Exploring Brazilian Biodiversity for novel omega transaminases for aromatic amine production
(2020) KMBM05 20201Applied Microbiology
- Abstract
- Aromatic amines are important chemical building blocks used in the pharmaceutical, food and agricultural industries. Greener methods, such as biocatalysis, using renewable feedstocks are needed to replace current synthesis strategies that rely on petrochemicals to achieve a more sustainable amine production. Aromatic amines may be produced from lignin, which is an abundant source of renewable aromatic substrates. Transaminases are a class of enzymes that have been successfully used for amine synthesis from carbonyl compounds. There is a need to find TAs that are able to convert lignin-derived aromatic ketones and aldehydes.
This project aimed to find novel transaminases capable of producing aromatic amines from lignin-derived precursors... (More) - Aromatic amines are important chemical building blocks used in the pharmaceutical, food and agricultural industries. Greener methods, such as biocatalysis, using renewable feedstocks are needed to replace current synthesis strategies that rely on petrochemicals to achieve a more sustainable amine production. Aromatic amines may be produced from lignin, which is an abundant source of renewable aromatic substrates. Transaminases are a class of enzymes that have been successfully used for amine synthesis from carbonyl compounds. There is a need to find TAs that are able to convert lignin-derived aromatic ketones and aldehydes.
This project aimed to find novel transaminases capable of producing aromatic amines from lignin-derived precursors with higher activity and wider substrate spectrum than known transaminases. To that end, several previously constructed metagenomic libraries of Brazilian origin were screened for novel transaminases that can be used for production of aromatic amines from lignin, using bioinformatics and a growth-based method was developed based on Pseudomonas putida as screening host. The bioinformatic screening resulted in one putative transaminase. The synthetic gene of this TA was cloned in Escherichia coli and evaluated experimentally. However, no activity could be detected. The activity-based method was demonstrated using a transaminase from Chromobacterium violaceum (CV-TA) that is known to have the desired activity. When applied to metagenomic libraries, no novel transaminases were found. However, a background activity in P. putida was observed.
The genome of P. putida was searched for putative transaminases using BLASTp with CV-TA as query sequence. Three candidate transaminases, PP-TA1, PP-TA2, PP-TA3, were identified and subsequently over-expressed in P. putida KT2440. The constructed strains were evaluated for vanillylamine production by whole-cell bioconversion of vanillin using growing and resting cells. Over-expression of PP-TA1 resulted in production of 0.70 mM (7.2 % yield) from 10 mM vanillin whereas PP-TA2 resulted in production of 0.18 mM (1.9 % yield), and PP-TA3 in 0.36 mM (3.6 % yield) vanillylamine. By comparison, over-expression of CV-TA resulted in production of 0.91 mM (9.6 % yield).
In summary, three new transaminases with activity towards vanillylamine were found and successfully over-expressed in P. putida. Furthermore, P. putida’s potential as a platform host for aromatic amine production was demonstrated although, the titer, yield, and rate of reaction achieved were low. Further reaction optimization by strain and process engineering are hence required prior to industrial implementation. (Less) - Popular Abstract
- Can we produce chemicals from wood?
Did you know that many medicines are produced using fossil resources? As we move towards a more sustainable society, we need to find renewable resources to reduce our reliance on fossil ones. Lignin is a complex molecule that is present in all plants as one of the main components of the plant cell wall giving it mechanical support. Every year, millions of tons of lignin are produced in paper factories around the world. It is mostly treated as a low value product, mainly used for heating purposes, when in fact it is a polymer of interesting molecules that, through the use of enzymes, can be used to produce chemical building blocks for pharmaceuticals in a sustainable way.
This project focused on one... (More) - Can we produce chemicals from wood?
Did you know that many medicines are produced using fossil resources? As we move towards a more sustainable society, we need to find renewable resources to reduce our reliance on fossil ones. Lignin is a complex molecule that is present in all plants as one of the main components of the plant cell wall giving it mechanical support. Every year, millions of tons of lignin are produced in paper factories around the world. It is mostly treated as a low value product, mainly used for heating purposes, when in fact it is a polymer of interesting molecules that, through the use of enzymes, can be used to produce chemical building blocks for pharmaceuticals in a sustainable way.
This project focused on one particular building block, vanillylamine which is of great interest because it can be used in the manufacturing of pharmaceuticals and polymers. Currently, it is produced using petrochemicals of fossil origins along with harsh organic salts and metal catalysts.
In this project, the vast biodiversity of Brazil was explored to find new enzymes to be used in the production of vanillylamine from vanillin, that is one of the aromatic compounds present in depolymerized lignin.
Using computational and experimental methods, four enzymes were found that could potentially be used for vanillylamine production. Three of these enzymes were introduced into the bacteria Pseudomonas putida to create cell factories that were able to produce small amounts of vanillylamine.
The successful use of these cell factories represents a step towards greener methods for producing vanillylamine, and amines in general. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9028169
- author
- Lund, Fredrik LU
- supervisor
- organization
- course
- KMBM05 20201
- year
- 2020
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Applied microbiology, biocatalysis, teknisk mikrobiologi
- language
- English
- id
- 9028169
- date added to LUP
- 2020-09-15 12:59:04
- date last changed
- 2020-09-15 12:59:04
@misc{9028169,
abstract = {{Aromatic amines are important chemical building blocks used in the pharmaceutical, food and agricultural industries. Greener methods, such as biocatalysis, using renewable feedstocks are needed to replace current synthesis strategies that rely on petrochemicals to achieve a more sustainable amine production. Aromatic amines may be produced from lignin, which is an abundant source of renewable aromatic substrates. Transaminases are a class of enzymes that have been successfully used for amine synthesis from carbonyl compounds. There is a need to find TAs that are able to convert lignin-derived aromatic ketones and aldehydes.
This project aimed to find novel transaminases capable of producing aromatic amines from lignin-derived precursors with higher activity and wider substrate spectrum than known transaminases. To that end, several previously constructed metagenomic libraries of Brazilian origin were screened for novel transaminases that can be used for production of aromatic amines from lignin, using bioinformatics and a growth-based method was developed based on Pseudomonas putida as screening host. The bioinformatic screening resulted in one putative transaminase. The synthetic gene of this TA was cloned in Escherichia coli and evaluated experimentally. However, no activity could be detected. The activity-based method was demonstrated using a transaminase from Chromobacterium violaceum (CV-TA) that is known to have the desired activity. When applied to metagenomic libraries, no novel transaminases were found. However, a background activity in P. putida was observed.
The genome of P. putida was searched for putative transaminases using BLASTp with CV-TA as query sequence. Three candidate transaminases, PP-TA1, PP-TA2, PP-TA3, were identified and subsequently over-expressed in P. putida KT2440. The constructed strains were evaluated for vanillylamine production by whole-cell bioconversion of vanillin using growing and resting cells. Over-expression of PP-TA1 resulted in production of 0.70 mM (7.2 % yield) from 10 mM vanillin whereas PP-TA2 resulted in production of 0.18 mM (1.9 % yield), and PP-TA3 in 0.36 mM (3.6 % yield) vanillylamine. By comparison, over-expression of CV-TA resulted in production of 0.91 mM (9.6 % yield).
In summary, three new transaminases with activity towards vanillylamine were found and successfully over-expressed in P. putida. Furthermore, P. putida’s potential as a platform host for aromatic amine production was demonstrated although, the titer, yield, and rate of reaction achieved were low. Further reaction optimization by strain and process engineering are hence required prior to industrial implementation.}},
author = {{Lund, Fredrik}},
language = {{eng}},
note = {{Student Paper}},
title = {{Exploring Brazilian Biodiversity for novel omega transaminases for aromatic amine production}},
year = {{2020}},
}