Microbial production of isoprenoid using the archaeal mevalonate
pathway
Hisashi HEMMI
Graduate School of Bioagricultural Sciences, Nagoya University
Research objective
Isoprenoid is the collective name of natural compounds that are biosynthesized fromisopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) and contain C5
isoprene units in their structures. Tens of thousands of isoprenoid compounds have beenreported so far, making them the biggest family of natural compounds. Hormones and nutrientssuch as steroid, lipid soluble vitamins (vitamin A, D, E, and K), and ubiquinone (coenzymeQ), which are essential for human health, are included in the group. Moreover, isoprenoid
contains important natural drugs and lead compounds for synthetic drugs, as represented byan anti-malarial drug, artemisinin (whose discoverer Dr. Tu Youyou received the Nobel prizein Physiology or Medicine in 2015), and a cancer chemotherapy drug, paclitaxel (taxol). Inaddition, many isoprenoid compounds are utilized as perfumes or industrial materials.
There are two pathways discovered so far for the biosynthesis of IPP and DMAPP.
One is the mevalonate (MVA) pathway, which has been reported in eukaryotes, archaea, andsome bacteria. The pathway starts from acetyl-CoA, proceeds via MVA, produces IPP first,and then isomerizes it to form DMAPP. Another is the methylerythritol phosphate (MEP)
pathway, which has been reported in major bacteria and in the chloroplast in plants. It starts
from pyruvate and glyceraldehyde 3-phosphate, proceeds via MEP, and yields IPP or DMAPPin the final step. We have worked on the MVA pathway in archaea, and recently discovered amodified MVA pathway that proceeds via a novel compound, trans-anhydromevalonate
phosphate (tAHMP) 1. Since this pathway is conserved in most archaea, it was named “thearchaeal MVA pathway”. The prominent feature of the pathway is that it consumes only two
ATP molecules for the production of one IPP, whereas the usual MVA pathway requires three
ATP molecules. This is because a dehydration/decarboxylation step, which is catalyzed only
by an ATP-dependent decarboxylase in the usual MVA pathway, is catalyzed by two novel
enzymes, i.e., phosphomevalonate dehydratase (PMDh) and tAHMP decarboxylase (AMPD),in an ATP-independent manner. From this property, the archaeal MVA pathway is expected
to be applicable to isoprenoid bioproduction as an energy saving metabolic pathway.
The objectives of this study are to show the usefulness of the archaeal MVA pathway
by constructing an isoprenoid production system involving the pathway in the hostEscherichia coli cells 2 and to search for enzymes of the pathway, which are more suitable forisoprenoid production, by taking advantage of the system.
Methods
The genes of MVA kinase (MVK), PMDh, AMPD, isopentenyl phosphate kinase(IPK), IPP isomerase (IDI), which catalyze the biosynthetic reactions from MVA to IPP andDMAPP, and that of prenylated FMN synthase that catalyzes the production of a coenzyme
for AMPD, were inserted into pBAD18, a plasmid for E. coli, to construct pBAD-mMP7 andpBAD-aMP7. MVK and IPK genes from a mesophilic methanogen Methanosarcina mazei
and the IDI gene from E. coli were used. The other genes for the construction of pBAD-mMP7
were from M. mazei, and from A. pernix for pBAD-aMP7. Each of the plasmids wasintroduced into E. coli harboring a plasmid, pACYC-IBE, containing carotenoid biosynthetic
genes (Figure 1A). The transformants were cultivated in 5 mL of LB medium containing 5.0
mg/mL of mevalonolactone (MVL). In addition to general aerobic shaking cultivation, a semianaerobic cultivation was performed in a medium overlayered with paraffin. After harvesting,lycopene, a colored carotenoid, was extracted from the cells with acetone and then quantified
based on absorption.
Results
When cultivated aerobically, E. coli strains harboring pBAD-mMP7 or pBAD-aMP7
decreased lycopene production compared with the negative control strain harboring pBAD18.
Therefore, no positive effect of the introduction of the archaeal MVA pathway was observed.
In contrast, when cultivated semi-anaerobically, the strain harboring pBAD-mMP7
significantly increased lycopene production, while that harboring pBAD-aMP7 only showeda slight increase (Figure 1B). The removal of MVL from the culture medium caused a decreasein lycopene production at a level comparable with that of the negative control. Since E. coli
originally exhibits the MEP pathway but not the MVA pathway, the increased lycopene
production in the presence of MVL was considered to be derived from IPP/DMAPP
biosynthesized from the introduced archaeal MVA pathway. The reason for the difference
between the culture conditions could be because the ATP-saving archaeal MVA pathway took
advantage of the semi-anaerobic conditions that result in low ATP production, or because theoxygen-sensitive enzymes in the pathway could not show enough activities under the aerobicconditions.
When deficient plasmids, which were constructed by removing the PMDh or AMPD
gene from pBAD-mMP7, were used, lycopene production did not increase. The lycopene
production was recovered when the removed gene was inserted back into each of the deficient
plasmids. Therefore, the deficient plasmids can be used to detect the activity of PMDh andAMPD. Using this system, we have identified PMDh and AMPD from multiple organisms

and are now trying to search for enzymes that have superior properties such as oxygenresistance. In addition, we are constructing an evaluation system for isoprenoid production
using volatile terpene as an index, instead of carotenoid which is hydrophobic and thus reaches
maximum production rapidly.
Conclusion
The archaeal MVA pathway is applicable for the bioproduction of isoprenoid whenconditions allow. It has a high potential especially under low oxygen.
References
1） Hayakawa, H. et al. (2018) Modified mevalonate pathway of the archaeon Aeropyrum pernix
proceeds via trans-anhydromevalonate 5-phosphate. Proc. Natl. Acad. Sci. U. S. A. 115:10034-10039.
2） Yoshida, R., Yoshimura, T., and Hemmi, H. (2020) Reconstruction of the “archaeal”
mevalonate pathway from the methanogenic archaeon Methanosarcina mazei in Escherichia
coli cells. Appl. Environ. Microbiol. 86: e02889-19.

Figure 1 Lycopene production system involving the archaeal MVA pathway(A) Scheme of the system. CrtI, CrtB, and CrtE are carotenogenic enzymes encoded in pACYC-IBE.
(B) E. coli cells producing lycopene. mMP7, the strain harboring pBAD-mMP7; aMP7, the strain
harboring pBAD-aMP7; NC, the strain harboring pBAD18. Copyright @ American Society for
Microbiology, Appl. Environ. Microbiol. 86: e02889-19 doi: 10.1128/AEM.02889-19, 2020.