# The Small of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast
## 5 Future Outlook
cerevisiae_[29], thus an avenue to follow is to test several similar enzymes from different organisms to assess their compatibility within the pathway. The “build” part encompasses all the aspects of constructing the strains that need to be tested. Multiplex CRISPR-based techniques for yeast have been developed and could easily be adapted to introduce combinations of the recombinant raspberry ketone pathway genes [76; 77]. CRISPR-based techniques enjoy the benefit of its precision in editing the genome that would lead to minor modifications being made to the genome. The system can also allow for the strain to be ‘marketless’—free of any antibiotic resistance genes normally used in conventional genetic manipulations. Additional mutagenesis, ranging from chemical mutagenesis to using a strain in which one could induce SCRaMbLE, could be included which will add more genetic diversity to the strains that need to be tested. The ‘testing’ part would, in most cases, involve some level of culturing of the strains coupled with determining the levels of the metabolite of interest. This would require high-throughput screening machinery. Raspberry ketone titres will need to be measured with mass spectrometry-based techniques. Eventual sequencing of high performers would be needed to ascertain what mutagenesis allowed for the superior production levels. Once the testing is concluded, the ‘learning’ part—where interpretation of the data (in this case identifying strains with superior raspberry ketone titres) and assessment of how one could improve upon the titres based on which strain performed better—is executed before commencing with a new cycle of DBTL. This is a simple example of a single iteration of DBTL with each step reliant on human intervention. The eventual goal—especially with the advent of genome or biofoundries—is to curtail human input and allow for automation and machine learning to dominate proceedings. Adapted from [87; 83]. As has been discussed in this review, nearly all of the reported metabolic engineering approaches for improvement in aroma compound production have been by way of rational design principles. This is because the pathways of the precursors have been largely elucidated and known bottlenecks or rate-limiting steps have been identified. Rational design will continue to be a potent pursuit as we learn more about relevant pathways and the regulation thereof. However, for fast-tracking improvements in aroma compound production in yeast, applying a DBTL-type approach holds a lot of potential. Indeed, it is perfectly suited for such an approach to target the production levels of a single metabolite. This is in contrast, however, to recent work in aroma compound development in yeast as part of an alcoholic beverage set-up, where hops flavour (a combination of geraniol and linalool) was introduced in yeast for beer production [87]. An elegant DBTL approach was followed to build strains with ideal hops flavour (similar to commercial beer). Although the researchers were eventually successful in obtaining strains that could ferment malt and impart hops flavour, many of their initial strain building attempts failed as strains selected for ideal hops flavour could not completely consume the malt sugars. This was unintentional and not easily explained but emphasised the limitations of employing DBTL on strains with multiparametric purposes. Nevertheless, to date, no DBTL-like approach has been performed on yeast for the sole purpose of producing enhanced levels of an aroma compound. A key element complementary to metabolic engineering within the synthetic biology realm, is that of individual enzyme engineering in order to alter the catalytic activity and increase the flux toward the production pathway of a given aroma compound [88]. Few cases exist where mutants of the enzymes within certain pathways were examined for improved production. Site-directed mutagenesis in enzymes involved in the MVA pathway has led to the general increase in terpenoids [89] or just specific classes of terpenoids [90].