Rational design of a bacterial import system for new-to-nature molecules.

Integration of novel compounds into biological processes holds significant potential for modifying or expanding existing cellular functions. However, the cellular uptake of these compounds is often hindered by selectively permeable membranes. We present a novel bacterial transport system that has been rationally designed to address this challenge. Our approach utilizes a highly promiscuous sulfonate membrane transporter, which allows the passage of cargo molecules attached as amides to a sulfobutanoate transport vector molecule into the cytoplasm of the cell. These cargoes can then be unloaded from the sulfobutanoyl amides using an engineered variant of the enzyme γ-glutamyl transferase, which hydrolyzes the amide bond and releases the cargo molecule within the cell. Here, we provide evidence for the broad substrate specificity of both components of the system by evaluating a panel of structurally diverse sulfobutanoyl amides. Furthermore, we successfully implement the synthetic uptake system in vivo and showcase its functionality by importing an impermeant non-canonical amino acid.

Combinatorial biosynthesis in yeast leads to over 200 diterpenoids.

Diterpenoids form a diverse group of natural products, many of which are or could become pharmaceuticals or industrial chemicals. The modular character of diterpene biosynthesis and the promiscuity of the enzymes involved make combinatorial biosynthesis a promising approach to generate libraries of diverse diterpenoids. Here, we report on the combinatorial assembly in yeast of ten diterpene synthases producing (+)-copalyl diphosphate-derived backbones and four cytochrome P450 oxygenases (CYPs) in diverse combinations. This resulted in the production of over 200 diterpenoids. Based on literature and chemical database searches, 162 of these compounds can be considered new-to-Nature. The CYPs accepted most substrates they were given but remained regioselective with few exceptions. Our results provide the basis for the systematic exploration of the diterpenoid chemical space in yeast using sequence databases.

Advances in Noncanonical Amino Acid Incorporation for Enzyme Engineering Applications.

Incorporation of noncanonical amino acids (ncAAs) via genetic code expansion (GCE) opens up new possibilities for chemical biology. The technology has led to the development of novel xenobiotic enzymes with tailored properties which can serve as entry points into a multitude of applications, including protein conjugation, immobilization, or labeling. In this review, we discuss recent progress in the use of GCE to create biocatalysts possessing reaction repertoires that lie beyond what is achievable with canonical amino acids (cAAs). Furthermore, we highlight how GCE enables to gain mechanistic insights into protein function by the incorporation of judiciously selected ncAAs. As the amino acid alphabet continues to grow and improved tools for ncAA incorporation are being developed, we anticipate the creation of additional powerful biological catalysts for synthetic application which merge the chemical versatility of anthropogenic building blocks with the exquisite selectivities of enzymes.

Artificial metalloenzymes in a nutshell: the quartet for efficient catalysis.

Artificial metalloenzymes combine the inherent reactivity of transition metal catalysis with the sophisticated reaction control of natural enzymes. By providing new opportunities in bioorthogonal chemistry and biocatalysis, artificial metalloenzymes have the potential to overcome certain limitations in both drug discovery and green chemistry or related research fields. Ongoing advances in organometallic catalysis, directed evolution, and bioinformatics are enabling the design of increasingly powerful systems that outperform conventional catalysis in a growing number of cases. Therefore, this review article collects challenges and opportunities in designing artificial metalloenzymes described in recent review articles. This will provide an equitable insight for those new to and interested in the field.

Opening a Novel Biosynthetic Pathway to Dihydroxyacetone and Glycerol in Escherichia coli Mutants through Expression of a Gene Variant (fsaAA129S) for Fructose 6-Phosphate Aldolase.

Phosphofructokinase (PFK) plays a pivotal role in glycolysis. By deletion of the genes pfkA, pfkB (encoding the two PFK isoenzymes), and zwf (glucose 6-phosphate dehydrogenase) in Escherichia coli K-12, a mutant strain (GL3) with a complete block in glucose catabolism was created. Introduction of plasmid-borne copies of the fsaA wild type gene (encoding E. coli fructose 6-phosphate aldolase, FSAA) did not allow a bypass by splitting fructose 6-phosphate (F6P) into dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (G3P). Although FSAA enzyme activity was detected, growth on glucose was not reestablished. A mutant allele encoding for FSAA with an amino acid exchange (Ala129Ser) which showed increased catalytic efficiency for F6P, allowed growth on glucose with a µ of about 0.12 h-1. A GL3 derivative with a chromosomally integrated copy of fsaAA129S (GL4) grew with 0.05 h-1 on glucose. A mutant strain from GL4 where dhaKLM genes were deleted (GL5) excreted DHA. By deletion of the gene glpK (glycerol kinase) and overexpression of gldA (of glycerol dehydrogenase), a strain (GL7) was created which showed glycerol formation (21.8 mM; yield approximately 70% of the theoretically maximal value) as main end product when grown on glucose. A new-to-nature pathway from glucose to glycerol was created.

C-H functionalization reactions catalyzed by artificial metalloenzymes.

CH functionalization, a promising frontier in modern organic chemistry, facilitates the direct conversion of inert CH bonds into many valuable functional groups. Despite its merits, traditional homogeneous catalysis, often faces challenges in efficiency, selectivity, and sustainability towards this transformation. In this context, artificial metalloenzymes (ArMs), resulting from the incorporation of a catalytically-competent metal cofactor within an evolvable protein scaffold, bridges the gap between the efficiency of enzymatic transformations and the versatility of transition metal catalysis. Accordingly, ArMs have emerged as attractive tools for various challenging catalytic transformations. Additionally, the coming of age of directed evolution has unlocked unprecedented avenues for optimizing enzymatic catalysis. Taking advantage of their genetically-encoded protein scaffold, ArMs have been evolved to catalyze various CH functionalization reactions. This review delves into the recent developments of ArM-catalyzed CH functionalization reactions, highlighting the benefits of engineering the second coordination sphere around a metal cofactor within a host protein.

Data-driven enzyme engineering to identify function-enhancing enzymes.

Identifying function-enhancing enzyme variants is a 'holy grail' challenge in protein science because it will allow researchers to expand the biocatalytic toolbox for late-stage functionalization of drug-like molecules, environmental degradation of plastics and other pollutants, and medical treatment of food allergies. Data-driven strategies, including statistical modeling, machine learning, and deep learning, have largely advanced the understanding of the sequence-structure-function relationships for enzymes. They have also enhanced the capability of predicting and designing new enzymes and enzyme variants for catalyzing the transformation of new-to-nature reactions. Here, we reviewed the recent progresses of data-driven models that were applied in identifying efficiency-enhancing mutants for catalytic reactions. We also discussed existing challenges and obstacles faced by the community. Although the review is by no means comprehensive, we hope that the discussion can inform the readers about the state-of-the-art in data-driven enzyme engineering, inspiring more joint experimental-computational efforts to develop and apply data-driven modeling to innovate biocatalysts for synthetic and pharmaceutical applications.

Integration of discovery and engineering in plant alkaloid research: Recent developments in elucidation, reconstruction, and repurposing biosynthetic pathways.

Plants synthesize tens of thousands of bioactive nitrogen-containing compounds called alkaloids, including some clinically important drugs in modern medicine. The discovery of new alkaloid structures and their metabolism in plants have provided ways to access these rich sources of bioactivities including new-to-nature compounds relevant to therapeutic and industrial applications. This review discusses recent advances in alkaloid biosynthesis discovery, including complete pathway elucidations. Additionally, the latest developments in the production of new and established plant alkaloids based on either biosynthesis or semisynthesis are discussed.

Challenges and opportunities in bringing nonbiological atoms to life with synthetic metabolism.

The relatively narrow spectrum of chemical elements within the microbial 'biochemical palate' limits the reach of biotechnology, because several added-value compounds can only be produced with traditional organic chemistry. Synthetic biology offers enabling tools to tackle this issue by facilitating 'biologization' of non-canonical chemical atoms. The interplay between xenobiology and synthetic metabolism multiplies routes for incorporating nonbiological atoms into engineered microbes. In this review, we survey natural assimilation routes for elements beyond the essential biology atoms [i.e., carbon (C), hydrogen (H), nitrogen (N), oxygen (O), phosphorus (P), and sulfur (S)], discussing how these mechanisms could be repurposed for biotechnology. Furthermore, we propose a computational framework to identify chemical elements amenable to biologization, ranking reactions suitable to build synthetic metabolism. When combined and deployed in robust microbial hosts, these approaches will offer sustainable alternatives for smart chemical production.

New Additions to the Arsenal of Biocatalysts for Noncanonical Amino Acid Synthesis.

Noncanonical amino acids (ncAAs) merge the conformational behavior and native interactions of proteinogenic amino acids with nonnative chemical motifs and have proven invaluable in developing modern therapeutics. This blending of native and nonnative characteristics has resulted in essential drugs like nirmatrelvir, which comprises three ncAAs and is used to treat COVID-19. Enzymes are appearing prominently in recent syntheses of ncAAs, where they demonstrate impressive control over the stereocenters and functional groups found therein. Here we review recent efforts to expand the biocatalyst arsenal for synthesizing ncAAs with natural enzymes. We also discuss how new-to-nature enzymes can contribute to this effort by catalyzing reactions inspired by the vast repertoire of chemical catalysis and acting on substrates that would otherwise not be used in synthesizing ncAAs. Abiotic enzyme-catalyzed reactions exploit the selectivity afforded by a macromolecular catalyst to access molecules not available to natural enzymes and perhaps not even chemical catalysis.

Directed Biosynthesis of New to Nature Alkaloids in a Heterologous Nicotiana benthamiana Expression Host.

Plants produce a wide variety of pharmacologically active molecules classified as natural products. Derivatization of these natural products can modulate or improve the bioactivity of the parent compound. Unfortunately, chemical derivatization of natural products is often difficult or impractical. Here we use the newly discovered biosynthetic genes for two monoterpene indole alkaloids, alstonine and stemmadenine acetate, to generate analogs of these compounds. We reconstitute these biosynthetic genes in the heterologous host Nicotiana benthamiana along with an unnatural starting substrate to produce the corresponding new-to-nature alkaloid product.

Prospecting Biochemical Pathways to Implement Microbe-Based Production of the New-to-Nature Platform Chemical Levulinic Acid.

Levulinic acid is a versatile platform molecule with potential to be used as an intermediate in the synthesis of many value-added products used across different industries, from cosmetics to fuels. Thus far, microbial biosynthetic pathways having levulinic acid as a product or an intermediate are not known, which restrains the development and optimization of a microbe-based process envisaging the sustainable bioproduction of this chemical. One of the doors opened by synthetic biology in the design of microbial systems is the implementation of new-to-nature pathways, that is, the assembly of combinations of enzymes not observed in vivo, where the enzymes can use not only their native substrates but also non-native ones, creating synthetic steps that enable the production of novel compounds. Resorting to a combined approach involving complementary computational tools and extensive manual curation, in this work, we provide a thorough prospect of candidate biosynthetic pathways that can be assembled for the production of levulinic acid in Escherichia coli or Saccharomyces cerevisiae. Out of the hundreds of combinations screened, five pathways were selected as best candidates on the basis of the availability of substrates and of candidate enzymes to catalyze the synthetic steps (that is, those steps that involve conversions not previously described). Genome-scale metabolic modeling was used to assess the performance of these pathways in the two selected hosts and to anticipate possible bottlenecks. Not only does the herein described approach offer a platform for the future implementation of the microbial production of levulinic acid but also it provides an organized research strategy that can be used as a framework for the implementation of other new-to-nature biosynthetic pathways for the production of value-added chemicals, thus fostering the emerging field of synthetic industrial microbiotechnology.

Deploying Microbial Synthesis for Halogenating and Diversifying Medicinal Alkaloid Scaffolds.

Plants produce some of the most potent therapeutics and have been used for thousands of years to treat human diseases. Today, many medicinal natural products are still extracted from source plants at scale as their complexity precludes total synthesis from bulk chemicals. However, extraction from plants can be an unreliable and low-yielding source for human therapeutics, making the supply chain for some of these life-saving medicines expensive and unstable. There has therefore been significant interest in refactoring these plant pathways in genetically tractable microbes, which grow more reliably and where the plant pathways can be more easily engineered to improve the titer, rate and yield of medicinal natural products. In addition, refactoring plant biosynthetic pathways in microbes also offers the possibility to explore new-to-nature chemistry more systematically, and thereby help expand the chemical space that can be probed for drugs as well as enable the study of pharmacological properties of such new-to-nature chemistry. This perspective will review the recent progress toward heterologous production of plant medicinal alkaloids in microbial systems. In particular, we focus on the refactoring of halogenated alkaloids in yeast, which has created an unprecedented opportunity for biosynthesis of previously inaccessible new-to-nature variants of the natural alkaloid scaffolds.

Nicotiana benthamiana as a Transient Expression Host to Produce Auxin Analogs.

Plant secondary metabolites have applications for the food, biofuel, and pharmaceutical industries. Recent advances in pathway elucidation and host expression systems now allow metabolic engineering of plant metabolic pathways to produce "new-to-nature" derivatives with novel biological activities, thereby amplifying the range of industrial uses for plant metabolites. Here we use a transient expression system in the model plant Nicotiana benthamiana to reconstitute the two-step plant-derived biosynthetic pathway for auxin (indole acetic acid) to achieve accumulation up to 500 ng/g fresh mass (FM). By expressing these plant-derived enzymes in combination with either bacterial halogenases and alternative substrates, we can produce both natural and new-to-nature halogenated auxin derivatives up to 990 ng/g FM. Proteins from the auxin synthesis pathway, tryptophan aminotransferases (TARs) and flavin-dependent monooxygenases (YUCs), could be transiently expressed in combination with four separate bacterial halogenases to generate halogenated auxin derivatives. Brominated auxin derivatives could also be observed after infiltration of the transfected N. benthamiana with potassium bromide and the halogenases. Finally, the production of additional auxin derivatives could also be achieved by co-infiltration of TAR and YUC genes with various tryptophan analogs. Given the emerging importance of transient expression in N. benthamiana for industrial scale protein and product expression, this work provides insight into the capacity of N. benthamiana to interface bacterial genes and synthetic substrates to produce novel halogenated metabolites.

Xenobiology: State-of-the-Art, Ethics, and Philosophy of New-to-Nature Organisms.

The basic chemical constitution of all living organisms in the context of carbon-based chemistry consists of a limited number of small molecules and polymers. Until the twenty-first century, biology was mainly an analytical science and has now reached a point where it merges with engineering science, paving the way for synthetic biology. One of the objectives of synthetic biology is to try to change the chemical compositions of living cells, that is, to create an artificial biological diversity, which in turn fosters a new sub-field of synthetic biology, xenobiology. In particular, the genetic code in living systems is based on highly standardized chemistry composed of the same "letters" or nucleotides as informational polymers (DNA, RNA) and the 20 amino acids which serve as basic building blocks for proteins. The universality of the genetic code enables not only vertical gene transfer within the same species but also horizontal gene transfer across biological taxa, which require a high degree of standardization and interconnectivity. Although some minor alterations of the standard genetic code are found in nature (e.g., proteins containing non-conical amino acids exist in nature, and some organisms use alternated coding systems), all structurally deep chemistry changes within living systems are generally lethal, making the creation of artificial biological system an extremely difficult challenge.In this context, one of the great challenges for bioscience is the development of a strategy for expanding the standard basic chemical repertoire of living cells. Attempts to alter the meaning of the genetic information stored in DNA as an informational polymer by changing the chemistry of the polymer (i.e., xeno-nucleic acids) or by changes in the genetic code have already yielded successful results. In the future this should enable the partial or full redirection of the biological information flow to generate "new" version(s) of the genetic code derived from the "old" biological world.In addition to the scientific challenges, the attempt to increase biochemical diversity also raises important ethical and philosophical issues. Although promotors of this branch of synthetic biology highlight the many potential applications to come (e.g., novel tools for diagnostics and fighting infection diseases), such developments could also bring risks affecting social, political, and other structures of nearly all societies.