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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - BIOSUSAMIN (The design and development of efficient biocatalytic cascades and biosynthetic pathways for the sustainable production of amines)

Teaser

Summary

The increasing quality of life and longevity of human beings stem from impressive developments in all fields of science and, in particular, from the remarkable achievements of synthetic chemistry over the last century. However, the current industrial manufacture of chemical compounds is based on processes that require mainly non-renewable resources and generate copious amount of waste.
The consequent progressive depletion of carbon fossil feedstock, the global climate change along with the continuous growth of the world population require the urgent transition to more efficient and sustainable routes for the conversion of feedstock (both petrol and bio-based) into pharmaceuticals, agrochemicals, fine chemicals and bulk chemicals. In fact, although there is public concern regarding energy supply and saving, approximately half of the demand of oil is employed for the production of building blocks and advanced intermediates by the chemical industry. Therefore, increasing resource efficiency is fundamental for the protection of the environment as well as for maintaining (and possibly improving) the quality of life and wealth of the European population (and worldwide). In the concept of green chemistry, resource efficiency means chemical and biotechnological processes that can convert starting material as feedstock into the final products with: i) the minimum number of (bio)chemical steps; ii) the generation of the minimum amount of waste; iii) the minimisation of energy consumption; iv) the preferential use of catalysts that are innocuous for the environment and the population, and possibly biodegradable. In the jargon of green chemistry, such ideal (bio)chemical processes possess a â€œhigh atom efficiencyâ€ and a low â€œenvironmental factorâ€. â€œHigh atom efficiencyâ€ means that most (or even all) the atoms that constitute the starting material will be found in the final product, with consequent minor (or even no) generation of waste. Furthermore, all the additional atoms in the final product must derive - as much as possible - from innocuous molecular oxygen (air) or other small molecules such as ammonia, molecular hydrogen, etc.
In this context, enzymes provide the opportunity for the development of a new generation of chemical processes, which can maximise â€œatom efficiencyâ€ and minimise environmental impact. First, enzymes fulfil all the twelve principles of green chemistry, that are: i) waste prevention, ii) elevated atom economy, iii) no hazardous chemical synthesis, iv) lower toxicity, v) compatibility with the use of green solvents such as water, vi) lower energy consumption, vii) compatibility with the use of renewable feedstock, viii) no requirement for protection and deprotection steps due to elevated enzymatic chemo-, regio- and stereoselectivity, ix) use of catalytic processes (i.e. enzymes are biocatalysts by definition); x) design for degradation (i.e. enzymes are intrinsically biodegradable) xi) compatibility for real-time analysis for pollution prevention; xii) safer chemistry for accident prevention. Second, enzymes are intrinsically compatible with each other and, therefore, they can be combined to carry out sequential multi-step processes in a single flask (or reactor, vessel) without the need for isolation and purification of the intermediates. Finally, enzymes originated from different organisms can be expressed in a single microbial host cell hence generating new metabolic pathways that do not exist in Nature. Such biocatalytic cascades (using isolated enzymes) and metabolic pathways (using microbial cells) are characterised by the highest atom efficiency and therefore potentially the lowest environmental factor. Furthermore, protein engineering techniques allow for the creation of new enzymes that are capable of catalysing chemical reactions that are not known in Nature. These variants can be integrated into artificial pathways to further shorten synthetic routes and improve efficiency.
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Work performed

During the first 21 months of the BioSusAmin project, the research team has developed the first asymmetric biocatalytic hydrogen-borrowing amination. This biocatalytic process enables the direct conversion of alcohols (even racemic) into enantiopure amines. The method relies on two enzyme classes, namely alcohol dehydrogenases and amine dehydrogenases, which work in tandem. This process is called â€œhydrogen-borrowingâ€ because the electrons liberated in the first oxidative step are internally recycled in the second reductive step. As a consequence, the overall cycle is redox self-sufficient: no additional oxidant or reductant is required and no additional waste is generated. The biocatalytic method possesses the highest possible atom efficiency because it requires ammonia as the nitrogen source and generates water as the sole by-product. It is noteworthy that the dual-enzyme hydrogen-borrowing amination can give direct access to highly valuable enantiopure amines. Most of these molecules constitute the active core of a large number of pharmaceuticals and fine chemical. The results were published in the prestigious journal Science, (IF 37.205; DOI: 10.1126/science.aac9283) and a world patent has been filed.
However, the successful implementation of this process in the near future from industry requires to further improve its efficiency and expand the substrate scope. The current major limitation is the relatively narrow substrate scope of the known amine dehydrogenases. Thus, the BioSusAmin team has studied the properties of the available amine dehydrogenases from a bio-organic, i.e. synthetic, point of view. The substrate scope for the reductive amination of a panel of structurally diverse prochiral ketones has been elucidated. The work was published in Green Chemistry (IF 9.125; DOI: 10.1039/C6GC01987K). Currently, the BioSusAmin is working towards the generation of a tool-box of amine dehydrogenase that will complement the enzymes already available. The team is using the so called rational-guide enzyme evolution or semi-rational enzyme engineering. That is a powerful combination between computational modelling of enzymes, generation of focused libraries via molecular biology and further screening with high-throughput assays or analytical tools. Positive hits are then characterised and analysed at molecular level in order to understand the influence of the mutations. The team has successfully engineered complementary amine dehydrogenases as well as novel alcohol dehydrogenases that will be applied in the artificial biocatalytic pathways of the BioSusAmin project.

Final results

The BioSusAmin project will contribute to the development of a new generation of (bio)chemical processes that will make our society less dependent on non-renewable resources and also allow for the reduction of energy consumption. Thus, the project will contribute to address one of the priorities set out by the European 2020 strategy concerning the delivery of growth that is smart and sustainable through a decisive move towards a resource-efficient, low-carbon economy. Continuing our current pattern of exploitation of the non-renewable natural resources is not possible in the medium to long term. Increasing resource efficiency is fundamental for securing growth and jobs. It will bring major economic opportunities, improve productivity, drive down costs and boost competitiveness. Additionally, the work underpins quality of life in many areas such as the sustainable manufacture of chemicals, healthcare, energy, environment, creation of new job opportunities, etc.
The training of the new generation of scientists to make them prepare to work in a multidisciplinary environment will also have an impact in this context.
Finally, policy makers may use the results to their advantage in developing arguments to support the technology base for renewable manufacture of chemicals through the creation of new artificial pathways in vitro and in microbial host organisms.