Rational design of an engineered alcohol dehydrogenase towards C6 or higher alcohols.
The invention/creation presented is a result of a protein engineering approach. This research area has facilitated the discovery and synthesis of novel proteins or improved enzymes with desired properties, surpassing the limitations of native enzymes (e.g., low thermostability and enzyme activity). Rational design of proteins is one of the strategies addressed by protein engineering. In our technology, AdhP from Escherichia coli was engineered to produce higher alcohols, namely octanol. Native adhP is an alcohol dehydrogenase (ADH), NADH-dependent enzyme, being easily expressed in E. coli, and exhibits high catalytic efficiency, namely to the reduction of small aldehydes (acetaldehyde and butanal). However, aldehydes higher than butanal are poorly reduced, which prompt the need to use rational protein engineering tools to improve the AdhP kinetic parameters of large substrates, especially octanal. The existence of structural data for AdhP allows the use of structural bioinformatics techniques in order to achieve this goal.
The analysis and the comparison of several alcohol dehydrogenases through structural alignment and kinetic data are crucial to characterize the catalytic center, identify the target amino acids and create a correlation between structural properties and substrate affinity. Due to existence of three-dimensional structure of native AdhP, it was possible to use several structural computational tools contributing for the rational design of an artificial enzyme with the desired characteristics. ADHs with more affinity for small aldehydes (e.g., acetaldehyde and butanal) have threonine and tryptophan residues at the catalytic center, while ADHs with high affinity for bulky substrates have smaller amino acids at the same positions.
Taking this in consideration, the distinctive characteristic of our technology relates with the ability to generate from enzymes poorly efficient to octanal reduction and consequently octanol production, the development of a new enzyme with two mutations, namely in the residues 39 and 84 (Thr39Ser and Trp84A). This enzyme exhibits high catalytic levels for octanal.
The existence of three-dimensional structure and the use of different computational tools improves protein engineering by allowing to study individual properties of a molecule as well as the interaction between receptor and ligands, which can indicate favourable mutations and consequently generate an enzyme to be used in an effective bioprocess, with new functionalities and/or high catalytic levels (Km, Kcat and Kcat/Km) and stability.
Briefly, we started by using the MODELLER software was used to align and compare three- dimensional structures of multiple ADHs and it was also used to build, in silico, the AdhP mutants. The volume of substrate binding pocket, to evaluate the impact of mutations, was determined by the trj_cavity tool. Additionally, molecular docking was performed to predict the productive binding mode of linear aldehydes within the binding site of the AdhP, as well as evaluate the impact of the mutations through the binding energy.
The multiple structural alignments of AdhP enzyme with well characterised alcohol dehydrogenases and identified 2 target amino acids (tryptophan (Trp84) and threonine (Thr39)) at the active site. The in silico results, substrate binding pocket volume calculations and molecular docking predictions, demonstrated that the replacement of these amino acids by smaller ones has a positive effect. The enlargement of the substrate binding pocket provoked by double mutations improved the accommodation and fitting of large substrates, allowing these substrates to take productive binding poses with low binding energies. In vitro characterization was performed with engineered and native adhP. A kinetic study was performed for several linear aldehydes and ethanol. Additionally, the thermostability of wild- type and mutant was also evaluated using different techniques, as differential scanning fluorometry and circular dichroism. Additionally, in vivo validation of this new enzyme was carried out. The in vitro experiments showed that the Thr39Ser:Trp84Ala mutant is 364-times more catalytically efficient regarding octanal reduction than the wild-type enzyme. Octanal is well accepted as substrate, with the lowest Km, 0.08 mM, when compared with the other aldehydes. The mutation implemented does not affect the thermostability of the protein and exhibits maximum activity profile plateaus between 25-30 ̊C. The new enzyme is also more active between pH 4-7.5, displaying the maximum activity at 7.
We developed a microbial cell factory to produce octanol through engineering the fatty acid biosynthesis pathway to allow the production of this alcohol from glucose. The last step of this pathway is mediated by an alcohol dehydrogenase. The octanol titers obtained by modified and wild-type AdhP were compared to validate the efficiency of the redesigned enzyme. The strain with the modified AdhP produced around 58 mg/L of octanol, while the strain harbouring the native enzyme achieved 22 mg/L. Overall, the results obtained proved that modified AdhP is very efficient, not only in vitro, but also in vivo.
STAGE OF DEVELOPMENT
Technology Readiness Level (TRL): 3 – Laboratory Prototype.
BENEFITS & APPLICATIONS
The utilization of higher alcohols to replace the conventional fuels has been promoted in order to minimize the drawbacks of short alcohols (e.g., hygroscopicity, corrosiveness, vapor pressure). AdhP is an alcohol dehydrogenase, catalysing the reduction of aldehyde into the corresponding alcohol. The enzyme engineered exhibits high specificity and catalytic efficiency to octanal, improving its reduction and consequently the octanol titers. Additionally, this enzyme is easily expressed in industrial workhorse Escherichia coli. Additionally, the enzyme reveals to be much more adaptable regarding pH and temperature ranges than the native protein, which is a valuable feature for industrial purposes.
Currently, the octanol market can be segmented into two different areas: industrial grade and food grade. In 2015, approximately 81 % of octanol market corresponded to industrial grade. In a report by FiorMarkets, the global market for octanol is forecasted to be € 243 million by 2024 with a Compound Annual Growth Rate (CAGR) of 4.7 %. Some of these growing markets for octanol include the following:
- Biofules – Due to octanol physical-chemical properties. The use of petrochemical processes, namely Zielger process, to produce an alcohol stream where octanol corresponds only to 17 % of total product. Therefore, the increase of alcohol carbon chain length increases the calorific value and the cetane number, approximating to the ones of diesel.
- Detergents- Octanol is a relevant industrial product being commonly used in coating, detergents and surfactants production, as well as perfumery/fragrances.
- Manufacturing of plasticizers- Octanol is the precursor for octene synthesis, an important monomer for linear low-density polyethylene.
- Cosmetics and personal care products: Octanol is also a common ingredient in cosmetics and personal care products, such as lotions, creams, shampoos, and conditioners. It is used as a solvent and emollient, and it helps to improve the skin’s texture and appearance.
- Chemical intermediates: Octanol is used to produce a variety of other chemicals, such as esters, ethers, and polymers. These chemicals have a wide range of applications, including in the production of plastics, textiles, and other products.
- Solvents: Octanol is a versatile solvent that can be used to dissolve a variety of materials, including oils, fats, and waxes. It is used in the manufacturing of paints, varnishes, and other products.
- Flavourings and fragrances: Octanol is used as a flavourings agent in some foods and beverages, such as ice cream, candy, and perfumes. It has a sweet, fruity odor and can be used to enhance the flavours of other ingredients.
- Pharmaceuticals: Octanol is used in the production of some pharmaceuticals, such as antibiotics and anti-inflammatory drugs. It is also used as a solvent in the manufacturing of other pharmaceutical products, such as tablets and capsules.
- EP23154653.2 (Priority date: 01/02/2023).
- Available for exclusive and non-exclusive licensing.
- Seeking co-development partners and/or Sponsored Research.