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Final Report Summary - HOTRAM (Thermostable transaminases for the synthesis of pharmaceutical building blocks)

Enantiomerically pure chiral amines are compounds that can be used for the preparation of pharmaceutical agents that span a range of therapeutic areas including antihypertensives, antibiotics, antidepressants, antihistamines, and antidiabetics. Unfortunately, efficient methods for their preparation are very rare. In the last two decades, biocatalysis, i.e. the use of natural catalysts, such as enzymes, to perform chemical transformations on organic compounds, has emerged as an interesting alternative to conventional chemical methods in generating chiral amine compounds.
In particular, transaminases (TAs), enzymes that catalyze the transfer of an amino group from a donor amine to an amine acceptor molecule, represent an attractive option for chiral amine synthesis and have been demonstrated as suitable tools for application on industrial scale. Many transaminases have been described in the literature and some of them are already commercialized and used in industrial processes, such as in the synthesis of the antidiabetic compound sitagliptin. Generally speaking, thermostable enzymes are of industrial interest because most of the industrial processes require high temperatures and the exploitation of thermostable enzymes would help avoiding cooling and reheating steps, thus making the whole process cheaper and less time- and energy-consuming. Moreover, thermostable biocatalysts are usually more stable under the process conditions, e.g., in the presence of organic solvents which could be used to improve the solubility of the substrates, and can be either used at lower specific activity for an extended reaction time or reused if immobilized. The overall concept of this project is to exploit metagenomics for the discovery of a new generation of thermostable TA from the hot terrestrial environments. The use of metagenomics for enzyme discovery is well established. Metagenomic is a very powerful tool as it allows to access the so far uncharacterized majority of microbes that in any habitat may constitute up to 99% of the entire population. Objectives of the HOTRAM project are:
• Objective 1: to enrich hot spring cultures with Transaminase activities.
• Objective 2: to screen and obtain novel thermostable Transaminases from the metagenomes of (hyper)thermophiles
• Objective 3: to carry out the biochemical and structural characterization of the novel Transaminases
• Objective 4: to investigate the biocatalytical application of the novel Transaminases

Specifically, the project (objectives 1 and 2) includes three complementary approaches that has to be exploited to discover new thermostable transaminases: 1) Enrichment of hot spring cultures with transaminase activities, generation of expression libraries using the genome of the microorganisms isolated during the enrichment process and library screening for transaminase activities. 2) Construction of expression libraries using metagenomic DNA extracted from hot terrestrial environment and library screening for transaminase activities. 3) In silico screening of new thermostable TAs from (hyper)-thermophiles metagenomes in a datebase of sequences generated sequencing DNA extracted from hot terrestrial environments.
Moreover, according to objectives 3 and 4, the project includes the cloning and overexpression in E. coli of the discovered enzymes, the characterization from a biochemical and structural point of view of the new enzymes and the investigation of their substrate specificity.

During the first part of the project (March 2014-February 2015) enrichment cultures were prepared starting from 9 environmental samples collected in Iceland at temperatures ranging from 50 to 90 °C and using as substrates 4 different amines (sec-butylamine, β-phenylalanine, amino-n-butyric acid, alpha-methylbenzyl amine). 2 microorganisms were successfully isolated growing on β-phenylalanine and the corresponding expression libraries were constructed.
Additionally metagenomic DNA was successfully extracted from an environmental sample collected in Iceland at 90°C and the corresponding expression library was constructed.
Moreover the in silico screening for new omega-TA in a database of sequences generated starting from the DNA extracted from hot terrestrial environments in Italy, China and Iceland was performed. 3 ORFs showing similarity to known omega-TA sequences were found in the metagenome of samples collected from hot springs in Iceland I) B3-TA, 85°C, pH7.0, II) Is3-TA at 90°C, pH 3.5 and in Italy, III) It1-TA, 76°C, pH3.5. The corresponding genes were successfully cloned in a suitable expression vector and over-expressed in E. coli.

In the second part of the project (March 2015-February 2016) an activity assay for the screening of transaminase activities in expression libraries was developed. Metagenomic expression libraries constructed during the first year of the project were screened using the developed assay, but to date no positive clone was detected.
The over-expression of the three transaminases found with the in silico screening was optimized and the three proteins were purified.
The activity of the three enzymes was evaluated at different temperatures using a spectrophotometric assay. The 3 transaminases showed high optimal activity at high temperatures, specifically both IT1-TA and Is3-TA showed optimal activity at temperatures around 50°C, and >10% relative activity at 60 and 70°C, respectively. Interestingly, the activity of B3-TA increases constantly with temperature up to 90°C, determination of activity at higher temperature values was technically unfeasible. Concerning the stability at different temperatures, proteins were incubated at different temperatures for 3 hours and the residual activity was estimated by spectrophotometric analysis. It resulted that IS3-TA and IT1-TA mantained about 40% of the starting activity after 3h at 40°C, while B3-TA retains 100 % activity after 3h thermal treatment at temperatures comprised between 20 and 80°C. Long-term stability of B3-TA was also investigated. The protein was incubated at 80°C for 3 weeks and the residual activity was estimated by spectrophotometric analysis at different times. The thermostability of the three enzymes emerged also by the evaluation of their melting temperatures (Tm) performed by using circular dichroism (CD) analysis. It resulted that all the three enzymes undergo to unfolding at high temperature values, specifically: Tm Is3-TA: 76.91 °C, Tm It1-TA: 57.32 °C, Tm B3-TA: 88.19 °C. These structural data are in agreement with those previously shown on the influence of temperature on the activity and stability of the novel enzymes. Finally, substrate specificity of the three enzymes was investigated. Is3-TA and It1-TA showed a broad substrate specificity, while B3-TA was active only in the conversion of one of the tested substrates.
The structural analysis of the 3 new TAs is under investigation at the University of Exeter.

The outcome of the project is successful, three new TAs with interesting features, such as thermostability and broad substrate specificity, were discovered and demonstrated to be valuable tools for the synthesis of chiral amines and potentially they can be used on industrial scale. The outcome of the project has a strong environmental impact. In fact, it is worth noting that chiral amines are currently produced by chemical synthesis using polluting substances, i.e. metal catalysts. The introduction of biocatalysis methods in place of the metal-catalysed processes in the synthesis of these valuable compounds will contribute to reduce the environmental impact of the synthetic process. Moreover the project will have an economic impact as the stable performance of the enzymes at higher than conventional temperatures will improve the industrial processes. In addition the estabilishment of sustainable chemical processes strongly contribute to European excellence and competitiveness. In fact the European Commission, CEFIC and Biotech Industries Association through the SusChem Platform considers biocatalysis a key component for the development of a sustainable chemistry in Europe.

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