| dc.description.abstract | The use of methanol as a carbon source for industrial biotechnology is a tantalizing prospect. Methanol is highly abundant, and has a long history of efficient, large-scale production. It can be produced abiotically from methane, coal, gasified biomass or CO2 and is stable as a liquid at atmospheric pressures and temperatures which makes it easy to store and transport. Methanol production does not depend on modern agriculture to supply the raw materials and is an environmentally friendly alternative to sugars as a feedstock. Interest in using methylotrophs as bacterial cell factories is steadily increasing, and one of the most promising candidates is Bacillus methanolicus.
B. methanolicus is a natural overproducer of L-glutamate, and much of the metabolic engineering in this organism has focused on producing alternative amino acids such as L-lysine or derivatives of amino acids such as cadaverine or γ-aminobutyric acid. In order to expand the production capability and diversity of this bacterium, in this project, B. methanolicus was genetically engineered to overproduce acetoin (3-hydroxy-2-butanone). B. methanolicus was able to produce acetoin in titers up to 0.26 g/L from methanol in shake flask cultivations by heterologous expression of acetolactate synthase (AlsS) and acetolactate decarboxylase (AlsD) from Bacillus subtilis using a single (θ-replicating) plasmid and a single promoter system. By exploiting the in-creased diversity of genetic tools recently available to B. methanolicus, the acetoin titers were increased to 0.42 g/L in shake flask cultivations by heterologous overexpression of malic enzyme from Geobacillus stearothermophilus and native enzymes from the glyoxylate shunt of B. methanolicus under the control of a secondpromoter on a second (rolling circle replicating) plasmid.
To explore the potential of B. methanolicus as an overproducer of recombinant proteins, its ability to secrete recombinant proteins into culture supernatants was investigated. Production and secretion of recombinant proteins from methanol is a valuable addition to the production capabilities of B. methanolicus, but so far no investigations into this system have been performed. Heterologous and homologous α-amylases and proteases, and the thermostable reporter protein sfGFP were fused to signal peptide sequences (both natitve and non-native), and extracellular enzyme activities and fluoresence were determined to confirm secretion of functional proteins. A total of three recombinant amylases and one recombinant protease were shown to be secreted outside the cells when fused to their native signal peptides, and sfGFP was successfully secreted when fused to three of the four amylase signal peptides
These experiments confirmed that B. methanolicus can use signal peptides from other organisms (G. stearothermophilus, B. licheniformis, B. subtilis), as well as native signal peptides to direct secretion of functional, recombinant proteins when growing on methanol. As expected, our results showed that the different signal peptides have varied effectiveness when fused to different proteins, and that no one signal sequence gives the best performance across all proteins. We also showed that placing the protein coding genes under the control of the xylose inducible promoter from Bacillus megaterium enabled controlled production of the target proteins. Finally, recombinant production and secretion of a thermostable α-amylase from G. stearothermophilus allowed B. methanolicus to grow on potato starch as its sole carbon and energy source for the first time, expanding the substrate range of this organism. | en_US |
