Elsevier

Metabolic Engineering

Volume 73, September 2022, Pages 26-37
Metabolic Engineering

Efficient production of retinol in Yarrowia lipolytica by increasing stability using antioxidant and detergent extraction

https://doi.org/10.1016/j.ymben.2022.06.001Get rights and content

Highlights

  • Retinol was produced in Yarrowia lipolytica by metabolic engineering.

  • First, a β-carotene-overproducing strain was generated from a lipid-overproducer.

  • Retinol was produced by introducing Mb.Blh into the β-carotene-overproducer.

  • Retinol stabilization by antioxidant and detergent extraction increased production.

  • 4.86 g/L retinol was produced with a 95% selectivity over retinal.

Abstract

The demand for bio-based retinol (vitamin A) is currently increasing, however its instability represents a major bottleneck in microbial production. Here, we developed an efficient method to selectively produce retinol in Yarrowia lipolytica. The β-carotene 15,15′-dioxygenase (BCO) cleaves β-carotene into retinal, which is reduced to retinol by retinol dehydrogenase (RDH). Therefore, to produce retinol, we first generated β-carotene-producing strain based on a high-lipid-producer via overexpressing genes including heterologous β-carotene biosynthetic genes, GGS1F43I mutant of endogenous geranylgeranyl pyrophosphate synthase isolated by directed evolution, and FAD1 encoding flavin adenine dinucleotide synthetase, while deleting several genes previously known to be beneficial for carotenoid production. To produce retinol, 11 copies of BCO gene from marine bacterium 66A03 (Mb.Blh) were integrated into the rDNA sites of the β-carotene overproducer. The resulting strain produced more retinol than retinal, suggesting strong endogenous promiscuous RDH activity in Y. lipolytica. The introduction of Mb.Blh led to a considerable reduction in β-carotene level, but less than 5% of the consumed β-carotene could be detected in the form of retinal or retinol, implying severe degradation of the produced retinoids. However, addition of the antioxidant butylated hydroxytoluene (BHT) led to a >20-fold increase in retinol production, suggesting oxidative damage is the main cause of intracellular retinol degradation. Overexpression of GSH2 encoding glutathione synthetase further improved retinol production. Raman imaging revealed co-localization of retinol with lipid droplets, and extraction of retinol using Tween 80 was effective in improving retinol production. By combining BHT treatment and extraction using Tween 80, the final strain CJ2104 produced 4.86 g/L retinol and 0.26 g/L retinal in fed-batch fermentation in a 5-L bioreactor, which is the highest retinol production titer ever reported. This study demonstrates that Y. lipolytica is a suitable host for the industrial production of bio-based retinol.

Introduction

All-trans retinol (vitamin A) is an essential micronutrient required in various biological processes such as embryo development, vision, learning and memory, immune system, reproduction, and skin function (Alvarez et al., 2014; Dollé and Niederreither, 2015). In human cells, retinol is oxidized into the bioactive forms, retinal and retinoic acid, or converted to retinyl ester as a storage form (Alvarez et al., 2014; Bonet et al., 2015; Dollé and Niederreither, 2015; O'Byrne and Blaner, 2013). 11-Cis-retinaldehyde functions as a visual chromophore, whereas all-trans- and 9-cis-retinoic acid regulate gene expression by binding to the nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR) (Alvarez et al., 2014; Bonet et al., 2015; Dollé and Niederreither, 2015). Retinol and its derivatives, collectively called retinoids, are widely used in food and beverages, pharmaceuticals, dietary supplements, animal feed, and skin care (Ferreira et al., 2020; Imhof and Leuthard, 2021; Mayo-Wilson et al., 2011). Currently, commercial retinol production is primarily based on chemical synthesis using petroleum-derived raw materials, and has been developed since the 1930s (Eggersdorfer et al., 2012; Parker et al., 2016). However, there is a growing demand for environmentally friendly bio-based retinol production.

Animals cannot biosynthesize retinoids, however, they can naturally produce retinal via cleavage of dietary β-carotene into two molecules of all-trans-retinal by β-carotene 15,15′-dioxygenase (BCO) (dela Sena et al., 2014). Retinal can be reversibly reduced to retinol by retinol dehydrogenases (RDHs) and alcohol dehydrogenases, or irreversibly oxidized to retinoic acid by retinal dehydrogenases (RALDHs) (Alvarez et al., 2014; Dollé and Niederreither, 2015). Other enzymes showing BCO activity, such as bacterio-opsin-related protein (Brp) and bacteriorhodopsin-related protein-like homolog protein (Blh), have been identified in certain halophilic archaea and bacteria (Kim et al., 2009; Peck et al., 2001). Retinal functions as a covalently bound cofactor for bacteriorhodopsin or proteorhodopsin, a light-driven proton transporter, in these organisms (Lanyi, 2004; Peck et al., 2001).

To date, few attempts have been made to synthesize retinoids in Escherichia coli and Saccharomyces cerevisiae by introducing heterologous β-carotene biosynthetic pathway and blh gene from the marine bacterium 66A03 (Mb.blh) or halophilic bacterium Salinibacter ruber (Sr.blh) (Choi et al., 2020; Jang et al., 2011; Sun et al., 2019). In both E. coli and S. cerevisiae, retinal, produced via cleavage of β-carotene, can be reduced to retinol via endogenous promiscuous RDH activity, resulting in the production of a mixture of retinal and retinol (Jang et al., 2011; Sun et al., 2019). Retinol is highly sensitive to light and oxidation owing to the conjugated double bonds present in the isoprenoid chain (Failloux et al., 2004). To overcome such instability and the limited intracellular storage capacity of hydrophobic retinoids, two-phase culture systems using hydrophobic solvent, including dodecane, mineral oil, or plant oil, have been applied in the in situ extraction of retinoids (Jang et al., 2011, 2014; Sun et al., 2019). In Mb.blh-expressing E. coli, up to 136 mg/L retinoids, consisting of retinol, retinal, and retinyl acetate at concentrations of 54, 67, and 15 mg/L, respectively, have been produced from glycerol using a two-phase flask culture with dodecane (Jang et al., 2011). A relatively more selective retinol production has been achieved in E. coli by overexpressing the endogenous ybbO gene and removing the plasmid-borne cat gene involved in retinol and retinyl acetate formation, respectively (Jang et al., 2015). The resulting strain produces 76 mg/L retinol as the major product, along with 5 mg/L retinal and retinyl acetate each (Jang et al., 2015). In addition, 69.96 mg/L retinyl palmitate is produced in E. coli by expressing Sr.blh, human lecithin retinol acyltransferase (LRAT), and cytoplasmic retinol-binding protein (Choi et al., 2020). In contrast, 8.2 mg/L retinoic acid was produced via the introduction of human RALDH (Han and Lee, 2021).

Although E. coli cell growth is inhibited by terpenoid pathway precursors, such as isopentenyl pyrophosphate (IPP) (George et al., 2018), yeasts have proven to be robust hosts for producing various terpenoids (Li et al., 2021; Zhang et al., 2017). Retinoid production has been achieved in a xylose-fermenting strain of S. cerevisiae by introducing Mb.blh and β-carotene biosynthetic genes from Xanthophyllomyces dendrorhous encoding geranylgeranyl pyrophosphate (GGPP) synthase (crtE), bifunctional lycopene cyclase/phytoene synthase (crtYB), and phytoene desaturase (crtI) (Sun et al., 2019). Due to the Crabtree effect, the engineered strain SR8A produces more retinoids from xylose than those obtained using glucose, producing up to 3.35 g/L retinoids (2.09 g/L retinal and 1.26 g/L retinol) in xylose fed-batch fermentation with in situ dodecane extraction (Sun et al., 2019). Recently, the SR8A strain has been further engineered to selectively produce retinol by introducing RDH12 gene encoding the human RDH and noxE derived from Lactococcus lactis encoding a water-forming NADH oxidase for redox balancing, achieving 123.1 mg/L of retinol production from xylose via in situ dodecane extraction (Lee et al., 2022).

Yarrowia lipolytica is an oleaginous yeast with high cytosolic acetyl-CoA pool, which is a common precursor for lipid and terpenoid production. Therefore, Y. lipolytica, a generally recognized as safe strain, is a promising industrial strain for the production of various lipid derivatives and terpenoids (Abdel-Mawgoud et al., 2018; Li et al., 2021; Liu et al., 2020; Xu et al., 2020). Y. lipolytica has an endogenous mevalonate pathway and produces enzymes that synthesize GGPP (Fig. 1A). Consequently, β-carotene has been successfully produced in Y. lipolytica by introducing heterologous genes encoding phytoene dehydrogenase and bifunctional lycopene cyclase/phytoene synthase from Mucor circinelloides or X. dendrorhous, and reinforcing GGPP synthase (GGPPS) activity (Gao et al., 2017; Larroude et al., 2018; Liu et al., 2021). Recently, by suppressing the lycopene substrate inhibition of CarRP, a bifunctional lycopene cyclase/phytoene synthase from M. circinelloides, 39.5 g/L of β-carotene production has been achieved in fed-batch fermentation (Ma et al., 2022). However, retinoid production in Y. lipolytica has not yet been reported.

In this study, we achieved retinol production in Y. lipolytica by increasing β-carotene production, introducing multiple copies of Mb.blh, and alleviating oxidative degradation of retinol, which was found to be a major bottleneck in efficient retinol production. By supplementing the antioxidant, butylated hydroxytoluene (BHT), to the medium and performing extraction using Tween 80, a nonionic detergent, we achieved selective production of up to 4.86 g/L retinol, suggesting that Y. lipolytica has great potential in the commercial production of bio-based retinol.

Section snippets

Culture conditions

Y. lipolytica cells were grown overnight on YPD medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose) and then inoculated in 20 mL YPDUL40 medium [10 g/L yeast extract, 20 g/L peptone, 40 g/L glucose, 1 g/L uracil, 1 g/L leucine, and 100 mM potassium phosphate (pH 7.0)] in a 250-mL baffled flask to an initial OD600 of 2. To select cells containing plasmid with URA3 marker, YLMM medium [40 g/L glucose, 1.25 g/L yeast nitrogen base without amino acids and ammonium sulfate, 3 g/L

Generation of β-carotene-producing Y. lipolytica strains

To produce retinol in Y. lipolytica, we first generated β-carotene-producing strains. Owing to the lipophilic nature of β-carotene, high-lipid-producers can produce relatively more β-carotene by providing high storage capacity (Larroude et al., 2018). Therefore, we used CJ0125 as a parent strain, which shows high lipid production capability (Fig. 1b and Table 1). In CJ0125, lipid production was improved by the PTEFINt- or PTEF-controlled overexpression of SLC1, DGA1, and LRO1, involved in

Discussion

Retinoids have a wide range of cellular functions, and represent valuable molecules with multiple applications as nutrients, pharmaceuticals, and cosmetic ingredients (Alvarez et al., 2014; Dollé and Niederreither, 2015; Ferreira et al., 2020; Imhof and Leuthard, 2021; Mayo-Wilson et al., 2011). Retinoic acid is mainly developed as a drug for the treatment of cancers and skin pathologies such as acne and eczema (Ferreira et al., 2020). In contrast, retinol is a commercially important retinoid

Competing financial interests

The authors declare no competing interest.

Author statement

Hyemin Park, Dongpil Lee: Investigation, Writing-Review & Editing Jae-Eung Kim, Seonmi Park, Joo Hyun Park, Kwang Hyun Park: Investigation Cheol Woong Ha: Project administration Minji Baek, Seok-Hwan Yoon: Funding acquisition Peter Lee: Conceptualization, Investigation, Writing-Original Draft, Supervision Ji-Sook Hahn: Writing - Original Draft, Visualization, Supervision.

Acknowledgements

This work was supported by the CJ CheilJedang Corporation. The authors thank for the support from CJ BLOSSOM PARK.

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