Introduction of Heterologous Mevalonate Pathway in Methylobacterium Extorquens AM1 for High Production of Value-Added Compounds from Methanol

Industrial biotechnology plays an important role in meeting growing demand for green economy. However the feedstock being used is mainly sugar, which limits the development of industrial biotechnology in terms of relative high price, seasonal fluctuation of supply of feedstock and policy restriction on guarantee for food. Methylotrophic bacteria, which utilize reduced carbon substrates (such as methane, methanol, and other methylated compounds) as sole sources of carbon and energy, provide a potential way to solve such problems under the glut of methane and methanol. To produce desirable value-added products by methylotrophic synthesis, we focused on mevalonate pathway, from which about 50,000 terpenoid compounds in nature are derived, including artemisinin, paclitaxel, isoprene, etc.

By introducing heterologous 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase (HMGS) and HMG-CoA reductase (HMGR) into Methylobacterium extorquens AM1, the top portion of mevalonate pathway can act in this strain, and convert acetyl-CoA to mevalonate acid. AM1-mve consisting of both HMGS and HMGR from Enterococcus Faecalis; AM1-mvh consisting of HMGS from Blattella Germanica and HMGR from Trypanosoma cruzi (either of which showed the highest enzymatic activity compared to other species), were first constructed. In flask cultivation, mevalonate production of AM1-mve was 0.30g/L, and that of AM1-mvh was 0.10g/L. Heterologous acetoacetyl-CoA thiolase was then introduced into AM1-mve and AM1-mvh respectively to construct engineered strains AM1-mva and AM1-mvt. With overexpression of acetoacetyl-CoA thiolase, mevalonate production of AM1-mvt increased 2.5 times over AM1-mvh, from 0.10g/L to 0.35g/L. However, AM1-mva showed a little decrease on production than AM1-mve, from 0.30g/L to 0.25g/L, and appeared severe cell clumps. Such results showed that acetoacetyl-CoA thiolase played a key role in balancing and broadening metabolic flux of heterologous mevalonate pathway. Therefore, 6 engineered strains carrying a series of plasmids with different RBS strengths in front of heterologous phaAgene (encoding acetoacetyl-CoA thiolase) of AM1-mvt were further constructed. With different levels of acetoacetyl-CoA thiolase expression, those strains showed significant differences on mevalonate production. By fermenting the best performed strain, a production of 3.2g/L in a 3L-bioreactor batch was achieved.

This study was the first time that mevalonate pathway was successfully expressed in methylotrophic strains. With the engineered chassis strain, we can easily synthesize desirable value-added terpenoids by introducing some heterologous gene further, which bright the future of large-scale production of value-added compounds from methanol.

Acknowledgement：

National Natural Science Foundation 21376137