Abstract
Substrate inhibition of enzymes can be a major obstacle to the production of valuable chemicals in engineered microorganisms. Here, we show substrate inhibition of lycopene cyclase as the main limitation in carotenoid biosynthesis in Yarrowia lipolytica. To overcome this bottleneck, we exploit two independent approaches. Structure-guided protein engineering yields a variant, Y27R, characterized by complete loss of substrate inhibition without reduction of enzymatic activity. Alternatively, establishing a geranylgeranyl pyrophosphate synthase-mediated flux flow restrictor also prevents the onset of substrate inhibition by diverting metabolic flux away from the inhibitory metabolite while maintaining sufficient flux towards product formation. Both approaches result in high levels of near-exclusive β-carotene production. Ultimately, we construct strains capable of producing 39.5 g/L β-carotene at a productivity of 0.165 g/L/h in bioreactor fermentations (a 1441-fold improvement over the initial strain). Our findings provide effective approaches for removing substrate inhibition in engineering pathways for efficient synthesis of natural products.
Highlights
Substrate inhibition of enzymes can be a major obstacle to the production of valuable chemicals in engineered microorganisms
Geranylgeranyl diphosphate synthase (GGPPS) should be considered as it controls the flux directed towards carotenoid instead of sterol synthesis (Fig. 1a and Supplementary Fig. 1)
Since Y. lipolytica already harbors a native copy of geranylgeranyl pyrophosphate synthase (GGPPS), we started by introducing gene expression cassettes encoding phytoene dehydrogenase and the bi-functional phytoene synthase/lycopene cyclase from X. dendrorhous (CrtI and CrtYB, respectively)[17,18] or M. circinelloides (CarB and CarRP, respectively)[19,20] into the Y. lipolytica po1f strain with TRP1 disruption (Supplementary Fig. 2 and Table 1)
Summary
Substrate inhibition of enzymes can be a major obstacle to the production of valuable chemicals in engineered microorganisms. In the second approach, similar titers and selectivity of β-carotene are obtained by reducing the carbon flow through the carotenoid pathway and preventing inhibitory metabolite accumulation to inhibitory levels, contrary to the traditional paradigms of pathway engineering This is achieved by establishing a geranylgeranyl pyrophosphate synthase (GGPPS)mediated metabolic flow restrictor that regulates the substrate lycopene formation rate, thereby effectively alleviating substrate inhibition. While this approach reduces flux through the pathway of interest, the gains from suppressing substrate levels and maintaining high enzymatic activity overcompensate for any losses in productivity suffered from the flux diversion. Our findings highlight the importance of and provide methods for abolishing substrate inhibition in engineering cell factories for biotechnological production of high-value compounds
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