In this study, we engineered fatty acid synthases (FAS) for the biosynthesis of short-chain fatty acids and polyketides, guided by a combined in vitro and in silico approach. Along with exploring the synthetic capability of FAS, we aim to build a foundation for efficient protein engineering, with the specific goal of harnessing evolutionarily related megadalton-scale polyketide synthases (PKS) for the tailored production of bioactive natural compounds.
We designed a synthetic route consisting of two engineered FAS modules, the first (module 1) optimized to produce octanoyl-CoA (C8-CoA), and the second (module 2) to nonreductively elongate this intermediate, yielding 6-HHP. While short-chain acyl-CoAs produced by module 1 are valuable precursors for short FA and short alkanes for biofuel production the final lactone 6-HHP or its derivatives are interesting platform chemicals. We also developed FAS into an experimental and theoretical testbed system, culminating in an in silico model of the FAS catalytic network.
Figure. Two engineered fatty acid synthases (FASs), termed module 1 and module 2, work in sequence to synthesize C8-CoA (FAS1) and the final 6-HHP product (FAS2).
Besides bioengineering of FAS, our study was also largely encouraged by exploration of FAS as a model for evaluating an integrative approach for engineering FAS–PKS reaction networks. PKS are evolutionarily and mechanistically related to FAS and are responsible for important natural compounds. Achievements in engineering PKS notwithstanding, progress in developing PKS into versatile tools for compound synthesis has remained slow. A combined in vitro and in silico approach may serve as a blueprint for making PKS more amenable to pathway design. Likewise, the kinetic model for describing iterative FAS can be adapted to represent even complex modular PKS with each layer of the model describing the chemistry of a separate module. Development of such a model will depend on the collection of quantitative data from in-depth enzymological characterization of PKS.
Figure. (a) Product distributions of engineered FAS1 mutated in KS. Data refer to means of technical replicates (n = 1; three measurements with s.d. below ± 0.76 μM for this sample set). For more information on statistics, see Online Methods. (b) Product distributions of FAS1 variants mutated in MPT (n = 1; four measurements with s.d. below ± 2.8% (share of total acyl-CoA detected)), except for Q1349V and Q1349L, which were determined in a single measurement owing to limited protein supply. (c) 6-HHP as produced in the coupled reaction by FAS1G2599S-M2600W-R1408K right arrow FAS2S126A-Y2227F at different substrate concentrations (conc.) (n = 1). For more information on statistics, see Online Methods. (d) Percentage yield in reference to the limiting substrate, calculated from c. (e) Reaction network of a simplified FAS. Underlying this representation is an array of kinetic rate constants, including the elongation reaction (dashed arrows, yielding C(n+2)) (left). Abstraction of chain elongations from C4 to C18, with each layer in the stack representing the simplified model for a different chain length Cn (right). ACP, acyl carrier protein. (f) Binding free energy change for acyl chains C4–C16 in the mutant FAS1G1250S-M1251W with respect to wild-type FAS, as calculated from molecular dynamics simulations. Negative values correspond to more favorable surroundings for the introduction of respective ethyl groups in the mutant protein compared to wild type. (g) Snapshots from two molecular dynamics simulations, representing respectively C6- (pink) and C8-ACP (cyan) binding to the modified KS domain. Acyl-ACP was modeled in truncated form, indicated by (*).