Dataset Information

Dissolved-oxygen feedback control fermentation for enhancing ?-carotene in engineered Yarrowia lipolytica.

ABSTRACT: The DO-stat fed-batch fermentation was carried out to explore the volumetric productivity of ?-carotene in engineered Yarrowia lipolytica C11 strain. Using DO-stat fed-batch fermentation, we achieved 94 g/L biomass and 2.01 g/L ?-carotene. Both biomass and ?-carotene were about 1.28-fold higher than that in fed-batch fermentation. The ATP, NADP⁺/NADPH, and gene expression levels of tHMG, GGS1, carRA, and carB were promoted as compared to that in fed-batch fermentation. As for as the kinetic parameters in DO-stat fed-batch fermentation, ?_m', Y_x/s', and Y_p/s' was 0.527, 0.353, and 0.158, respectively. The ?_m' was elevated 4.66-fold than that in fed-batch fermentation. These data illustrate that more dissolved oxygen increased the biomass. The Y_x/s' and Y_p/s' were increased 1.15 and 22.57-fold, which suggest that the DO-stat fed-batch fermentation reduced the Crabtree effect and improved the utilization rate of glucose. Therefore, DO-stat fed-batch fermentation is a promising strategy in the industrialized production of ?-carotene.

SUBMITTER: Lv PJ

PROVIDER: S-EPMC7555900 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Dissolved-oxygen feedback control fermentation for enhancing β-carotene in engineered Yarrowia lipolytica.

Lv Peng Jun PJ Qiang Shan S Liu Liang L Hu Ching Yuan CY Meng Yong Hong YH

Scientific reports 20201013 1

The DO-stat fed-batch fermentation was carried out to explore the volumetric productivity of β-carotene in engineered Yarrowia lipolytica C11 strain. Using DO-stat fed-batch fermentation, we achieved 94 g/L biomass and 2.01 g/L β-carotene. Both biomass and β-carotene were about 1.28-fold higher than that in fed-batch fermentation. The ATP, NADP<sup>+</sup>/NADPH, and gene expression levels of tHMG, GGS1, carRA, and carB were promoted as compared to that in fed-batch fermentation. As for as the k ...[more]

PMID: 33051539

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Project description:β-Carotene is a provitamin A precursor and an important antioxidant that is used widely in the aquaculture, food, cosmetic, and pharmaceutical industries. Oleaginous Yarrowia lipolytica has been demonstrated as a competitive producer microorganism for the production of hydrophobic β-carotene through rational engineering strategies. However, the limited understanding of the complexity of the metabolic network between carotenoid biosynthesis and other cellular processes has hampered further advancement. Genome-scale mutagenesis and high-throughput screening of mutagenesis libraries have been extensively employed in gene mining or in the identification of key targets associated with particular phenotypes. In this study, we developed a fluorescence-activated cell-sorting approach for the effective high-throughput screening of high-β-carotene-producing strains. Using this approach, millions of mutants were screened rapidly, and new gene targets involved in lipid metabolism, sterol metabolism, signal transduction, and stress response were identified. The disruption of the genes affecting fatty acid oxidation, lipid composition, and sterol transcriptional regulation (4CL-8, GCS, and YIsterTF) increased β-carotene significantly. By engineering these targets in a high-β-carotene production, a strain that produced 9.4 g/L β-carotene was constructed. Here, we used a flow cytometry approach to improve screening efficiency and eliminate the interference of intermediate metabolites. The targets obtained in this study can be used in studies focusing on metabolic engineering in the future for improving carotenoid production. IMPORTANCE β-Carotene is a high-value-added product that is widely used in the aquaculture, food, cosmetic, and pharmaceutical industries. In our previous study, Yarrowia lipolytica has been engineered extensively to produce β-carotene. To further improve its production, high-throughput screening and the identification of new beneficial gene targets are required. Herein, we developed a fluorescence-activated cell-sorting approach for the effective high-throughput screening of high-β-carotene-producing strains. Using this approach, millions of mutants were screened rapidly, and new gene targets involved in lipid metabolism, sterol metabolism, signal transduction, and stress response were identified. The disruption of the genes affecting fatty acid oxidation, lipid composition, and sterol transcriptional regulation (4CL-8, GCS, and YIsterTF) increased β-carotene significantly. By engineering these targets in a high-β-carotene production, a strain that produced 9.4 g/L β-carotene was constructed.

Project description:Background:Xylose is the most prevalent sugar available in hemicellulose fraction of lignocellulosic biomass (LCB) and of great interest for the green economy. Unfortunately, most of the cell factories cannot inherently metabolize xylose as sole carbon source. Yarrowia lipolytica is a non-conventional yeast that produces industrially important metabolites. The yeast is able to metabolize a large variety of substrates including both hydrophilic and hydrophobic carbon sources. However, Y. lipolytica lacks effective metabolic pathway for xylose uptake and only scarce information is available on utilization of xylose. For the economica feasibility of LCB-based biorefineries, effective utilization of both pentose and hexose sugars is obligatory. Results:In the present study, succinic acid (SA) production from xylose by Y. lipolytica was examined. To this end, Y. lipolytica PSA02004 strain was engineered by overexpressing pentose pathway cassette comprising xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulose kinase (XK) gene. The recombinant strain exhibited a robust growth on xylose as sole carbon source and produced substantial amount of SA. The inhibition of cell growth and SA formation was observed above 60 g/L xylose concentration. The batch cultivation of the recombinant strain in a bioreactor resulted in a maximum biomass concentration of 7.3 g/L and SA titer of 11.2 g/L with the yield of 0.19 g/g. Similar results in terms of cell growth and SA production were obtained with xylose-rich hydrolysate derived from sugarcane bagasse. The fed-batch fermentation yielded biomass concentration of 11.8 g/L (OD600: 56.1) and SA titer of 22.3 g/L with a gradual decrease in pH below 4.0. Acetic acid was obtained as a main by-product in all the fermentations. Conclusion:The recombinant strain displayed potential for bioconversion of xylose to SA. Further, this study provided a new insight on conversion of lignocellulosic biomass into value-added products. To the best of our knowledge, this is the first study on SA production by Y. lipolytica using xylose as a sole carbon source.

Project description:As a valuable carotenoid, ?-carotene is commercially used in food, cosmetics, animal feeds, and other industries. Metabolic engineering of microorganisms has been widely explored to improve the production of ?-carotene. Compared with the traditional genetic modifications mainly focused on the pathways of mevalonate (MVA) and ?-carotene biosynthesis, this study aims to increase the ?-carotene production through promoting the synthesis of precursor substances by overexpressing hexokinase and hydroxymethylglutaryl-CoA synthase in an engineered Yarrowia lipolytica. In this study, we investigated the effect of the unique hexokinase gene (Hxk) overexpression on ?-carotene accumulation and glucose consumption. The Hxk gene was introduced into a ?-carotene producing strain Y.L-1 to generate strain Y.L-2, and this increased the ?-carotene content by 98%. Overexpression of the Hxk gene led to increasing in hexokinase activity (329% higher), glucose-6-phosphate content (92% higher), and improvement of the transcriptional level of Hxk (315% higher) compared to the control Y.L-1 strain. Moreover, Hxk overexpression accelerated the utilization rate of glucose. The gene erg13 encoding hydroxymethylglutaryl-CoA synthase was also overexpressed to increase the precursor supply for ?-carotene biosynthesis. Recombinant Y.L-4 harboring two copies of erg13 produced 8.41 mg/g dry cell weight (DCW) of ?-carotene, which was 259% higher than Y.L-1. The ?-carotene content of 9.56 mg/g DCW was achieved in strain Y.L-6 by integrating erg13 into the chromosome and Hxk overexpression. The 3-Hydroxy-3-Methylglutaryl-CoA content in the cells was increased by overexpressing two copies of the erg13 gene. Finally, the titer of ?-carotene reached 2.4 g/L using a 50 L bioreactor by the engineered strain, and the fermentation cycle was shortened from 144 to 120 h. Overall, overexpression of Hxk and erg13 could improve ?-carotene production and successfully overcoming the bottleneck of precursor generation to support a more efficient pathway for the production of the target product. Our results revealed a novel strategy to engineer the pathway of ?-carotene synthesis.

Dataset Information

Dissolved-oxygen feedback control fermentation for enhancing ?-carotene in engineered Yarrowia lipolytica.

Publications

Dissolved-oxygen feedback control fermentation for enhancing β-carotene in engineered Yarrowia lipolytica.

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