Background: Yarrowia lipolytica, a non-traditional oil yeast, has been widely used as a platform for lipid production. However, the production of other chemicals such as terpenoids in engineeredY. lipolyticais still low. α-Farnesene, a sesquiterpene, can be used in medicine, bioenergy and other fields, and has very high economic value. Here, we used α-farnesene as an example to explore the potential ofY. lipolyticafor terpenoid production.Results: We constructed libraries of strains overexpressing mevalonate pathway and α-farnesene synthase genes by non-homologous end-joining (NHEJ) mediated integration into theY. lipolyticachromosome. First, a mevalonate overproduction strain was selected by overexpressing relevant genes and changing the cofactor specificity. Based on this strain, the downstream α-farnesene synthesis pathway was overexpressed by iterative integration. Culture conditions were also optimized. A strain that produced 25.55 g/L α-farnesene was obtained. This is the highest terpenoid titer reported inY. lipolytica.Conclusions: Yarrowia lipolyticais a potentially valuable species for terpenoid production, and NHEJ-mediated modular integration is effective for expression library construction and screening of high-producer strains.

Microbial production of monoterpenes provides a promising substitute for traditional chemical-based methods, but their production is lagging compared with sesquiterpenes. Geraniol, a valuable monoterpene alcohol, is widely used in cosmetic, perfume, pharmaceutical and it is also a potential gasoline alternative. Previously, we constructed a geraniol production strain by engineering the mevalonate pathway together with the expression of a high-activity geraniol synthase. In this study, we further improved the geraniol production through reducing the endogenous metabolism of geraniol and controlling the precursor geranyl diphosphate flux distribution. The deletion of OYE2 (encoding an NADPH oxidoreductase) or ATF1 (encoding an alcohol acetyltransferase) both involving endogenous conversion of geraniol to other terpenoids, improved geraniol production by 1.7-fold or 1.6-fold in batch fermentation, respectively. In addition, we found that direct down-regulation of ERG20 expression, the branch point regulating geranyl diphosphate flux, does not improve geraniol production. Therefore, we explored dynamic control of ERG20 expression to redistribute the precursor geranyl diphosphate flux and achieved a 3.4-fold increase in geraniol production after optimizing carbon source feeding. Furthermore, the combination of dynamic control of ERG20 expression and OYE2 deletion in LEU2 prototrophic strain increased geraniol production up to 1.69 g/L with pure ethanol feeding in fed-batch fermentation, which is the highest reported production in engineered yeast. An efficient geraniol production platform was established by reducing the endogenous metabolism of geraniol and by controlling the flux distribution of the precursor geranyl diphosphate. The present work also provides a production basis to synthesis geraniol-derived chemicals, such as monoterpene indole alkaloids.

Succinic acid (SA) is an important C4-dicarboxylic acid. Microbial production of SA at low pH results in low purification costs and hence good overall process economics. However, redox imbalances limited SA biosynthesis from glucose via the reductive tricarboxylic acid (TCA) cycle in yeast. Here, we engineer the strictly aerobic yeast Yarrowia lipolytica for efficient SA production without pH control. Introduction of the reductive TCA cycle into the cytosol of a succinate dehydrogenase-disrupted yeast strain causes arrested cell growth. Although adaptive laboratory evolution restores cell growth, limited NADH supply restricts SA production. Reconfiguration of the reductive SA biosynthesis pathway in the mitochondria through coupling the oxidative and reductive TCA cycle for NADH regeneration results in improved SA production. In pilot-scale fermentation, the engineered strain produces 111.9 g/L SA with a yield of 0.79 g/g glucose within 62 h. This study paves the way for industrial production of biobased SA.