Honeybees are important crop pollinators, but they increasingly face pollen starvation as a result of agricultural intensification and climate change¹. Frequent flowering dearth periods and high-density rearing conditions weaken colonies, which often leads to their demise². Beekeepers provide colonies with pollen substitutes, but these feeds do not sustain brood production because they lack essential sterols found in pollen^3,4. Here we describe a technological advance in honeybee nutrition with wide-reaching impacts on global food security. We first measured the quantity and proportion of sterols present in honeybee tissues. Using this information, we genetically engineered a strain of the oleaginous yeast Yarrowia lipolytica to produce a mixture of essential sterols for bees and incorporated this yeast strain into an otherwise nutritionally complete diet. Colonies exclusively fed with this diet reared brood for significantly longer than those fed diets without suitable sterols. The use of this method to incorporate sterol supplements into pollen substitutes will enable honeybee colonies to produce brood in the absence of floral pollen. Optimized diets created using this yeast strain could also reduce competition between bee species for access to natural floral resources and stem the decline in wild bee populations.

Engineering a new metabolic function in a microbial host can be limited by the availability of the relevant cofactor. For instance, in Yarrowia lipolytica, the expression of a functional nitrate reductase is precluded by the absence of molybdenum cofactor (Moco) biosynthesis. In this study, we demonstrated that the Ogataea parapolymorpha Moco biosynthesis pathway combined with the expression of a high affinity molybdate transporter could lead to the synthesis of Moco in Y. lipolytica. The functionality of Moco was demonstrated by expression of an active Moco-dependent nitrate assimilation pathway from the same yeast donor, O. parapolymorpha. In addition to 11 heterologous genes, fast growth on nitrate required adaptive laboratory evolution which, resulted in up to 100-fold increase in nitrate reductase activity and in up to 4-fold increase in growth rate, reaching 0.13h-1. Genome sequencing of evolved isolates revealed the presence of a limited number of non-synonymous mutations or small insertions/deletions in annotated coding sequences. This study that builds up on a previous work establishing Moco synthesis in S. cerevisiae demonstrated that the Moco pathway could be successfully transferred in very distant yeasts and, potentially, to any other genera, which would enable the expression of new enzyme families and expand the nutrient range used by industrial yeasts.