A curcumin direct protein biosensor for cell-free prototyping.

In synthetic biology, biosensors are routinely coupled with a gene expression system for detecting small molecules and physical signals. We reveal a fluorescent complex, based on the interaction of an coli double bond reductase (CurA), as a detection unit with its substrate curcumin-we call this a direct protein (DiPro) biosensor. Using a cell-free synthetic biology approach, we use the CurA DiPro biosensor to fine tune 10 reaction parameters (cofactor, substrate, and enzyme levels) for cell-free curcumin biosynthesis, assisted through acoustic liquid handling robotics.

Sequestration of P fractions in the soils of an incipient ferralisation chronosequence on a humid tropical volcanic island.

Background: Phosphorus (P) is the limiting nutrient in many mature tropical forests. The ecological significance of declining P stocks as soils age is exacerbated by much of the remaining P being progressively sequestered. However, the details of how and where P is sequestered during the ageing in tropical forest soils remains unclear.

High-yield 'one-pot' biosynthesis of raspberry ketone, a high-value fine chemical.

Cell-free extract and purified enzyme-based systems provide an attractive solution to study biosynthetic strategies towards a range of chemicals. 4-(4-hydroxyphenyl)-butan-2-one, also known as raspberry ketone, is the major fragrance component of raspberry fruit and is used as a natural additive in the food and sports industry. Current industrial processing of the natural form of raspberry ketone involves chemical extraction from a yield of ∼1-4 mg kg of fruit.

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications.

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a natural producer of chloramphenicol, jadomycin, and pikromycin, which makes it an attractive candidate as a next-generation synthetic biology chassis.

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.

A Cell-Free Toolkit for Synthetic Biology.

Prokaryotic cell-free coupled transcription-translation (TX-TL) systems are emerging as a powerful tool to examine natural product biosynthetic pathways in a test tube. The key advantages of this approach are the reduced experimental time scales and controlled reaction conditions. To realize this potential, it is essential to develop specialized cell-free systems in organisms enriched for biosynthetic gene clusters.

Developing synthetic biology for industrial biotechnology applications.

Since the beginning of the 21st Century, synthetic biology has established itself as an effective technological approach to design and engineer biological systems. Whilst research and investment continues to develop the understanding, control and engineering infrastructural platforms necessary to tackle ever more challenging systems - and to increase the precision, robustness, speed and affordability of existing solutions - hundreds of start-up companies, predominantly in the US and UK, are already translating learnings and potential applications into commercially viable tools, services and products. Start-ups and SMEs have been the predominant channel for synthetic biology commercialisation to date, facilitating rapid response to changing societal interests and market pull arising from increasing awareness of health and global sustainability issues.

Model-based optimization of antibody galactosylation in CHO cell culture.

Exerting control over the glycan moieties of antibody therapeutics is highly desirable from a product safety and batch-to-batch consistency perspective. Strategies to improve antibody productivity may compromise quality, while interventions for improving glycoform distribution can adversely affect cell growth and productivity. Process design therefore needs to consider the trade-off between preserving cellular health and productivity while enhancing antibody quality.

Functionalizing cell-mimetic giant vesicles with encapsulated bacterial biosensors.

The design of vesicle microsystems as artificial cells (bottom-up synthetic biology) has traditionally relied on the incorporation of molecular components to impart functionality. These cell mimics have reduced capabilities compared with their engineered biological counterparts (top-down synthetic biology), as they lack the powerful metabolic and regulatory pathways associated with living systems. There is increasing scope for using whole intact cellular components as functional modules artificial cells, as a route to increase the capabilities of artificial cells.

Synthetic gene regulation for independent external induction of the pseudohyphal growth phenotype.

Pseudohyphal growth is a multicellular phenotype naturally performed by wild budding yeast cells in response to stress. Unicellular yeast cells undergo gross changes in their gene regulation and elongate to form branched filament structures consisting of connected cells. Here, we construct synthetic gene regulation systems to enable external induction of pseudohyphal growth in .

Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics.

The polyhydroxyalkanoates (PHAs) are microbially-produced biopolymers that could potentially be used as sustainable alternatives to oil-derived plastics. However, PHAs are currently more expensive to produce than oil-derived plastics. Therefore, more efficient production processes would be desirable.

EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology.

Development of advanced synthetic biology tools is always in demand since they act as a platform technology to enable rapid prototyping of biological constructs in a high-throughput manner. EcoFlex is a modular cloning (MoClo) kit for Escherichia coli and is based on the Golden Gate principles, whereby Type IIS restriction enzymes (BsaI, BsmBI, BpiI) are used to construct modular genetic elements (biological parts) in a bottom-up approach. Here, we describe a collection of plasmids that stores various biological parts including promoters, RBSs, terminators, ORFs, and destination vectors, each encoding compatible overhangs allowing hierarchical assembly into single transcription units or a full-length polycistronic operon or biosynthetic pathway.

Rapid acquisition and model-based analysis of cell-free transcription-translation reactions from nonmodel bacteria.

Native cell-free transcription-translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, , is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters.

A systematic analysis of the expression of the anti-HIV VRC01 antibody in Pichia pastoris through signal peptide optimization.

Pichia pastoris (Komagataella phaffi) has been used for recombinant protein production for over 30 years with over 5000 proteins reported to date. However, yields of antibody are generally low. We have evaluated the effect of secretion signal peptides on the production of a broadly neutralizing antibody (VRC01) to increase yield.

Construction of hybrid regulated mother-specific yeast promoters for inducible differential gene expression.

Engineered promoters with predefined regulation are a key tool for synthetic biology that enable expression on demand and provide the logic for genetic circuits. To expand the availability of synthetic biology tools for S. cerevisiae yeast, we here used hybrid promoter engineering to construct tightly-controlled, externally-inducible promoters that only express in haploid mother cells that have contributed a daughter cell to the population.

Inactivation of the dnaK gene in Clostridium difficile 630 Δerm yields a temperature-sensitive phenotype and increases biofilm-forming ability.

Clostridium difficile infection is a growing problem in healthcare settings worldwide and results in a considerable socioeconomic impact. New hypervirulent strains and acquisition of antibiotic resistance exacerbates pathogenesis; however, the survival strategy of C. difficile in the challenging gut environment still remains incompletely understood.

A streamlined cloning workflow minimising the time-to-strain pipeline for Pichia pastoris.

Although recent advances in E. coli self-assembly have greatly simplified cloning, these have not yet been harnessed for the high-throughput generation of expression strains in the early research and discovery phases of biopharmaceutical production. Here, we have refined the technique and incorporated it into a streamlined workflow for the generation of Pichia pastoris expression strains, reducing the timeline by a third and removing the reliance on DNA editing enzymes, which often require troubleshooting and increase costs.

A transatlantic perspective on 20 emerging issues in biological engineering.

Advances in biological engineering are likely to have substantial impacts on global society. To explore these potential impacts we ran a horizon scanning exercise to capture a range of perspectives on the opportunities and risks presented by biological engineering. We first identified 70 potential issues, and then used an iterative process to prioritise 20 issues that we considered to be emerging, to have potential global impact, and to be relatively unknown outside the field of biological engineering.

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application.

NAD(P)H-dependent oxidoreductases catalyze the reduction or oxidation of a substrate coupled to the oxidation or reduction, respectively, of a nicotinamide adenine dinucleotide cofactor NAD(P)H or NAD(P). NAD(P)H-dependent oxidoreductases catalyze a large variety of reactions and play a pivotal role in many central metabolic pathways. Due to the high activity, regiospecificity and stereospecificity with which they catalyze redox reactions, they have been used as key components in a wide range of applications, including substrate utilization, the synthesis of chemicals, biodegradation and detoxification.

Recombinant expression and characterisation of the oxygen-sensitive 2-enoate reductase from Clostridium sporogenes.

'Ene'-reductases have attracted significant attention for the preparation of chemical intermediates and biologically active products. To date, research has been focussed primarily on Old Yellow Enzyme-like proteins, due to their ease of handling, whereas 2-enoate reductases from clostridia have received much less attention, because of their oxygen sensitivity and a lack of suitable expression systems. A hypothetical 2-enoate reductase gene, fldZ, was identified in Clostridium sporogenes DSM 795.

Synthetic biology in the UK - An outline of plans and progress.

Synthetic biology is capable of delivering new solutions to key challenges spanning the bioeconomy, both nationally and internationally. Recognising this significant potential and the associated need to facilitate its translation and commercialisation the UK government commissioned the production of a national Synthetic Biology Roadmap in 2011, and subsequently provided crucial support to assist its implementation. Critical infrastructural investments have been made, and important strides made towards the development of an effectively connected community of practitioners and interest groups.

The Interplay between Feedback and Buffering in Cellular Homeostasis.

Buffering, the use of reservoirs of molecules to maintain concentrations of key molecular species, and negative feedback are the primary known mechanisms for robust homeostatic regulation. To our knowledge, however, the fundamental principles behind their combined effect have not been elucidated. Here, we study the interplay between buffering and negative feedback in the context of cellular homeostasis.