The in vitro characterization of heterologously expressed enzymes to inform in vivo biofuel production optimization
- Author(s): Garcia, David Ernest
- Advisor(s): Keasling, Jay D
- Wemmer, David E
- et al.
The mevalonate pathway is of critical importance to cellular function as it is the conduit for the production of terpenoids, hormones, and steroids. These molecules are also valuable on an industrial scale because they are used as antibiotics, flavoring agents, and fragrances, examples of which are clerocidin, cinnamon, and sandalwood, respectively. Because the biosynthetic route for the production of these compounds is the deoxyxylulose 5-phosphate pathway in E. coli, by engineering the heterologous mevalonate pathway into E. coli the native forms of pathway regulation were overcome to successfully create a bio-industrial route for the production of artemisinin, an antimalarial drug. Advanced biofuels can also be biosynthesized via the mevalonate pathway with some minor alterations to the final enzymatic conversions.
However, the toxicity of intermediates and products, as well as regulation internal to the pathway, limited our ability to increase production. The mevalonate pathway enzymes we used were native to S. cerevisiae, two of which had not been kinetically characterized. We set out to determine the nature of these enzymes and how they might be regulated by mechanisms internal to the mevalonate pathway, such as feedback inhibition. Because both mevalonate kinase (MK) and phosphomevalonate kinase (PMK) were derived from S. cerevisiae they had to be codon-optimized for production in E. coli, particularly because they had already been identified as expressing poorly in laboratory production strains through targeted protein studies. By cloning the codon-optimized sequences into high copy expression vectors with a six-histidine tag at the C- or N-terminus (PMK and MK, respectively) we were able to produce large enough amounts of the active proteins to purify on a Ni2+ resin column.
Kinetic characterization of MK revealed a KMATP of 315 ± 21 μM and a vmax of 50 ± 3 μM/min/μgE (or Kcat = 41 ± 3 s-1). Additionally, substrate inhibition of MK by mevalonate was determined to exist at concentrations above 2.5 mM, and Ki values for farnesyl pyrophosphate (79 ± 11 nM), geranyl pyrophosphate (147 ± 6 nM), geranylgeranyl pyrophosphate (303 ± 64 nM), dimethylallyl pyrophosphate, (29 ± 12 μM), and isopentenyl pyrophosphate (36 ± 5 μM) were determined.
Kinetic characterization of PMK revealed that maximum activity occurs at pH = 7.2 and [Mg2+] = 10 mM. KMATP was determined to be 98.3 μM and 74.3 μM at 30 °C and 37 °C, respectively. KMmev-p was determined to be 885 μM and 880 μM at 30 °C and 37 °C, respectively. vmax was determined to be 45.1 μmol/min/μgE and 53.3 μmol/min/μgE at 30 °C and 37 °C, respectively. From the high KMmev-p value it appears as if PMK might have very poor activity at normal cellular concentrations of mevalonate-5-phosphate, indicating that MK and PMK might play a very coordinated role in balancing intermediate levels within the pathway.