Microorganisms continue to gain traction as viable hosts for overproducing free fatty acids, which are useful starting points for building renewable oleochemicals. To date, most studies have focused on engineering native or heterologous enzymes to adjust fatty acid production in genetically tractable hosts. This work explores kinetically guided approaches for making similar adjustments without new enzymes. In brief, we combined a detailed kinetic model of the E. coli fatty acid synthase (FAS), a fully reconstituted in vitro system, and several engineered strains to investigate the influence of changes in the ratios of four native FAS components (i.e., β-ketoacyl-ACP synthases [KAS] I, II and III and a thioesterase) on FAS outputs. At the outset of the project, our in silico and in vitro analyses suggested that high levels of ‘TesA and low levels of KAS I and II, which compete for acyl-acyl carrier protein (ACP) substrates, could reduce the average length of fatty acids, while the opposite ratio increases length. We applied this “ratiometric” tuning strategy in vivo by using a multi-plasmid system to overexpress or inhibit the expression of target enzymes; indeed, changes in FAS compositions could shift profiles toward shorter or longer chains. Intriguingly, strategies for reducing chain lengths (which included overexpression of FabH and/or CRISPRi-mediated depletion of FabF or FabB) were more limited, perhaps, as a result of the toxicity of these adjustments to growing cells. Ultimately, this work provides a kinetically guided approach (one highly complementary to protein engineering efforts) for fine-tuning the titer and product distribution of FASs in engineered strains.

Changes in intracellular enzyme ratios enable fine-tuning of fatty acid profiles in Escherichia coli

ACS Fall 2021

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