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Background: Crocetin is a multifunctional apocarotenoid natural product derived from saffron, holding significant promises for protection against various diseases and other nutritional applications. Historically, crocetin has been extracted from saffron stigmas, but this method is hindered by the limited availability of high-quality raw materials and complex extraction processes. To overcome these challenges, metabolic engineering and synthetic biology can be applied to the sustainable production of crocetin. Results: We constructed a Yarrowia lipolytica strain using hybrid promoters and copy number adjustment, which was able to produce 2.66 g/L of β-carotene, the precursor of crocetin. Next, the crocetin biosynthetic pathway was introduced, and we observed both the production and secretion of crocetin. Subsequently, the metabolite profiles under varied temperatures were studied and we found that low temperature was favorable for crocetin biosynthesis in Y. lipolytica. Therefore, a two-step temperature-shift fermentation strategy was adopted to optimize yeast growth and biosynthetic enzyme activity, bringing a 2.3-fold increase in crocetin titer. Lastly, fermentation media was fine-tuned for an optimal crocetin output of 30.17 mg/L, bringing a 51% higher titer compared with the previous highest report in shake flasks. Concomitantly, we also generated Y. lipolytica strains capable of achieving substantial zeaxanthin production, yielding 1575.09 mg/L, doubling the previous highest reported titer. Conclusions: Through metabolic engineering and fermentation optimization, we demonstrated the first de novo biosynthesis of crocetin in the industrial yeast Yarrowia lipolytica. In addition, we achieved a higher crocetin titer in flasks than all our known reports. This work not only represents a high production of crocetin, but also entails a significant simultaneous zeaxanthin production, setting the stage for sustainable and cost-effective production of these valuable compounds. [ABSTRACT FROM AUTHOR]

Sphingolipids are vital membrane components in in mammalian cells, plants, and various microbes. We aimed to explore and exploit the sphingolipid biosynthesis pathways in an oleaginous and dimorphic yeast Yarrowia lipolytica by constructing and characterizing mutant strains with specific gene deletions and integrating exogenous genes to enhance the production of long-chain bases (LCBs) and glucosylceramides (GlcCers). To block the fungal/plant-specific phytosphingosine (PHS) pathway, we deleted the SUR2 gene encoding a sphinganine C4-hydroxylase, resulting in a remarkably elevated secretory production of dihydrosphingosine (DHS) and sphingosine (So) without acetylation. The Y. lipolytica SUR2 deletion (Ylsur2 Δ) strain displayed retarded growth, increased pseudohyphal formation and stress sensitivity, along with the altered profiles of inositolphosphate-containing ceramides, GlcCers, and sterols. The subsequent disruption of the SLD1 gene, encoding a fungal/plant-specific Δ8 sphingolipid desaturase, restored filamentous growth in the Ylsur2 Δ strain to a yeast-type form and further increased the production of human-type GlcCers. Additional introduction of mouse alkaline ceramidase 1 (maCER1) into the Ylsur2 Δ sld1 Δ double mutants considerably increased DHS and So production while decreasing GlcCers. The production yields of LCBs from the Ylsur2 Δ sld1 Δ/ maCER1 strain increased in proportion to the C/N ratio in the N-source optimized medium, leading to production of 1.4 g/L non-acetylated DHS at the 5 L fed-batch fermentation with glucose feeding. This study highlights the feasibility of using the engineered Y. lipolytica strains as a cell factory for valuable sphingolipid derivatives for pharmaceuticals, cosmeceuticals, and nutraceuticals. [Display omitted] • Yarrowia lipolytica was engineered for production of sphingolipid derivatives. • SUR2 deletion enhanced secretory production of sphingoid bases without acetylation. • Subsequent SLD1 deletion recovered yeast-type growth and boosted human-type GlcCers. • Mouse alkaline ceramidase 1 expression increased the production of DHS and sphingosine. • Fed-batch fermentation with glucose feeding produced 1.4 g/L non-acetylated DHS. [ABSTRACT FROM AUTHOR]

Abstract Background Crocetin is a multifunctional apocarotenoid natural product derived from saffron, holding significant promises for protection against various diseases and other nutritional applications. Historically, crocetin has been extracted from saffron stigmas, but this method is hindered by the limited availability of high-quality raw materials and complex extraction processes. To overcome these challenges, metabolic engineering and synthetic biology can be applied to the sustainable production of crocetin. Results We constructed a Yarrowia lipolytica strain using hybrid promoters and copy number adjustment, which was able to produce 2.66 g/L of β-carotene, the precursor of crocetin. Next, the crocetin biosynthetic pathway was introduced, and we observed both the production and secretion of crocetin. Subsequently, the metabolite profiles under varied temperatures were studied and we found that low temperature was favorable for crocetin biosynthesis in Y. lipolytica. Therefore, a two-step temperature-shift fermentation strategy was adopted to optimize yeast growth and biosynthetic enzyme activity, bringing a 2.3-fold increase in crocetin titer. Lastly, fermentation media was fine-tuned for an optimal crocetin output of 30.17 mg/L, bringing a 51% higher titer compared with the previous highest report in shake flasks. Concomitantly, we also generated Y. lipolytica strains capable of achieving substantial zeaxanthin production, yielding 1575.09 mg/L, doubling the previous highest reported titer. Conclusions Through metabolic engineering and fermentation optimization, we demonstrated the first de novo biosynthesis of crocetin in the industrial yeast Yarrowia lipolytica. In addition, we achieved a higher crocetin titer in flasks than all our known reports. This work not only represents a high production of crocetin, but also entails a significant simultaneous zeaxanthin production, setting the stage for sustainable and cost-effective production of these valuable compounds.

Unconventional oleaginous yeast Yarrowia lipolytica has gained widespread applications as a microbial cell factory for synthesizing various chemicals and natural products. The construction of efficient cell factories requires intricate metabolic engineering. However, multi-gene expression in Y. lipolytica is labor-intensive. To facilitate multi-gene expression, we developed the polycistronic expression tool using 2A peptides. We first compared different 2A peptides in Y. lipolytica and identified two 2A peptides with high cleavage efficiency: P2A and ERBV-1. The effect of 2A peptides on the expression level of upstream and downstream genes was then determined. Ultimately, we applied the identified 2A peptides to express four genes in canthaxanthin biosynthetic pathway within one expression cassette for canthaxanthin production. This study enriches the multi-gene expression tools of Y. lipolytica, which will facilitate the cell factory construction of Y. lipolytica.

Abscisic acid (ABA) is an important phytohormone with diverse applications. It currently relies on the fermentation of Botrytis cinerea, which suffers from limited availability of genetic engineering tools. Here, Yarrowia lipolytica was engineered to enable de novo biosynthesis of ABA. To overcome the rate-limiting P450 enzymes, systematic engineering strategies were implemented. Firstly, the dissolved oxygen was increased to boost the activity of P450 enzymes. Secondly, the expansion of endoplasmic reticulum was implemented to improve the functional expression of P450 enzymes. Lastly, rate-limiting enzymes were assembled to facilitate substrate trafficking. Moreover, ABA production was further improved by strengthening the mevalonate pathway. Finally, the engineered strain produced 1221.45 mg/L of ABA in a 5-L bioreactor. The study provides effective approaches for alleviating rate-limiting P450 enzymes to enhance ABA production and achieve competitive industrial-level ABA production in Y. lipolytica.

[Display omitted] • Yarrowia lipolytica was engineered and evolved for improved xylose assimilation. • The resulting Y. lipolytica efficiently converted xylose into lipids. • Genetic variations eliciting enhanced xylose assimilation were identified. • Sorghum hydrolysate was efficiently converted into lipids by engineered Y. lipolytica. Yarrowia lipolytica , an oleaginous yeast, shows promise for industrial fermentation due to its robust acetyl-CoA flux and well-developed genetic engineering tools. However, its lack of an active xylose metabolism restricts the conversion of cellulosic sugars to valuable products. To address this, metabolic engineering, and adaptive laboratory evolution (ALE) were applied to the Y. lipolytica PO1f strain, resulting in an efficient xylose-assimilating strain (XEV). Whole-genome sequencing (WGS) of the XEV followed by reverse engineering revealed that the amplification of the heterologous oxidoreductase pathway and a mutation in the GTPase-activating protein gene (YALI0B12100g) might be the primary reasons for improved xylose assimilation in the XEV strain. When a sorghum hydrolysate was used, the XEV strain showed superior xylose consumption and lipid production compared to its parental strain (X123). This study advances our understanding of xylose metabolism in Y. lipolytica and proposes effective metabolic engineering strategies for optimizing lignocellulosic hydrolysates. [ABSTRACT FROM AUTHOR]

Succinic acid is an essential bulk chemical with wide-ranging applications in materials, food, and pharmaceuticals. With the advancement of biotechnology, there has been a surge in focus on low-carbon sustainable microbial synthesis methods for producing biobased succinic acid. Due to its high intrinsic acid tolerance, Yarrowia lipolytica has gained recognition as a competitive chassis for the industrial manufacture of succinic acid. This review summarizes the research progress on succinic acid biomanufacturing using Y. lipolytica . First, it introduces the major metabolic routes for succinic acid biosynthesis and the pertinent engineering approaches for building efficient cell factories. Subsequently, we offer a review of methods employed for succinic acid synthesis by Y. lipolytica utilizing alternative substrates as well as the relevant optimization strategies for the fermentation process. Finally, future research directions for improving succinic acid biomanufacturing in Y. lipolytica are delineated in light of the recent progress, obstacles, and trends in this area.

7-Dehydrocholesterol (7-DHC) is an important precursor of vitamin D 3 . The microbial synthesis of 7-DHC has attracted substantial attention. In this study, multiple strategies were developed to create a sustainable green route for enhancing 7-DHC yield from kitchen waste by engineered Yarrowia lipolytica . Y. lipolytica strains were engineered and combined with various Δ 24 -dehydrocholesterol reductases. Overexpressing all the genes in the mevalonate pathway improved the precursor pool, increasing the 7-DHC titer from 21.8 to 145.6 mg/L. Additionally, optimizing medium components using the response surface method significantly raised the 7-DHC titer to 391.0 mg/L after shake flask cultivation. The engineered strain yielded a record 7-DHC titer of 3.5 g/L in a 5-L bioreactor when kitchen waste was used as a carbon source. Overall, these results demonstrate that engineered Y. lipolytica efficiently synthesizes 7-DHC from waste lipid feedstock, offering a promising route for its bioproduction.

Utilization of microbial hosts to produce natural plant products is regarded as a promising and sustainable approach. However, achieving highly efficient production of terpenoids using microorganisms remains a significant challenge. Here, mevalonate, a building block of terpenoids, was used as a demo product to explore the potential metabolic constraints for terpenoid biosynthesis in Yarrowia lipolytica . First, by regulation of the expression of ERG12 and HMGR , the mevalonate titer was improved by 7660%. Subsequently, the native mevalonate pathway (MVA pathway) was enhanced, and the production of mevalonate increased to 4.16 g/L. To ensure a sufficient supply of acetyl-CoA, the citrate route and TCA cycle were simultaneously engineered, and the mevalonate titer was further improved to 5.25 g/L in shake flasks. Ultimately, the citrate overflow metabolism of Y. lipolytica was eliminated by deleting CEX1 , resulting in the highest mevalonate titer of 101 g/L with a yield of 0.255 g/g of glucose in eukaryotes. These insights could be applied to the effective production of terpenoids and biochemicals derived from central carbon metabolic pathways.