Sustainable production of oleochemicals requires establishment of cell factory platform strains. The yeast Saccharomyces cerevisiae is an attractive cell factory as new strains can be rapidly implemented into existing infrastructures such as bioethanol production plants. Here we show high-level production of free fatty acids (FFAs) in a yeast cell factory, and the production of alkanes and fatty alcohols from its descendants. The engineered strain produces up to 10.4 g l−1 of FFAs, which is the highest reported titre to date. Furthermore, through screening of specific pathway enzymes, endogenous alcohol dehydrogenases and aldehyde reductases, we reconstruct efficient pathways for conversion of fatty acids to alkanes (0.8 mg l−1) and fatty alcohols (1.5 g l−1), to our knowledge the highest titres reported in S. cerevisiae. This should facilitate the construction of yeast cell factories for production of fatty acids derived products and even aldehyde-derived chemicals of high value.

Baker’s yeast Saccharomyces cerevisiae is one of the most important and widely used cell factories for recombinant protein production. Many strategies have been applied to engineer this yeast for improving its protein production capacity, but productivity is still relatively low, and with increasing market demand, it is important to identify new gene targets, especially targets that have synergistic effects with previously identified targets. Despite improved protein production, previous studies rarely focused on processes associated with intracellular protein retention. Here we identified genetic modifications involved in the secretory and trafficking pathways, the histone deacetylase complex, and carbohydrate metabolic processes as targets for improving protein secretion in yeast. Especially modifications on the endosome-to-Golgi trafficking was found to effectively reduce protein retention besides increasing protein secretion. Through combinatorial genetic manipulations of several of the newly identified gene targets, we enhanced the protein production capacity of yeast by more than fivefold, and the best engineered strains could produce 2.5 g/L of a fungal α-amylase with less than 10% of the recombinant protein retained within the cells, using fed-batch cultivation.

Baker's yeast Saccharomyces cerevisiae is an attractive cell factory for production of chemicals and biofuels. Many different products have been produced in this cell factory by reconstruction of heterologous biosynthetic pathways; however, endogenous metabolism by itself involves many metabolites of industrial interest, and de-regulation of endogenous pathways to ensure efficient carbon channelling to such metabolites is therefore of high interest. Furthermore, many of these may serve as precursors for the biosynthesis of complex natural products, and hence strains overproducing certain pathway intermediates can serve as platform cell factories for production of such products. Here we implement a modular pathway rewiring (MPR) strategy and demonstrate its use for pathway optimization resulting in high-level production of L-ornithine, an intermediate of L-arginine biosynthesis and a precursor metabolite for a range of different natural products. The MPR strategy involves rewiring of the urea cycle, subcellular trafficking engineering and pathway re-localization, and improving precursor supply either through attenuation of the Crabtree effect or through the use of controlled fed-batch fermentations, leading to an L-ornithine titre of 1,041±47 mg l−1 with a yield of 67 mg (g glucose)−1 in shake-flask cultures and a titre of 5.1 g l−1 in fed-batch cultivations. Our study represents the first comprehensive study on overproducing an amino-acid intermediate in yeast, and our results demonstrate the potential to use yeast more extensively for low-cost production of many high-value amino-acid-derived chemicals.

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Keratinases are becoming biotechnologically important since they have shown potential in hydrolysis of recalcitrant keratins with highly rigid and strongly cross-linked structures. However, the large-scale application of keratinases has been limited by the inefficient expression level and low enzyme activity. In this work, we employed pro-peptide engineering and saturation mutagenesis to construct excellent keratinase variants with improved activities. It turned out that amino acid substitutions at the pro-peptide cleavage site (P1) could accelerate the release of active mature enzymes, resulting in a 3-fold activity increase. Eighteen sites of the pro-peptide area were targeted for codon mutagenesis, and a multisite saturation mutagenesis library of the six potential sites was generated, achieving a significant improvement of keratinase activity from 179 to 1114 units/mL. Also, the mutants exhibited alterant catalytic properties. Finally, fermentation for keratinase production in a 15 L fermenter was carried out, and the enzyme activity reached up to over 3000 units/mL. Our results demonstrated that propeptide engineering played a crucial role in high expression and engineering of proteases. This study provides a universal route toward improvement of industrial enzymes that were first synthesized as precursors in the form of pre-pro-protein.

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