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Construction of Gene Libraries from Environmental Isolates for expression in Pseudomonas putida

Helgadóttir, Helga LU (2018) KMBM01 20181
Applied Microbiology
Biotechnology
Abstract
Currently most aromatic compounds used in industry are derived from unsustainable petro-chemical feedstock. However the depolymerization of lignocellulosic biomass generates a wide range of aromatic compounds that could be further valued through microbial conversion with microorgansims such as the bacterium Pseudomonas putida. The aim of the present project was to develop a reproducible methodology for the construction of expression gene libraries from environmental bacterial isolates capable of degrading aromatic compounds derived from depolymerization of lignin and to use this library for expanding the range of aromatic compounds that P. putida can use. For this purpose, bacterial strains were isolated from an environmental sample... (More)
Currently most aromatic compounds used in industry are derived from unsustainable petro-chemical feedstock. However the depolymerization of lignocellulosic biomass generates a wide range of aromatic compounds that could be further valued through microbial conversion with microorgansims such as the bacterium Pseudomonas putida. The aim of the present project was to develop a reproducible methodology for the construction of expression gene libraries from environmental bacterial isolates capable of degrading aromatic compounds derived from depolymerization of lignin and to use this library for expanding the range of aromatic compounds that P. putida can use. For this purpose, bacterial strains were isolated from an environmental sample submitted to the presence of lignin-derived aromatic compounds and DNA was extracted, cut and used for cloning in a P. putida compatible vector. However the construction of a gene library that would cover the whole genome of the environmental isolates was unsuccessful, as coverage of only 0.3-13% of the genomes was achieved. This resulted from a low transformation efficiency, most likely because the digestion and extraction of fragmented DNA had low yields. Therefore the digestion and extraction of fragmented genomic DNA will require further optimisation. (Less)
Popular Abstract
Lignin is a part of plant biomass and is one of the most common polymers on earth. Lignin has a complex structure and its depolymerisation (the breakdown into smaller units) results in heterologous mixture of compounds. The composition of depolymerized lignin depends both on the depolymerisation method used and the plant source. Because of its complexity the use of lignin today is mostly limited to generating heat and electricity. Lignin is the only large-volume renewable source of aromatics (very stable ring-shaped compounds) and many other compounds. Currently most aromatics and many other compounds that are found in depolymerized lignin are derived from petroleum. Since petroleum is an unsustainable source and the depletion of natural... (More)
Lignin is a part of plant biomass and is one of the most common polymers on earth. Lignin has a complex structure and its depolymerisation (the breakdown into smaller units) results in heterologous mixture of compounds. The composition of depolymerized lignin depends both on the depolymerisation method used and the plant source. Because of its complexity the use of lignin today is mostly limited to generating heat and electricity. Lignin is the only large-volume renewable source of aromatics (very stable ring-shaped compounds) and many other compounds. Currently most aromatics and many other compounds that are found in depolymerized lignin are derived from petroleum. Since petroleum is an unsustainable source and the depletion of natural reserves resulting in higher prices it is advantageous to find a way to use the sustainable source that lignin is for production of valuable compounds.
In nature lignin is mainly broken down by fungi but the breakdown results in large amount of aromatics and therefore many microorganism have evolved a mechanism to degrade them. Due to the heterogeneity of depolymerized lignin and the microorganism preference of substrates no microorganism can degrade all types of depolymerized lignin. By using the diverse mechanism various microorganism already have for degradation of aromatics combined with genetic engineering it is possible to engineer a microorganism capable of using variety of aromatics found in depolymerized lignin and turn them into valuable compounds.
Making a gene library from an organism with interesting abilities has proven to be good way to find the genes coding for the enzymes responsible. A gene library from a bacterium is made by cutting its genome into pieces and then transfer the pieces into a vector. This vector can be placed in another bacteria (host) that express the gene or genes the piece codes for into functional proteins. This results in a lot of vectors containing pieces of the genome, that is a gene library. The gene library can be screened for activities e.g. by growing the bacteria containing the vectors on a medium were the only carbon source is a compound of interest. If the vector doesn´t have the mechanism capable of degrade the compound the bacteria will not grow. With this method it is possible to find genes that code for enzymes that can degrade aromatics. These gene or genes can then be incorporated into the host bacteria genome; making the host bacteria capable of degrading more compounds than it can naturally. One of the most promising hosts is a bacterium called Pseudomonas putida that can naturally degrade some of the aromatics from lignin.
To find bacteria capable of breaking down aromatics derived from lignin a sample of compost, containing soil and branches was used. The sample was cultivated on aromatics and the bacteria that grew were capable of breaking down the aromatics. Six bacterial strains were isolated and identified but the attempt to make a gene library that covers the whole genome of the bacteria was not successful. However this is a work in progress and the main hurdles of the method were identified and future work will be to overcome them. (Less)
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author
Helgadóttir, Helga LU
supervisor
organization
course
KMBM01 20181
year
type
H2 - Master's Degree (Two Years)
subject
keywords
applied microbiology, teknisk mikrobiologi
language
English
id
8963409
date added to LUP
2019-01-02 15:06:12
date last changed
2019-01-02 15:06:19
@misc{8963409,
  abstract     = {Currently most aromatic compounds used in industry are derived from unsustainable petro-chemical feedstock. However the depolymerization of lignocellulosic biomass generates a wide range of aromatic compounds that could be further valued through microbial conversion with microorgansims such as the bacterium Pseudomonas putida. The aim of the present project was to develop a reproducible methodology for the construction of expression gene libraries from environmental bacterial isolates capable of degrading aromatic compounds derived from depolymerization of lignin and to use this library for expanding the range of aromatic compounds that P. putida can use. For this purpose, bacterial strains were isolated from an environmental sample submitted to the presence of lignin-derived aromatic compounds and DNA was extracted, cut and used for cloning in a P. putida compatible vector. However the construction of a gene library that would cover the whole genome of the environmental isolates was unsuccessful, as coverage of only 0.3-13% of the genomes was achieved. This resulted from a low transformation efficiency, most likely because the digestion and extraction of fragmented DNA had low yields. Therefore the digestion and extraction of fragmented genomic DNA will require further optimisation.},
  author       = {Helgadóttir, Helga},
  keyword      = {applied microbiology,teknisk mikrobiologi},
  language     = {eng},
  note         = {Student Paper},
  title        = {Construction of Gene Libraries from Environmental Isolates for expression in Pseudomonas putida},
  year         = {2018},
}