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.
Submitter: Jonathan Greenhalgh
Submission Date: July 20, 2021, 11:09 a.m.
|Number of data points||2355|
|Proteins||MT-ACR ; MB-ACR ; MA-ACR|
|Assays/Quantities/Protocols||Experimental Assay: C6 ; Experimental Assay: C8 ; Experimental Assay: C10 ; Experimental Assay: C12 ; Experimental Assay: C14 ; Experimental Assay: C16 ; Experimental Assay: Sum of C6-C16 ; Experimental Assay: OD ; Experimental Assay: Expression _Level _(µg/mL at OD 20) ; Experimental Assay: SD of OD ; Experimental Assay: Km (Palmitoyl-ACP) ; Experimental Assay: kcat (Palmitoyl-ACP) ; Experimental Assay: Km (Palmitoyl-CoA) ; Experimental Assay: kcat (Palmitoyl-CoA) ; Derived Quantity: SD of C6 ; Derived Quantity: SD of C8 ; Derived Quantity: SD of C10 ; Derived Quantity: SD of C12 ; Derived Quantity: SD of C14 ; Derived Quantity: SD of C16 ; Derived Quantity: SD of Sum of C6-C16 ; Derived Quantity: SD of Expression _Level _(µg/mL at OD 20) ; Derived Quantity: SD of Km (Palmitoyl-ACP) ; Derived Quantity: SD of kcat (Palmitoyl-ACP) ; Derived Quantity: SD of Km (Palmitoyl-CoA) ; Derived Quantity: SD of kcat (Palmitoyl-CoA)|
|Libraries||Fatty alcohol titers in BL21 (DE3) assay. Variants were cloned onto a pET 28 backbone and transformed into BL21 (DE3) cells. 5 mL cultures were started from individual colonies and grown overnight for 16-20 hours in LB. Then they were diluted 100-fold into 5 mL of fresh LB and grown for 2.5-3 hours. Protein expression was induce by adding IPTG (final concentration 100 µM) and the expression cultures were incubated for 24 hours at 20 ˚C. Fatty alcohols were extracted from the cultures using 1 mL hexane with 5 µM nonanol and 5 µM heptadecanol as internal standards. The hexane/culture was mixed by vortexing and then centrifuged to separate the phases. The organic layer was extracted and analyzed by GC-FID. ; Fatty alcohol titers in CM24. Sequences were cloned into the CM24 E. Coli strain. 5 mL cultures were grown for 16-20 hours and then 600 µL of culture was diluted in 30 mL of LB + 1% Glucose + 20% dodecane in a serum vial. The serum vial was sealed and the culture was grown for 2 hours at 30 ˚C. IPTG (final concentration 100 µM) was added to the vials using a syringe and the cultures were incubated at 30 ˚C for 48 hours. 5 mL of the culture was used to perform an extraction with 1 mL of hexane, and the organic layer was analyzed by GC-FID. ; Kinetics on Palmitoyl-ACP ; Fatty alcohol titers in RL08ara. 5 mL cultures started from individual colonies were grown overnight for 16-20 hours. The OD was measured and culture was added to 50 mL LB + 1% glycerol + 20 % dodecane such that the starting OD would be 0.01. The culture was grown for 45 minutes at 37 C at which point it was induced with IPTG (final concentration 100 µM) and incubated for 18 hours at 30 ˚C. Then an internal standard was added and fatty alcohols were extracted using hexane and quantified by GC-FID.|