• Amer M, Wojcik E, Sun C, Hoeven R, Hughes J, Faulkner M, Yunus IS, Tait S, Johannissen L, Hardman S, Heyes D, Chen G-Q, Smith MH, Jones PR, Toogood H, Scrutton N. (2020). Low Carbon Strategies for Sustainable Bio-alkane Gas Production and Renewable Energy. Energy Environ. Sci., 13, 1818-31

Propane and butane are the main constituents of liquefied petroleum gas and are used extensively for transport and domestic use. They are clean burning fuels, suitable for the development of low carbon footprint fuel and energy policies. Here, we present blueprints for the production of bio-alkane gas (propane and butane) through the conversion of waste volatile fatty acids by bacterial culture. We show that bio-propane and bio-butane can be produced photo-catalytically by bioengineered strains of E. coli and Halomonas (in non-sterile seawater) using fatty acids derived from biomass or industrial waste, and by Synechocystis (using carbon dioxide as feedstock).

• Bommareddy RR, Wang Y, Pearcy N, Hayes M, Lester E, Minton NP, Conradie AV. (2020). A Sustainable Chemicals Manufacturing Paradigm Using CO2 and Renewable H2. iScience, 23: 101218.

The chemical industry must decarbonize to align with UN Sustainable Development Goals. A shift toward circular economies makes CO₂ an attractive feedstock for producing chemicals, provided renewable H₂ is available through technologies such as supercritical water (scH₂O) gasification. Furthermore, high carbon and energy efficiency is paramount to favorable techno-economics, which poses a challenge to chemo-catalysis. This study demonstrates continuous gas fermentation of CO₂ and H₂ by the cell factory, Cupriavidus necator, to (R,R)-2,3-butanediol and isopropanol as case studies. Although a high carbon efficiency of 0.75 [(C-mol product)/(C-mol CO₂)] is exemplified, the poor energy efficiency of biological CO₂ fixation requires ∼8 [(mol H₂)/(mol CO₂)], which is techno-economically infeasible for producing commodity chemicals. Heat integration between exothermic gas fermentation and endothermic scH₂O gasification overcomes this energy inefficiency. This study unlocks the promise of sustainable manufacturing using renewable feedstocks by combining the carbon efficiency of bio-catalysis with energy efficiency enforced through process engineering.

• Finnigan W, Citoler J, Cosgrove SC, Turner NJ. (2020). Rapid model-based optimization of a two-enzyme system for continuous reductive amination in flow. Org. Process Res. Dev., 24: 1969 – 77.

Enzymes are increasingly combined into multienzyme systems for cost and productivity benefits. Further advantages can be gained through the use of immobilized enzymes, allowing continuous biotransformations in flow. However, the optimization of such multienzyme systems is challenging, particularly where immobilized enzymes are used. Here, we meet this challenge using both mechanistic and empirical modeling to optimize a reaction involving a reductive aminase and a glucose dehydrogenase for continuous biocatalytic reductive amination in flow. Crucially, the construction of the mechanistic model was achieved quickly, with only a few important parameters determined experimentally, and ensemble modeling used to facilitate the use of estimates or literature values.

• Messiha Hanan L, Payne Karl AP, Scrutton Nigel S, Leys David (2021). A Biological Route to Conjugated Alkenes: Microbial Production of Hepta-1,3,5-triene. ACS Synthetic Biology, 10: 228.

Conjugated alkenes such as dienes and polyenes have a range of applications as pharmaceutical agents and valuable building blocks in the polymer industry. Development of a renewable route to these compounds provides an alternative to fossil fuel derived production. The enzyme family of the UbiD decarboxylases offers substantial scope for alkene production, readily converting poly unsaturated acids. However, biochemical pathways producing the required substrates are poorly characterized, and UbiD-application has hitherto been limited to biological styrene production. Herein, we present a proof-of-principle study for microbial production of polyenes using a bioinspired strategy employing a polyketide synthase (PKS) in combination with a UbiD-enzyme. Deconstructing a bacterial iterative type II PKS enabled repurposing the broad-spectrum antibiotic andrimid biosynthesis pathway to access the metabolic intermediate 2,4,6-octatrienoic acid, a valuable chemical for material and pharmaceutical industry. Combination with the fungal ferulic acid decarboxylase (Fdc1) led to a biocatalytic cascade-type reaction for the production of hepta-1,3,5-triene in vivo. Our approach provides a novel route to generate unsaturated hydrocarbons and related chemicals and provides a blue-print for future development and application.

• Roberts AD, Payne KAP, Cosgrove S, Tilakaratna V, Penafiel I, Finnigan W, Turner NJ, Scrutton NS. (2021). Enzyme immobilisation on wood-derived cellulose scaffoldsviacarbohydrate-binding module fusion constructs. Green Chemistry, 23: 4716-32.

The development of cost-effective and green enzyme immobilisation techniques will facilitate the adoption of continuous flow biocatalysis (CFB) by industry and academia. In this work, a relatively mild sulfite pulping process was employed to remove lignin and hemicellulose from wood with minimal disruption of its native porous structure, resulting in aligned macroporous cellulosic monoliths termed cellulose scaffolds (CSs). By engineering carbohydrate-binding modules (CBMs) onto the termini of recombinant proteins, the CSs could be employed as low-cost, renewable and biodegradable materials for enzyme immobilisation without any further chemical functionalisation. CBM-tagged fluorescent proteins were initially employed to demonstrate proof-of-principle and to optimise immobilisation conditions; this resulted in initial protein loadings as high as 5.24 wt% and immobilisation efficiencies as high as 97.1%. The process was then translated to a CBM-tagged ω-transaminase (ωTA) from Bacillus megaterium, obtaining enzyme loadings and immobilisation efficiencies as high as 3.99 wt% and 82.4%, respectively. A demonstrative CFB reaction with the immobilised CBM-tagged ωTA displayed ca. 95 ± 5% conversion efficiency relative to the free enzyme in solution under analogous conditions, suggesting that CBM-tagged recombinant enzymes immobilised on wood-derived CSs could potentially compete with other, more complex and costly enzyme immobilisation technologies.

• Roberts AD, Whittal DR, Breitling R, Takano E, Blaker JJ, Hay S, Scrutton NS. (2021). Blood, sweat and tears: extraterrestrial regolith biocomposites with in vivo binders. Materials Today Bio, 12: 100136.

The proverbial phrase ‘you can’t get blood from a stone’ is used to describe a task that is practically impossible regardless of how much force or effort is exerted. This phrase is well-suited to humanity’s first crewed mission to Mars, which will likely be the most difficult and technologically challenging human endeavor ever undertaken. The high cost and significant time delay associated with delivering payloads to the Martian surface means that exploitation of resources in situ — including inorganic rock and dust (regolith), water deposits, and atmospheric gases — will be an important part of any crewed mission to the Red Planet. Yet there is one significant, but chronically overlooked, source of natural resources that will — by definition — also be available on any crewed mission to Mars: the crew themselves. In this work, we explore the use of human serum albumin (HSA) — a common protein obtained from blood plasma — as a binder for simulated Lunar and Martian regolith to produce so-called ‘extraterrestrial regolith biocomposites (ERBs).’ In essence, HSA produced by astronauts in vivo could be extracted on a semi-continuous basis and combined with Lunar or Martian regolith to ‘get stone from blood’, to rephrase the proverb. Employing a simple fabrication strategy, HSA-based ERBs were produced and displayed compressive strengths as high as 25.0 MPa. For comparison, standard concrete typically has a compressive strength ranging between 20 and 32 MPa. The incorporation of urea — which could be extracted from the urine, sweat, or tears of astronauts — could further increase the compressive strength by over 300% in some instances, with the best-performing formulation having an average compressive strength of 39.7 MPa. Furthermore, we demonstrate that HSA-ERBs have the potential to be 3D-printed, opening up an interesting potential avenue for extraterrestrial construction using human-derived feedstocks. The mechanism of adhesion was investigated and attributed to the dehydration-induced reorganization of the protein secondary structure into a densely hydrogen-bonded, supramolecular β-sheet network — analogous to the cohesion mechanism of spider silk. For comparison, synthetic spider silk and bovine serum albumin (BSA) were also investigated as regolith binders — which could also feasibly be produced on a Martian colony with future advancements in biomanufacturing technology.

• Russell M, Currin A, Rowe W, Cheng, G, Barran P, Scrutton NS. (2022). Baseline proteomics characterisation of the emerging host biomanufacturing organism Halomonas bluephagenesis. Sci Data 9, 492.

Despite its greener credentials, biomanufacturing remains financially uncompetitive compared with the higher carbon emitting, hydrocarbon-based chemical industry. Replacing traditional chassis such as E. coli with novel robust organisms, are a route to cost reduction for biomanufacturing. Extremophile bacteria such as the halophilic Halomonas bluephagenesis TD01 exemplify this potential by thriving in environments inherently inimical to other organisms, so reducing sterilisation costs. Novel chassis are inevitably less well annotated than established organisms. Rapid characterisation along with community data sharing will facilitate adoption of such organisms for biomanufacturing. The data record comprises a newly sequenced genome for the organism and evidence via LC-MS based proteomics for expression of 1160 proteins (30% of the proteome) including baseline quantification of 1063 proteins (27% of the proteome), and a spectral library enabling re-use for targeted LC-MS proteomics assays. Protein data are annotated with KEGG Orthology, enabling rapid matching of quantitative data to pathways of interest to biomanufacturing.

• Sun C, Theodoropoulos C, Scrutton N. (2020). Techno-economic assessment of microbial limonene production. Bioresour. Technol., 300: 122666-73.

To satisfy the growing demand for limonene, novel pathways for microbial production of limonene have been sought. A techno-economic analysis is carried out for one such process producing limonene from sugar at an industrial plant scale to assess potential economic viability. A conceptual design of the process is developed, in which a gas stripping-solvent scrubbing method is chosen for recovering limonene from bioreactors based on consideration of payback time and process operability. Minimum limonene selling prices are estimated over a range of fermentation productivity based on the calculation of net present value using discounted cash flow method. Under 45% of the maximum theoretical yield, the selling price reaches $19.9/kg, which could be competitive with established production processes when fermentation productivity is above 0.7 kg/(m3·h). Reduction of cost could be realised through improvement of microbial strains, utilisation of cheaper feedstocks, reduction in capital investment and strategic business planning.

• Thompson MP, Peñafiel I, Cosgrove SC, Turner NJ. (2019). Biocatalysis Using Immobilized Enzymes in Continuous Flow for the Synthesis of Fine Chemicals. Organic Process Research & Development, 23: 9-18.

Biocatalysis has emerged as one of the most promising technologies to enable green synthesis of important chemicals, due to the ambient conditions generally applied for these reactions. Nonetheless, a general uptake of enzymatic transformations has been hindered by the perceived high cost of recombinant proteins. Recent interest in continuous flow from the synthetic chemistry community has now begun to spread to biotransformations, with protein immobilization playing a key part. As a consequence, continuous biotransformations using immobilized enzymes are becoming more accessible to nonexperts. This review will discuss several recent examples of continuous biotransformations that use immobilization, with a focus on examples involving fine chemical synthesis.