Metabolic engineering of hyperthermophilic archaeon thermococcus onnurineus for 3-hydroxypropionate and 3-hydroxyisobutyrate production

Abstract

Hyperthermphilic microorganisms has advantages for biochemical production such as reduced risk of bacterial contamination and reduced cooling requirement. Thermococcus onnurineus is a sulfur-reducing hyperthermophilic archaeon which was isolated from a deep-sea hydrothermal vent region. For biochemical production on this beneficial hyperthermophilic microbial platform, T. onnurineus was engineered using transformation with heterogeneous genes. 3-Hydroxypropionate (3-HP) and 3-Hydroxyisobutyrate (3-HIBA) were chosen as model products. For the construction of 3-HP platform, malonyl-CoA reductase (MCR) and malonate semialdehyde reductase (MSR) were introduced on the pyruvate degradation pathway of the host. T. onnurineus has several advantages as biochemical production platform. Cell mass and 3-HP could be produced simultaneously since the optimal temperatures for cell growth and related enzyme activity are close to each other and NAD(P)H, the common cofactor for both reductases, can be sufficiently generated by well-developed redox pathways. A recombinant strain for 3-HP platform was constructed successfully and $140 mg liter^{-1}$ of 3-HP was synthesized in fed-batch fermentation, Continuous fed-batch process increased 3-HP titer up to $191 mg liter^{-1}$ with a productivity was $25.3 mg liter^{-1} h^{-1}$. For the construction of 3-HIBA platform, propionate utilization pathways of the host was engineered with two heterogeneous genes of MCR and 3-Hydroxyisobutyrate dehydrogenase (HIBADH). By fed-batch fermentation, $2.36 mg liter^{-1}$ of 3-HIBA was synthesized. It is the first example of genetically engineered biochemical production platform based on T. onnurineus. However, further study is necessary to increase the productivity and to develop more valuable biochemical compounds.