Chemical synthesis links a series of chemical reactions together to yield a target product. This is the most common way to produce high value industrial chemicals. An emerging strategy to improve the sustainability of chemical production is to employ biosynthetic methods. Biosynthetic methods have the potential to deliver value-added chemicals from renewable feedstocks at mild temperatures, in aqueous solvent conditions, and in a single pot. Because of these features, biosynthetic processes minimize energy usage and waste streams and, ideally, obviate the need to isolate chemical intermediates. However, despite major advances in metabolic engineering and synthetic biology, the rapid engineering of microbes to deliver high yields and titers of target compounds remains as a challenge. For instance, alteration of one metabolic pathway may have unexpected ramifications in other pathways or the metabolic intermediates and end products build up and become toxic to organisms or trigger feedback inhibition. In-situ product recovery, which is used to extract target products, is appealing but requires that the desired chemical products effectively partition into biocompatible extractants.
To address the aforementioned system challenges, a team of researchers from the Department of Chemistry at Colorado School of Mines, including Ph.D. candidate Kelsey Stewart and Ms. Emily Hicks and led by Professor Dylan Domaille developed a new strategy for biosynthesis. Rather than using overexpressed enzymes to drive flux to a target product, they hypothesized that biocompatible chemical catalysts could play the role of overexpressed enzymes to intercept metabolic intermediates and redirect flux toward target chemicals. Their work is currently published in the research journal, ACS Sustainable Chemistry & Engineering.
In their reported system, a Gram negative non-pathogenic bacteria, Gluconobacter oxidans, was used to oxidize aliphatic alcohols to aliphatic aldehydes. When lysine, a biocompatible aldol catalyst, was added to the culture media, the aldehyde was dimerized to its aldol condensation product. A feedstock substrate of n-butanol delivered good yields of the high-volume chemical, 2-ethyl-2-hexenal (2-EH), in aqueous buffer at mild temperature. This is in sharp contrast to the existing method of 2-EH production, which involves high temperatures and creates large volumes of basic waste streams. A biocompatible isooctane extractant selectively removed the C8 2-EH product as it formed, driving flux toward the target product.
Product analysis of aqueous and organic phases revealed that in the absence of catalyst, only n-butanoic acid, which is the product of continued microbial oxidation, was produced. This analysis revealed that the in-situ lysine catalyst redirected metabolic flux to 2-EH, minimizing production of the undesirable n-butanoic acid. Moreover, the authors were able to show that their approach upgraded a range of C2−C6 n-alkyl alcohols in both self- and crossed aldol-type reactions.
In summary, the study presented a mild, aqueous synthesis approach of the high-volume chemical 2-EH from n-butanol. Further, it also demonstrated that the presented methodology was applicable to the self- and crossed aldol type couplings of C2−C6 substrates. In a statement to Advances in Engineering, Professor Dylan W. Domaille pointed out that their work revealed an exciting new strategy of flux redirection that can be used to expand the scope of products from biosynthetic processes.
Kelsey N. Stewart, Emily G. Hicks, and Dylan W. Domaille. Merger of Whole Cell Biocatalysis with Organocatalysis Upgrades Alcohol Feedstocks in a Mild, Aqueous, One-Pot Process. ACS Sustainable Chemistry & Engineering 2020, volume 8, page 4114−4119.