Host and Pathway Engineering for Enhanced Lycopene Biosynthesis in Yarrowia lipolytica

Abstract

Carotenoids are a class of molecules with commercial value as food and feed additives with nutraceutical properties. Shifting carotenoid synthesis from petrochemical-based precursors to bioproduction from sugars and other biorenewable carbon sources promises to improve process sustainability and economics. In this work, we engineered the oleaginous yeast Yarrowia lipolytica to produce the carotenoid lycopene. To enhance lycopene production, we tested a series of strategies to modify host cell physiology and metabolism, the most successful of which were mevalonate pathway overexpression and alleviating auxotrophies previously engineered into the PO1f strain of Y. lipolytica. The beneficial engineering strategies were combined into a single strain, which was then cultured in a 1-L bioreactor to produce 21.1 mg/g DCW. The optimized strain overexpressed a total of eight genes including two copies of HMG1, two copies of CrtI, and single copies of MVD1, EGR8, CrtB, and CrtE. Recovering leucine and uracil biosynthetic capacity also produced significant enhancement in lycopene titer. The successful engineering strategies characterized in this work represent a significant increase in understanding carotenoid biosynthesis in Y. lipolytica, not only increasing lycopene titer but also informing future studies on carotenoid biosynthesis.

Changing Form and Function through Carotenoids and Synthetic Biology

“…Elucidating the functional organization of the multienzyme complex in plants is challenging. Fortunately, reconstruction of the plant carotenoid biosynthetic pathway can be achieved in Escherichia coli or yeast, which both synthesize the isoprenoid precursors (Chen et al, 2010;Pollmann et al, 2017;Ren et al, 2017;Schwartz et al, 2017;Zhang et al, 2018). These heterologous systems are excellent test beds for rapid characterization of carotenoid biosynthetic enzymes of the present and synthetic future, as well as for study of enzyme interactions and biosynthetic complex organization.…”

Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica

“…Herein, we demonstrate a methodology to measure and validate the activity of each guide RNA in a genome-wide library for screening in the oleaginous yeast Yarrowia lipolytica. We selected this non-conventional yeast because it has value as a bioprocessing host for the conversion of biomass derived sugars and industrial waste streams (e.g., glycerol, alkanes, and fatty acids) into value added chemicals and fuels (Qiao et al, 2017;Schwartz et al, 2017a;Wagner et al, 2018). Unlike the model yeast Saccharomyces cerevisiae, DNA repair in Y. lipolytica and most other eukaryotes is dominated by nonhomologous end-joining (NHEJ) (Lobs et al, 2017).…”

EngineeringYarrowia lipolyticaas a chassis forde novosynthesis of five aromatic-derived natural products and chemicals

“…Y. lipolytica, as a ‘generally regarded as safe’ (GRAS) yeast 1 has been extensively engineered for the production of oleochemicals, fuels and commodity chemicals 2–5 . The abundant acetyl-CoA and malonyl-CoA precursors in Y. lipolytica have been harnessed for synthesizing plant secondary metabolites in recent years, including flavonoids 6–8 , polyketides 9, 10 , polyunsaturated fatty acids 11, 12 and isoprenoids 13–15 . A large collection of customized genetic toolkits, including Golden-gate cloning 16–18 , genome integration 6, 19, 20 , CRISPR-Cas9/Cpf1 genome editing 21–23 , transposons 24 , auxotrophic markers 25 and promoter libraries 26–28 have accelerated our ability to perform targeted genetic manipulations.…”