Exploring the potential of Methylomicrobium album BG8 as a platform for the bioconversion of methane and methanol to isoprenoids
Creators: Das, Shibashis
Publisher: name: University of Alberta Library
Year: 2024

<p>Methane and methanol are inexpensive and sustainable carbon feedstocks for<br>
bioconversion to value-added products. Methane being a greenhouse gas, its conversion has the<br>
added benefit of mitigating emissions that affect climate change. Methanotrophic bacteria<br>
consume methane and methanol as sole sources of carbon and energy and can convert them into<br>
value-added products such as bioplastics, biofuels, and platform chemicals. The methanotroph<br>
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<br>
tools and processing methods to expand the range of potential bioproducts synthesized by this<br>
bacterium. Towards this goal, three broad host range plasmids based on the IncP, IncQ and pBBR1<br>
incompatibility families were developed. These vectors were introduced into M. album BG8<br>
through conjugation and were successfully propagated. Two expression vectors based on the<br>
IncP replicon and IncQ replicon were validated with expression of a luciferase protein under the<br>
PMMA (Mxa type methanol dehydrogenase) constitutive promoter. pBBR1 and IncQ plasmids<br>
were propagated simultaneously by M. album BG8, suggesting plasmids from different<br>
incompatibility families can be co-stabilized in this strain. Three different promoters were used<br>
in these expression vectors to demonstrate expression levels of heterologous genes. These results<br>
open the door towards production of commercially useful products in a modified M. album BG8<br>
strain. To improve strain production, batch, sequential batch and fed-batch strategies were<br>
developed for M. album BG8 cultures in bioreactors. In the fed-batch operation of M. album<br>
BG8, maximum optical density reached 16.56, a value comparable to that obtained with more<br>
widely studied methylotrophic strains that grow on methanol. This culturing study further lays<br>
the basic framework for advanced bioreactor operations aimed at achieving high density growth<br>
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<br>
precursors for aviation fuel, and thus represent a promising class of value-added products for<br>
methane and methanol bioconversion. Genetically modified M. album BG8 has the potential to<br>
synthesize certain types of isoprenoids, some of which are toxic to microbes. Based on their<br>
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<br>
pinene isomers than other isoprenoid classes tested. The addition of a top layer of n-dodecane<br>
increased LD50 of all isoprenoids. Thus, the inclusion of an n-dodecane overlayer during<br>
production can facilitate industrial biosynthesis of isoprenoids by M. album BG8. Together, the results from this thesis lay a framework for future isoprenoid production<br>
from methane and methanol by a genetically modified strain of M.album BG8</p>