Alcohol-forming fatty acyl reductases (FARs) catalyze the reduction of thioesters to alcohols and are key enzymes for microbial production of fatty alcohols. Many metabolic engineering strategies utilize FARs to produce fatty alcohols from intracellular acyl-CoA and acyl-ACP pools; however, enzyme activity, especially on acyl-ACPs, remains a significant bottleneck to high-flux production. Here, we engineer FARs with enhanced activity on acyl-ACP substrates by implementing a machine learning (ML)-driven approach to iteratively search the protein fitness landscape. Over the course of ten design-test-learn rounds, we engineer enzymes that produce over twofold more fatty alcohols than the starting natural sequences. We characterize the top sequence and show that it has an enhanced catalytic rate on palmitoyl-ACP. Finally, we analyze the sequence-function data to identify features, like the net charge near the substrate-binding site, that correlate with in vivo activity. This work demonstrates the power of ML to navigate the fitness landscape of traditionally difficult-to-engineer proteins.
Fatty acyl reductases (FARs) catalyze the reduction of thioesters to alcohols and are key enzymes for the microbial production of fatty alcohols. Many existing metabolic engineering strategies utilize these reductases to produce fatty alcohols from intracellular acyl-CoA pools; however, acting on acyl-ACPs from fatty acid biosynthesis has a lower energetic cost and could enable more efficient production of fatty alcohols. Here we engineer FARs to preferentially act on acyl-ACP substrates and produce fatty alcohols directly from the fatty acid biosynthesis pathway. We implemented a machine learning-driven approach to iteratively search the protein fitness landscape for enzymes that produce high titers of fatty alcohols in vivo. After ten design-test-learn rounds, our approach converged on engineered enzymes that produce over twofold more fatty alcohols than the starting natural sequences. We further characterized the top identified sequence and found its improved alcohol production was a result of an enhanced catalytic rate on acyl-ACP substrates, rather than enzyme expression or KM effects. Finally, we analyzed the sequence-function data generated during the enzyme engineering to identify sequence and structure features that influence fatty alcohol production. We found an enzyme's net charge near the substrate-binding site was strongly correlated with in vivo activity on acyl-ACP substrates. These findings suggest future rational design strategies to engineer highly active enzymes for fatty alcohol production.
Understanding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interacts with different mammalian angiotensin-converting enzyme II (ACE2) cell entry receptors elucidates determinants of virus transmission and facilitates development of vaccines for humans and animals. Yeast display-based directed evolution identified conserved ACE2 mutations that increase spike binding across multiple species. Gln42Leu increased ACE2-spike binding for human and four of four other mammalian ACE2s; Leu79Ile had an effect for human and three of three mammalian ACE2s. These residues are highly represented, 83% for Gln42 and 56% for Leu79, among mammalian ACE2s. The above findings can be important in protecting humans and animals from existing and future SARS-CoV-2 variants.
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