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Papers by Ahir Pushpanath
Current Research in Chemical Biology
Biocatalysis
Johnson Matthey (JM) is a specialty chemicals company, committed to the development of value addi... more Johnson Matthey (JM) is a specialty chemicals company, committed to the development of value adding sustainable technologies by using our expertise in chemistry and its applications. As one aspect of our business we offer both services and (chiral) catalyst technologies to the pharmaceutical, agrochemical, flavour and fragrance industries. Increasingly, JM is applying and developing biocatalysts for chiral applications alongside chemocatalysis. In this chapter, we highlight the dual role of JM as a service and a product provider in biocatalysis, depending on the two types of customers. For the first type of customer, we offer an entire solution for the requested chemistry by offering catalyst screening, process optimisation and manufacturing. For the second type of customer, we offer specialised enzyme selections to maximise their chances of finding suitable catalysts for their processes. Certain key commercial and technical considerations need to be addressed in order to provide biocatalytic solutions that make both economic and technical sense for either type of customer. Here, these considerations are analysed with specific examples that demonstrate the challenges but also the successes that can be achieved within rigid timelines and budgets. Examples ranging from streamlining computational approaches for biocatalyst recruitment to achieving process scalability are showcased in this chapter.
The concept of a protein's fitness landscape – an abstract space in which related sequences a... more The concept of a protein's fitness landscape – an abstract space in which related sequences are close together and matched with their fitness – is a useful tool to visualize core principles of protein evolution. Acquiring a new function, for example the laboratory evolution of an enzyme to convert an industrially relevant substrate, can be understood as a stepwise climb through a fitness landscape, reaching higher fitness (or activity) with each step (or mutation). The valleys of such a space relate to the starting points of protein engineering campaigns. Understanding this area could enlighten principles of how proteins quickly adapt in nature and help to identify starting points with a high potential for evolution, a high 'evolvability', speeding up protein engineering. In this study, high-throughput technologies will be developed that enable the read-out of directed evolution on a large scale, tracking the exploration of the valley of a fitness landscape: the conversi...
Organic Process Research & Development, 2020
Reduction of aromatic nitro compounds to anilines is of great interest to the chemical industry. ... more Reduction of aromatic nitro compounds to anilines is of great interest to the chemical industry. Biocatalytic reduction of nitroarenes has made it possible to effectively produce anilines by applying nitroreductase enzymes (NR) in combination with vanadium pentoxide. Herein, the NR-catalyzed reduction of 2-methyl-5-nitro-pyridine (2) to give the desired aniline (1) was studied as a model reaction. It demonstrates the importance of process development and enzyme engineering as key approaches to overcome scale-up issues and improve yield and productivity. Moving to fed-batch allowed controlling the feeding rate of 2 to prevent the accumulation of intermediates and formation of undesired side products. Starting with a substrate (2) concentration of 200 mM (28 g/L) and enzyme loading of 5 mg/mL (18% w/w), it was possible to achieve complete conversion and 1 in 95% yield by highperformance liquid chromatography (89.1% isolated yield) over 18 h, whereas, with 500 mM (69 g/L) 2 and an enzyme loading of 10 mg/mL (14.5% w/w), the same conversion and yield were achieved in 26 h. A rational engineering of NR-4 yielded faster variants, including NR-5, in only one round. The improved rate of the new variants allowed increasing the feeding rate of 2 to shorten the reaction time to less than a day as well as decreasing the enzyme loading to 3.6%.
ChemBioChem, 2021
The exploration of large DNA libraries of metagenomic or synthetic origin is greatly facilitated ... more The exploration of large DNA libraries of metagenomic or synthetic origin is greatly facilitated by ultrahigh-throughput assays that use monodisperse water-in-oil emulsion droplets as sequestered reaction compartments. Millions of samples can be generated and analysed in microfluidic devices at kHz speeds, requiring only micrograms of reagents. The scope of this powerful platform for the discovery of new sequence space is, however, hampered by the limited availability of assay substrates, restricting the functions and reaction types that can be investigated. Here, we broaden the scope of detectable biochemical transformations in droplet microfluidics by introducing the first fluorogenic assay for alcohol dehydrogenases (ADHs) in this format. We have synthesized substrates that release a pyranine fluorophore (8-hydroxy-1,3,6-pyrenetrisulfonic acid, HPTS) when enzymatic turnover occurs. Pyranine is well retained in droplets for > 6 weeks (i. e. 14-times longer than fluorescein), avoiding product leakage and ensuring excellent assay sensitivity. Product concentrations as low as 100 nM were successfully detected, corresponding to less than one turnover per enzyme molecule on average. The potential of our substrate design was demonstrated by efficient recovery of a bona fide ADH with an > 800-fold enrichment. The repertoire of droplet screening is enlarged by this sensitive and direct fluorogenic assay to identify dehydrogenases for biocatalytic applications.
Science Advances, 2020
Sequence function relationships help to understand how imine reductase enzymes perform reductive ... more Sequence function relationships help to understand how imine reductase enzymes perform reductive amination in water.
Lab on a Chip, 2021
Successful screening of enzyme libraries in functional metagenomics and directed evolution become... more Successful screening of enzyme libraries in functional metagenomics and directed evolution becomes more likely after uniform cell growth in droplets amplifies genotype and phenotype.
Nature Communications, 2020
The success of protein evolution campaigns is strongly dependent on the sequence context in which... more The success of protein evolution campaigns is strongly dependent on the sequence context in which mutations are introduced, stemming from pervasive non-additive interactions between a protein’s amino acids (‘intra-gene epistasis’). Our limited understanding of such epistasis hinders the correct prediction of the functional contributions and adaptive potential of mutations. Here we present a straightforward unique molecular identifier (UMI)-linked consensus sequencing workflow (UMIC-seq) that simplifies mapping of evolutionary trajectories based on full-length sequences. Attaching UMIs to gene variants allows accurate consensus generation for closely related genes with nanopore sequencing. We exemplify the utility of this approach by reconstructing the artificial phylogeny emerging in three rounds of directed evolution of an amine dehydrogenase biocatalyst via ultrahigh throughput droplet screening. Uniquely, we are able to identify lineages and their founding variant, as well as non...
Johnson Matthey Technology Review, 2020
The asymmetric reduction of C=C double bonds is a sought-after chemical transformation to obtain ... more The asymmetric reduction of C=C double bonds is a sought-after chemical transformation to obtain chiral molecules used in the synthesis of fine chemicals. Biocatalytic C=C double bond reduction is a particularly interesting transformation complementary to more established chemocatalytic methods. The enzymes capable of catalysing this reaction are called ene-reductases (ENEs). For the reaction to take place, ENEs need an electron withdrawing group (EWG) in conjugation with the double bond. Especially favourable EWGs are carbonyls and nitro groups; other EWGs, such as carboxylic acids, esters or nitriles, often give poor results. In this work, a substrate engineering strategy is proposed whereby a simple transformation of the carboxylic acid into a fluorinated ester or a cyclic imide allows to increase the ability of ENEs to reduce the conjugated double bond. Up to complete conversion of the substrates tested was observed with enzymes ENE-105 and *ENE-69.
Organic Process Research & Development, 2019
Imine reductases (IREDs) have attracted increasing attention as novel biocatalysts for the synthe... more Imine reductases (IREDs) have attracted increasing attention as novel biocatalysts for the synthesis of various cyclic and acyclic amines. Herein, a number of guidelines and considerations towards the development and scale-up of IRED catalyzed reactions have been determined based on the reductive amination of cyclohexanone (1) with cyclopropylamine (2). A Design of Experiments (DoE) strategy has been followed to study the different reaction parameters, facilitating resourceefficient and informative screening. Enzyme stability was identified to be the limiting factor. By moving from batch to fed-batch it was possible to double the concentration of the substrate and turnover number (TON). Kinetic studies revealed that IRED-33 was the best enzyme for the reaction with respect to both activity and stability. Under the optimal reaction conditions, it was possible to react 1 and 2 at 750 mM concentration and reach 100% conversion to the desired amine (>90% isolated yield) in the space of 8 h. Hence, excellent volumetric productivity of 12.9 g L-1 h-1 and TON above 48000 were achieved.
Catalysis Science & Technology, 2016
An industrial-scale method employing a phenylalanine ammonia lyase enzyme.
ACS Catalysis, 2017
The direct asymmetric reductive amination of ketones using ammonia as the sole amino donor is a g... more The direct asymmetric reductive amination of ketones using ammonia as the sole amino donor is a growing field of research in both chemo-and biocatalysis. Recent research has focused on enzyme engineering of amino acid dehydrogenases (to obtain amine dehydrogenases) and this technology promises to be a potentially exploitable route for chiral amine synthesis. However, the use of these enzymes in industrial biocatalysis has not yet been demonstrated with substrate loadings above 80 mM, owing to the enzymes' generally low turnover numbers (k cat <0.1 s-1) and variable stability in reaction conditions. In this work, a newly engineered amine dehydrogenase from a phenylalanine dehydrogenase (PheDH) from Caldalkalibacillus thermarum was recruited and compared against an existing amine dehydrogenase (AmDH) from Bacillus badius for both kinetic and thermostability parameters, with the former showing an Page 1 of 17 ACS Paragon Plus Environment ACS Catalysis 2 increased thermostability (T m) of 83.5 °C compared to 56.5 °C of the latter. The recruited enzyme was further used in the reductive amination of up to 400 mM of phenoxy-2-propanone (c = 96 %, ee (R) <99 %) in a biphasic reaction system utilizing a lyophilized whole-cell preparation. Finally, we performed computational docking simulations to rationalize the generally lower turnover numbers of AmDHs compared to their PheDH counterparts.
Applied Biocatalysis, 2020
Current Research in Chemical Biology
Biocatalysis
Johnson Matthey (JM) is a specialty chemicals company, committed to the development of value addi... more Johnson Matthey (JM) is a specialty chemicals company, committed to the development of value adding sustainable technologies by using our expertise in chemistry and its applications. As one aspect of our business we offer both services and (chiral) catalyst technologies to the pharmaceutical, agrochemical, flavour and fragrance industries. Increasingly, JM is applying and developing biocatalysts for chiral applications alongside chemocatalysis. In this chapter, we highlight the dual role of JM as a service and a product provider in biocatalysis, depending on the two types of customers. For the first type of customer, we offer an entire solution for the requested chemistry by offering catalyst screening, process optimisation and manufacturing. For the second type of customer, we offer specialised enzyme selections to maximise their chances of finding suitable catalysts for their processes. Certain key commercial and technical considerations need to be addressed in order to provide biocatalytic solutions that make both economic and technical sense for either type of customer. Here, these considerations are analysed with specific examples that demonstrate the challenges but also the successes that can be achieved within rigid timelines and budgets. Examples ranging from streamlining computational approaches for biocatalyst recruitment to achieving process scalability are showcased in this chapter.
The concept of a protein's fitness landscape – an abstract space in which related sequences a... more The concept of a protein's fitness landscape – an abstract space in which related sequences are close together and matched with their fitness – is a useful tool to visualize core principles of protein evolution. Acquiring a new function, for example the laboratory evolution of an enzyme to convert an industrially relevant substrate, can be understood as a stepwise climb through a fitness landscape, reaching higher fitness (or activity) with each step (or mutation). The valleys of such a space relate to the starting points of protein engineering campaigns. Understanding this area could enlighten principles of how proteins quickly adapt in nature and help to identify starting points with a high potential for evolution, a high 'evolvability', speeding up protein engineering. In this study, high-throughput technologies will be developed that enable the read-out of directed evolution on a large scale, tracking the exploration of the valley of a fitness landscape: the conversi...
Organic Process Research & Development, 2020
Reduction of aromatic nitro compounds to anilines is of great interest to the chemical industry. ... more Reduction of aromatic nitro compounds to anilines is of great interest to the chemical industry. Biocatalytic reduction of nitroarenes has made it possible to effectively produce anilines by applying nitroreductase enzymes (NR) in combination with vanadium pentoxide. Herein, the NR-catalyzed reduction of 2-methyl-5-nitro-pyridine (2) to give the desired aniline (1) was studied as a model reaction. It demonstrates the importance of process development and enzyme engineering as key approaches to overcome scale-up issues and improve yield and productivity. Moving to fed-batch allowed controlling the feeding rate of 2 to prevent the accumulation of intermediates and formation of undesired side products. Starting with a substrate (2) concentration of 200 mM (28 g/L) and enzyme loading of 5 mg/mL (18% w/w), it was possible to achieve complete conversion and 1 in 95% yield by highperformance liquid chromatography (89.1% isolated yield) over 18 h, whereas, with 500 mM (69 g/L) 2 and an enzyme loading of 10 mg/mL (14.5% w/w), the same conversion and yield were achieved in 26 h. A rational engineering of NR-4 yielded faster variants, including NR-5, in only one round. The improved rate of the new variants allowed increasing the feeding rate of 2 to shorten the reaction time to less than a day as well as decreasing the enzyme loading to 3.6%.
ChemBioChem, 2021
The exploration of large DNA libraries of metagenomic or synthetic origin is greatly facilitated ... more The exploration of large DNA libraries of metagenomic or synthetic origin is greatly facilitated by ultrahigh-throughput assays that use monodisperse water-in-oil emulsion droplets as sequestered reaction compartments. Millions of samples can be generated and analysed in microfluidic devices at kHz speeds, requiring only micrograms of reagents. The scope of this powerful platform for the discovery of new sequence space is, however, hampered by the limited availability of assay substrates, restricting the functions and reaction types that can be investigated. Here, we broaden the scope of detectable biochemical transformations in droplet microfluidics by introducing the first fluorogenic assay for alcohol dehydrogenases (ADHs) in this format. We have synthesized substrates that release a pyranine fluorophore (8-hydroxy-1,3,6-pyrenetrisulfonic acid, HPTS) when enzymatic turnover occurs. Pyranine is well retained in droplets for > 6 weeks (i. e. 14-times longer than fluorescein), avoiding product leakage and ensuring excellent assay sensitivity. Product concentrations as low as 100 nM were successfully detected, corresponding to less than one turnover per enzyme molecule on average. The potential of our substrate design was demonstrated by efficient recovery of a bona fide ADH with an > 800-fold enrichment. The repertoire of droplet screening is enlarged by this sensitive and direct fluorogenic assay to identify dehydrogenases for biocatalytic applications.
Science Advances, 2020
Sequence function relationships help to understand how imine reductase enzymes perform reductive ... more Sequence function relationships help to understand how imine reductase enzymes perform reductive amination in water.
Lab on a Chip, 2021
Successful screening of enzyme libraries in functional metagenomics and directed evolution become... more Successful screening of enzyme libraries in functional metagenomics and directed evolution becomes more likely after uniform cell growth in droplets amplifies genotype and phenotype.
Nature Communications, 2020
The success of protein evolution campaigns is strongly dependent on the sequence context in which... more The success of protein evolution campaigns is strongly dependent on the sequence context in which mutations are introduced, stemming from pervasive non-additive interactions between a protein’s amino acids (‘intra-gene epistasis’). Our limited understanding of such epistasis hinders the correct prediction of the functional contributions and adaptive potential of mutations. Here we present a straightforward unique molecular identifier (UMI)-linked consensus sequencing workflow (UMIC-seq) that simplifies mapping of evolutionary trajectories based on full-length sequences. Attaching UMIs to gene variants allows accurate consensus generation for closely related genes with nanopore sequencing. We exemplify the utility of this approach by reconstructing the artificial phylogeny emerging in three rounds of directed evolution of an amine dehydrogenase biocatalyst via ultrahigh throughput droplet screening. Uniquely, we are able to identify lineages and their founding variant, as well as non...
Johnson Matthey Technology Review, 2020
The asymmetric reduction of C=C double bonds is a sought-after chemical transformation to obtain ... more The asymmetric reduction of C=C double bonds is a sought-after chemical transformation to obtain chiral molecules used in the synthesis of fine chemicals. Biocatalytic C=C double bond reduction is a particularly interesting transformation complementary to more established chemocatalytic methods. The enzymes capable of catalysing this reaction are called ene-reductases (ENEs). For the reaction to take place, ENEs need an electron withdrawing group (EWG) in conjugation with the double bond. Especially favourable EWGs are carbonyls and nitro groups; other EWGs, such as carboxylic acids, esters or nitriles, often give poor results. In this work, a substrate engineering strategy is proposed whereby a simple transformation of the carboxylic acid into a fluorinated ester or a cyclic imide allows to increase the ability of ENEs to reduce the conjugated double bond. Up to complete conversion of the substrates tested was observed with enzymes ENE-105 and *ENE-69.
Organic Process Research & Development, 2019
Imine reductases (IREDs) have attracted increasing attention as novel biocatalysts for the synthe... more Imine reductases (IREDs) have attracted increasing attention as novel biocatalysts for the synthesis of various cyclic and acyclic amines. Herein, a number of guidelines and considerations towards the development and scale-up of IRED catalyzed reactions have been determined based on the reductive amination of cyclohexanone (1) with cyclopropylamine (2). A Design of Experiments (DoE) strategy has been followed to study the different reaction parameters, facilitating resourceefficient and informative screening. Enzyme stability was identified to be the limiting factor. By moving from batch to fed-batch it was possible to double the concentration of the substrate and turnover number (TON). Kinetic studies revealed that IRED-33 was the best enzyme for the reaction with respect to both activity and stability. Under the optimal reaction conditions, it was possible to react 1 and 2 at 750 mM concentration and reach 100% conversion to the desired amine (>90% isolated yield) in the space of 8 h. Hence, excellent volumetric productivity of 12.9 g L-1 h-1 and TON above 48000 were achieved.
Catalysis Science & Technology, 2016
An industrial-scale method employing a phenylalanine ammonia lyase enzyme.
ACS Catalysis, 2017
The direct asymmetric reductive amination of ketones using ammonia as the sole amino donor is a g... more The direct asymmetric reductive amination of ketones using ammonia as the sole amino donor is a growing field of research in both chemo-and biocatalysis. Recent research has focused on enzyme engineering of amino acid dehydrogenases (to obtain amine dehydrogenases) and this technology promises to be a potentially exploitable route for chiral amine synthesis. However, the use of these enzymes in industrial biocatalysis has not yet been demonstrated with substrate loadings above 80 mM, owing to the enzymes' generally low turnover numbers (k cat <0.1 s-1) and variable stability in reaction conditions. In this work, a newly engineered amine dehydrogenase from a phenylalanine dehydrogenase (PheDH) from Caldalkalibacillus thermarum was recruited and compared against an existing amine dehydrogenase (AmDH) from Bacillus badius for both kinetic and thermostability parameters, with the former showing an Page 1 of 17 ACS Paragon Plus Environment ACS Catalysis 2 increased thermostability (T m) of 83.5 °C compared to 56.5 °C of the latter. The recruited enzyme was further used in the reductive amination of up to 400 mM of phenoxy-2-propanone (c = 96 %, ee (R) <99 %) in a biphasic reaction system utilizing a lyophilized whole-cell preparation. Finally, we performed computational docking simulations to rationalize the generally lower turnover numbers of AmDHs compared to their PheDH counterparts.
Applied Biocatalysis, 2020