Astaxanthin is a high-value red pigment and antioxidant used by pharmaceutical, cosmetics, and food industries. The astaxanthin produced chemically is costly and is not approved for human consumption due to the presence of by-products. The astaxanthin production by natural microalgae requires large open areas and specialized equipment, the process takes a long time, and results in low titers. Recombinant microbial cell factories can be engineered to produce astaxanthin by fermentation in standard equipment. In this work, an oleaginous yeast Yarrowia lipolytica was engineered to produce astaxanthin at high titers in submerged fermentation. First, a platform strain was created with an optimised pathway towards β-carotene. The platform strain produced 331 ± 66 mg/L of β-carotene in small-scale cultivation, with the cellular content of 2.25% of dry cell weight. Next, the genes encoding β-ketolase and β-hydroxylase of bacterial (Paracoccus sp. and Pantoea ananatis) and algal (Haematococcus pluvialis) origins were introduced into the platform strain in different copy numbers. The resulting strains were screened for astaxanthin production, and the best strain, containing algal β-ketolase and β-hydroxylase, resulted in astaxanthin titer of 44 ± 1 mg/L. The same strain was cultivated in controlled bioreactors, and a titer of 285 ± 19 mg/L of astaxanthin was obtained after seven days of fermentation on complex medium with glucose. Our study shows the potential of Y. lipolytica as the cell factory for astaxanthin production.

The yeast Saccharomyces cerevisiae is widely used in industrial biotechnology for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals. To obtain high‐performing strains for such bioprocesses, it is often necessary to test tens or even hundreds of metabolic engineering targets, preferably in combinations, to account for synergistic and antagonistic effects. Here, we present a method that allows simultaneous perturbation of multiple selected genetic targets by combining the advantage of CRISPR/Cas9, in vivo recombination, USER assembly and RNA interference. CRISPR/Cas9 introduces a double‐strand break in a specific genomic region, where multiexpression constructs combined with the knockdown constructs are simultaneously integrated by homologous recombination. We show the applicability of the method by improving cis,cis‐muconic acid production in S. cerevisiae through simultaneous manipulation of several metabolic engineering targets. The method can accelerate metabolic engineering efforts for the construction of future cell factories.

We present a teaching protocol suitable for demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of industrially relevant yeasts Saccharomyces cerevisiae and Yarrowia lipolytica, using β-carotene production as a case study. The protocol details all steps required to generate DNA parts, transform and genotype yeast, and perform a phenotypic screen to determine β-carotene production. The protocol is intended to be used as an instruction manual for a two-week practical course aimed at MSc and PhD students. The protocol details all necessary steps for students to engineer yeast to produce β-carotene and serves as a practical introduction to the principles of metabolic engineering including the concepts of boosting native precursor supply and alleviating rate-limiting steps. It also highlights key differences in the metabolism and heterologous production capacity of two industrially relevant yeast species. The protocol is divided into daily experiments covering a two week period and provides detailed instructions for every step meaning this protocol can be used ‘as is’ for a teaching course or as a case study for how yeast can be engineered to produce value-added molecules.

β-Carotene is a yellow–orange–red pigment used in food, cosmetics and pharmacy. There is no commercial yeast-based process for β-carotene manufacturing. In this work, we engineered the baker's yeast Saccharomyces cerevisiae by expression of lipases and carotenogenic genes to enable the production of β-carotene on hydrophobic substrates. First, the extracellular lipase (LIP2) and two cell-bound lipases (LIP7 and LIP8) from oleaginous yeast Yarrowia lipolytica were expressed either individually or in combination in S. cerevisiae. The engineered strains could grow on olive oil and triolein as the sole carbon source. The strain expressing all three lipases had ∼40% lipid content per dry weight. Next, we integrated the genes encoding β-carotene biosynthetic pathway, crtI, crtYB and crtE from Xanthophyllomyces dendrorhous. The resulting engineered strain bearing the lipases and carotenogenic genes reached a titer of 477.9 mg/L β-carotene in yeast peptone dextrose (YPD) medium supplemented with 1% (v/v) olive oil, which was 12-fold higher than an analogous strain without lipases. The highest β-carotene content of 46.5 mg/g DCW was obtained in yeast nitrogen base (YNB) medium supplemented with 1% (v/v) olive oil. The study demonstrates the potential of applying lipases and hydrophobic substrate supplementation for the production of carotenoids in S. cerevisiae.