Construction and Characterization of 3rd Generation Saccharomyces cerevisiae Strains for PHB production form Xylose
(2018) KMBM01 20181Applied Microbiology
Biotechnology
- Abstract
- Poly-3-D-hydroxybutyrate (PHB) is a biopolymer naturally produced by a range of bacterial species. PHB production both from glucose and xylose has also been enabled by engineering the robust baker’s yeast Saccharomyces cerevisiae, however yields remain low. In the present study, additional modifications were carried out in S. cerevisiae strains already expressing functional enzymatic pathways for PHB production from xylose. The first approach was to improve the efficiency of acetyl-CoA conversion to acetoacetyl-CoA in the PHB pathway by introducing enzymes possessing lower Km for acetyl-CoA than the original expressed enzyme from Cupriavidus necator. Two distinct acetyl-CoA acetyltransferases were introduced, Acat1 from Rattus norvegicus... (More)
- Poly-3-D-hydroxybutyrate (PHB) is a biopolymer naturally produced by a range of bacterial species. PHB production both from glucose and xylose has also been enabled by engineering the robust baker’s yeast Saccharomyces cerevisiae, however yields remain low. In the present study, additional modifications were carried out in S. cerevisiae strains already expressing functional enzymatic pathways for PHB production from xylose. The first approach was to improve the efficiency of acetyl-CoA conversion to acetoacetyl-CoA in the PHB pathway by introducing enzymes possessing lower Km for acetyl-CoA than the original expressed enzyme from Cupriavidus necator. Two distinct acetyl-CoA acetyltransferases were introduced, Acat1 from Rattus norvegicus and Erg10p from yeast using the CRISPR-Cas9 system; however no improvement in PHB production was obtained. The second approach was to increase the acetaldehyde pool so that the introduced heterologous acetylating acetaldehyde dehydrogenases (EutE) encoded by eutE would enhance the flux from acetaldehyde towards acetyl-CoA in one step using NAD+, which should increase PHB production, when combined with deletion of the main acetaldehyde dehydrogenase (ALD6) gene. Obtained results showed no PHB production in any strains expressing the EutE and therefore it was assumed that EutE was catalysing the reaction in the reverse direction resulting in increased acetaldehyde, which led to further investigations of the enzyme involving activity and overall functionality. Deletion of the eutE gene was also carried out in (EutE carrying) non-PHB producing strains to see if the PHB production would be restored. The eutE was also re-integrated in PHB producing strains. However in both cases no PHB was produced. These results indicated that the EutE gene integration was inactivating PHB production since removal of eutE did not restore PHB production. Therefore, the negative effect of EutE might be at the genetic level on either one or several of the PHB genes phaA, phaB or phaC. (Less)
- Popular Abstract
- Over the past years, concerns have increased worldwide regarding hazardous compounds, such as water- and air pollutants and waste disposal being spread to the environment. Solutions towards sustainability involve the replacement of non-renewable resources by renewable ones for the generation of bio-based and biodegradable materials. The microorganism Saccharomyces cerevisiae, also known as baker´s yeast, has been well studied and is commonly used for the production of such compounds. S. cerevisiae is very efficient for the utilization of 6-carbon sugars (also called “hexose” sugars) such as glucose as carbon source but in industrial applications it is preferable to use cheap and non-edible renewable feedstock for the production of biofuels... (More)
- Over the past years, concerns have increased worldwide regarding hazardous compounds, such as water- and air pollutants and waste disposal being spread to the environment. Solutions towards sustainability involve the replacement of non-renewable resources by renewable ones for the generation of bio-based and biodegradable materials. The microorganism Saccharomyces cerevisiae, also known as baker´s yeast, has been well studied and is commonly used for the production of such compounds. S. cerevisiae is very efficient for the utilization of 6-carbon sugars (also called “hexose” sugars) such as glucose as carbon source but in industrial applications it is preferable to use cheap and non-edible renewable feedstock for the production of biofuels and other chemicals. However non-edible biomass, such as wood or agricultural waste, often contain a large fraction of 5-carbon sugars (aka pentoses), notably xylose that cannot be naturally assimilated by S. cerevisiae so this yeast has been genetically engineered to enable xylose utilization, notably for producing the so-called 2nd generation bioethanol. Nowadays, S. cerevisiae is being foreseen as a putative platform organism for the generation of biodegradable plastic precursors from biomass as there is a strong will to reduce the production of petroleum-based plastics. Among bioplastics, the biopolymer poly-3-hydroxybutyrate (PHB) is a promising substitute for non-renewable polymers since it can be completely degraded under both anaerobic and aerobic conditions by means of various microorganisms. In this study, S. cerevisiae that already was genetically modified to produce PHB from xylose was further engineered to improve PHB levels. The strategy consisted in increasing, inside the cells, the pool of a precursor of PHB called acetyl-CoA. This was done by modifying the levels of some enzymes as well as adding more efficient enzymes. There was no improvement in PHB production; instead the study indicated that one of the introduced gene negatively impacted the correct function of some of the PHB genes. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/8957316
- author
- Haraldsdóttir, Pálína LU
- supervisor
- organization
- course
- KMBM01 20181
- year
- 2018
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Applied microbiology, Teknisk mikrobiologi
- language
- English
- id
- 8957316
- date added to LUP
- 2018-08-31 14:48:25
- date last changed
- 2019-03-01 03:45:49
@misc{8957316,
abstract = {{Poly-3-D-hydroxybutyrate (PHB) is a biopolymer naturally produced by a range of bacterial species. PHB production both from glucose and xylose has also been enabled by engineering the robust baker’s yeast Saccharomyces cerevisiae, however yields remain low. In the present study, additional modifications were carried out in S. cerevisiae strains already expressing functional enzymatic pathways for PHB production from xylose. The first approach was to improve the efficiency of acetyl-CoA conversion to acetoacetyl-CoA in the PHB pathway by introducing enzymes possessing lower Km for acetyl-CoA than the original expressed enzyme from Cupriavidus necator. Two distinct acetyl-CoA acetyltransferases were introduced, Acat1 from Rattus norvegicus and Erg10p from yeast using the CRISPR-Cas9 system; however no improvement in PHB production was obtained. The second approach was to increase the acetaldehyde pool so that the introduced heterologous acetylating acetaldehyde dehydrogenases (EutE) encoded by eutE would enhance the flux from acetaldehyde towards acetyl-CoA in one step using NAD+, which should increase PHB production, when combined with deletion of the main acetaldehyde dehydrogenase (ALD6) gene. Obtained results showed no PHB production in any strains expressing the EutE and therefore it was assumed that EutE was catalysing the reaction in the reverse direction resulting in increased acetaldehyde, which led to further investigations of the enzyme involving activity and overall functionality. Deletion of the eutE gene was also carried out in (EutE carrying) non-PHB producing strains to see if the PHB production would be restored. The eutE was also re-integrated in PHB producing strains. However in both cases no PHB was produced. These results indicated that the EutE gene integration was inactivating PHB production since removal of eutE did not restore PHB production. Therefore, the negative effect of EutE might be at the genetic level on either one or several of the PHB genes phaA, phaB or phaC.}},
author = {{Haraldsdóttir, Pálína}},
language = {{eng}},
note = {{Student Paper}},
title = {{Construction and Characterization of 3rd Generation Saccharomyces cerevisiae Strains for PHB production form Xylose}},
year = {{2018}},
}