively, using a Spectramax i3x plate reader with Softmax Pro 7.0.3 software.

[0202] Table 6. Aldehyde chemistries used in Example 3

[0203] Table 7. Strains and Plasmids used in Example 3 2. Results

[0204] Our goal was to produce non-standard amino acids (nsAAs) from aryl aldehyde precursors using a lysate-based biocatalytic cascade. After supplementation of 5 mM of 14 different aldehyde precursors (denoted ald-1 through ald-14) to a reaction mixture containing s-ObiH and s-RpPSDH clarified lysates for 24 h, we observed production of each of the corresponding phenylalanine derivatives by LC-MS. We expanded the screen to include precursors 4-Isopropoxybenzaldehyde and 2-Chloro-4- nitrobenzaldehyde, confirming production of the corresponding nsAAs.

[0205] Next, we sought to incorporate the biocatalytically produced nsAAs from the lysate-based reactions into a target protein (green fluorescent protein, GFP), without the need to first isolate the nsAA from the reaction mixture. In the presence of supplemented nsAA or biosynthesized nsAA (as in the case of our lysate-based reaction), the result will be full length translation of the protein, and therefore a fluorescent output. In the absence of nsAA, the result will be a truncated protein product, terminated at the UAG stop codon, and therefore no fluorescence. We designed a culture media formulation consisting of MOPS EZ rich media lacking the aromatic amino acids (Y, F, W) with 25% of the volume, typically consisting of water, replaced with a reaction mixture (e.g. a lysate-based reaction 24 h after precursor addition to produce the final target nsAA). We then incubated cells containing the OTSs and GFP reporter in the culture media containing each of the 14 different reaction mixtures (corresponding to 14 different biosynthesized nsAAs). We observed an increase in fluorescence normalized by the optical density of the cells at 600 nm (FL / OD) of cells in the presence of 13 of the 14 different reaction mixtures, as compared to the negative control consisting of a reaction mixture lacking an aldehyde precursor across the aaRSs screened (FIG. 9). These results indicate this platform can be used to rapidly screen for the production and incorporation of novel nsAAs.

[0206] Additionally, we sought to test the efficacy of our biosynthetic pathway in resting whole cells, which can further simplify reaction preparation by obviating the need for cell lysis or for ATP or NADPH supplementation (or a cofactor regeneration system). We demonstrated the efficacy of the cascade in resting whole cells for our 3- enzyme pathway (s-ObiH, s-RpPSDH, EcAT(s)), as well as for our 4-enzyme pathway (MavCAR, s-ObiH, s-RpPSDH, EcAT(s)). We therefore can use a resting whole cell catalysis platform for produce nsAAs from aryl aldehyde or carboxylic acid precursors and supplement the reaction mixture to growing cells for subsequent nsAA incorporation. We demonstrate an increase in the FL / OD for conditions in which we supplemented resting whole cell-based reaction mixtures supplied 4-Azidobenzoic acid, 4-Benzoylbenzoic acid, MHET, and 4-(Phenylazo)benzoic acid (FIG. 10). 3. Conclusions

[0207] We have developed a platform to directly incorporate nsAAs produced via biocatalytic reactions without the need for nsAA isolation. This protocol may facilitate the rapid screening of novel and / or expensive nsAA targets by harnessing a highly polyspecific and high yield enzymatic cascade for phenylalanine derivative production. Further, the implementation of the cascade as a lysate-based or resting whole-cell based reaction can greatly simplify reaction preparation and cost. These nsAAs can then be directly screened for incorporation into a target protein by a panel or library of engineered orthogonal translation systems.

[0208] Example 4. Expanding access to nsAAs from alcohol precursors

[0209] 1. Materials & Methods

[0210] 1.1 Resting whole cell preparation

[0211] Cultures were grown in LB-Lennox medium (LB: 10 g / L bacto tryptone, 5 g / L sodium chloride, 5 g / L yeast extract). To prepare cells for resting cell assays, confluent overnight cultures of f. coli strains were used to inoculate 200 mL cultures in LB media in 1 L baffled shake flasks with appropriate antibiotics (AOs: 50 pg / mL kanamycin, TTA-PSDH : 34 pg / mL chloramphenicol). The cultures were grown at 37 °C until midexponential phase (OD600 = 0.5-0.8) and then induced (AO: 0.1 pg / mL aTc, TTA- PSDH : 1 mM IPTG). After induction, the cultures were dropped to 18 °C overnight for 18 h. Cells were then pelleted and used or frozen at -80 °C. Cells used in this study were stored at -80 for less than 24 h.

[0212] 1.2 Resting whole cell assay for nsAA preparation

[0213] To prepare cells for resting cell assays, an alcohol oxidase (AO) variant from multispecies of Burkholderia (BuAO) was cloned into a pZE vector. The cloned vectors, pZE-BuAO, and previously described pCola-s-PSDH-s-ObiH and pCola-s-PSDH-s-PbTTA were transformed into the E. coli ROAR strain. Resting cells were prepared as previously mentioned and were washed with 200 mM HEPES, pH 7.5 buffer prior to use. To assay AO in resting cells, cells were resuspended in buffer with 200 mM HEPES at a pH 7.5. To assay TTA-PSDH and AO + TTA-PSDH coupled in resting cells, cells were resuspended in buffer with 200 mM HEPES, 1 mM PLP, 10 mM MgCk, 200 mM L- Thr and 50 mM L-Glu at a pH of 7.5. The resuspended resting cells were then aliquoted into 96-deep-well plates at a wet cell weight of 50 mg / mL for AO and TTA-PSDH assays and a total wet cell weight of 100 mg / mL for AO and TTA-PSDH coupled assays. For AO and AO+TTA-PSDH coupled assays, reaction mixtures were supplemented with 5 mM of 4-methoxybenyl alcohol (prepared in 100 mM stocks in DMSO) and for TTA assays, reaction mixtures were supplemented with 5 mM of 4-methoxybenzaldehyde (prepared in 100 mM stocks in DMSO). Assays were carried out at a reaction volume of 400 pL. Resting cells were then incubated at 30 °C with shaking at 1000 RPM and an orbital radius of 3 mm. Samples were taken by pipetting 100 pL from the cultures, centrifuging in a round bottom plate, and collecting the extracellular broth. Compounds were quantified using HPLC with samples collected at 4 h.

[0214] 2. Results

[0215] Our goal was to produce non-standard amino acids (nsAAs) from aryl alcohol precursors using a resting cell biocatalytic cascade. The reaction first consists of alcohol oxidation via an alcohol oxidase (AO). We specifically chose a variant from a multispecies Burkholderia ( ietnamiensis, pseudomallei, cenocepacia) which belongs to the GMC family oxidoreductase and capable of producing aryl aldehydes utilizing a FAD cofactor. The AO can be coupled with our engineering cascade (TTA, PSDH, AT) to produce nsAAs from aryl alcohols.

[0216] We will prepare our cascade in resting cells, which offer unique advantages within our enzymatic cascade. First, AOs utilize oxygen as a terminal electron acceptor to produce a toxic byproduct of hydrogen peroxide. However, we can utilize endogenous catalases in E. coll for the depletion of hydrogen peroxide, thus improving cascade efficiency. Second, resting cells allow us to decouple cellular growth and protein production from product production.

[0217] We sought to test the efficacy of our biocatalytic cascade in resting cells. We tested our system by splitting up the pathway into an aldehyde generating cell (AO) and a nsAA producing cell (TTA+PSDH). We observed very high conversions of nsAA production when AO and TTA+PSDH cells were coupled together. Further, we increased the ratio of TTA+PSDH cells to AO cells and achieved full conversion to the nsAA o- methyltyrosine (FIGS. 11A-11B).

[0218] 3. Conclusions

[0219] We have developed a platform to directly produce nsAAs starting with aryl alcohols using an AO and TTA-PSDH coupled cascade. This protocol allows the production of a wide range of potential nsAAs through a cheaper alcohol starting substrate compared to an aldehyde starting substrate. Further, this cascade highlights a less redox intensive approach than starting with a carboxylic acid and utilizing a carboxylic acid reductase (CAR) to produce the aldehyde intermediate. The CAR reaction utilizes an ATP and NADPH cofactor for aldehyde formation as well as expression of a coenzyme for enzyme activation. Thus, alcohol oxidation via a regenerative FAD cofactor presents a potentially cheaper and less redox intensive alternative to produce nsAAs without direct supplementation of an expensive aldehyde substrate. Table 8. Sequences All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and / or other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

WHAT IS CLAIMED:

1. A recombinant cell expressing a first enzyme, a second enzyme and a third enzyme and producing a nonstandard amino acid (nsAA), wherein the first enzyme is a L-threonine transaldolase (L-TTA) or a threonine aldolase (TAs), the second enzyme is a phenylserine dehydratase or a threonine deaminase (TD), and the third enzyme is an aminotransferase (AT), a L-amino acid dehydrogenase (L-AADH) or a D-amino acid aminotransferase, wherein at least one of the first enzyme, the second enzyme and the third enzyme is heterologous to the recombinant cell.

2. The recombinant cell of claim 1, wherein the L-TTA is from Pseudomonas fluorescens (ObiH) (SEQ ID NO: 1) or from Parachlamydiales bacterium (PbTTA) (SEQ ID NO: 2).

3. The recombinant cell of claim 1, wherein the phenylserine dehydratase is from Ralstonia pickettii PS22 (RpPSDH) (SEQ ID NO: 3).

4. The recombinant cell of claim 1, wherein the AT is a tyrosine aminotransferase from E. coli (TyrB) (SEQ ID NO: 4).

5. The recombinant cell of claim 1, further expressing a carboxylic acid reductase (CAR) and a phosphopantetheinyl transferase.

6. The recombinant cell of claim 5, wherein the CAR is from Segniliparus rotundus (SrCAR) (SEQ ID NO: 5) or from Mycobacterium avium (MavCAR) (SEQ ID NO: 6).

7. The recombinant cell of claim 5, wherein the phosphopantetheinyl transferase is from Bacillus subtilis (sfp) (SEQ ID NO: 7).

8. The recombinant cell of claim 1, further expressing an alcohol oxidase (AO) or alcohol dehydrogenase (ADH).

9. The recombinant cell of claim 1, further expressing a target protein and comprising an orthogonal translation system (OTS), wherein the target protein comprises a site for amber stop codon suppression, and wherein the OTS comprises an aminoacyl-tRNA synthetase and a tRNA pair decoding the amber stop codon.

10. The recombinant cell of claim 9, wherein the nsAA is incorporated into the target protein at the site.

11. The recombinant cell of any one of claims 1-10, wherein the recombinant cell is E. coli.

12. A method for producing a nonstandard amino acid (nsAA) by a recombinant cell, comprising :(a) growing a recombinant cell in a culture medium, wherein the culture medium comprises an aldehyde, a L-amino acid and an amine donor, wherein the L- amino acid is L-threonine (L-Thr) or L-glycine (L-Gly);(b) expressing a first enzyme, a second enzyme and a third enzyme by the recombinant cell, wherein the first enzyme is a L-threonine transaldolase (L-TTA) when the L-amino acid is L-Thr or a threonine aldolase (TA) when the L-amino acid is L-Gly, the second enzyme is a phenylserine dehydratase or a threonine deaminase (TD), and the third enzyme is an aminotransferase (AT), a L-amino acid dehydrogenase (L-AADH) or a D-amino acid aminotransferase, wherein at least one of the first enzyme, the second enzyme and the third enzyme is heterologous to the recombinant cell;(c) reacting the aldehyde with the L-Thr or L-Gly, whereby a p-hydroxy a- amino acid (0-OH AA) is generated;(d) converting the p-OH AA to a keto acid; and(e) reacting the keto acid with the amine donor, whereby a nonstandard amino acid (nsAA) is produced by the recombinant cell.

13. The method of claim 12, excluding supplying the p-OH AA and / or the keto acid into the culture medium.

14. The method of claim 12, wherein the culture medium further comprises a carboxylic acid, the method further comprising expressing a carboxylic acid reductase (CAR) and a phosphopantetheinyl transferase by the recombinant cell, activating the CAR and converting the carboxylic acid to the aldehyde.

15. The method of claim 14, excluding supplying the aldehyde into the culture medium.

16. The method of claim 12, wherein the culture medium further comprises an alcohol, the method further comprising expressing an alcohol oxidase (AO) or an alcohol dehydrogenase (ADH), and converting the alcohol to the aldehyde.

17. The method of claim 16, excluding supplying the aldehyde into the culture medium.

18. The method of claim 12, wherein the recombinant cell further expresses a target protein and comprises an orthogonal translation system (OTS), wherein the target protein comprises a site for amber stop codon suppression, and wherein the OTS comprises an aminoacyl-tRNA synthetase and a tRNA pair decoding the amber stop codon, the method further comprising incorporating the nsAA into the target protein at the site, whereby the target protein comprising the nsAA is produced by the recombinant cell.

19. The method of claim 12, wherein the aldehyde is an aryl aldehyde, the nsAA is a L-phenylalanine derivative, and the AT is an aromatic amino acid aminotransferase.

20. A method for producing a nonstandard amino acid (nsAA) in a container, wherein the container comprises an aldehyde, an L-amino acid, an amine donor, a firstenzyme, a second enzyme and a third enzyme, wherein the L-amino acid is L-threonine (L-Thr) or L-glycine (L-Gly), the first enzyme is a L-threonine transaldolase (L-TTA) when the L-amino acid is L-Thr or a threonine aldolase (TAs) when the L-amino acid is L-Gly, the second enzyme is a phenylserine dehydratase or threonine deaminase (TD), and the third enzyme is an aminotransferase (AT), a L-amino acid dehydrogenase (L- AADH), or a D-amino acid aminotransferase, the method comprising:(a) reacting the aldehyde with the L-Thr or the L-Gly in the container, whereby a |3-hydroxy a-amino acid (0-OH AA) is generated in the container;(b) converting the p-OH AA to a keto acid in the container; and(c) reacting the keto acid with the amine donor, whereby a nonstandard amino acid (nsAA) is produced in the container.

21. The method of claim 20, excluding supplying the p-OH AA and / or the keto acid into the container.

22. The method of claim 20, wherein the container further comprises a carboxylic acid and a carboxylic acid reductase (CAR), a phosphopantetheinyl transferase, ATP, and a NADPH and / or cofactor regeneration system, wherein the NADPH and / or cofactor regeneration system comprises an a type 2-III polyphosphate kinase from Erysipelotrichaceae organism (PPK12) (SEQ ID NO: 8), a Bacillus megaterium glucose dehydrogenase (bmGDH) (SEQ ID NO: 9), and an E. coll inorganic pyrophosphatase (ecPPase) (SEQ ID NO: 10), the method further comprising activating the CAR, and converting the carboxylic acid to the aldehyde in the container.

23. The method of claim 22, excluding supplying the aldehyde into container.

24. The method of claim 20, wherein the container further comprises an alcohol and either an alcohol oxidase (AO) or an alcohol dehydrogenase (ADH), the method further comprising converting the alcohol to the aldehyde in the container.

25. The method of claim 24, excluding supplying the aldehyde into the container.

26. The method of any one of claims 20, wherein the container further comprises a recombinant cell, wherein the recombinant cell comprises a target protein and an orthogonal translation system (OTS), wherein the target protein comprises a site for amber stop codon suppression, and wherein the OTS comprises an aminoacyl- tRNA synthetase and a tRNA pair decoding the amber stop codon, the method further comprising incorporating the nsAA into the target protein at the site, whereby a target protein comprising the nsAA is produced by the recombinant cell.

27. The method of claim 20, wherein the aldehyde is an aryl aldehyde, the nsAA is a L-phenylalanine derivative, and the AT is an aromatic amino acid aminotransferase.

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