Refactoring of a synthetic raspberry ketone pathway with EcoFlex.

BACKGROUND:  A key focus of synthetic biology is to develop microbial or cell-free based biobased routes to value-added chemicals such as fragrances. Originally, we developed the EcoFlex system, a Golden Gate toolkit, to study genes/pathways flexibly using Escherichia coli heterologous expression. In this current work, we sought to use EcoFlex to optimise a synthetic raspberry ketone biosynthetic pathway. Raspberry ketone is a high-value (~ £20,000 kg-1) fine chemical farmed from raspberry (Rubeus rubrum) fruit. RESULTS:  By applying a synthetic biology led design-build-test-learn cycle approach, we refactor the raspberry ketone pathway from a low level of productivity (0.2 mg/L), to achieve a 65-fold (12.9 mg/L) improvement in production. We perform this optimisation at the prototype level (using microtiter plate cultures) with E. coli DH10ß, as a routine cloning host. The use of E. coli DH10ß facilitates the Golden Gate cloning process for the screening of combinatorial libraries. In addition, we also newly establish a novel colour-based phenotypic screen to identify productive clones quickly from solid/liquid culture. CONCLUSIONS:  Our findings provide a stable raspberry ketone pathway that relies upon a natural feedstock (L-tyrosine) and uses only constitutive promoters to control gene expression. In conclusion we demonstrate the capability of EcoFlex for fine-tuning a model fine chemical pathway and provide a range of newly characterised promoter tools gene expression in E. coli.

Metabolic engineering to improve the biomanufacturing efficiency of acetic acid bacteria: advances and prospects.

With the high tolerance for acetic acid and abundant multifunctional enzymes, acetic acid bacteria (AAB), as valuable biocatalysts, exhibit great advantages during industrial acetic acid production and value-added chemical fermentation. However, low biomass and a low production rates arising from acid stress remains major hurdles in industrial processes. Engineering AAB with excellent properties is expected to obtain economically viable production and facilitates their biotechnological applications. Here, the investigation of acetic acid-tolerance mechanisms and metabolic features is discussed, and effective targets are provided for the metabolic engineering of AAB. Next, we review the advances in improving AAB and compare these advances with improvement to other model acid-tolerant microorganisms. Furthermore, future directions involving the combination of systems biology and synthetic biology to achieve efficient biomanufacturing in AAB are highlighted.

Toward Genome-Based Metabolic Engineering in Bacteria.

Prokaryotes modified stably on the genome are of great importance for production of fine and commodity chemicals. Traditional methods for genome engineering have long suffered from imprecision and low efficiencies, making construction of suitable high-producer strains laborious. Here, we review the recent advances in discovery and refinement of molecular precision engineering tools for genome-based metabolic engineering in bacteria for chemical production, with focus on the λ-Red recombineering and the clustered regularly interspaced short palindromic repeats/Cas9 nuclease systems. In conjunction, they enable the integration of in vitro-synthesized DNA segments into specified locations on the chromosome and allow for enrichment of rare mutants by elimination of unmodified wild-type cells. Combination with concurrently developing improvements in important accessory technologies such as DNA synthesis, high-throughput screening methods, regulatory element design, and metabolic pathway optimization tools has resulted in novel efficient microbial producer strains and given access to new metabolic products. These new tools have made and will likely continue to make a big impact on the bioengineering strategies that transform the chemical industry.

Recent advances in biotechnological applications of alcohol dehydrogenases.

Alcohol dehydrogenases (ADHs), which belong to the oxidoreductase superfamily, catalyze the interconversion between alcohols and aldehydes or ketones with high stereoselectivity under mild conditions. ADHs are widely employed as biocatalysts for the dynamic kinetic resolution of racemic substrates and for the preparation of enantiomerically pure chemicals. This review provides an overview of biotechnological applications for ADHs in the production of chiral pharmaceuticals and fine chemicals.

SYNBIOCHEM-a SynBio foundry for the biosynthesis and sustainable production of fine and speciality chemicals.

The Manchester Synthetic Biology Research Centre (SYNBIOCHEM) is a foundry for the biosynthesis and sustainable production of fine and speciality chemicals. The Centre's integrated technology platforms provide a unique capability to facilitate predictable engineering of microbial bio-factories for chemicals production. An overview of these capabilities is described.

Industrial systems biology and its impact on synthetic biology of yeast cell factories.

Engineering industrial cell factories to effectively yield a desired product while dealing with industrially relevant stresses is usually the most challenging step in the development of industrial production of chemicals using microbial fermentation processes. Using synthetic biology tools, microbial cell factories such as Saccharomyces cerevisiae can be engineered to express synthetic pathways for the production of fuels, biopharmaceuticals, fragrances, and food flavors. However, directing fluxes through these synthetic pathways towards the desired product can be demanding due to complex regulation or poor gene expression. Systems biology, which applies computational tools and mathematical modeling to understand complex biological networks, can be used to guide synthetic biology design. Here, we present our perspective on how systems biology can impact synthetic biology towards the goal of developing improved yeast cell factories. Biotechnol. Bioeng. 2016;113: 1164-1170. © 2015 Wiley Periodicals, Inc.

Guyparkeria halophila: Novel cell platform for the efficient valorization of carbon dioxide and thiosulfate into ectoine.

Utilizing carbon dioxide (CO2) for valuable chemical production is key to a circular economy. Current processes are costly due to limited microorganism use, low-value products, and the need for affordable energy. This study addresses these challenges by using industrial contaminants like thiosulfate (S2O32-) for CO2 conversion into ectoines. Ectoines, are important ingredients as pharmaceuticals and cosmetics. Here, six microbial genomes were identified as potential candidates to valorize CO2 and S2O32- into ectoine. After laboratory validation at 3 % NaCl, the fastest-growing strain, Guyparkeria halophila, was optimized at 6 %, 9 %, and 15 % NaCl, showing the highest specific ectoine contents (mgEct gbiomass-1) at 15 %. Batch bioreactors, combining optimal conditions, achieved maximum specific ectoine contents of 47 %. These results not only constitute the highest ectoine content so far reported by autotrophs and most of heterotrophs, but also the first proof of a novel valorization platform for CO2 and S2O32-, focused on pharmaceuticals production.

Transition-metal-catalyzed laboratory-scale carbon-carbon bond-forming reactions of ethylene.

Ethylene, the simplest alkene, is the most abundantly synthesized organic molecule by volume. It is readily incorporated into transition-metal-catalyzed carbon-carbon bond-forming reactions through migratory insertions into alkylmetal intermediates. Because of its D2h symmetry, only one insertion outcome is possible. This limits byproduct formation and greatly simplifies analysis. As described within this Minireview, many carbon-carbon bond-forming reactions incorporate a molecule (or more) of ethylene at ambient pressure and temperature. In many cases, a useful substituted alkene is incorporated into the product.

Photoenzymatic decarboxylation: A promising way to produce sustainable aviation fuels and fine chemicals.

As one of the fastest-growing carbon emission sources, the aviation sector is severely restricted by carbon emission reduction targets. Sustainable aviation fuel (SAF) has emerged as the most potential alternative to traditional aviation fuel, but harsh production technologies limit its commercialization. Fatty acids photodecarboxylase from Chlorella variabilis NC64A (CvFAP), the latest discovered photoenzyme, provides promising approaches to produce various carbon-neutral biofuels and fine chemicals. This review highlights the state-of-the-art strategies to enhance the application of CvFAP in carbon-neutral biofuel and fine chemicals production, including supplementing alkane as decoy molecular, screening efficient CvFAP variants with directed evolution, constructing genetic strains, employing biphasic catalytic system, and immobilizing CvFAP in an efficient photobioreactor. Furthermore, future opportunities are suggested to enhance photoenzymatic decarboxylation and explore the catalytic mechanism of CvFAP. This review provides a broad context to improve CvFAP catalysis and advance its potential applications.

Recent Advances in the Multistep Continuous Preparation of APIs and Fine Chemicals.

Over the last two decades, with the advent of continuous flow technologies, continuous processes have emerged as a major area in organic synthesis. In this context, continuous flow processes have been increasing in the preparation of Active Pharmaceutical Ingredients (APIs) and fine chemicals, such as complex synthetic intermediates, agrochemicals, and fragrances. Thus, the development of multi-step protocols has attracted special interest from the academic and industrial chemistry communities. In addition to the beneficial aspects intrinsically associated with continuous processes (e.g., waste reduction, optimal heat transfer, improved safety, and the possibility to work under harsh reaction conditions and with more dangerous reagents), these protocols also allow a rapid increase in molecular complexity. Moreover, in telescoped multi-step processes, isolation and purification steps are generally avoided or, if necessary, carried out in-line, presenting an important economy of time, solvents, reagents, and labor. Last, important synthetic strategies such as photochemical and electrochemical reactions are compatible with flow processes and are delivering relevant advances to the synthetic approaches. In this review, a general overview of the fundamentals of continuous flow processes is presented. Recent examples of multi-step continuous processes for the preparation of fine chemicals, including telescoped and end-to-end processes, are discussed, pointing out the possible advantages and/or limitations of each of these methodologies.

Multicomponent Reactions Applied to Total Synthesis of Biologically Active Molecules: A Short Review.

Multicomponent reactions (MCRs) are processes in which three or more starting materials are combined in the same reaction vessel, forming an adduct that contains all or most of the atoms of the starting materials. MCRs are one-pot processes that provide attractive advantages for the total synthesis of target molecules. These reactions allow rapid access to structurally complex adducts from particularly simple starting materials. Moreover, MCRs are generally intrinsically associated with principles of green syntheses, such as atom economy, minimization of isolation, and purification of synthetic intermediates, leading to large solvent economies and avoiding the production of large amounts of reaction waste. Thus, synthetic routes employing multicomponent reactions are generally more convergent, economical and often allow higher overall yields. In total synthesis, the use of MCRs has been mainly applied in the preparation of key advanced intermediates. Progress in the use of MCRs in total synthesis has been described over the last decades, including not only classical MCRs reactions (e.g. isocyanide-based transformations), but also non-traditional multicomponent reactions. Furthermore, reports concerning stereoselective multicomponent transformations are still scarce and present further development opportunities. This review aims to provide a general overview of the application of MCRs as key steps in the rapid preparation of structurally complex derivatives and fine chemicals. In special, some selected examples have been successfully applied for medicinal purposes. Finally, in some representative cases, either key intermediates formed during the reaction vessel or corresponding transition states have been disclosed in order to provide insights into the reaction mechanisms.

Shape and Stability Matter: Enhanced Catalytic Reactions via Sol-gel-Entrapped Catalysts.

The possibility to tune the solid catalyst morphology and the unique chemical and physical stability of organosilica-entrapped sol-gel catalysts allow the application of these catalysts to the synthesis of a wide variety of valued molecules, including polymers, manufactured by the fine-chemical industry. Referring to selected independent research achievements, we provide a practice oriented insight on these materials that will hopefully be useful in new, unified catalysis education aimed to foster the uptake of heterogeneous catalysis in the fine and specialty chemical industry.

Sustainable utilization of food waste for bioenergy production: A step towards circular bioeconomy.

The population growth, along with lifestyle changes, has resulted in unprecedented levels of food waste at all phases of the supply chain, including harvest, packing, transportation, and consumption. Conventional practices involve dumping of food waste with municipal garbage. However, these methods have serious environmental and health consequences. Food waste has a great recycling perspective due to its high biodegradability and water content, making it an ideal substrate for the production of biofuels and other industrially important chemicals including pigments, enzymes, organic acids, and essential oils. This review extensively covers conversion of food waste to generate bioenergy which will help to reduce environmental pollution and facilitate implementation of a circular bioeconomy. Moreover, review also highlights novel technologies like supercritical fluid extraction, ultra-sonication, pressurized liquid extraction, and microwave assisted extractions that are being employed in food waste management to increase the efficiency of value-added product recovery in an economically viable manner. Metabolic engineering of microorganisms for specificity of product would be a future breakthrough in food waste valorization/management.

Using different cultivation strategies and methods for the production of microalgal biomass as a raw material for the generation of bioproducts.

Microalgal biomass and its fine chemical production from microalgae have pioneered algal bioprocess technology with few limitations such as lab-to-industry. However, laboratory-scale transitions and industrial applications are hindered by a plethora of limitations comprising expensive in culturing methods. Therefore, to emphasize the profitable benefits, the algal culturing techniques appropriately employed for large-scale microalgal biomass yield necessitates intricate assessment to emphasize the profitable benefits. The present review holistically compiles the culturing strategies for improving microalgal biomass production based on appropriate factors like designing better bioreactor designs. On the other hand, synthetic biology approaches for abridging the effective industrial transition success explored recently. Prospects in synthetic biology for enhanced microalgal biomass production based on cultivation strategies and various mechanistic modes approach to enrich cost-effective and viable output are discussed. The State-of-the-art culturing techniques encompassing enhancement of photosynthetic activity, designing bioreactor design, and potential augmenting protocols for biomass yield employing indoor cultivation in both (Open and or/closed) methods are enumerated. Further, limitations hindering the microalgal bioproducts development are critically evaluated for improving culturing techniques for microalgal cell factories, subsequently escalating the cost-benefit ratio in bioproducts synthesis from microalgae. The comprehensive analysis could provide a rational and deeper detailed insight for microalgal entrepreneurs through alternative culturing technology viz., synthetic biology and genome engineering in an Industrial perspective arena.

Pyrolysis of cajuput (Melaleuca leucadendron) twigs and rice (Oryza sativa) husks to produce liquid smoke-containing fine chemicals for antibacterial agent application.

Pyrolysis of cajuput (Melaleuca leucadendron) twigs and rice (Oryza sativa) husks to produce liquid smoke and antibacterial activities of the liquid smoke fractions were investigated. The liquid smoke was produced by pyrolysis at 500 °C for 8 h and contained fine chemicals, such as acetic acid, carbonyl, cyclic ketones, and phenolic compounds with pH 2.1-2.9. The liquid smoke was separated by vacuum evaporation under vacuum conditions at low temperatures (40 °C, 50 °C, and 60 °C) to recover three fractions. The composition of each fraction influenced its antibacterial activities. Antibacterial activities of the liquid smoke fractions were tested against Gram-positive bacteria (Listeria monocytogenes, Bacillus subtilis, and Staphylococcus aureus) and Gram-negative bacteria (Salmonella typhimurium, Pseudomonas aeruginosa, and Escherichia coli). Whole fractions of the liquid smoke inhibited the six pathogenic bacteria, with the inhibition zone larger or smaller than the positive control. Among the liquid smoke fractions, the liquid recovered at 60 °C for the cajuput twigs and rice husks demonstrated a stronger inhibitory effect on bacterial growth than the other fractions.

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.

Self-Sufficient Class VII Cytochromes P450: From Full-Length Structure to Synthetic Biology Applications.

Members of class VII cytochromes P450 are catalytically self-sufficient enzymes containing a phthalate dioxygenase reductase-like domain fused to the P450 catalytic domain. Among these, CYP116B46 is the first enzyme for which the 3D structure of the whole polypeptide chain has been solved, shedding light on the interaction between its domains, which is crucial for catalysis. Most of these enzymes have been isolated from extremophiles or detoxifying bacteria that can carry out regio- and enantioselective oxidation of compounds of biotechnological interest. Protein engineering has generated mutants that can perform challenging organic reactions such as the anti-Markovnikov alkene oxidation. This potential, combined with the detailed 3D structure, forms the basis for further directed evolution studies aimed at widening their biotechnological exploitation.

Direct observation of endoglucanase fibrillation and rapid thickness identification of cellulose nanoplatelets using constructive interference.

Better understanding through direct observation of the mechanisms involved in chemical and enzymatic hydrolysis of biomass is of great importance, to implement a substitute for the common cellulose standards. We report the hydrolysis of biomass, using exclusively the parenchyma, to isolate cellulose nanoplatelets using a less harsh pretreatment. Then, we show direct evidence of the effect of endoglucanase on the structure of cellulose nanoplatelets, finding that amorphous cellulose is exclusively digested, loosening the cellulose nanofibrils in the process. The analysis of micrographs demonstrates that when cellulose nanoplatelets are deposited on a silicon wafer, its thickness can be qualitatively measured by the interference color detected using an optical microscope. This finding facilitates further studies of mechanisms involved in lignin removal and cellulose nanofibrils production by specific enzymatic digestion.

Biocatalytic derivatization of proteinogenic amino acids for fine chemicals.

Amino acids (AAs), which contain various functional groups including α-amino group, α-carboxyl group, and certain specific side-chains, exhibit a wide range of chemical properties. The derivatization of these functional groups of AAs via biocatalysis represents a sustainable approach for producing various chemicals, such as α-keto acids, α-hydroxyl acids, non-proteinogenic AAs, amines, peptides, higher alcohols, and phenylpropanoids, that are valuable in the pharmaceutical, chemical synthesis, cosmetic, and food industries. Here, we review recent advances in the derivatization of AAs to describe the design of derivatization reactions, how to overcome technical bottlenecks, and the potential range of chemicals that may be obtained. Furthermore, we highlight future prospects and address the challenges that remain in the derivatization of AAs for the production of fine chemicals.

Characterization of a novel marine unicellular alga, Pseudoneochloris sp. strain NKY372003 as a high carbohydrate producer.

Production of biofuels and fine chemicals from biomass-derived carbohydrates through biorefinery attracts much attention because it is recognized as an environmentally friendly process. Microalgae can serve as promising carbohydrate producers for biorefinery rather than woody and crop biomass due to high biomass productivity, high CO2 fixation, and no competition with food production. However, microalgae with high carbohydrate productivity have not been well investigated despite intensive studies of microalgal lipid production. In this study, the carbohydrate production of Pseudoneochloris sp. strain NKY372003 isolated as a high carbohydrate producer, was investigated. Cultivation conditions with various combinations of nutrient contents and photon flux density were examined to maximize the biomass and carbohydrate productivities. At the optimal condition, the biomass and carbohydrate production of this strain reached 8.11 ± 0.37 g/L and 5.5 ± 0.2 g/L, respectively. As far as we know, this is the highest carbohydrate production by microalgae among ever reported. Cell staining with Lugol's solution visualized intracellular starch granules. Because algal starch can be converted to biofuels and building blocks of fine chemicals, Pseudoneochloris sp. NKY372003 will be a promising candidate for production of fermentable carbohydrates towards biofuels and fine chemicals production.