In which fields of chemical industry does biocatalysis present a growing interest?
Historically, significant advancements within the chemical industry have revolved around improving the production of pharmaceuticals, and due to excellent chemoand stereospecificity biocatalysis has taken its place amongst this progress. It is now a vital part of the modern manufacturing of many drug products and a cross-section of the specialty chemicals industry, including food and feed, agrochemicals, flavour and fragrances segments, and many others. Additionally, the commodity chemicals and polymer sectors are increasingly utilising biocatalysis to address the need for cleaner, sustainable, and more efficient processes to ensure competitiveness within a rapidly changing market that is increasingly aware of the need to address climate change. A recent
example in the polymer industry is the highly efficient biocatalytic production of 1,5-diaminopentane (cadaverine) produced from bio-based lysine applying a decarboxylase catalysed process with substrate load of 500 g/L and complete conversion within 24 hours (1).
What are the most recent and promising biocatalysis applications?
Although biocatalysis has already been well-established in several commercial markets, rapid advancements in various aspects of the technology continue. For example, breakthroughs have been made to accelerate enzyme engineering methodologies and the successful integration of bioinformatic technology
in the optimisation and innovation process. Furthermore, enzyme cascades are increasingly a focus of process development for producing active pharmaceutical ingredients (APIs). A prominent example of enzyme cascade advancement is the reaction developed to produce the API for molnupiravir, an antiviral agent for the treatment of COVID-19, developed by Merck & Co. in cooperation with an enzyme engineering service provider. The synthetic route for molnupiravir includes a novel cascade of engineered enzymes that incorporates an innovative phosphate recycling system and a dehydration reaction that facilitates high yield and purity. The new route leveraging an engineered enzyme cascade is 70 percent shorter compared to the initially developed route (2). Another dominant trend is that more enzymes from different classes outside the well-established esterases/lipases and ketoreductases are maturing to the point of being successfully utilised in multi-tonne-scale processes. Examples are numerous, but we would like to highlight transaminases and carboligases. For several years now, it has become common to use transaminases for key conversions in the synthesis of chiral amines. One prominent illustration is sitagliptin, developed about ten years ago. However, recently engineered transaminases are used in a neat substrate reaction set-up to produce the cinacalcet intermediate (R)-naphthylethylamine (3). Similarly, carboligase applications are rapidly emerging due to their high efficiency for enantioselective carbon-carbon bond formation, a key reaction in organic synthesis. For example, the synthesis of the metaraminol intermediate (R)-1-Hydroxy-1-(3-hydroxyphenyl)-2-propanone has been scaled-up successfully and demonstrates clear advantages in effi ciency and selectivity compared to the historical baker’s yeast catalysed process. Moreover, metaraminol can be produced in a two-step enzymatic cascade combining carboligases and transaminases with high stereo-selectivities and high purities. Just adding a third step (subsequent Pictet–Spengler reaction catalysed by a norcoclaurine synthase), 1,3,4-trisubstituted tetrahydroisoquinolines (THIQs) with three chiral centers can be produced in a step-efficient and selective manner without intermediate purification (4).
What is the future of the development chemist: A comprehensive toolbox of chemo- and bio-catalysts to cover all applications? What should be in the toolbox then?
Based on our work, we believe biocatalysis can replace conventional chemical catalysis steps for a considerable range of reactions. It is probably well-known that regioselective esterifi cation and ester hydrolysis performed by enzymes are superior to chemical approaches where protection groups are required if a molecule has several functional groups that could be affected. Another example are L-threonine aldolase (LTA) catalysed reactions, where two chiral centers are created in one reaction step from achiral precursors (5). However, biocatalysis should not be viewed as merely replacing already established chemical processes. The creation of chemo-enzymatic synthesis pathways, like the one for levetiracetam, already demonstrated that a combination of both catalytic approaches is superior to their individual strengths (6).
What are the (perceived) hurdles to step into biocatalysis?
Overall, we expect the application of biocatalysis to continue to grow. From interactions with customers, we’ve realised that, although the historical limitations of engineered enzymes have in many cases been overcome, numerous synthetic chemists are unaware of the progress that has been made. Therefore, a signifi cant driver for the future growth of biocatalysis relies on busting myths and sharing current understandings. One of the most prominent myths is the view that enzymes are not stable under process conditions. Many believe enzymes only perform under natural conditions in aqueous solutions, leading to low solubilities of bulky and hydrophobic substrates, which prevents high conversion rates, yield, and easy catalyst recycling. It is important to note that these limitations typically only apply to natural enzymes.
Modern, advanced computer-aided enzyme engineering and innovative reaction engineering have paved the way for biocatalysis under even harsh temperature, pH, and organic solvent process conditions. Notably, enzyme engineering advancements have signifi cantly reduced the costs and time to develop industrialapplicable enzymes. Today, engineered enzyme development takes a few months rather than years.
Additionally, a major concern of many not currently leveraging biocatalysis is hesitation relating to the security of their intellectual property (IP) and the attachment of onerous IP strings when working with enzyme engineering partners. Understandably, most companies seeking enzyme engineering service providers value fair and fl exible IP arrangements and tend to prefer accommodating fee-for-service business models.
Sourcing of the enzymes for biocatalysis or producing them in house?
Finally, enzyme sourcing is crucial for large-scale manufacturing as well as research purposes. Several vendors offer enzyme kits used primarily for initial enzyme screening. However, having large-scale, dedicated fermentation sites is paramount for commercial-scale enzyme production. Fortunately, many CMOs worldwide now offer extensive fermentation expertise and capacities lessening the need for chemical companies to establish in-house fermentation capabilities.
References and notes
1. Unpublished results from Enzymaster
2. McIntosh J. A., et al., ACS Cent. Sci., 7 (12), 1980-1985, 2021
3. Cai B., et al., Org. Process Res. Dev., XXXX, XXX, XXX-XXX, Articles ASAP, 2021 (https://doi.org/10.1021/acs.oprd.1c00409)
4. Erdmann V., et al., Angew. Chem. Int. Ed., 56, 12503-12507, 2017
5. Chen H., et al., Patent WO2018219107A1, 2018
6. Arndt S., et al., Green Chem., 23, 388, 2021
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