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Deconstruction of polyethylene terephthalate (PET) plastic waste generates opportunities for valorization to alternative products. We recently designed an enzymatic cascade that could produce terephthalaldehyde (TPAL) from terephthalic acid. Here, we showed that the addition of TPAL to growing cultures ofEscherichia coliwild-type strain MG1655 and an engineered strain for reduced aromaticaldehyderection (RARE) strain resulted in substantial reduction. We then investigated if we could mitigate this reduction using multiplex automatable genome engineering (MAGE) to create anE. colistrain with 10 additional knockouts in RARE. Encouragingly, we found this newly engineered strain enabled a 2.5-fold higher retention of TPAL over RARE after 24h. We applied this new strain for the production ofpara-xylylenediamine (pXYL) and observed a 6.8-fold increase in pXYL titer compared to RARE. Overall, our study demonstrates the potential of TPAL as a versatile intermediate in microbial biosynthesis of chemicals that derived from waste PET.

Plastics are an extremely important class of materials that are prevalent in all facets of society; however, their widespread use over time, combined with limited end-of-life strategies, has led to increasing levels of waste accumulation. Although currently considered a burden, plastics waste is potentially an untapped feedstock for numerous chemical and manufacturing processes. In this review, we discuss the state of the art of approaches for valorization of plastics waste from a materials research perspective, including previous efforts to utilize plastics waste and recent innovations that have opportunities to add significant value. Although additional progress is necessary, we present several diverse capabilities and strategies for valorization that, when brought together, address end-of-life challenges for plastics at every stage of design and product consumption. In short, a materials research–based framework offers a unique perspective to address the urgent issues posed by plastics, unlocking the potential of polymers and plastics waste.

Aldehydes are attractive chemical targets given applications as end products in the flavors and fragrances industry and as intermediates due to their propensity for C-C bond formation. While biosynthetic routes to diverse aldehydes have been designed, a common challenge is the stability of these aldehydes in the presence of microbial hosts of engineered pathways. Here, we identify and address unexpected oxidation of a model collection of aromatic aldehydes, including many that originate from biomass degradation, in the presence ofEscherichia colistrains that were engineered to minimize aldehyde reduction. Of heightened interest to us were resting cell conditions as they offer numerous advantages for the bioconversion of toxic metabolites. Surprisingly, when diverse aldehydes are supplemented toE. coliRARE cells grown under aerobic conditions, they remain stabilized on the timescale of days, whereas when these same aldehydes are supplemented to resting cell preparations ofE. coliRARE that had been grown under the same conditions, we observe substantial oxidation. By performing combinatorial inactivation of six candidate aldehyde dehydrogenase genes in theE. coligenome using multiplexed automatable genome engineering (MAGE), we demonstrate that this oxidation can be substantially slowed, with greater than 50% retention of 6 out of 8 aldehydes when assayed 4 hours after their addition. Given that our newly engineered strain exhibitsReducedOxidationAndReduction of aromatic aldehydes, we dubbed it theE. coliROAR strain. Seeking to apply this new strain to resting cell biocatalysis, we compared the capability to synthesis the aldehyde furfural from 2-furoic acid via the carboxylic acid reductase enzyme fromNocardia iowensis. Here, we found that use of ROAR resting cells achieved 2-fold enhancement in furfural titer after 4 h and nearly 9-fold enhancement after 20 h as compared to resting cells of the RARE strain. Moving forward, the use of this strain to generate resting cells should allow aldehyde product isolation, further enzymatic conversion, or chemical reactivity under cellular contexts that better accommodate aldehyde toxicity.HighlightsWhen genes that encode aldehyde reductases are knocked out inEscherichia colistrains, supplemented aldehydes can experience oxidation instead of reduction, which is catalyzed by a different set of endogenous enzymes.Interestingly, we show for a collection of aromatic aldehydes that this oxidation is far more substantial when using resting cell preparations than during aerobic fermentation.We investigate the identity of the responsible genes by performing combinatorial gene inactivation using multiplex automatable genome engineering.The strain that we engineer exhibits Reduced Oxidation And Reduction (theE. coliROAR strain) and thereby enables design of more efficient aldehyde bioconversion processes under diverse formats.

To better incentivize the collection of plastic wastes, new chemical transformations must be developed that add value to plastic deconstruction products. Polyethylene terephthalate (PET) is a common plastic whose deconstruction through chemical or biological means has received much attention. However, a limited number of alternative products have been formed from PET deconstruction, and only a small share could serve as building blocks for alternative materials or therapeutics. Here, we demonstrate the production of useful mono-amine and diamine building blocks from known PET deconstruction products. We achieve this by designing one-pot biocatalytic transformations that are informed by the substrate specificity of an ω-transaminase and diverse carboxylic acid reductases (CAR) towards PET deconstruction products. We first establish that an ω-transaminase fromChromobacterium violaceum(cvTA) can efficiently catalyze amine transfer to potential PET-derived aldehydes to form the mono-aminepara-(aminomethyl)benzoic acid (pAMBA) or the diaminepara-xylylenediamine (pXYL). We then identified CAR orthologs that could perform the bifunctional reduction of TPA to terephthalaldehyde (TPAL) or the reduction ofmono-(2-hydroxyethyl) terephthalic acid (MHET) to its corresponding aldehyde. After characterizing 17 CARsin vitro, we show that the CAR fromSegniliparus rotundus(srCAR) had the highest observed activity on TPA. Given these newly elucidated substrate specificity results, we designed modular enzyme cascades based on coupling srCAR and cvTA in one-pot with enzymatic co-factor regeneration. When we supply TPA, we achieve a 69 ± 1% yield of pXYL, which is useful as a building block for materials. When we instead supply MHET and subsequently perform base-catalyzed ester hydrolysis, we achieve 70 ± 8% yield of pAMBA, which is useful for therapeutic applications and as a pharmaceutical building block. This work expands the breadth of products derived from PET deconstruction and lays the groundwork for eventual valorization of waste PET to higher-value chemicals and materials.GRAPHICAL ABSTRACT

Bacillus subtilis is a model gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, here we report broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms., B. subtilis is valuable both as a model for cell biology and as an industrial organism. Here the authors use genetic code expansion to enable functional tools for exploring cell division dynamics.

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.

Nitroaromatic functional groups can impart valuable properties to chemicals and to biological macromolecules including polypeptides. Para-nitro-L-phenylalanine (pN-Phe) is a nitroaromatic amino acid with uses including immune stimulation and fluorescence quenching. As the chemical synthesis of pN-Phe does not follow green chemistry principles and impedes provision of pN-Phe to engineered bacterial cells in some contexts, we sought to design a de novo biosynthetic pathway for pN-Phe in Escherichia coli. To generate the nitro chemical functional group, we identified natural diiron monooxygenases with measurable in vitro and in vivo activity on envisioned amine-containing precursors of para-amino-L-phenylalanine (pA-Phe) and para-aminophenylpyruvate. By expressing one of these N-oxygenase genes together with previously characterized genes for the biosynthesis of pA-Phe, we achieved the synthesis of pN-Phe from glucose. Through further optimization of the chassis, plasmid constructs, and media conditions, we were able to improve the selectivity of pN-Phe biosynthesis, resulting in a maximum titer of 819 µM in rich defined media under shake-flask conditions. These results provide a foundation for the biosynthesis of related nitroaromatic chemicals and for downstream biological applications that could utilize pN-Phe as a building block.HighlightsPara-nitro-L-phenylalanine (pN-Phe) is a valuable small molecule for its applications in genetic code expansion.We establish de novo biosynthesis of pN-Phe from glucose in E. coli, which is also the first example of a de novo pathway design for an unnatural but commonly used non-standard amino acid.We show the first use of an N-oxygenase enzyme in the de novo synthesis of a nitroaromatic product.Screening of natural N-oxygenases and strain engineering resulted in final pN-Phe titers of 820 ± 130 µM in shake flask experiments with rich defined media.

Aromatic compounds have broad applications and have been the target of biosynthetic processes for several decades. New biomolecular engineering strategies have been applied to improve production of aromatic compounds in recent years, some of which are expected to set the stage for the next wave of innovations. Here, we will briefly complement existing reviews on microbial production of aromatic compounds by focusing on a few recent trends where considerable work has been performed in the last 5 years. The trends we highlight are pathway modularization and compartmentalization, microbial co-culturing, non-traditional host engineering, aromatic polymer feedstock utilization, engineered ring cleavage, aldehyde stabilization, and biosynthesis of non-standard amino acids. Throughout this review article, we will also touch on unmet opportunities that future research could address.

Understanding the evolutionary stability and possible context-dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While batch cultures of synthetic auxotrophic Escherichia coli previously exhibited undetectable escape throughout 14 days of monitoring, the long-term effectiveness of synthetic auxotrophy is unknown. Here, we report automated continuous evolution of a synthetic auxotroph using custom chemostats that supply a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution in three separate trials, populations exhibit no observable escape and are capable of normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction of three proteins implicated in small molecule transport reveals their contribution to increased fitness at low BipA concentrations. Mutations do not appear in orthogonal translation machinery nor in synthetic auxotrophic markers. Based on its evolutionary stability, we introduce the progenitor synthetic auxotroph directly to mammalian cell culture. We observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on timescales and in contexts that enable diverse applications.One Sentence SummaryTo ascertain whether life inevitably finds a way, we continuously evolve an Escherichia coli strain that was not able to escape from engineered biocontainment before, and we find that it does not escape even after 100 days of evolution, nor does it escape when added to mammalian cell culture.

Recombination-mediated genetic engineering, also known as recombineering, is the genomic incorporation of homologous single-stranded or double-stranded DNA into bacterial genomes. Recombineering and its derivative methods have radically improved genome engineering capabilities, perhaps none more so than multiplex automated genome engineering (MAGE). MAGE is representative of a set of highly multiplexed single-stranded DNA-mediated technologies. First described in Escherichia coli, both MAGE and recombineering are being rapidly translated into diverse prokaryotes and even into eukaryotic cells. Together, this modern set of tools offers the promise of radically improving the scope and throughput of experimental biology by providing powerful new methods to ease the genetic manipulation of model and non-model organisms. In this Primer, we describe recombineering and MAGE, their optimal use, their diverse applications and methods for pairing them with other genetic editing tools. We then look forward to the future of genetic engineering. This Primer by Wannier and colleagues summarizes the methodology, analysis and utility of recombineering and multiplex automated genome engineering (MAGE) in microbial species. In addition, this Primer examines advanced techniques that pair MAGE with other tools to improve editing efficiency.

Engineering at microscopic scales has an immense effect on the modern bioeconomy. Microbes contribute to such disparate markets as chemical manufacturing, fuel production, crop optimization, and pharmaceutical synthesis, to name a few. Due to new and emerging synthetic biology technologies, and the sophistication and control afforded by them, we are on the brink of deploying engineered microbes to not only enhance traditional applications but also to introduce these microbes to sectors, contexts, and formats not previously attempted. In microbially managed medicine, microbial engineering holds promise for increasing efficacy, improving tissue penetration, and sustaining treatment. In the environment, the most effective areas for deployment are in the management of crops and protection of ecosystems. However, caution is warranted before introducing engineered organisms to new environments where they may proliferate without control and could cause unforeseen effects. We summarize ideas and data that can inform identification and assessment of the risks that these tools present to ensure that realistic hazards are described and unrealistic ones do not hinder advancement. Further, because modes of containment are crucial complements to deployment, we describe the state of the art in microbial biocontainment strategies, current gaps, and how these gaps might be addressed through technological advances in synthetic engineering. Collectively, this work highlights engineered microbes as a foundational and expanding facet of the bioeconomy, projects their utility in upcoming deployments outside the laboratory, and identifies knowns and unknowns that will be necessary considerations and points of focus in this endeavor.

Since the elucidation of its structure, DNA has been at the forefront of biological research. In the past half century, an explosion of DNA-based technology development has occurred with the most rapid advances being made for DNA sequencing. In parallel, dramatic improvements have also been made in the synthesis and editing of DNA from the oligonucleotide to the genome scale. In this Review, we will summarize four different subfields relating to DNA technologies following this trajectory of smaller to larger scale. We begin by talking about building materials out of DNA which in turn can act as delivery vehicles in vivo. We then discuss how altering microbial genomes can lead to novel methods of production for industrial biologics. Next, we talk about the future of writing whole genomes as a method of studying evolution. Lastly, we highlight the ways in which barcoding biological systems will allow for their three-dimensional analysis in a highly multiplexed fashion.

Efforts are underway to construct several recoded genomes anticipated to exhibit multivirus resistance, enhanced nonstandard amino acid (nsAA) incorporation, and capability for synthetic biocontainment. Although our laboratory pioneered the first genomically recoded organism (Escherichia coli strain C321.∆A), its fitness is far lower than that of its nonrecoded ancestor, particularly in defined media. This fitness deficit severely limits its utility for nsAA-linked applications requiring defined media, such as live cell imaging, metabolic engineering, and industrial-scale protein production. Here, we report adaptive evolution of C321.∆A for more than 1,000 generations in independent replicate populations grown in glucose minimal media. Evolved recoded populations significantly exceeded the growth rates of both the ancestral C321.∆A and nonrecoded strains. We used next-generation sequencing to identify genes mutated in multiple independent populations, and we reconstructed individual alleles in ancestral strains via multiplex automatable genome engineering (MAGE) to quantify their effects on fitness. Several selective mutations occurred only in recoded evolved populations, some of which are associated with altering the translation apparatus in response to recoding, whereas others are not apparently associated with recoding, but instead correct for off-target mutations that occurred during initial genome engineering. This report demonstrates that laboratory evolution can be applied after engineering of recoded genomes to streamline fitness recovery compared with application of additional targeted engineering strategies that may introduce further unintended mutations. In doing so, we provide the most comprehensive insight to date into the physiology of the commonly used C321.∆A strain.

Significance Accurate incorporation of nonstandard amino acids (nsAAs) is central for genetic code expansion to increase the chemical diversity of proteins. However, aminoacyl-tRNA synthetases are polyspecific and facilitate incorporation of multiple nsAAs. We investigated and repurposed a natural protein degradation pathway, the N-end rule pathway, to devise an innovative system for rapid assessment of the accuracy of nsAA incorporation. Using this tool to monitor incorporation of the nsAA biphenylalanine allowed the identification of tyrosyl-tRNA synthetase (TyrRS) variants with improved amino acid specificity. The evolved TyrRS variants enhanced our ability to contain unwanted proliferation of genetically modified organisms. This posttranslational proofreading system will aid the evolution of orthogonal translation systems for specific incorporation of diverse nsAAs., Incorporation of nonstandard amino acids (nsAAs) leads to chemical diversification of proteins, which is an important tool for the investigation and engineering of biological processes. However, the aminoacyl-tRNA synthetases crucial for this process are polyspecific in regard to nsAAs and standard amino acids. Here, we develop a quality control system called “posttranslational proofreading” to more accurately and rapidly evaluate nsAA incorporation. We achieve this proofreading by hijacking a natural pathway of protein degradation known as the N-end rule, which regulates the lifespan of a protein based on its amino-terminal residue. We find that proteins containing certain desired N-terminal nsAAs have much longer half-lives compared with those proteins containing undesired amino acids. We use the posttranslational proofreading system to further evolve a Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) variant and a tRNATyr species for improved specificity of the nsAA biphenylalanine in vitro and in vivo. Our newly evolved biphenylalanine incorporation machinery enhances the biocontainment and growth of genetically engineered Escherichia coli strains that depend on biphenylalanine incorporation. Finally, we show that our posttranslational proofreading system can be designed for incorporation of other nsAAs by rational engineering of the ClpS protein, which mediates the N-end rule. Taken together, our posttranslational proofreading system for in vivo protein sequence verification presents an alternative paradigm for molecular recognition of amino acids and is a major advance in our ability to accurately expand the genetic code.

Genetically encoded small molecule sensors can facilitate metabolic engineering by enabling high-throughput detection of metabolite concentrations, directed evolution of host and pathway enzymes, and dynamic regulation. The engineered de novo vanillin biosynthesis pathway assembled in Escherichia coli is industrially relevant and ideal for biosensor deployment given that the pathway requires only three heterologous enzyme-catalyzed reactions, generates naturally occurring metabolites, and may benefit from dynamic regulation. However, pathway flux is stalled and diverted by the activity of the Homo sapiens catechol O-methyltransferase, which is intended to catalyze the conversion of protocatechuate to vanillate. To confront this challenge, we constructed and applied a vanillate sensor based on the Caulobacter crescentus VanR-VanO system. Using components from a previously characterized E. coli promoter library, we achieved greater than 14-fold dynamic range in our best rationally constructed sensor. We characterized sensor substrate specificity and found that this construct and an evolved variant are remarkably selective, exhibiting no detectable response to the regioisomer byproduct isovanillate. We then harnessed the evolved biosensor to conduct rapid bioprospecting of natural catechol O-methyltransferases. We identified eight that appear to have greater desired activity than the originally used variant, including three previously uncharacterized O-methyltransferases. Collectively, these efforts enrich our knowledge of how biosensing can aid metabolic engineering and constitute the foundation for future improvements in vanillin pathway productivity.

Carboxylic acid reductases (CARs) catalyze the conversion of carboxylic acids to aldehydes, which are a valuable class of chemicals for many consumer and industrial applications. CARs generally exhibit broad substrate specificity that encompasses aromatic, aliphatic, and di/tri-carboxylic acids, enabling the development of biosynthetic pathways to a wide array of potential aldehyde products. De novo synthetic pathways implementing CARs have enabled the production of sustainable aldehyde products or utilized highly reactive aldehydes as intermediates in the production of chemicals including amines, alcohols, and alkanes. Recent determination of crystal structures of the domains of three CARs has provided insight into the substrate binding and domain dynamics of CARs, which could enable future engineering efforts to both alter the specificity of CAR and expand its potential in future synthetic pathways. In this review, we summarize the current structural and mechanistic understanding of CARs including substrate and catalytic scope, their potential for future engineering, and the advantages and challenges of their application in de novo synthesis.

Bio-sourced macromolecules such as cyanophycin are an attractive source for alternative, sustainable plastics. While the chemical structure and biological function of cyanophycin have been previously investigated, its material properties remain largely unexplored. This study investigates the structural, thermal, mechanical, and solution properties of cyanophycin produced from recombinant Escherichia coli . Unplasticized, it has an elastic compression modulus of about 560 MPa and undergoes brittle failure at 78 MPa. Cyanophycin exhibits thermal stability in air up to 200 °C and does not undergo glass transition within its limit of thermal stability. The polypeptide is amorphous and has no long-range ordering in the solid state. In solution, water-soluble cyanophycin is thermoresponsive, exhibiting both upper and lower critical solution temperatures. Because the feasibility of industrial scale cyanophycin production through fermentation has been well studied, an expanded understanding of its materials properties should contribute to the development of new applications for this biopolymer.

Carboxylic acid reductases (CARs) have been harnessed in metabolic pathways to produce aldehydes in engineered organisms. However, desired aldehyde products inhibit cell growth and limit product titers currently achievable from fermentative processes. Aldehyde toxicity can be entirely circumvented by performing aldehyde biosynthesis in non-cellular systems. Use of purified CARs for preparative-scale aldehyde synthesis has been limited by in vitro turnover of model CARs, such as Car Ni from Nocardia iowensis , despite robust conversion of substrates associated with expression in heterologous hosts such as E. coli and yeast. In this study, we report that in vitro activity of Car Ni is inhibited by formation of the co-product pyrophosphate, and that pairing of an inorganic pyrophosphatase (Ppa Ec ) with Car Ni substantially improves the rate and yield of aldehyde biosynthesis. We demonstrate that, in the presence of Ppa Ec , Michaelis–Menten kinetic models based on initial rate measurements accurately predict Car Ni kinetics within an in vitro pathway over longer timescales. We rationalize our novel observations for Car Ni by examining previously posed arguments for pyrophosphate hydrolysis made in the context of other adenylate-forming enzymes. Overall, our findings may aid in increasing adoption of CARs for cell-free in vitro aldehyde biosynthetic processes.

In experimental evolution, scientists evolve organisms in the lab, typically by challenging them to new environmental conditions. How best to evolve a desired trait? Should the challenge be applied abruptly, gradually, periodically, sporadically? Should one apply chemical mutagenesis, and do strains with high innate mutation rate evolve faster? What are ideal population sizes of evolving populations? There are endless strategies, beyond those that can be exposed by individual labs. We therefore arranged a community challenge, Evolthon, in which students and scientists from different labs were asked to evolve Escherichia coli or Saccharomyces cerevisiae for an abiotic stress—low temperature. About 30 participants from around the world explored diverse environmental and genetic regimes of evolution. After a period of evolution in each lab, all strains of each species were competed with one another. In yeast, the most successful strategies were those that used mating, underscoring the importance of sex in evolution. In bacteria, the fittest strain used a strategy based on exploration of different mutation rates. Different strategies displayed variable levels of performance and stability across additional challenges and conditions. This study therefore uncovers principles of effective experimental evolutionary regimens and might prove useful also for biotechnological developments of new strains and for understanding natural strategies in evolutionary arms races between species. Evolthon constitutes a model for community-based scientific exploration that encourages creativity and cooperation., This Community Page article describes Evolthon; a first-of-its-kind community-based effort, involving about 30 participant labs around the world, aiming to explore the best strategy for evolving microorganisms to cope with an environmental challenge.

Typical renewable liquid fuel alternatives to gasoline are not entirely compatible with current infrastructure. We have engineered Escherichia coli to selectively produce alkanes found in gasoline (propane, butane, pentane, heptane, and nonane) from renewable substrates such as glucose or glycerol. Our modular pathway framework achieves carbon-chain extension by two different mechanisms. A fatty acid synthesis route is used to generate longer chains heptane and nonane, while a more energy efficient alternative, reverse-β-oxidation, is used for synthesis of propane, butane, and pentane. We demonstrate that both upstream (thiolase) and intermediate (thioesterase) reactions can act as control points for chain-length specificity. Specific free fatty acids are subsequently converted to alkanes using a broad-specificity carboxylic acid reductase and a cyanobacterial aldehyde decarbonylase (AD). The selectivity obtained by different module pairings provides a foundation for tuning alkane product distribution for desired fuel properties. Alternate ADs that have greater activity on shorter substrates improve observed alkane titer. However, even in an engineered host strain that significantly reduces endogenous conversion of aldehyde intermediates to alcohol byproducts, AD activity is observed to be limiting for all chain lengths. Given these insights, we discuss guiding principles for pathway selection and potential opportunities for pathway improvement., United States. Army Research Office (Institute for Collaborative Biotechnologies. Grant W911NF-09-0001), Shell Global Solutions (US) Inc., National Science Foundation (U.S.). Graduate Research Fellowship Program, United States. Dept. of Energy. Office of Science Graduate Fellowship Program

Efforts are underway to construct several recoded genomes anticipated to exhibit multi-virus resistance, enhanced non-standard amino acid (NSAA) incorporation, and capability for synthetic biocontainment. Though we succeeded in pioneering the first genomically recoded organism (Escherichia colistrain C321.ΔA), its fitness is far lower than that of its non-recoded ancestor, particularly in defined media. This fitness deficit severely limits its utility for NSAA-linked applications requiring defined media such as live cell imaging, metabolic engineering, and industrial-scale protein production. Here, we report adaptive evolution of C321.ΔA for more than 1,000 generations in independent replicate populations grown in glucose minimal media. Evolved recoded populations significantly exceed the growth rates of both the ancestral C321.ΔA and non-recoded strains, permitting use of the recoded chassis in several new contexts. We use next-generation sequencing to identify genes mutated in multiple independent populations, and we reconstruct individual alleles in ancestral strains via multiplex automatable genome engineering (MAGE) to quantify their effects on fitness. Several selective mutations occur only in recoded evolved populations, some of which are associated with altering the translation apparatus in response to recoding, whereas others are not apparently associated with recoding, but instead correct for off-target mutations that occurred during initial genome engineering. This report demonstrates that laboratory evolution can be applied after engineering of recoded genomes to streamline fitness recovery compared to application of additional targeted engineering strategies that may introduce further unintended mutations. In doing so, we provide the most comprehensive insight to date into the physiology of the commonly used C321.ΔA strain.Significance StatementAfter demonstrating construction of an organism with an altered genetic code, we sought to evolve this organism for many generations to improve its fitness and learn what unique changes natural selection would bestow upon it. Although this organism initially had impaired fitness, we observed that adaptive laboratory evolution resulted in several selective mutations that corrected for insufficient translation termination and for unintended mutations that occurred when originally altering the genetic code. This work further bolsters our understanding of the pliability of the genetic code, it will help guide ongoing and future efforts seeking to recode genomes, and it results in a useful strain for non-standard amino acid incorporation in numerous contexts relevant for research and industry.

Progress in genetic code expansion requires accurate, selective, and high-throughput detection of non-standard amino acid (NSAA) incorporation into proteins. Here, we discover how the N-end rule pathway of protein degradation applies to commonly used NSAAs. We show that several NSAAs are N-end stabilizing and demonstrate that other NSAAs can be made stabilizing by rationally engineering the N-end rule adaptor protein ClpS. We use these insights to engineer a synthetic quality control method, termed “Post-Translational Proofreading” (PTP). By implementing PTP, false positive proteins resulting from misincorporation of structurally similar standard amino acids or undesired NSAAs rapidly degrade, enabling high-accuracy discrimination of desired NSAA incorporation. We illustrate the utility of PTP during evolution of the biphenylalanine orthogonal translation system used for synthetic biocontainment. Our new OTS is more selective and confers lower escape frequencies and greater fitness in all tested biocontained strains. Our approach presents a new paradigm for molecular recognition of amino acids in target proteins.

Aromatic aldehydes are useful in numerous applications, especially as flavors, fragrances, and pharmaceutical precursors. However, microbial synthesis of aldehydes is hindered by rapid, endogenous, and redundant conversion of aldehydes to their corresponding alcohols. We report the construction of an Escherichia coli K-12 MG1655 strain with reduced aromatic aldehyde reduction (RARE) that serves as a platform for aromatic aldehyde biosynthesis. Six genes with reported activity on the model substrate benzaldehyde were rationally targeted for deletion: three genes that encode aldo-keto reductases and three genes that encode alcohol dehydrogenases. Upon expression of a recombinant carboxylic acid reductase in the RARE strain and addition of benzoate during growth, benzaldehyde remained in the culture after 24 h, with less than 12% conversion of benzaldehyde to benzyl alcohol. Although individual overexpression results demonstrated that all six genes could contribute to benzaldehyde reduction in vivo, additional experiments featuring subset deletion strains revealed that two of the gene deletions were dispensable under the conditions tested. The engineered strain was next investigated for the production of vanillin from vanillate and succeeded in preventing formation of the byproduct vanillyl alcohol. A pathway for the biosynthesis of vanillin directly from glucose was introduced and resulted in a 55-fold improvement in vanillin titer when using the RARE strain versus the wild-type strain. Finally, synthesis of the chiral pharmaceutical intermediate L-phenylacetylcarbinol (L-PAC) was demonstrated from benzaldehyde and glucose upon expression of a recombinant mutant pyruvate decarboxylase in the RARE strain. Beyond allowing accumulation of aromatic aldehydes as end products in E. coli, the RARE strain expands the classes of chemicals that can be produced microbially via aldehyde intermediates.

Background Vanillin is an industrially valuable molecule that can be produced from simple carbon sources in engineered microorganisms such as Saccharomyces cerevisiae and Escherichia coli. In E. coli, de novo production of vanillin was demonstrated previously as a proof of concept. In this study, a series of data-driven experiments were performed in order to better understand limitations associated with biosynthesis of vanillate, which is the immediate precursor to vanillin. Results Time-course experiments monitoring production of heterologous metabolites in the E. coli de novo vanillin pathway revealed a bottleneck in conversion of protocatechuate to vanillate. Perturbations in central metabolism intended to increase flux into the heterologous pathway increased average vanillate titers from 132 to 205 mg/L, but protocatechuate remained the dominant heterologous product on a molar basis. SDS-PAGE, in vitro activity measurements, and l-methionine supplementation experiments suggested that the decline in conversion rate was influenced more by limited availability of the co-substrate S-adenosyl-l-methionine (AdoMet or SAM) than by loss of activity of the heterologous O-methyltransferase. The combination of metJ deletion and overexpression of feedback-resistant variants of metA and cysE, which encode enzymes involved in SAM biosynthesis, increased average de novo vanillate titers by an additional 33 % (from 205 to 272 mg/L). An orthogonal strategy intended to improve SAM regeneration through overexpression of native mtn and luxS genes resulted in a 25 % increase in average de novo vanillate titers (from 205 to 256 mg/L). Vanillate production improved further upon supplementation with methionine (as high as 419 ± 58 mg/L), suggesting potential for additional enhancement by increasing SAM availability. Conclusions Results from this study demonstrate context dependency of engineered pathways and highlight the limited methylation capacity of E. coli. Unlike in previous efforts to improve SAM or methionine biosynthesis, we pursued two orthogonal strategies that are each aimed at deregulating multiple reactions. Our results increase the working knowledge of SAM biosynthesis engineering and provide a framework for improving titers of metabolic products dependent upon methylation reactions., National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (Grant EEC-0540879), National Science Foundation (U.S.). Graduate Research Fellowship

This review paper describes systems used to cultivate microalgae for biofuel production. It addresses general design considerations pertaining to reactors that use natural light and photosynthetic growth mechanisms, with an emphasis on large-scale reactors. Important design aspects include lighting, mixing, water consumption, CO2 consumption, O2 removal, nutrient supply, temperature, and pH. Though open pond reactors are the most affordable option, they provide insufficient control of nearly all growth conditions. In contrast, a variety of closed reactors offer substantial control, but few feature the likelihood for levels of productivity that offset their high cost. One of the greatest challenges of closed photobioreactor design is how to increase reactor size in order to benefit from economy of scale and produce meaningful quantities of biofuel. This paper also highlights the concept of combining open and closed systems and concludes with a discussion regarding a possible optimal reactor configuration.

Increasingly complex metabolic pathways have been engineered by modifying natural pathways and establishing de novo pathways with enzymes from a variety of organisms. Here we apply retro-biosynthetic screening to a modular pathway design to identify a redox neutral, theoretically high yielding route to a branched C6 alcohol. Enzymes capable of converting natural E. coli metabolites into 4-methyl-pentanol (4MP) via coenzyme A (CoA)-dependent chemistry were taken from nine different organisms to form a ten-step de novo pathway. Selectivity for 4MP is enhanced through the use of key enzymes acting on acyl-CoA intermediates, a carboxylic acid reductase from Nocardia iowensis and an alcohol dehydrogenase from Leifsonia sp. strain S749. One implementation of the full pathway from glucose demonstrates selective carbon chain extension and acid reduction with 4MP constituting 81% (90±7 mg l(-1)) of the observed alcohol products. The highest observed 4MP titre is 192±23 mg l(-1). These results demonstrate the ability of modular pathway screening to facilitate de novo pathway engineering.