Engineering MVK to Relieve Biosynthetic Bottlenecks

Mevalonate Kinase Is a Biosynthetic Bottleneck

Biosynthesis is a powerful tool to convert cheap feed material into complex high-value chemicals using living cells. The advantage of this approach is that enzymes can be produced by a single cell to work in concert, allowing for a sequence of targeted chemical reactions that ultimately produce a desired product. Optimization of such a cascade involves ensuring that each enzyme is sufficiently active and specific to allow for production of the desired final product. One well-known biosynthetic bottleneck is mevalonate kinase (MVK), an enzyme natively found in eukaryotes and many prokaryotes that converts mevalonic acid (“mevalonate”) to mevalonate-5-phosphate (Fig. 1). MVK is known to be inhibited by downstream products of central metabolism, which creates a bottleneck when these products are part of a biosynthetic cascade to drive production of non-native compounds.

Microfluidic Droplet Screening to Rank MVK Mutants

We targeted MVK for activity engineering, to enable higher production of non-native biosynthetic products downstream of mevalonate-5-phosphate. We constructed a scanning library with a theoretical diversity of nearly 2000 MVK variants. This library was subjected to a single round of microfluidic droplet sorting using multiple fluorescence thresholds to select increasingly active pools of MVK variants (Fig. 2).

Relieving the Bottleneck With Improved Enzymes

We selected for further study those variants that showed the highest enrichment as a function of pool activity, and then cloned these variants into strains with a complete biosynthetic pathway that included MVK. We observed that overexpression of our top engineered MVK variant increased final product biosynthesis by 26%, compared to overexpression of wildtype MVK (Fig. 3).

Conclusion

In a single round of directed evolution, we screened a library with a theoretical diversity of ~2000 MVK variants at increasingly stringent production thresholds. We then cloned top performers from this screen into a host that contained a multi-enzyme biosynthetic cascade of which MVK was a bottleneck. Overexpression of our top MVK variant increased production of the final biosynthetic product by 26%, compared to overexpression of the wildtype MVK gene. This allowed us to relieve the biosynthetic bottleneck of MVK and produce higher quantities of our desired product.