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Exploring the potential of Methylomicrobium album BG8 as a platform for the bioconversion of methane and methanol to isoprenoids
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- Author / Creator
- Das, Shibashis
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Methane and methanol are inexpensive and sustainable carbon feedstocks for
bioconversion to value-added products. Methane being a greenhouse gas, its conversion has the
added benefit of mitigating emissions that affect climate change. Methanotrophic bacteria
consume methane and methanol as sole sources of carbon and energy and can convert them into
value-added products such as bioplastics, biofuels, and platform chemicals. The methanotroph
Methylomicrobium album BG8 is notable for its fast and robust growth and rich genetic potential, making it an excellent candidate for bioproduction. The aim of this work was to develop genetic
tools and processing methods to expand the range of potential bioproducts synthesized by this
bacterium. Towards this goal, three broad host range plasmids based on the IncP, IncQ and pBBR1
incompatibility families were developed. These vectors were introduced into M. album BG8
through conjugation and were successfully propagated. Two expression vectors based on the
IncP replicon and IncQ replicon were validated with expression of a luciferase protein under the
PMMA (Mxa type methanol dehydrogenase) constitutive promoter. pBBR1 and IncQ plasmids
were propagated simultaneously by M. album BG8, suggesting plasmids from different
incompatibility families can be co-stabilized in this strain. Three different promoters were used
in these expression vectors to demonstrate expression levels of heterologous genes. These results
open the door towards production of commercially useful products in a modified M. album BG8
strain. To improve strain production, batch, sequential batch and fed-batch strategies were
developed for M. album BG8 cultures in bioreactors. In the fed-batch operation of M. album
BG8, maximum optical density reached 16.56, a value comparable to that obtained with more
widely studied methylotrophic strains that grow on methanol. This culturing study further lays
the basic framework for advanced bioreactor operations aimed at achieving high density growth
of M. album BG8 towards an industrial platform for methanol bioconversion. Isoprenoids are a class of molecules with a broad range of applications, including as
precursors for aviation fuel, and thus represent a promising class of value-added products for
methane and methanol bioconversion. Genetically modified M. album BG8 has the potential to
synthesize certain types of isoprenoids, some of which are toxic to microbes. Based on their
potential toxicity, a study was conducted with six isoprenoids: α-pinene (+), α-pinene (-), β- pinene (-), D-limonene, sabinene and farnesene. M. album BG8 was more tolerant towards
pinene isomers than other isoprenoid classes tested. The addition of a top layer of n-dodecane
increased LD50 of all isoprenoids. Thus, the inclusion of an n-dodecane overlayer during
production can facilitate industrial biosynthesis of isoprenoids by M. album BG8. Together, the results from this thesis lay a framework for future isoprenoid production
from methane and methanol by a genetically modified strain of M.album BG8 -
- Subjects / Keywords
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- Graduation date
- Spring 2024
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.