University of Groningen
Metabolic engineering of Bacillus subtilis for terpenoids production Xue, Dan
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Publication date: 2018
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Xue, D. (2018). Metabolic engineering of Bacillus subtilis for terpenoids production. University of Groningen.
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Introduction and scope of the thesis
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Chapter 1
The structural diversity of nature products is of great importance for the biological activity of pharmaceutical substances, and the complexity of naturally active compounds is not easy to reproduce chemically. A large group of interesting naturally occurring compounds are the isoprenoids. Isoprenoids fulfill a vast array of functions throughout nature. They serve as pigments, reproductive hormones, defensive agents and constituents of the cell membrane amongst many other functions. Industrially, isoprenoids like menthol, citronella and lycopene are used as flavorings and spices, ingredients in perfumes, insecticides and as a raw material for the production of rubber and many other products. Some isoprenoids possess important medicinal properties. Well known examples of valuable isoprenoid drugs are the antimalarial drug artemisinin and the chemotherapeutic agent paclitaxel (Taxol®).
Several strategies are available for the industrial production of these compounds. Molecules can be produced chemically, or isolated from their natural resources. However, these approaches have their limitations in industrial scale production of these pharmaceuticals. Chemical synthesis is complicated by the complex structure of these compounds. The isolation from natural resources mostly results in low yields and puts a considerable strain on the availability of these species, some of which are rare. Additionally, the total yield of isolation depends on environmental conditions, which affects the growth of natural plant recourses. Thus, an alternative approach to produce these pharmaceuticals is microbial engineering, in which the synthetic pathway for the production of the target molecules from the original resource is transferred to microbial host organisms.
Bacillus subtilis has been the model organism for Gram-positive bacteria for decades.
The availability of many genetic tools and databases on genomic and proteomic data and the fact that B. subtilis is known for its high protein secretion capabilities provides researchers an excellent platform for building a cell factory for the production of biochemicals. It has been a choice of host for roughly 50% of worldwide production of interesting enzymes and vitamins. Because of its availability of many classical mutants
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Introduction and scope of the thesis
and genetically engineered strains, there are unlimited possibilities for building cell factories to produce small molecules (riboflavin) and secreted proteins (lipases).
In addition, B. subtilis has an inherent methylerythritol phosphate (MEP) pathway, which could produce C5 building blocks for terpenoids synthesis. Thus, in
order to establish a stable and efficient “cell factory” for the production of terpenoids, the potential of B. subtilis was elucidated in this thesis.
The research on metabolic engineering of B. subtilis for terpenoid production and encountered challenges were reviewed in Chapter 2. This chapter summarized some major advances and outline future directions for exploiting the potential of B. subtilis as a desired “cell factory” to produce terpenoids.
In Chapter 3, the effect of systematic overexpression of the genes involved in the MEP pathway on isoprenoid production in B. subtilis was investigated. It is shown that the production of carotenoids, a C30 terpenoid, can be increased
significantly by overexpressing the MEP pathway enzymes.
Then in Chapter 4 the segregational and structural stability of the B. subtilis host overexpressing the MEP pathway enzymes were further evaluated. A strain overexpressing eight genes of the MEP pathway on a plasmid clearly produced the highest level of carotenoids. The level of transcription for each gene in the operon was analyzed by RT-qPCR analysis a very sensitive method using the polymerase chain reaction. This is the first report of merging and stably expressing this large size operon (complete MEP pathway) from a plasmid-based system in B. subtilis.
Chapter 5 demonstrates the possibility to express the terpene cyclase, amorphadiene
synthase (ADS), in B. subtilis. Furthermore, with overexpression of MEP pathway which upregulates the building block for terpenoid compounds, isoprene,
B. subtilis strain could produce considerable yield of amorphadiene, the precursor of
artemisinine.
