Four-Step Pathway from Phenylpyruvate to Benzylamine, an Intermediate to the High-Energy Propellant CL-20.

Benzylamine is a commodity chemical used in the synthesis of motion-sickness treatments and anticonvulsants, in dyeing textiles, and as a precursor to the high-energy propellant CL-20. Because chemical production generates toxic waste streams, biosynthetic alternatives have been explored, recently resulting in a functional nine-step pathway from central metabolism (phenylalanine) in E. coli. We report a novel four-step pathway for benzylamine production, which generates the product from cellular phenylpyruvate using enzymes from different sources: a mandelate synthase (Amycolatopsis orientalis), a mandelate oxidase (Streptomyces coelicolor), a benzoylformate decarboxylase (Pseudomonas putida), and an aminotransferase (Salicibacter pomeroyi). This pathway produces benzylamine at 24 mg/L in 15 h (4.5% yield) in cultures of unoptimized cells supplemented with phenylpyruvate. Because the yield is low, supplementation with pathway intermediates is used to troubleshoot the design. This identifies conversion inefficiencies in the mandelate synthase-mediated synthesis of (S)-mandelic acid, and subsequent genome mining identifies a new mandelate synthase (Streptomyces sp. 1114.5) with improved yield. Supplementation experiments also reveal native redirection of ambient phenylpyruvate away from the pathway to phenylalanine. Overall, this work illustrates how retrosynthetic design can dramatically reduce the number of enzymes in a pathway, potentially reducing its draw on cellular resources. However, it also shows that such benefits can be abrogated by inefficiencies of individual conversions. Addressing these barriers can provide an alternative approach to green production of benzylamine, eliminating upstream dependence on chlorination chemistry.

Organism Engineering for the Bioproduction of the Triaminotrinitrobenzene (TATB) Precursor Phloroglucinol (PG).

Organism engineering requires the selection of an appropriate chassis, editing its genome, combining traits from different source species, and controlling genes with synthetic circuits. When a strain is needed for a new target objective, for example, to produce a chemical-of-need, the best strains, genes, techniques, software, and expertise may be distributed across laboratories. Here, we report a project where we were assigned phloroglucinol (PG) as a target, and then combined unique capabilities across the United States Army, Navy, and Air Force service laboratories with the shared goal of designing an organism to produce this molecule. In addition to the laboratory strain Escherichia coli, organisms were screened from soil and seawater. Putative PG-producing enzymes were mined from a strain bank of bacteria isolated from aircraft and fuel depots. The best enzyme was introduced into the ocean strain Marinobacter atlanticus CP1 with its genome edited to redirect carbon flux from natural fatty acid ester (FAE) production. PG production was also attempted in Bacillus subtilis and Clostridium acetobutylicum. A genetic circuit was constructed in E. coli that responds to PG accumulation, which was then ported to an in vitro paper-based system that could serve as a platform for future low-cost strain screening or for in-field sensing. Collectively, these efforts show how distributed biotechnology laboratories with domain-specific expertise can be marshalled to quickly provide a solution for a targeted organism engineering project, and highlights data and material sharing protocols needed to accelerate future efforts.

A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology.

Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

BASIC: A Simple and Accurate Modular DNA Assembly Method.

Biopart Assembly Standard for Idempotent Cloning (BASIC) is a simple, accurate, and robust DNA assembly method. The method is based on linker-mediated DNA assembly and provides highly accurate DNA assembly with 99 % correct assemblies for four parts and 90 % correct assemblies for seven parts [1]. The BASIC standard defines a single entry vector for all parts flanked by the same prefix and suffix sequences and its idempotent nature means that the assembled construct is returned in the same format. Once a part has been adapted into the BASIC format it can be placed at any position within a BASIC assembly without the need for reformatting. This allows laboratories to grow comprehensive and universal part libraries and to share them efficiently. The modularity within the BASIC framework is further extended by the possibility of encoding ribosomal binding sites (RBS) and peptide linker sequences directly on the linkers used for assembly. This makes BASIC a highly versatile library construction method for combinatorial part assembly including the construction of promoter, RBS, gene variant, and protein-tag libraries. In comparison with other DNA assembly standards and methods, BASIC offers a simple robust protocol; it relies on a single entry vector, provides for easy hierarchical assembly, and is highly accurate for up to seven parts per assembly round [2].

Bricks and blueprints: methods and standards for DNA assembly.

DNA assembly is a key part of constructing gene expression systems and even whole chromosomes. In the past decade, a plethora of powerful new DNA assembly methods - including Gibson Assembly, Golden Gate and ligase cycling reaction (LCR) - have been developed. In this Innovation article, we discuss these methods as well as standards such as the modular cloning (MoClo) system, GoldenBraid, modular overlap-directed assembly with linkers (MODAL) and PaperClip, which have been developed to facilitate a streamlined assembly workflow, to aid the exchange of material between research groups and to create modular reusable DNA parts.

BASIC: A New Biopart Assembly Standard for Idempotent Cloning Provides Accurate, Single-Tier DNA Assembly for Synthetic Biology.

The ability to quickly and reliably assemble DNA constructs is one of the key enabling technologies for synthetic biology. Here we define a new Biopart Assembly Standard for Idempotent Cloning (BASIC), which exploits the principle of orthogonal linker based DNA assembly to define a new physical standard for DNA parts. Further, we demonstrate a new robust method for assembly, based on type IIs restriction enzyme cleavage and ligation of oligonucleotides with single stranded overhangs that determine the assembly order. It allows for efficient, parallel assembly with great accuracy: 4 part assemblies achieved 93% accuracy with single antibiotic selection and 99.7% accuracy with double antibiotic selection, while 7 part assemblies achieved 90% accuracy with double antibiotic selection. The linkers themselves may also be used as composable parts for RBS tuning or the creation of fusion proteins. The standard has one forbidden restriction site and provides for an idempotent, single tier organization, allowing all parts and composite constructs to be maintained in the same format. This makes the BASIC standard conceptually simple at both the design and experimental levels.

R2oDNA designer: computational design of biologically neutral synthetic DNA sequences.

R2oDNA Designer is a web application that stochastically generates orthogonal sets of synthetic DNA sequences designed to be biologically neutral. Biologically neutral sequences may be used for directing efficient DNA assembly by overlap-directed methods, as a negative control for functional DNA, as barcodes, or potentially as spacer regions to insulate biological parts from local context. The software creates optimized sequences using a Monte Carlo simulated annealing approach followed by the elimination of sequences homologous to host genomes and commonly used biological parts. An orthogonal set is finally determined by using a network elimination algorithm. Design constraints can be defined using either a web-based graphical user interface (GUI) or uploading a file containing a set of text commands.

One-pot DNA construction for synthetic biology: the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy.

Overlap-directed DNA assembly methods allow multiple DNA parts to be assembled together in one reaction. These methods, which rely on sequence homology between the ends of DNA parts, have become widely adopted in synthetic biology, despite being incompatible with a key principle of engineering: modularity. To answer this, we present MODAL: a Modular Overlap-Directed Assembly with Linkers strategy that brings modularity to overlap-directed methods, allowing assembly of an initial set of DNA parts into a variety of arrangements in one-pot reactions. MODAL is accompanied by a custom software tool that designs overlap linkers to guide assembly, allowing parts to be assembled in any specified order and orientation. The in silico design of synthetic orthogonal overlapping junctions allows for much greater efficiency in DNA assembly for a variety of different methods compared with using non-designed sequence. In tests with three different assembly technologies, the MODAL strategy gives assembly of both yeast and bacterial plasmids, composed of up to five DNA parts in the kilobase range with efficiencies of between 75 and 100%. It also seamlessly allows mutagenesis to be performed on any specified DNA parts during the process, allowing the one-step creation of construct libraries valuable for synthetic biology applications.