Identification of the fatty alcohol oxidase FAO1 from Starmerella bombicola and improved novel glycolipids production in an FAO1 knockout mutant

Takahashi, Fumiya; Igarashi, Kazuaki; Hagihara, Hiroshi

doi:10.1007/s00253-016-7702-6

Cited by 17 publications

(17 citation statements)

References 14 publications

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“…Two putative FAOs were identified from an in‐house genomic database of S. bombicola . The first one ( fao1) (GenBank: AB907775) was already proven to have alcohol oxidase activity by Takahashi, Igarashi, and Hagihara (). When this gene was blasted against the available S. bombicola genome, a second putative fatty alcohol oxidase was found, with 32% identity to the FAO1 enzyme.…”

Section: Resultsmentioning

confidence: 99%

From lab to market: An integrated bioprocess design approach for new‐to‐nature biosurfactants produced by Starmerella bombicola

Renterghem

Roelants

Baccile

et al. 2018

Biotech & Bioengineering

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Glycolipid microbial biosurfactants, such as sophorolipids (SLs), generate high industrial interest as 100% biobased alternatives for traditional surfactants. A well-known success story is the efficient SL producer Starmerella bombicola, which reaches titers well above 200 g/L. Recent engineering attempts have enabled the production of completely new types of molecules by S. bombicola, e.g. the bolaform SLs. Scale-up of bolaform SL production was performed at 150 L scale. The purified product was evaluated in detergent applications, as classic SLs are mostly applied in eco-friendly detergents. In this paper, we show that they can be used as green and non-irritant surfactants in for example (automatic) dishwashing applications. However, due to the presence of an ester function in the biosurfactant molecule a limited chemical stability at higher pH values (>6.5) was noticed, (therefore called 'non-symmetrical' (nsBola)) which, is a major drawback that will most likely inhibit market introduction. An integrated bioprocess design (IBPD) strategy was thus applied to resolve this issue. The strategy was to replace the fed fatty acids with fatty alcohols, to generate so-called "symmetrical bolaform (sBola) sophorosides (SSs)," containing two instead of one glycosidic bond. Next to a change in feeding strategy, the blocking of the fatty alcohols from metabolizing/oxidizing through the suggested ω-oxidation pathway was necessary. For the latter, two putative fatty alcohol oxidase genes (fao1 and fao2) were identified in the S. bombicola genome and deleted in the bolaform SL producing strain (ΔatΔsble). Shake flask experiments for these new strains (ΔatΔsbleΔfao1 and ΔatΔsbleΔfao2) were performed to evaluate if the fed fatty alcohols were directly implemented into the SL biosynthesis pathway. Indeed, sBola sophorosides (SSs) production up to 20 g/L was observed for the ΔatΔsbleΔfao1 strain. Unexpectedly, the ΔatΔsbleΔfao2 strain only produced minor amounts of sBola sophorosides (SSs), and mainly nsBola SLs (alike the parental ΔatΔsble strain). The sBola sophorosides (SSs) were purified and their symmetrical structure was confirmed by NMR. They were found to be significantly more stable at higher pH, opening up the application potential of the biosurfactant by enhancing its stability properties.

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Section: Resultsmentioning

confidence: 99%

From lab to market: An integrated bioprocess design approach for new‐to‐nature biosurfactants produced by Starmerella bombicola

Renterghem

Roelants

Baccile

et al. 2018

Biotech & Bioengineering

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“…The strains used in this study are shown in Table 1. Starmerella bombicola KSM36, an industrial strain for sophorolipid production (Inoue and Ito 1982), ura3-auxotrophic mutant KSM- ura3 Δ and FAO1-negative mutant KSM- fao1 Δ (Takahashi, Igarashi and Hagihara 2016) were used as the parent strains. The methods for plasmid maintenance, cultivation of S. bombicola and preparation of cell extracts were reported earlier without hygromycin (Takahashi, Igarashi and Hagihara 2016).…”

Section: Methodsmentioning

confidence: 99%

“…We previously reported that S. bombicola fatty alcohol oxidase 1 (FAO1) is a primary alcohol oxidase essential in the primary alcohol utilization pathway of S. bombicola (Takahashi, Igarashi and Hagihara 2016). It has been reported that S. bombicola has another fatty alcohol oxidase (FAO), which is a gene having homology with FAO1 (Van Renterghem et al .…”

Section: Introductionmentioning

confidence: 99%

Elucidation of secondary alcohol metabolism in Starmerella bombicola and contribution of primary alcohol oxidase FAO1

Takahashi

Igarashi

Takimura

et al. 2019

FEMS Yeast Research

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The yeast Starmerella bombicola NBRC10243 is an excellent producer of sophorolipids, which are among the most useful biosurfactants. The primary alcoholic metabolic pathway of S. bombicola has been elucidated using alcohol oxidase FAO1, but the secondary alcohol metabolic pathway remains unknown. Although the FAO1 mutant was unable to grow with secondary alcohols and seemed to be involved in the secondary alcohol metabolism pathway of S. bombicola , it had very low activity toward secondary alcohols. By analyzing the products of secondary alcohol metabolism, alkyl polyglucosides hydroxylated at the ω position in the alkyl chain of the secondary alcohol were observed in the FAO1 mutant, but not in the wild-type yeast. In the double mutant of FAO1 and UGTA1, accumulation of 1,13-tetradecandiol and 2,13-tetradecandiol was observed. The above results indicated that hydroxylation occurred first at the ω and ω–1 positions in the secondary alcohol metabolism of S. bombicola , followed by primary alcohol oxidation.

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“…[26,27] Also -oxidization and fatty alcohol oxidation deficient strains favored direct incorporation of fatty alcohols into the SLs. [28,29] The goal of this study was the evaluation of S. bombicola and C. kuoi for production of long-chain SL emulsifiers according to the structure proposal of Tulloch and Spencer. [23] Especially for C. kuoi, which solely incorporates terminally hydroxylated fatty acids into SLs, the direct incorporation of a primary fatty alcohol was assumed an equally probable synthesis route.…”

Section: Introductionmentioning

confidence: 99%

Microbial Synthesis of Nonionic Long‐Chain Sophorolipid Emulsifiers Obtained from Fatty Alcohol and Mixed Lipid Feeding

Zerhusen

Bollmann

Gödderz

et al. 2019

Euro J Lipid Sci & Tech

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Starmerella bombicola is known to produce sub-terminally hydroxylated lactonic sophorolipids (SLs), while Candida kuoi synthesizes acidic open chain SLs with terminally hydroxylated fatty acids. Upon feeding glucose and fatty alcohols both strains form long-chain nonionic SLs. According to structure elucidation the SLs consist of a hydroxylated fatty acid esterified with fatty alcohol and linked via a glycoside bond to the diacetylated sophorose unit. Palmityl, stearyl, and oleyl alcohols lead to products with lipid chain lengths of C32 or C36. Oleyl alcohol is the preferred substrate leading to 45 g L −1 of the double unsaturated C36 SL with S. bombicola and 20 g L −1 with C. kuoi. Scale up from shake flask to 1.5 L fermentations is possible and 65 g L −1 long-chain SLs are obtained with S. bombicola within 7 days. Mixed feeding of oleic acid and a variety of fatty alcohols leads to new long-chain SLs. In the presence of oleic acid the yeasts do not oxidize the fatty alcohol and thus the production of biosurfactants with tailored chain length is possible. The long-chain SLs show good emulsification ability of water/paraffin oil mixtures at low energy input and reduced interfacial tension significantly. Practical Applications: Sophorolipids are produced by fermentation on industrial scale focusing on cleaning and detergent applications. Mainly lactonic or anionic open-chain forms are used today. The new long-chain SLs presented in this manuscript are accessible with existing production technology and can be produced with high titers from cost-efficient renewable raw materials. In contrast to the commercial products the long-chain SLs are more hydrophobic and exhibit a strong emulsification behavior. Therefore they have the potential to broaden the application range of SLs in future. They may be useful as novel emulsifiers for cosmetic creams and lotions, pharmaceutical ointments and food products or may find application in oil spill remediation.

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Identification of the fatty alcohol oxidase FAO1 from Starmerella bombicola and improved novel glycolipids production in an FAO1 knockout mutant

Cited by 17 publications

References 14 publications

From lab to market: An integrated bioprocess design approach for new‐to‐nature biosurfactants produced by Starmerella bombicola

From lab to market: An integrated bioprocess design approach for new‐to‐nature biosurfactants produced by Starmerella bombicola

Elucidation of secondary alcohol metabolism in Starmerella bombicola and contribution of primary alcohol oxidase FAO1

Microbial Synthesis of Nonionic Long‐Chain Sophorolipid Emulsifiers Obtained from Fatty Alcohol and Mixed Lipid Feeding

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