The Pathway Less Traveled: Engineering Biosynthesis of Nonstandard Functional Groups

TIBTEC 1872 No. of Pages 14

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Review

The Pathway Less Traveled: Engineering Biosynthesis of Nonstandard Functional Groups Morgan Sulzbach1,2 and Aditya M. Kunjapur

1,2, ,@

*

The field of metabolic engineering has achieved biochemical routes for conversion of renewable inputs to structurally diverse chemicals, but these products contain a limited number of chemical functional groups. In this review, we provide an overview of the progression of uncommon or ‘nonstandard’ functional groups from the elucidation of their biosynthetic machinery to the pathway optimization framework of metabolic engineering. We highlight exemplary efforts from primarily the last 5 years for biosynthesis of aldehyde, ester, terminal alkyne, terminal alkene, fluoro, epoxide, nitro, nitroso, nitrile, and hydrazine functional groups. These representative nonstandard functional groups vary in development stage and showcase the pipeline of chemical diversity that could soon appear within customized, biologically produced molecules.

Highlights Nonstandard functional groups that are derived from natural products face many hurdles prior to mainstream integration into engineered products, including pathway discovery, heterologous expression, and product stability. Metabolic engineers have achieved control over formerly uncommon groups such as aldehydes, esters, terminal alkynes, terminal alkenes, and fluorinated chemicals, by addressing the challenges above, as well as by increasing protein activity and expanding product range.

Advancements in Metabolic Engineering

Recently discovered pathways for formation of nonstandard groups such as nitro, nitrile, epoxide, nitroso, and hydrazine could benefit from more investigation of activity towards nonnative substrates for eventual integration into valuable products in agriculture, materials, pharmaceuticals, and more.

The field of metabolic engineering is devoted to achieving biological production of diverse chemicals by perturbing organisms at the DNA level to alter their metabolism [1]. Research efforts have predominantly focused on the redirection of carbon flux obtained from microbial breakdown of inputs such as renewable sugars towards the production of fuels or value-added chemicals, with a shift towards higher value products such as fragrances, nutraceuticals, cosmetics, and pharmaceutically active ingredients during the last decade [2]. The success of these efforts in academic settings has created a flourishing and still nascent commercial ecosystem of biochemical manufacturers. Given that traditional metabolic engineering has proven its utility, what are the next set of technical obstacles that academic research efforts may seek to overcome? Among these major challenges is the attainment of chemical diversity at the level of functional groups. Metabolic engineers have historically focused on increasing carbon flux towards production of certain molecules or building blocks rather than prioritizing specific functional group chemistry. Based on considerations such as maximum theoretical yield, natural flux distributions, and product price/market size, initial product targets contained functional groups common to central metabolism. During the last two decades, more emphasis has been placed on achieving production of nonnatural substitutes for industrial chemicals [3]. Decreases in DNA synthesis cost and increases in genomic/metagenomic sequencing data have increased the percentage of synthetic DNA sequences appearing in repositories such as Addgene and facilitated heterologous expression of genes across larger phylogenetic distances [4]. Given the diversity of biochemical production reported, it may appear as though metabolic engineers can manipulate a well-studied microorganism to produce any desired chemical. Unfortunately, the reality is that metabolic engineers have a limited ability to integrate many of the chemical elements and functional groups used in synthetic chemistry [3,5]. Trends in Biotechnology, Month 2019, Vol. xx, No. xx

1

Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19711, USA 2 www.kunjapurlab.org

*Correspondence: [email protected] (A.M. Kunjapur). @ Twitter: @kunjapur

https://doi.org/10.1016/j.tibtech.2019.12.014 © 2019 Elsevier Ltd. All rights reserved.

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Here we loosely define ‘nonstandard’ functional groups as those that appear uncommonly within accumulated metabolites from central and secondary metabolism. In many cases, these functional groups are the most important feature of the chemicals within which they appear. In a few cases, one or two molecules could be exemplary of the class, such as the largemarket flavor additives vanillin and benzaldehyde for aldehydes. However, most often a functional group is desirable for a somewhat structure-independent property such as bio-orthogonal reactivity, electronegativity, or stimuli-response. Before engineers can control structurally uncommon motifs, several stages of research must be performed (Figure 1, Key Figure). These stages bridge

Key Figure

Research Stages for Nonstandard Functional Group Biosynthesis

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Figure 1. This illustration depicts the interdisciplinary stages of research required to achieve integration of nonstandard functional groups into biosynthesized products. Bioprospecting of organisms or metagenomic sequence analysis from unique habitats has revealed natural products that exhibit useful bioactivities, occasionally due to unusual chemistries. Researchers in the natural product community typically elucidate the biosynthetic machinery required for formation of nonstandard functional groups as well as the biochemical pathway that generates the desired bioactive molecule. The subsequent steps of metabolic and protein engineering, which are the focus of this review, rely on the fundamental characterization of the nonstandard functional group in its natural context and seek to broaden that context. Protein engineering is employed to increase production rate or to broaden substrate specificity. Metabolic engineering is harnessed to supply key biosynthetic machinery with sufficient precursor and cofactor supply, as well as to increase organismal tolerance and prevent byproduct formation from key metabolites.

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interdisciplinary fields of natural product discovery, metabolic and protein engineering, and chemical biology. This review focuses on exemplary nonstandard functional groups that are of bacterial origin and the challenges that metabolic engineers have or likely will face in generating high yields of diverse products. We transition from functional groups that have already been the target of many engineering efforts to recently elucidated biosynthetic machinery in bacterial natural products to illustrate trends in maturation (Figure 2).

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Figure 2. Overview of Functional Groups and Progress. Each row contains a functional group that spans milestones of pathway discovery and metabolic engineering progress. Heterologous expression in model organisms and basic biochemical characterization are often first steps in natural product discovery, followed by expanding the product scope and engineering higher activity. Here we consider functional groups that have yet to reach inclusion in de novo pathways as ‘recently discovered’, whereas functional groups that have been stabilized through cellular engineering, included in products where titers were optimized, detected via high-throughput screening, or produced using alternative carbon substrates were considered ‘established in metabolic engineering’. The fluoro group is an exception to the traditional framework, as engineering work focused on incorporating a fluorinated feedstock, whereas all other groups focus on formation and accumulation of the functional group.

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Established Nonstandard Functional Groups for Metabolic Engineering We begin by showcasing examples of two functional groups, aldehydes and esters, that are further along the stages of readiness for metabolic engineering. While present in diverse metabolic pathways en route to alcohols, aldehydes have only recently become stabilized in living cells after host modification [6]. In addition, biosynthetic machinery responsible for diverse aldehyde biosynthesis has been characterized recently. Esters are ubiquitous in natural metabolism and the promiscuity of their biosynthetic machinery enables transfer across diverse chemical structural templates at high rates [7]. In contrast, other functional groups that have received some engineering effort, including terminal alkynes, terminal alkenes, and halogens, are currently limited in applications due to biosynthetic machinery that is either insufficiently promiscuous, insufficiently active, or uncharacterized (Figure 2). However, the latter functional groups are exceedingly promising for the next-generation of metabolic engineering applications. Aldehydes Aldehydes are valuable as end-products in the flavors and fragrances industries and as intermediates in biological routes to fuels and pharmaceuticals [8]. Pathways to reach aldehydes are wellknown given that aldehydes are transient intermediates during reduction of carboxylic acids to alcohols [8]. While several enzyme classes generate specific aldehydes, the discovery of carboxylic acid reductases was notable given their broad specificity on aromatic and aliphatic substrates of diverse chain-lengths [9–11]. Due to the reactivity of aldehyde functional groups, cells evolved mechanisms to sense and respond to aldehydes broadly. One example is the yqhC transcriptional activator, which has recently been harnessed as an aldehyde biosensor [12]. However, endogenous enzymes within cells rapidly catalyze reduction of diverse aldehydes to alcohols. Pioneering work demonstrated that deletions of genes that encode aldehyde reductases in Escherichia coli enable accumulation of isobutyraldehyde [13]. Deletion of multiple genes encoding alcohol dehydrogenases and aldo keto reductases led to a strain that can accumulate diverse aromatic and aliphatic aldehydes [6,14]. Aldehydes such as vanillin, the key ingredient responsible for vanilla flavor, have been produced from several industrially relevant carbon sources, including ferulate derived from paper and pulp waste (Figure 3A) [15]. Though aldehyde toxicity remains a major challenge for scale-up of fermentations seeking to produce aldehydes as end-products [16–18], innovations in bioprocess engineering can mitigate negative effects on cell growth by efficiently separating products from cells [19–21]. Esters Compounds containing esters are commonly used as natural flavor additives, industrial solvents, plasticizers, lubricants, and more [7]. The primary in vivo ester production pathway is the enzymatic reaction of an alcohol and acyl-CoA facilitated by an alcohol acyltransferase (AAT) (Figure 3B) [7]. AATs have inherent diversity, are found across several domains of life, and have complementary and broad specificity towards both alcohols and acyl-CoAs. Given the broad substrate scope of AATs, efforts have focused on engineering metabolic pathways to overproduce the precursors needed for ester formation and on methods to mitigate potential toxicity. Early engineering efforts focused on increasing carbon flux through acetyl-CoA by supplementing CoA precursor [22], knocking out other major fermentative pathways to increase flux through pyruvate [23], and overexpressing pyruvate dehydrogenase which converts pyruvate to acetyl-CoA [24]. To achieve greater product diversity, metabolic engineers can use other sources for the acylCoA group in AAT condensation such as branched chain and aromatic acyl-CoAs, intermediates in amino acid degradation [23,25], longer chain acyl-CoAs available through fatty acid synthesis [26–30], and exogenously supplied chemicals such as carboxylic acids [31–33]. Yields for ester production can also be improved by managing chemical toxicity of the products, such as by creating a layer extraction that preferentially separates the product from your substrate and 4

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Figure 3. Pathways for Established Functional Groups in Metabolic Engineering. Circled enzymes are responsible for the functional group formation. (A) Conversion of ferulic acid to vanillin by feruloyl-CoA synthetase (Fcs) and enoyl-CoA hydratase/aldolase (Ech) from Amycolatopsis thermoflava N1165 [15]. (B) Production of isobutyl acetate from isobutanol and acetyl-CoA through an alcohol acyltransferase (AAT) [23]. (C) Pathways for terminal alkyne production. The first is conversion of hexenoic acid to hexynoic acid through JamABC [38], and the second is production of propargylglycine from lysine through the Bes pathway [40]. (D) Terminal alkene production from the fatty acid pathway, a thioesterase (TesA) acting on an acyl-ACP and UndA acting on the carboxylic acid to form 1-undecene [51]. (E) Pathway for the production of a fluoropolymer from fluoromalonate [63].

cells [23,30,31]. Given that strategies for diverse ester production have already been demonstrated, next steps for research include improving titer, incorporating more sustainable substrates, and more comprehensively addressing ester toxicity. Terminal Alkynes Terminal alkynes have diverse and intriguing applications in chemical biology focused around applications of their bio-orthogonal conjugation chemistry with azido groups [34–37]. These applications have motivated the discovery and engineering of two different pathways for the biosynthesis of terminal alkynes in E. coli. The first discovered pathway applies to polyketide synthesis, while the second, more recent, discovery applies to amino acid biosynthesis. The first pathway was found through investigation of jamaicamides and carmabins, where JamB, a membrane bound fatty acid desaturase was found to work in tandem with JamA, a fatty acylCoA ligase, and JamC, an acyl carrier protein, to produce a terminal alkyne in a polyketide

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synthetase (PKS) system (Figure 3C) [38]. Investigation of substrate specificity revealed that JamB acted on C6 terminal alkenoic acids in the presence of JamAC to form the JamC bound terminal alkyne. Incorporation of the terminal alkyne has been shown to successfully occur in conjugation with heterologously expressed PKS systems in starter units followed by malonyl CoA extension and in extender units following use of anthranilate-based starter units [38]. However, the 5-hexenoic acid supplied in the starter unit context outcompeted the terminal alkyne due to the low activity of JamB. To improve activity in E. coli, an azido fluorogenic probe, di-pegOF, was used in directed evolution through random mutagenesis that increased activity 20-fold on the two-step reaction to convert hexanoic acid to the terminal alkyne [39]. Therefore, while the current substrate specificity of JamB is limited [39], the proof-of-concept of the fluorogenic screen for terminal alkynes could be expanded to increase activity towards other substrates; meanwhile the ability of JamB to interface with various PKS systems maintains the possibility of product diversity. The second pathway is called the ‘Bes pathway’ (for production of Beta-ethynylserine) and was discovered in Streptomyces cattleya (Figure 3C) [40]. This pathway involves multiple interesting functional groups on the pathway to the terminal alkyne, including a 4-chlorinated lysine and chlorinated terminal alkene. Through gamma elimination of the chlorine on the terminal alkene, the terminal alkyne in propargylglycine is formed, and additional hydroxylation yields Bes. By choosing the subset of genes for heterologous expression, one can control which product is created in vivo [40]. The discovery of the initial machinery for production of terminal alkyne amino acids could lead to identifying homologs with altered substrate specificity, leading to a wider range of terminal alkyne amino acid products with applications in bio-orthogonal conjugation. Terminal Alkenes Multiple classes of enzymes have been shown to form a terminal alkene group, a functionality that is valuable in the sustainable fuels industry [41,42] as well as in bioconjugation efforts [43]. Commonly used enzymes are OleTJE, a cytochrome P450 enzyme from Jeotgalicoccus sp. ATCC 8456 [44], its homologs [45], UndA, a non-heme iron(II)-dependent oxidase from Pseudomonas putida [46], and UndB, a fatty acid desaturase or aldehyde decarboxylase also from P. putida [47]. The native substrate specificities of the different enzymes overlap, but OleTJE prefers longer chain fatty acid substrates (C12–C20) while UndA and UndB have higher activities on medium chain fatty acids (C10–C14 and C10–C16, respectively). The substrate specificity of OleTJE has since been expanded to include shorter chain fatty acids (C4–C9) by employing efficient electron transfer machinery and optimizing cofactor recycling [48] and to include aromatic carboxylic acids through targeted point mutations [49]. Each of the three enzymes has been heterologously expressed in different organisms for production of valuable compounds. OleTJE was expressed in E. coli alongside an oleate hydratase in order to produce isoprene from mevalonate [50]. UndA was coexpressed with a thioesterase in Acinetobacter baylyi ADP1 to produce 1-undecene exclusively from ferulate, a lignin-derived compound (Figure 3D) [51], and UndB was also expressed in Pseudomonas to produce 1-alkenes in an engineered lipolytic platform from hydrophobic substrates [52]. The established pathways all focused on fatty acid substrates and products valuable to the sustainable fuel industry, but future engineering could take advantage of the expanded substrate scope of OleTJE or explore the substrate scope of UndA and UndB to produce a larger variety of terminal alkene chemicals for other applications, including intermediates in biosynthesis of other functional groups like epoxides. Fluoro Groups Fluorinated products are widespread with applications in a variety of industries, including medicine, agriculture, and materials [53,54]. However, fluorine is scarcely presented in natural 6

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metabolism across all domains of life, especially when compared with the over 5000 organohalogens containing chlorine, bromine, or iodine [55]. Though fluoroacetate has been found in some plants, it is unknown whether the plants or their soil bacteria are responsible for the fluoroacetate production [54]. The origin of plant fluoroacetate is confounded by the discovery of C–F bond formation and fluoroacetate pathways in S. cattleya, a common soil bacteria that was found to create 5′-fluoro3′-deozyadenosine through the use of a fluorinase enzyme that acts on inorganic fluorine and S-adenosylmethionine (SAM) [56]. Other known pathway enzymes were found to produce fluoroacetate and fluorothreonine through a common intermediate, fluoroacetaldehyde [57–59]. Engineering efforts for fluorinated product biosynthesis focused on incorporating a fluorinated feedstock into natural product biosynthetic pathways [60,61]. Early efforts were not able to achieve incorporation of fluoroacetyl-CoA as an extender unit in PKS systems, most likely due to fluorine’s chemical properties disrupting the natural pathway chemistry. This issue was addressed by supplementation of fluoromalonate and overexpression of the malonyl-CoA synthetase MatB to form fluoromalonyl-CoA as a potential PKS extender unit [62]. Starting with just a single unit extension, they established NphT7 as a competent enzyme in reacting acetylCoA with a fluoromalonyl-CoA extender. This enzyme and an acetoacetyl-CoA reductase, PhaB, was used to incorporate fluorine into a more complex polyketide [62] as well as a fluorinated polyhydroxyalkanoate (Figure 3E) [63]. While the PKS system did not accept the fluorinated substrate as efficiently as its natural one, the polymerase was able to incorporate the fluorinated monomer up to 15% and at a ratio that reflects the contents of the culture media [63], giving the engineer control over the amount of fluorinated monomer. While toxicity, transport, and incorporation of fluorinated substrates have been at least partially addressed, formation of the carbon–fluorine bond remains an obstacle to constructing a fully de novo pathway for fluorinated products.

Recently Discovered Functional Groups of Interest Elucidation of natural product biosynthetic pathways is often the first step towards enabling metabolic engineers to confer a wider range of functional groups on societally relevant molecules. Discovery of enzymes with novel functionality is enabled by comparison of biosynthetic gene clusters (BGCs) contained within the producer microbe with known BGCs to identify unique candidates [64,65]. Once an enzyme responsible for generating a new functional group is identified, additional bioprospecting can be performed to obtain an enzyme with improved activity or substrate specificity. Advances in bioinformatics have accelerated elucidation of enzymes critical for novel functional group formation, leading the way for future research into expanding substrate specificity and product scope, improving product stability and accumulation, developing highthroughput detection methods, and ultimately integration into de novo pathways (Figure 2). Nitro Groups The nitro group provides valuable functionality to pharmaceuticals and explosives [66]. However, in known natural product biosynthesis, the current substrate scope is limited to aromatic molecules. The enzymes that form nitroaromatics have been characterized and fall under two methods of nitration. The first method is direct nitration, which occurs on an aromatic ring with nitric oxide and oxygen as co-substrates, as in the cases of TxtE from thaxtomin production in Streptomyces scabies [67] and RufO from rufomycin production in Streptomyces atratus ATCC 14046 (Figure 4A) [68]. The second method is sequential monooxygenation, or the conversion of an amine group to a nitro group by sequentially adding oxygen, as in the case of CmlI from chloramphenicol biosynthesis in Streptomyces venezuelae [69] or the better known AurF from aureothin production in Streptomyces thioluteus (Figure 4A) [70–72]. Only a few cases of aliphatic nitro addition are known and even fewer have been elucidated. Nitropropionic

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(See figure legend at the bottom of the next page.)

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acid and nitroglycine [73–76] are both naturally occurring metabolites, whose biosynthetic machineries are unknown. Biosynthetic machinery is known, however, for nitrosuccinic acid, which is generated as an intermediate in the Cremeomycin pathway in Streptomyces cremeus [77]. CreE, when paired with CreD, creates nitrous acid from aspartic acid, but in vitro assays showed nitrosuccinic acid production with the addition of just CreE. While the biosynthetic pathways of nitroaromatics are well-characterized, their stability in vivo currently limits product accumulation, as the nitro groups face potential reduction to amines which could hinder longterm engineering efforts [78,79]. Nitriles Nitrile production exists along a pathway called the aldoxime-nitrile pathway in the carbon and nitrogen metabolism interactions of plants and microbes [80]. Given the presence of the pathway at species interfaces, the nitrile group can be biosynthesized from diverse substrates based on pathway expression level and directionality. One starting point is an aldoxime using an aldoxime dehydratase such as the phenylacetaldoxime dehydratase found in Bacillus sp. strain OxB-1 (Figure 4B) [81] or the indolyl-3-acetaldoxim dehydratase from Sclerotinia sclerotiorum [82]. Another origin is the conversion of an amine to a nitro followed by nitrile generation, as shown in borrelidin biosynthesis in Streptomyces by BorI [83]. In addition to these pathways, a single enzyme has been found that catalyzes conversion directly from a carboxylic acid to a nitrile through the amine synthetase ToyM in Streptomyces rimosus (Figure 4B) [84]. The different pathways to the nitrile group can be tailored to the substrate and utilized to reach other groups such as aldoximes, amides, and carboxylic acids. Epoxides Epoxide natural products have valuable roles as synthetic intermediates for production of enantiomerically pure diols, halogens, and cyclized products [85]. Many enzymes downstream of epoxide formation have already been found and developed for specific applications [85–87]. Thus, elucidation and engineering of epoxide-forming enzymes would allow de novo biosynthesis of diverse products. There are four enzyme classes for epoxide biosynthesis that vary in substrate utilization [88]: (i) heme-dependent [89], (ii) flavin-dependent [90], (iii) cofactor-independent, and (iv) non-heme iron-dependent. Heme-dependent and flavin-dependent enzymes exclusively act on alkenes to form an epoxide like in epothilone biosynthesis in Sorangium cellulosum [91] and lasalocid biosynthesis in Streptomyces lasaliensis [92], respectively. Cofactor-independent enzymes, however, require a phenol substrate evidenced by the biosynthesis of LL-c10037α in Streptomyces (Figure 4C) [93]. Non-heme iron-dependent enzymes dehydrogenate a secondary alcohol to make the epoxide as in fosfomycin biosynthesis in Streptomyces (Figure 4C) [94]. While each case has different initial substrate requirements, characterization of substrate specificity towards nonnative substrates and epoxide stability on diverse chemical structures would provide valuable insights for engineering. Nitroso Groups Pathway engineering for nitroso group formation is relevant to the pharmaceutical industry because of their role as an important bioactive group in therapeutics [95,96]. A pathway for the N-nitroso group from the biosynthesis of streptozotocin, an N-nitrosourea natural product, Figure 4. Recently Discovered Pathways of Interest. Circled enzymes are responsible for the functional group formation. (A) Nitro group formation through direct nitration by RufO [68] and sequential monooxygenation by AurF [70]. (B) Nitrile group formation through an aldoxime intermediate [81], formed by a cytochrome P450 enzyme (CYP) and directly from a carboxylic acid by ToyM [84]. (C) Epoxide formation by cofactor-independent DHAE1 in the biosynthesis of LH-C10037α [93] and by the non-heme iron-dependent HppE in fosfomycin biosynthesis [94]. (D) N-nitroso biosynthesis by Szn-F through sequential hydroxylation and rearrangement of methylated arginine [97] and C-nitroso formation by NspEF through modification of a primary amine [98]. (E) Hydrazine biosynthesis by Spb40 to form hydrazinoacetic acid [101] and by the Fzm pathway to form a glutamic acid carrier [100].

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has recently been elucidated [97]. The enzyme SznF from Streptomyces achromogenes was found to be responsible the nitroso bond after sequential N-hydroxylation and rearrangement of Nω-methyl-L-arginine (Figure 4D). However, the product was unstable even in culture media devoid of cells and exhibited conversion of the nitroso group to nitric oxide unless reacted with another molecule [97]. Two enzymes that create a C-nitroso group have also been found with complementary substrate specificity on the five aromatic amine compounds tested: NspF in Streptomyces murayamaensis (Figure 4D) and GriF in Streptomyces griseus. Both enzymes are copper-containing monooxygenases that work on an amine group to form the C-nitroso [98]. While the inherent instability of this functional group could pose a challenge to metabolic engineers, the recent discovery of these pathways, and the possibility for several more homologs, could lead to engineering efforts to optimize the substrate, the enzyme, and the environment to improve the stability of the nitroso functional group for product accumulation. Hydrazines Several functional groups found in natural products have a nitrogen–nitrogen (N–N) bond at their core, such as diazo, hydrazide, and azoxy groups [73]. Recent efforts have begun to characterize these bond formations and a key observation was the formation of a diazo group in cremeomycin by CreM activation of nitrite in the later stages of biosynthesis in S. cremeus [99]. Given that the enzyme catalyzes the step prior to final product formation and is therefore downstream in the biosynthesis pathway, its substrate specificity may be narrow compared with enzymes that are found upstream of pathway branch points. Discovery of hydrazine functional group in early stages of two different biosynthetic pathways shows promise for future metabolic engineering efforts incorporating N–N bonds as hydrazine appears on carrier molecules in each pathway. In the first pathway, glutamic acid becomes a carrier for the hydrazine bond in the biosynthesis of kinamycin, an antibiotic containing a diazo group, and fosfazinomycin, an antibiotic containing a hydrazide group [100]. In the pathway, two enzymes react sequentially with aspartic acid to form nitrous acid that reacts with another N-containing intermediate to form the initial N–N bond. The pathway continues to form an intermediate of acetylhydrazine before reacting with glutamic acid (Figure 4E) and then branching out to the different biosynthetic pathways. The second carrier molecule was found by investigating the biosynthesis of s56-p1, a dipeptide with a hydrazone functional group, in Streptomyces sp. SoC090715LN17. The initial N–N bond is formed through an enzymatic reaction between glycine and an N-hydroxylated lysine, and the carrier molecule, hydrazinoacetic acid is formed through enzymatic oxidation of that product (Figure 4E) [101]. In both cases, the later stage enzymes that convert the hydrazine bond to the final diazo, hydrazide, or hydrazone bonds have not yet been identified, but the creation of the N–N bond in the early stage is promising to engineers who want to incorporate and modify the functional group in nonnative products.

Alternative Approaches to Novel Functional Group Biosynthesis Whether the goal is to achieve biosynthesis of molecules that contain standard or nonstandard functional groups, the options are not limited to bioprospecting from natural product pathways. Given that standard functional groups by definition occur more frequently in natural metabolic pathways, the default strategy is to harness naturally occurring genes from heterologous sources. However, because pathway enzymes may need to act on complex molecules and nonnatural chemical templates to form desired products, protein engineering may be required to broaden substrate specificity. Often protein engineering to increase activity of rate-limiting enzymes, cofactor supply engineering, and host tolerance engineering will be required for all traditional metabolic engineering efforts. 10

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One notable alternative of greater relevance to nonstandard functional group biosynthesis is directed evolution, most often of metalloenzymes, for novel reactivity (though it is very helpful if the progenitor enzyme already exhibits some activity). Enzyme libraries can be generated through rational or random mutagenesis and then screened to obtain new-to-nature reactivity. This approach was developed and repeatedly utilized by Francis Arnold and led to the biosynthesis of several functional groups using evolved metalloenzymes including cyclopropanes [102], strained carbocycles [103], silicon [104], and boron [105]. Beyond directed evolution, insertion of nonnatural cofactors [106] or nonstandard amino acids [107] into metalloenzymes can also confer different reactions than the native protein. These approaches all purposely diverge from natural pathways, requiring greater expertise in protein engineering rather than metabolic engineering to reach the desired functional group. However, a combination of approaches can be applied to enhance the substrate specificity of natural enzymes for inclusion of nonstandard functional groups onto more diverse chemical structures.

Concluding Remarks Metabolic engineering has been evolving since its origins in mathematical modeling towards production of nonnative and nonnatural metabolites by introducing heterologous proteins into model hosts and optimizing production through engineered protein activity, product formation, and product stability. The field continues to repurpose proteins and functional groups to provide sustainable routes for production of ever-diverse chemicals. Though additional nonstandard functional groups will be uncovered during the years ahead, challenges await researchers who aspire to endow diverse chemical templates with under-represented motifs using biosynthesis. Major obstacles in the discovery stages include the vast percentage of currently unculturable microbes, understudied clades, and cryptic pathways that require activation. The development of tools that enable expression within diverse hosts, such as recently developed chassisindependent recombinase-assisted genome engineering [108], should aid activation efforts. Improvements in computational tools across the entire discovery pipeline, ranging from metagenomic and gene cluster annotation and identification to chemical structure prediction, will further accelerate discovery efforts. Prediction of substrate promiscuity would increase the utility of these tools for engineering chemical diversification. Nonstandard functional group engineering will rely heavily on protein engineering to improve activity on nonnative substrates. Development of functional group-based high-throughput screening methods would accelerate advancement of many groups discussed here.

Outstanding Questions Which functional groups are most sought after by industry? Which groups will be discovered next in nature? Of the natural biosynthetic machinery discovered, what is the specificity and efficiency towards nonnative substrates? Do natural homologs have different specificity? Can the field develop quantitative criteria for when protein engineering is a better option than bioprospecting? What is the stability of these products in fermentation-relevant environments and alongside different host microbes? Does the functional group get modified by endogenous enzymes or chemically degrade? Are pathway enzymes functional upon heterologous expression in E. coli? Is a different host organism a better option for production? What tools are needed to improve enzyme or pathway production on nonnative substrates or in nonnative hosts? Is there potential for highthroughput screening based on chemical reactivity or genetically encoded biosensors? How can these strategies achieve integration of chemical elements (in addition to functional groups) that are poorly represented in nature?

Going forward, a major opportunity for innovation is in the development of generalizable and wellcharacterized strategies to treat unstable or toxic functional groups. For highly unstable groups that appear naturally as a transient intermediate, it can be difficult to deconvolute failure of expressed proteins in forming product from failure to retain product. For toxic groups, a variety of emerging strategies from synthetic biology could be used for mitigation at the cellular level, including subcellular compartmentalization and dynamic regulation of biosynthetic machinery. Implementation of these strategies can be pursued in parallel with determination of a fuller range of cellularly compatible chemistries. If chemistries are incompatible with living systems, then cell-free approaches can be pursued. Metabolic engineering of nonstandard functional groups faces many of the above challenges and more (see Outstanding Questions), but some of these challenges have already been overcome for functional groups farther along in the research pipeline. As nonstandard functional groups advance through the stages of research summarized earlier (Figure 1), the experience, while unique to the specific properties and challenges of the functional group, will lower the hurdle for the next and contribute to the overall advance of the discipline. Incorporating more functional Trends in Biotechnology, Month 2019, Vol. xx, No. xx

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groups into the metabolic engineer’s toolbox will soon allow the field to span the breadth of chemical synthesis while retaining benefits of biocatalysis such as stereospecificity, mild reaction conditions, and sustainable raw materials. Acknowledgments We are grateful to Neil Butler for helpful discussions during manuscript preparation.

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