ᗈᓥ኱ᏛᏛ఩ㄳồㄽᩥ

Study on long-chain alcohol utilization
pathways in the glycolipid-producing
yeast Starmerella bombicola㸦⢾⬡㉁⏕⏘⳦ Starmerella bombicola ࡟࠾ࡅࡿ
㛗㙐࢔ࣝࢥ࣮ࣝ㈨໬⤒㊰࡟㛵ࡍࡿ◊✲㸰㸮㸯㸶ᖺᗈᓥ኱Ꮫ኱Ꮫ㝔⌮Ꮫ◊✲⛉ᩘ⌮ศᏊ⏕࿨⌮Ꮫᑓᨷ㧘ᶫ ྐဨ┠ ḟ

1. ୺ㄽᩥ
Study on long-chain alcohol utilization pathways in the glycolipid-producing yeast
Starmerella bombicola⢾⬡㉁⏕⏘⳦ Starmerella bombicola ࡟࠾ࡅࡿ㛗㙐࢔ࣝࢥ࣮ࣝ㈨໬⤒㊰࡟㛵ࡍࡿ◊✲2㸬බ⾲ㄽᩥ
Takahashi, F., Igarashi, K., Hagihara, H. Identification of the fatty alcohol oxidase
FAO1 from Starmerella bombicola and improved novel glycolipids production in an
FAO1 knockout mutant. Applied Microbiology and Biotechnology 100: 9519-9528(2016)

3㸬ཧ⪃ㄽᩥ
1) Takahashi, F., Sumitomo, N., Hagihara, H., Ozaki, K. Increased dipicolinic acidproduction with an enhanced spoVF operon in Bacillus subtilis and medium
optimization. 2015 Bioscience, Biotechnology and Biochemistry 79: 505-511(2015)

2) Tanaka, T., Takahashi, F., Fukui, T., Fujiwara, S., Atomi, H., Imanaka, T.
Characterization

of

a

novel

glucosamine-6-phosphatedeaminasefrom

hyperthermophilic archaeon. Journal of Bacteriology 187: 7038-7044 (2005)a୺ㄽᩥStudy on long-chain alcohol utilization pathways
in the glycolipid-producing yeast Starmerellabombicola㸦⢾⬡㉁⏕⏘⳦ Starmerella bombicola ࡟࠾ࡅࡿ
㛗㙐࢔ࣝࢥ࣮ࣝ㈨໬⤒㊰࡟㛵ࡍࡿ◊✲㸧

Fumikazu Takahashi
2018Contents

General Introduction-2-

Chapter 1. Identification of the fatty alcohol oxidase FAO1 from
Starmerella bombicola and improved novel glycolipids
production in an FAO1 knockout mutant.

-5-

Chapter 2. Elucidation of secondary alcohol metabolism in Starmerellabombicola and contribution of primary alcohol oxidase-35-

FAO1.

General Conclusions-58-

Acknowledgements

-62-

1

General Introduction

Alkyl polyglycosides (APGs) are safe and superior detergents having both high foaming
properties and non-irritative to the skin, and can be blended with other detergents.
Industrial production of APGs is mainly carried out by chemical synthesis using glucose
and alcohol as raw materials, but requires large amounts of alcohol and suffers fromglucose is denaturation during high temperature reactions. In order to develop more
efficient methods, APG production at ambient temperature under atmospheric pressureusing microorganisms or enzymes has been studied. For instance, Starmerellabombicola produces alkyl sophoroside which is a type of APGs from glucose andsecondary fatty alcohols (Brakemeier et al., 1995).
S. bombicola is known as a sophorolipids-producing yeast. Our company was
the first in the world to undertake industrial production of sophorolipids, and formulatethem into cosmetic bases (Inoue and Ito, 1982; Kimura, 1992). In recent years, manyother companies (Evonik, Ecover, Saraya, Allied Carbon Solutions and so on) havebegun to commercialize sophorolipids, due to the rise in environmental awareness.
However, molecular biological research on sophorolipids has lagged behind theprogress in their commercial use. When I started this study, a genome analysis of S.
bombicola had not been done and only one group had published a report on atransformation system (Van Vogaert et al., 2008).
As mentioned above, S. bombicola can produce APGs from secondary alcohols,
however it is difficult to produce APGs from primary alcohols. There are no reports on
the APG production pathway and metabolic system for fatty alcohols in S. bombicola.
The NAD dehydrogenase pathway is generally known as the embolism pathway for2

fatty alcohols. The reaction proceeds from the alcohol dehydrogenase and aldehyde
dehydrogenase, and the corresponding fatty acid is produced (Iwama et al., 2015). On
the other hand, the fatty alcohol oxidase (FAO) pathway was reported in some Candida
yeasts and fungi (Cheng et al., 2005). In S. bombicola, intracellular FAO activity has
been confirmed (Hommel and Ratledge, 1990). However, the role of FAO in alcohol
assimilation, its amino acid and gene sequence of FAO remain unknown. The aims ofthis study are to elucidate the fatty alcohol utilization pathway in S. bombicola and toconstruct an efficient APG production method.
In Chapter 1, I describe a cDNA analysis of S. bombicola, construction ofrecombination system, and identification of primary alcohol oxidase FAO1. In addition,a high yield of APGs and a novel diol-type glycolipid production method using an
FAO1-deficient strain are constructed. In Chapter 2, I clarify the contribution of FAO1
to the secondary alcohol utilization pathway, and examine for the whole fatty alcohol
utilization pathway in S. bombicola.

References
Brakemeier, A., Lang, S., Wullbrandt, D., Merschel, L., Benninghoven, A., Buschmann,N., Wagner, F. (1995) Novel sophorose lipids from microbial conversion of 2-alkanols.
Biotechnol. Lett. 17: 1183–1188.

Cheng, Q., Sanglard, D., Vanhanen, S., Huan, T.L., Bombelli, P., Smith, A., Slabas, A.R.
(2005) Candida yeast long chain fatty alcohol oxidase is a c-type haemoprotein andplays an important role in long chain fatty acid metabolism. Biochim. Biophys. Acta
1735: 192–203.
3

Hommel, R., Ratledge, C. (1990) Evidence for two fatty alcohol oxidases in thebiosurfactant-producing yeast Candida (Torulopsis) bombicola. FEMS Microbiol. Lett.
70: 183–186.

Inoue, S., Ito, S. (1982) Sophorolipids from Torulopsis bombicola as microbial
surfactants in alkane fermentations. Biotechnol. Lett. 4: 3–8.

Iwama, R., Kobayashi, S., Ohta, A., Horiuchi, H., Fukuda, R. (2015) Alcohol
dehydrogenases and an alcohol oxidase involved in the assimilation of exogenous fatty
alcohols in Yarrowia lipolytica. FEMS Yeast Res. 15: 1–12.

Kimura, Y. (1992) Feature/Functions of metabolites produced by yeast and theirapplications to cosmetics and toiletries. A glycolipid surfactant and its application.
Fragr. J. 20: 22-29.

Van Bogaert, I.N.A., De Maeseneire, S.L., Develter, D., Soetaert, W., Vandamme, E.J.
(2008)Developmentofatransformationandselection

glycolipid-producing yeast Candida bombicola. Yeast 25: 273–278.

4

systemfortheChapter 1
Identification of the fatty alcohol oxidase FAO1 in Starmerellabombicola and improved novel glycolipids production in an
FAO1 knockout mutant strain

Summary
Alkyl polyglucosides (APGs), which were first commercialized in the 1990s, are mild,
non-ionic surfactants comprising fatty alcohols and glucose derived from recyclable
starch. APGs have good properties as cleaners, foaming agents, and emulsifiers, andthey do not undergo hydrolysis at an alkaline pH. In addition to their advantages overtraditional synthetic surfactants, APGs are low-irritant surfactants that are nontoxic andeasily degradable in the environment. Thus, APGs are considered to be environmentallyfriendly surfactants. Starmerella bombicola glycosylates long-chain ω or ω-1 hydroxy
fatty acids, and it also directly glycosylates secondary alcohols. Although it is generally
difficult to directly glycosylate primary alcohols, they are easily converted to thecorresponding fatty acids by S. bombicola because of its strong alcohol oxidase activity.
To redirect unconventional substrates toward APG synthesis, the long-chain alcoholoxidation pathway was blocked by knocking out the fatty alcohol oxidase gene. The
complete sequence of the S. bombicola FAO1 gene (2,046 bp) was cloned, and theobtained nucleotide sequence was used to construct a knockout cassette. An FAO1
knockout mutant with the correct genotype and phenotype was evaluated byfermentation on 1-tetradecanol. The mutant produced tetradecyl disaccharides andtetradecanediol tetrasaccharides. The APGs and diol polyglucosides (DPGs) production5

of the mutant was 27.3g/L ((APGs + DPGs) / de novo sophorolipids ratio was about
15:1), while the parent strain did not produce APG or DPG. These data indicate that thesubstrates had been redirected toward novel glycolipids synthesis in the mutant.

Introduction
The yeast Starmerella bombicola is a potent producer of economically relevantbiosurfactants, which are referred to as sophorolipids (Spencer et al., 1970).
Sophorolipids derived from S. bombicola are glycolipids that have a disaccharide
sophorose linked glycosidically to the hydroxyl group at the ω or ω-1 carbon of C16 toC18 chain fatty acids. Several approaches have been used to improve sophorolipid
productivity (>3.5 g L−1 h−1) and determine the sophorolipid biosynthetic pathway (Gaoet al., 2013; Van Bogaert et al., 2013). Novel sophorose lipids were successfully
produced by microbial conversion using S. bombicola from blends of glucose and2-alkanols as the carbon source, and 2-dodecanol and 2-tetradecanol were directly
attached without further metabolization to their corresponding disaccharides(Brakemeier et al., 1995). Compared with classical sophorose lipids, most of thesecompounds have better water solubility and more effectively decrease the surface
tension of water. The sophorolipid and alkyl polyglucoside (APG) biosynthesis
pathways in S. bombicola are shown in Fig. 1. The sophorolipid biosynthesis system
was clarified (Van Bogaert et al., 2013), and the group of enzymes that participate inhydroxylation, glycosylation, acetylation, and lactonization reactions were identified. In
contrast, APGs may be synthesized in a single step by S. bombicola. Unfortunately,
secondary alcohols are costly, and there are few commercial suppliers. However,primary alcohols are inexpensive and readily available. However, it is difficult for S.
6

bombicola to produce APGs from primary alcohols, because this strain has strong
alcohol oxidase (AO) activity (Hommel and Ratledge, 1990). A better understanding of
the APG synthesis pathway and subsequent genetic engineering of this yeast species
could provide methods for producing higher APG yields, as well as modifications of the
APG structure.
The fatty AO (FAO) pathway in Candida tropicalis has been characterized
extensively (Cheng et al., 2005). Three FAO genes were cloned from C. tropicalis, oneof which was designated FAO1, and two putative allelic genes were designated FAO2aand FAO2b. Both enzymes oxidize long-chain primary alcohols. FAO1 oxidizes
ω-hydroxy fatty acids, but not 2-alkanols, whereas FAO2 oxidizes 2-alkanols, but not
ω-hydroxy fatty acids. However, the FAO pathway in S. bombicola has not yet been
determined, although it is proposed to have multiple FAO enzyme activities (Hommel
and Ratledge, 1990). There is no information regarding the FAO gene sequences andwhether the main FAO enzyme is expressed when S. bombicola is grown on a primaryalcohol as the sole carbon source.
This study is the first to report the sequence of an FAO gene in S. bombicola,
and it demonstrates that this enzyme plays a major role in the long-chain alcoholoxidation pathway. Here we describe the disruption of the FAO gene of S. bombicola toachieve a strain that is deficient in the long-chain alcohol oxidation pathway. This strain
not only was used to increase the production of APGs, but also was able to produce
novel diolpolyglucosides (DPGs) from primary alcohols.

Materials and methods
Strains, fermentation, and glycolipids production
7

The strains used in this study are shown in Table 1. S. bombicola KSM36 (an industrial
strain for sophorolipid production (Inoue and Ito, 1982) was used as the parent strain. S.
bombicola NBRC10243 (a type strain) was used for cDNA analysis. Escherichia coli
DH5α (TaKaRa Bio Inc., Kusatsu, Japan) was used for plasmid maintenance, and E.
coli BL21(DE3) (Funakoshi Co., Ltd., Tokyo, Japan) was used for protein expression. S.
bombicola was cultured on yeast peptone dextrose (YPD) medium (1% yeast extract,
2% peptone, and 2% glucose) or on synthetic dextrose (SD) medium (0.67% yeast
nitrogen base without amino acids (Sigma-Aldrich, St. Louis, MO, USA) and 2%
glucose). To obtain better growth of the mutants, 0.03% uracil was added to the YPD
medium. The SD medium used for the selection of auxotrophic mutants was
supplemented with 0.03% uracil and 0.5% 5-fluoroorotic acid (5-FOA). Liquid yeast
shake-flask cultures were incubated at 30°C with shaking at 120 rpm. E. coli was grownin Luria–Bertani (LB) medium (1% tryptone, 0.5% yeast extract, and 0.5% NaCl)
supplemented with 0.01% sodium ampicillin if necessary. Liquid E. coli cultures wereincubated at 37°C with shaking at 120 rpm. S. bombicola was grown on a sophorolipid
production medium (10% glucose, 10% methyl palmitate, 0.5% yeast extract, and 1%
urea (the pH was set to 5.0 with HCl)), or on an APG production medium comprising
15% glucose, 0.5% sodium citrate·3H2O, 0.04% yeast extract, 0.0154% NH4Cl, 0.1%
KH2PO4, 0.07% MgSO4·7H2O, 0.05% NaCl, 0.027% CaCl2·2H2O, and 0.016%
K2HPO4·3H2O. The seed culture (5 ml of YPD in a 100-ml test tube) was incubated for
48 h at 30°C with shaking (250 rpm) on a rotary shaker. An aliquot (1 ml) of this culture
was added to 100 ml of production medium in a 500-ml Sakaguchi flask. After 48 h, 10g L−1 of a primary alcohol was added to the microbial culture in the case of APG
production medium. The pH was not adjusted, and the total incubation time was 120 h.
8Washing cells from cultures
Culture samples (20 mL) were centrifuged at approximately 5,000 × g for 10 min at 4°C
to pellet the cells. The pellet was washed by resuspension in 10 ml of 0.08% NaCl,followed by centrifugation at 5,000 × g at 4°C for 1 min. This washing procedure wasrepeated twice.

Preparation of cell extracts and microsomes
The washed cell pellet was resuspended in 10 ml of 1/15 M potassium buffer (pH7.4).
Phenylmethylsulfonyl fluoride was added to a final concentration of 1 mM prior to
breaking the cells by shaking vigorously three times at 2,700 rpm for 1 min with
0.5-mm glass beads and a Multi-beads shocker (Yasui Kikai, Osaka, Japan). Then, thebroken cell suspension was centrifuged at approximately 15,000 × g for 60 min at 4°C.
The supernatant consisted of the soluble cell extract. Microsomes were prepared fromthe cell extract by centrifugation at 100,000 × g for 1.5 h at 4°C. The supernatant wasremoved, and the microsomal pellet was resuspended in 200 μl of 1/15 M potassium
buffer (pH7.4) containing 1 mM phenylmethylsulfonyl fluoride, and it was assayed for
FAO activity. Cell extracts from E. coli were prepared in the same manner.

Nucleic acid extraction and cDNA synthesis
Yeast genomic DNA was isolated using the Mr. Gentle High Recovery Kit (TaKaRa
Bio). Plasmid DNA was isolated using a plasmid purification kit (Roche, Basel,
Switzerland). RNA was isolated using an RNeasy Mini Kit (Qiagen, Valencia, CA,
USA) according to the manufacturer’s protocol, except that in the cell lysis step,
zymolyase-20T (Seikagaku Corporation, Tokyo, Japan) was added to a final
9

concentration of 100 units L−1 in 1 mL of buffer Y1. A full-length cDNA library wassynthesized using the SMARTer cDNA Synthesis Kit (Clontech, Mountain View, CA,
USA) according to the manufacturer’s protocol. S. bombicola 10243 cells that werefermented for 48 h in sophorolipid production medium were used for RNA extraction
and cDNA sequencing (described below).

Library construction and 454 sequencing
A 454 library was constructed using the Roche GS FLX Titanium Rapid Library
Preparation Kit according to the manufacturer’s instructions. The library was sequenced
using a GS FLX Titanium Sequencing Kit XLR70 on a 454 FLX Titanium Genome
Sequencer (Roche).

Sequence assembly and functional annotation
To obtain unique transcripts, all high-quality reads were assembled de novo using thecDNA mode of Newbler v2.3 software (www.454.com) with default assemblyparameters. The amino acid sequence of C. tropicalis FAO2 (accession no.
AAS46880.1) was used to search for the putative AO gene in S. bombicola using thetranslated

nucleotide

Basic

Local

Alignment

SearchTool(TBLASTN)(ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/) with default parameters,
excluding –F F (low complexity filter = OFF).

Transformation
S. bombicola cells were transformed via electroporation (Wang et al., 2001) using a
Gene Pulser Xcell (Bio-Rad, Carlsbad, CA, USA) set to 25 μF, 350 Ω, and 2.5 kV.
10Transformants were selected on SD plates. E. coli cells were transformed as described
previously (Sambrook and Russell 2001).

Polymerase chain reaction (PCR) and sequencing analysis
The primers used in this study are shown in Table 2. The URA3 genes of the selectedmutants were amplified by yeast colony PCR with the primers Pura3F and ura3F. The
obtained PCR fragments were cloned into pUC118/HincII(BAP) (TaKaRa Bio), and oneof the resulting vectors was called pUC-URA3. The yeast colony PCR used KOD FX
Neo (TOYOBO, Osaka, Japan). PCR amplification was performed with an initialdenaturation of 94°C for 2 min, followed by 30 cycles of 98°C for 30 s, 55°C for 30 s,
and 72°C for 2 min, followed by a final 2-min elongation at 72°C. All DNA sequenceswere determined using a Big Dye ver. 3.1 (ABI) and 3500 Genetic Analyzer (Applied
Biosystems, Foster City, CA, USA).

Subcloning and expression of FAO1
The FAO1 F and FAO1 R primers that were used to PCR the open reading frame (ORF)
of FAO1 were designed to provide a 15-bp region that was complementary to pET21a in
the final PCR product. The primers for pET21a were pET21 F and pET21 R. The PCR
products were purified with a PCR Purification Kit (Roche). The FAO1 amplicon was
ligated into pET21a using the In-Fusion HD Cloning Kit (TaKaRa Bio). The FAO1
expression vector was designated pET-FAO1.

Production of FAO1 in E. coli
Overnight cultures of respective E. coli clones were grown at 30°C in 5 ml of LB
11

medium containing 0.01% sodium ampicillin with shaking at 250 rpm. Fifty milliliters
of LB medium containing 0.01% sodium ampicillin was placed in each of two 500-ml
Sakaguchi-flasks. The flasks were inoculated with overnight cultures and grown at 37°C
with shaking at 120 rpm. At an optical density at 600 nm of 0.3 to 0.4, the cultures were
induced with isopropyl β-D-1-thiogalactopyranoside (0.1 mM final concentration).
Then, the cultures were incubated at 16 h at 25°C and harvested by centrifugation.

FAO activity assay
The assay procedure was modified from the protocol of Kemp et al. (1988). The assay is
a two-enzyme, coupled reaction. 1-Dodecanol was used as the standard for the general
FAO assay. The final reaction mixture for the general AO assay comprised 100 μl of 100
mM sodium phosphate buffer (pH7.4), 50 μl of 2.8 mg ml−1 2,2’-azino-di[3-ethylbenzothiazoline-(6)-sulfonic acid] solution in deionized water, 30 μl of ahorseradish peroxidase (47 units mL−1) solution (Wako Pure Chemical Industries, Ltd.,
Osaka, Japan), 10 μl of microsomal protein or cell extract solutions, and 10 μl of a 5
mM substrate solution. FAO activity was measured at 30°C at 405 nm. FAO activity
was reported as specific activity units per milligram of protein (1 U = 1 μmol ofoxidized substrate min−1).

Protein determination
The protein concentration in the extracts was determined using a Bradford protein assay
kit (Bio-Rad). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was
performed according to the method of Laemmli (1970).

12

Creation of the knockout fragments
The strategy for knocking out the FAO1 gene is shown in Fig. 2. The 1,000-bp upstream
and 1,000-bp downstream fragments of the FAO1 gene were amplified with the primers
FAO1USinF and FAO1US-UraR, and Ura-FAO1DSF and FAO1DSinR, respectively.
The primers Pura3F and ura3R were designed according to the manufacturer's
guidelines and used to integrate the functional S. bombicola URA3 sequence into FAO1.
The primers pUC F and pUC R were used to amplify a linear DNA fragment of pUC118.
pUC-ΔFAO1 was constructed such that these fragments were ligated using the In-Fusion
HD Kit (TaKaRa Bio). The primer pair FAO1USinF and FAO1DSinR was used toamplify a 1,023-bp fragment, which contained the URA3 sequence and 1,000-bp of the
FAO1 upstream and downstream flanking sequences, from pUC-ΔFAO1. The fragment
was used to transform the ura3-auxotrophic KSM-ura3Δ strain, which was isolatedusing the 5-FOA selection method (Van Bogaert et al., 2008). We used this method toselect a spontaneous ura3-auxotrophic mutant, designated KSM-ura3Δ, of S. bombicola
KSM36.

Analysis of ura3-auxotrophic mutants
The sequences of the URA3 gene in NBRC 10243 (GenBank accession no.
DQ916828), KSM36, and KSM-ura3Δ were analyzed. The NBRC10243 and KSM36
URA3 genes were identical. KSM-ura3Δ had a single C54Y mutation in the URA3 gene(data not shown); thus, this strain requires ura3 for growth, and it is 5-FOA resistant.
The pUC-URA3 vector carries the functional URA3 gene of S. bombicola
NBRC10243, including the 789-bp coding sequence and a 234-bp 5′-untranslated
region. When 1 μg of pUC-URA3 vector was used in the transformation, over 100
13colonies appeared on minimal medium plates. Insertion of the wild-type URA3 gene
into the KSM-ura3Δ genome was verified by PCR. The primers Pura3 and ura3R
specifically amplified the URA3 gene, while pUC118F and pUC118R were specific forthe pUC118-containing fragment. Eleven randomly selected prototrophs that wereobtained after the transformation with linear DNA were tested. Ten of the tested
colonies resulted from a single crossover or non-homologous recombination event, thatis, they contained the pUC118 fragment. For one transformant, a double-crossover event
occurred in the URA3 region. This strain was designated KSM-ura3Δ::URA3, and it wasused as the comparative strain for the KS-ura3Δ strain. The KSM ura3Δ::URA3 strain
had the same ability to produce sophorolipids from oleic acid, as well as an alkyl
glucoside from 2-tetradecanol, as the wild-type strain (data not shown).

High-pressure liquid chromatography/charged aerosol detection (HPLC/CAD) andmass spectroscopy (MS) analysis
Glycolipids samples were analyzed by an Chromaster HPLC system (Hitachi, Tokyo,
Japan) and Corona CAD (Thermo Scientific, Waltham, MA, USA) using an Acclaim
Surfactant column (4.6 x 150 mm, 5 μm (Dionex, Sunnyvale, CA, USA)) at 40°C.
Parameters of CAD were set to the nitrogen gas pressure of 35 psi and the detector
response range of 100 pA. A gradient of two eluents, a 0.1% formic acid (w/w) aqueous
solution and acetonitrile, was used to separate the components. The gradient started at
20% acetonitrile for 3 min, and it increased linearly to 95% over 37 min and to 95%
acetonitrile for 5 min. The mixture was maintained at this composition for 30 min andthen brought back to 20% acetonitrile over 5 min. A flow rate of 0.5 ml min−1 wasapplied. A Prominence UFLC HPLC System (Shimadzu, Kyoto, Japan) and liquid
14chromatography (LC)-MS 2020 (Shimadzu) were used for the HPLC-MS analysis. A
scan analysis was performed for molecules with masses between 30 and 1,500 Da.

Sampling and analytical methods
Analytical glycolipids samples were prepared as follows. Five hundred microliters ofbutanol containing 0.05% 1-octadecanol as an internal standard was added to 500 μl of
culture broth and shaken vigorously for 5 min. After centrifugation at 3,000 × g for 5min, the upper solvent layer was removed, placed in fresh glass culture tubes (ST-13M,
Tokyo Rikakikai Co., Ltd., Tokyo, Japan), and dried using a centrifugal evaporator. For
alkaline hydrolysis of ester linkages, the solid sample was refluxed in 500 μl of a 1 M
NaOH solution for 4 h. After adjusting the pH to 4 with 1 M HCl, 1 ml of butanol wasadded and shaken vigorously for 5 min. After centrifugation at 3,000 × g for 5 min, theupper solvent layer was removed, placed in fresh glass culture tubes (ST-13M), anddried using a centrifugal evaporator. A 1-(trimethylsilyl)imidazole solution (500 μl, GL
Sciences Inc., Tokyo, Japan) was added and incubated at 60°C for 10 min. An equalvolume (500 μl) of hexane and saturated saline were added and shaken vigorously for 5
min. After centrifugation at 3,000 × g for 5 min, the upper solvent layer was analyzed
by gas chromatography/MS (GC-MS) on 7890A (Agilent Technologies, Santa Clara,
CA, USA) and 5975C Systems (Agilent Technology) using a DB-1 ms column (20 m ×
0.10 mm × 0.1 μm), helium gas as the mobile phase at a flow rate 0.3 ml min −1, and thefollowing time program: 100°C for 2 min, followed by an increase of 15°C min−1 to
300°C (20 min). n-Dodecyl-E-D-maltoside (Calbiochem, EMD Millipore, Billerica,
MA, USA) was used as a standard. Colony-forming units were determined by plating
serial dilutions on YPD agar plates, which were incubated at 30°C for 3 d.
15

Nucleotide sequence accession number
The nucleotide sequence described in this paper was deposited in the GenBank
nucleotide database under the accession number AB907775.

Results
Isolation of the FAO1 gene sequence
In this study, a cDNA sequence analysis was performed on the type strain of S.
bombicola NBRC10243. A putative AO gene was found that had weak homology(identity 35%) to C. tropicalis FAO2. The complete ORF of 2,046 bp encodes a putative
FAO comprising 681 amino acids, which we named FAO1 (Fig. 3). A single exon
encoding a genomic clone contained the full-length FAO1 coding 4688 sequence.
To isolate the complete FAO1 gene from the S. bombicola KSM36 genome, the2,787-bp FAO1 fragment was amplified by high-fidelity PCR of freshly isolated
genomic DNA. The DNA sequence of the FAO1 gene from the KSM36 strain was thesame as that of NBRC10243; thus, NBRC10243 and KSM36 are likely to be closely
related. The ORF encodes a protein with an estimated molecular mass of 75.0 kDa andan estimated isoelectric point of 8.48.

Sequence homology to other FAO proteins
Analysis of the putative FAO1 protein sequence via the Conserved Domain Databaseavailable

attheNational

Centerfor

Biotechnology

Information

website(Marchler-Bauer et al., 2009) indicated that this protein (EC 1.1.3.20) belongs to the
GMC oxidoreductase superfamily, which is a polyspecific family that harbors over 30
different inverting oxidases that use molecular O2. The other FAO, Candida cloacae
16

FAO1 (CAB75352.1, Vanhanen et al., 2000), has a consensus SKL amino acid sequence
motif

that

is

a

consensus

http://elm.eu.org/elms/TRG_PTS1).

peroxisome-targeting

sequence

corresponding

carboxyl-terminal

The

FAO1

(PTS,

sequence is TKL, which also may be a PTS.

Expression of FAO1 in E. coli, and its AO activity
FAO1 was expressed in E. coli BL21(DE3). FAO1 migrated as an ~75 kDa band in
sodium dodecyl sulfate-polyacrylamide gels (Fig. 4a), which corresponds well to its
calculated molecular mass (75 kDa). FAO1 had AO activity towards 1-dodecanol, while
E. coli BL21(DE3) did not (Fig. 4b). The substrate specificity of FAO1 was verified(Fig. 4c). FAO1 had oxidase activity toward primary alcohols with chain lengths of 4 toConstruction of an FAO1-negative S. bombicola strain
A linear FAO1 knockout cassette was used to transform the KSM ura3Δ strain, and thetransformants were selected by complementation as a result of the disruption of the
FAO1 gene by the insertion of the URA3 marker. Four days after the transformation,
many transformants were obtained on selective plates, and 10 of them were subjected tocolony PCR using the primers FAOUSinF and FAO1DSinR to check their genotype. Of
the 10 transformants, seven yielded the expected amplicon for double-crossover events.
One of the strains was designated KSM-fao1Δ. The other mutants probably arose from
non-homologous recombination events or recombination of the URA3 marker with the
KSM ura3Δ allele.

Growth of the KSM-fao1Δ strain in various long-chain alcohol substrates
17

S. bombicola strains were grown on a basal medium containing 1-tetradecanol as thesole carbon source. We determined the ability of the KSM-ura3Δ::URA3 andKSM-fao1Δ strains to use several carbon sources. As shown in Table 3, both strains
grew on glucose as the sole carbon source. The KSM-ura3Δ::URA3 strain, but not theKSM-fao1Δ strain, also grew on media containing 1-tetradecanol or 1-hexadecanol. The
AO activities of the KSM-ura3Δ::URA3 and KSM-fao1Δ strains were determined.
These strains were cultivated on SD medium containing 1% 1-hexadecanol for 48 h.
The specific FAO activities toward 1-dodecanol in the microsome fractions of the
KSM-ura3Δ::URA3 and KSM-fao1Δ strains were 70.6 ± 16.2 and 6.9 ± 3.9
U/g-protein, respectively. These data suggest that FAO1 is the main AO for theutilization of long-chain alcohols.

Structural analysis of glycolipids produced by the KSM-fao1Δ strain
The ability of the strains to produce glycolipids from 1-tetradecanol was tested.
Seventy-two hours after the addition of alcohol, glycolipids production by the
KSM-ura3Δ::URA3 and KSM-fao1Δ strains was determined. Tetradecanoic acid was
detected in strain KSM ura3Δ::URA3, but not in strain KSM-fao1Δ, via GC. This
indicated that alcohol oxidation was suppressed by deleting the FAO1 gene.

The chromatograms obtained for the KSM-fao1Δ strain, but not the KSM-ura3Δ::URA3
strain, showed a peak that corresponded to the mobility of dodecyl maltoside (18.9 min)(Fig. 5). The supernatant was then subjected to HPLC/CAD and HPLC/MS.
Monoacetylated and diacetylated tetradecyl disaccharide hydronium ions had m/z of
598.5 and 640.5, respectively. These ions were observed at 26.7 min (peak 2 in Fig. 6b)
18

and 28.3 min (peak 3 in Fig. 6b) in the culture of the KSM-fao1Δ strain. Based on aprevious study (Fleurackers et al., 2010), we estimated that the disaccharide backbone
wasasophoroside.

Monoacetylated

1-O-tetradecyl-(2-O-β-D-glucopyranosyl-

β-D-glucopyranoside (molecular weight 580 Da) and diacetylated 1-O-tetradecyl(2′-O-β-D-glucopyranosyl-β-D-glucopyranoside) (molecular weight 622 Da) were
contained in the mixture (Fig. 7).
The main product (peak 1) ion had an m/z of 1,064.7 in the positive ion mode
and an m/z of 1,045.6 in the negative ion mode. This ion corresponded to tetraacetylated
tetradecandiol tetrasaccharide (molecular weight 1,046 Da). The results of acidic
methanolysis of butanol extracts from the cultures and an additional GC analysis arepresented in Table 4. There was a very high 1,14-tetradecandiol content in theglycolipids, which was also true to some extent for 2,13-tetradecandiol and1-tetradecanol. Based on a previous study (Brakemeier et al., 1995), we estimated thatthe constituents of peak 1 were sophorolipids SL-C and SL-D (Fig. 7). The peaks from26 to 32 min (26.4, 27.1, 29.0, and 31.1 min) are de novo-synthesized sophorolipids(Fig. 6c).

Production of glycolipids by the KSM-fao1Δ strain
To determine the novel glycolipids yield, the same sample was analyzed by
HPLC/CAD. De novo sophorolipids production were 6.4 g/L/120 h and 1.8 g/L/120 h inthe

KSM-ura3Δ::URAand

KSM-fao1Δ

strains,

respectively.

While

the

KSM-ura3Δ::URA3 strain did not produce APGs or DPGs from primary alcohols (Fig.
8), 27.3g L−1 novel glycolipids were obtained from the KSM-fao1Δ strain. Thus, theKSM-fao1Δ strain efficiently converted primary alcohols to glycolipids. The ratios of19glycolipids were 0.78, 0.16, 0.06 in alkyldisophorosides (SL-C, D), alkylsophorosides(SL-A, B), and classical sophorolipids, respectively, in the culture of the KSM-fao1Δ
strain.

Discussion
A few studies that altered sophorolipid structures, such as their sugar or lipid
components, via gene recombination in S. bombicola have been reported. More recently,
the biosyntheses of sophorolipids and sophorolipid transporters were reported by Van
Bogaert et al. (2013), and host-modified research for sophorolipid production is
expected to progress rapidly (Van Bogaert et al., 2013). Moreover, Van Bogaert et al.
(2009) also reported that a higher production of medium-chain length sophorolipids wasachieved by blocking the beta-oxidation pathway. We attempted to determine the main
enzyme responsible for alcohol oxidation in S. bombicola, and to convert this strain to
one that produces tetradecyl disaccharide and novel diol tetrasaccharide by deleting its
AO gene. Alcohol oxidation enzymes may be categorized as alcohol dehydrogenases or
alcohol oxidases based on the mechanisms involved in the catalytic reactions. S.
bombicola was suggested to have multiple alcohol oxidases (Hommel and Ratledge,1990).
We identified FAO1, a novel gene encoding an AO, in the S. bombicola genome.
The deletion of FAO1 prevented the growth of S. bombicola in media containing
primary alcohols as the carbon source, and AO activity in the microsomal fraction
decreased by ~90% compared with that of the parent strain. Thus, FAO1 must be adominant factor in the assimilation of primary alcohols. Deletion of FAO1 improved theyields of tetradecanol-based sophorolipids, and tetradecanediol-based sophorolipids
20

from 0 to 16.9% and from 0 to 46.2%, respectively (Fig. 8). It was unexpected that
tetradecanediol-based sophorolipids were major products. S. bombicola should have ωand ω-1 hydroxylation activity toward alcohols. These glycolipids were not detected in
the parent strain because alcohols and diols could be easily oxidized to theircorresponding fatty acids by FAO1.
The KSM-fao1Δ strain efficiently produces novel glycolipids from primaryalcohols. Although sophorolipids are classified as low-foaming detergents, the culture
supernatant of the KSM-fao1Δ strain mutant clearly exhibited increased foam formationand stability compared with the supernatant containing wild-type yeast sophorolipids.
These strong foaming properties are of interest to several industries. With a yield ofapproximately 27g L−1, the FAO1 mutant offers an environmentally acceptable method
compared to chemical synthetic procedure at a moderate temperature, standard pressure,and in a water reaction field.

21

FiguresFig. 1 Sophorolipid biosynthesis and proposed APG biosynthesis in S. bombicola

22

Fig. 2 FAO1 gene knockout strategy.
A, S. bombicola wild-type FAO1 allele, B, knockout cassette, C, mutant allele.
Sites of homologous recombination between the genome and the cassette are indicated,
and all theprimers used to create the cassette and determine the genotype of the mutant
are indicated.

23

Fig. 3 Nucleotide and deduced amino acid sequence of the FAO1 gene of S. bombicola.
Possible promoter elements are boxed.
24Fig. 4 (a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of protein
profiles of E. coli BL21(DE3)-expressed FAO1. Samples were obtained 19 h after
induction. The induced protein is indicated by an arrow. Lane 1, cell-free extract of
BL21-pET21a; lane 2, cell-free extract of BL21-pET-FAO1. (b) Specific FAO activity incell-free extracts of E. coli BL21-pET21a and of BL21-pET-FAO1. The substrate for the
enzyme assay was 1-dodecnol. (c) Substrate specificity of FAO1. C4, 1-butanol; C6,
1-hexanol; C8, 1-octanol; C10, 1-decanol; C12, 1-dodecanol; C14, 1-tetradecanol; C16,
1-hexadecanol

25

Fig. 5 Gas chromatography analysis of alkyl polyglucosides from (a) S. bombicola theKSM-ura3Δ::URA3 and (b) KSM-fao1Δ strains grown on 1-tetradecanol.

26

Fig. 6 HPLC analysis of glycolipids from KSM-fao1Δ grown on 1-tetradecanol, (a) the
chromatogram of ESI MS, (b) the chromatogram of CAD. (c) HPLC/CAD analysis ofglycolipids from KSM-fao1Δ absent 1-tetradecanol. Panel D, E, F show a detail of thepositive ion mode mas spectrum of LC-ESI peak 1, 2, 3 in (a), respectively.

27

Fig. 7 Putative structure of the glycolipids from the KSM-fao1Δ strain grown on
1-tetradecanol. SL, sophorolipid.

28

Fig. 8 Glycolipid production of the parent and the KSM-fao1Δ strains grown on
1-tetradecanol. (a) The KSM-ura3Δ:URA3 strain, and (b) the KSM-fao1Δ strain. Filledbars

indicatealkyl

sophorosides;

gray

bars

indicated

tetradecanediol-based

sophorolipids; open bars indicate de novo-synthesized sophorolipids; and open circles
indicate the glucose concentration in the culture

TablesTable 1 Yeast and bacterial strains used or constructed in this study
StrainProperties

Source or reference

KSM36

S. bombicola KSM36

FERM-BP㻌 799

KSM-ura3Δ
KSM-ura3Δ::URA3

KSM36ΔURA3KSMΔURA3::URA3

This study
This study

KSM- fao1Δ

KSM36ΔURA3,
FAO1::URA3

This study

NBRC10243

Type strain

Escherichia coli
BL21(DE3)

NITE Biological Resource
Center, Japan
TaKaRa BIO

㻌29

Table 2 Primers used for isolating, cloning, expression and knocking out the S.
bombicola FAO1 gene.
PrimerSequence (5'-3')Purpose in this study

Pura3 FAGTACATATTTTTCGAAACAGCTCGCAA

Cloning of URA3

ura3 RCTAAGAAACTCATCTTGACTGAACTTTTC

FAO1 F

FAO1 RTTATCCTC
AGTGCGGCCGCAAGCTAGCTTAGTTTGA
AGCTTAG
GCTTGCGGCCGCACTCGAG

pET21 RATGTATATCTCCTTCTTAAAGFAO1US-ura㻌 R

Ura-FAO1DS㻌 F

FAO1DSin㻌 R

and KSM-ura3Δ::URA3

GAAGGAGATATACATATGACTGACGCAG

pET21 F

FAO1USin㻌 F

Construction of KSM- fao1Δ

Construction of pET-FAO1

AGCTTGCATGCCTGCTTTAAATCCAGAAAGAACTGCGAAAAATATGTACTGATAACTGCGTCAGTCATTGAGATGAGTTTCTTAGAAGCCTTATATCGAATACAC

Construction of KSM- fao1Δ

ATTCGAGCTCGGTACGACACTTCTCAGG
AACCCTC

pUC F

GTACCGAGCTCGAATTCGTpUC R

GCAGGCATGCAAGCTTGGC

pUC118㻌 F

GGCGAAAGGGGGATGTGC

pUC118㻌 R

GCACCCCAGGCTTTACAC

Checking

forrecombinationof pUC118 region

30

illegitimate

Table 3 Growth of KSM36 and mutant strains grown in primary alcohols.
Substrates/strains

KSM36

KSM-ura3Δ::URA3

KSM- fao1Δ

Glucose
1-dodecanol
1-tetradecanol
1-hexadecanol

+

+

+

䠉
+
+

䠉
+
+

䠉
䠉
䠉

'+: Growth, 䠉: no growth.

Table 4 GC analysis of lipid constituents after the methanolysis of glycolipids after
cultivation on glucose/1-tetradecanol

Lipid constituents

Lipid quantities in glycolipids afterof glycolipid

cultivation on 1-tetradecanol

1-tetradecanol

25.9%

㻌

1,13-tetradecanediol

22.4%

㻌

1,14-tetradecanediol

49.9%

㻌

17-hydroxyoleate

1.0%

㻌

17-hydroxystearate

0.8%

㻌

References
Brakemeier, A., Lang, S., Wullbrandt, D., Merschel, L., Benninghoven, A., Buschmann,N., Wagner, F. (1995) Novel sophorose lipids from microbial conversion of 2-alkanols.
Biotechnol. Lett. 17: 1183–1188.

Cheng, Q., Sanglard, D., Vanhanen, S., Huan, T.L., Bombelli, P., Smith, A., Slabas, A.R.
(2005) Candida yeast long chain fatty alcohol oxidase is a c-type haemoprotein and31

plays an important role in long chain fatty acid metabolism. Biochim. Biophys. Acta
1735: 192–203.

Fleurackers, S.J.J., Van Bogaert, I.N.A., Develter, D. (2010) On the production andidentification of medium-chained sophorolipids. Euro. J. Lipid Science Technol. 112:
655–662.

Gao, R., Falkeborg, M., Xu, X., Guo, Z. (2013) Production of sophorolipids withenhanced volumetric productivity by means of high cell density fermentation. Appl.
Microbiol. Biotechnol. 97: 1103–1111.

Hommel, R., Ratledge, C. (1990) Evidence for two fatty alcohol oxidases in thebiosurfactant-producing yeast Candida (Torulopsis) bombicola. FEMS Microbiol. Lett.
70: 183–186.

Inoue, S., Ito, S. (1982) Sophorolipids from Torulopsis bombicola as microbial
surfactants in alkane fermentations. Biotechnol. Lett. 4: 3–8.

Kemp, G.D., Dickinson, F.M., Ratledge, C. (1988) Inducible long chain alcohol oxidasefrom alkane-grown Candida tropicalis. Appl. Microbiol. Biotechnol. 29: 370–374.

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head ofbacteriophage T4. Nature 227: 680–685.

32

Marchler-Bauer, A., Anderson, J.B., Chitsaz, F., Derbyshire, M.K., DeWeese-Scott, C.,
Fong, J.H., Geer, L.Y., Geer, R.C., Gonzales, N.R., Gwadz, M., He, S., Hurwitz, D.I.,
Jackson, J.D., Ke, Z., Lanczycki, C.J., Liebert, C.A., Liu, C., Lu, F., Lu, S., Marchler,
G.H., Mullokandov, M., Song, J.S., Tasneem, A., Thanki, N., Yamashita, R.A., Zhang,D., Zhang, N., Bryant, S.H. (2009) CDD: specific functional annotation with theconserved domain database. Nucleic Acids Res. 37: 205–210.

Sambrook, J., Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Spencer, J.F.T., Gorin, P.A.J., Tulloch, A.P. (1970) Torulopsis bombicola sp. Antonie
Van Leeuwenhoek 36: 129–133.

Van Bogaert INA, De Maeseneire SL, Develter D, Soetaert W, Vandamme EJ (2008)
Development of a transformation and selection system for the glycolipid-producing
yeast Candida bombicola. Yeast 25: 273–278.

Van Bogaert, I.N.A., Sabiora, J., Develter, D., Soetaert, W., Vandamme, E.J. (2009)
Knocking out the MFE-2 gene of Candida bombicola leads to improved medium-chain
sophorolipid production. FEMS Yeast Res. 9: 610–617.

Van Bogaert INA, Holvoet K, Roelants SLKW, Li B, Lin Y-C, Van de Peer Y, Soetaert
W (2013) The biosynthetic gene cluster for sophorolipids: A biotechnologicalinteresting biosurfactant produced by Starmerella bombicola. Mol. Microbiol. 88: 501–
33

509.

Vanhanen S., West M., Kroon JT, Lindner N, Casey J, Cheng Q, Elborough KM andSlabas AR (2000) A consensus sequence for long-chain fatty-acid alcohol oxidases from
Candida identifies a family of genes involved in lipid ω-oxidation in yeast withhomologues in plants and bacteria. J. Biol. Chem. 275: 4445–4452.

Wang T-T, Choi Y-J, Lee BH (2001) Transformation systems of non-Saccharomyces
yeasts. Crit. Rev. Biotechnol. 21: 177–218.

34

Chapter 2
Elucidation of secondary alcohol metabolism in Starmerellabombicola and contribution of primary alcohol oxidase FAO1

Summary
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 thesecondary alcohol metabolic pathway remains unknown. Although the FAO1 mutant
was unable to grow with secondary alcohols and seemed to be involved in thesecondary 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,

accumulationof

1,13-tetradecandiol

and2,13-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.

Introduction
The yeast Starmerella bombicola is an excellent sophorolipid producer (Spencer et al.,1970). Sophorolipids derived from S. bombicola are glycolipids in which disaccharide
sophorose is linked glycosidically to the hydroxyl group at the Z or Z-1 carbons of35

C16–C18 fatty acids. Alkyl polyglucosides were also reported to be produced by S.
bombicola using secondary alcohols as a raw material (Brakemeier et al., 1995).
Compared with classical sophorolipids, sophorolipids derived from secondary alcohols
have better water solubility and more effectively decrease the surface tension of water.
We previously reported that S. bombicola fatty alcohol oxidase 1 (FAO1) is a primaryalcohol oxidase essential in the primary alcohol utilization pathway of S. bombicola(Takahashi et al., 2016). In contrast, the secondary alcohol metabolism pathway of S.
bombicola has not been clarified. The fatty alcohol oxidase (FAO) pathway has beencharacterized in Candida tropicalis, a yeast with high fatty alcohol and alkane
utilization (Cheng et al., 2005). Three FAO genes were identified in C. tropicalis,
namely FAO1 and two allelic genes designated as FAO2a and FAO2b. Both enzymes
oxidize long-chain primary alcohols. FAO1 oxidizes ω-hydroxy fatty acids, but not
2-alkanols, while FAO2 oxidizes 2-alkanols, but not ω-hydroxy fatty acids. In general
for alkane-utilizing microbes, the n-alkane terminus is oxidized to produce a primaryalcohol, which is further oxidized to an aldehyde by alcohol dehydrogenase or FAO,
and then to a fatty acid by aldehyde dehydrogenase. Furthermore, it has been suggested
that a subterminal oxidation pathway exists in some microorganisms. In the subterminal
oxidation pathway, n-alkanes are thought to be converted into secondary alcohols,
which are further oxidized to ketones by alcohol dehydrogenase or FAO and then toesters by Baeyer–Villiger monooxygenase, and then finally transformed into primaryalcohols and fatty acids by esterase (Van Beilen et al., 2003).
Herein, I report the secondary alcohol metabolism pathway in S. bombicola for
the first time, and show that S. bombicola FAO1 contributes to this metabolic pathway.
Furthermore, we demonstrate that the FAO1 mutants are not only deficient in the36secondary alcohol oxidation pathway, but also able to produce novel sophorose lipids
derived from 1,13-tetradecanediol.

Materials and methods
Strains, fermentation, and glycolipids production
The strains used in this study are shown in Table 1. S. bombicola KSM36, an industrial
strain for sophorolipid production (Inoue and Ito, 1982), ura3-auxotrophic mutant
KSM-ura3Δ, and FAO1-negative mutant KSM-fao1Δ (Takahashi et al., 2016) wereused as the parent strains. Escherichia coli DH5α (TaKaRa Bio Inc., Kusatsu, Japan)
was used for plasmid maintenance and E. coli BL21(DE3) (Funakoshi Co., Ltd., Tokyo,
Japan) was used for protein expression. S. bombicola was cultured on yeast peptone
dextrose (YPD) medium (1% yeast extract, 2% peptone, and 2% glucose) or on
synthetic dextrose (SD) medium (0.67% yeast nitrogen base without amino acids(Sigma-Aldrich, St. Louis, MO, USA) and 2% glucose). To obtain hygromycin resistantstrains, 0.05% hygromycin B (FUJIFILM Wako Pure Chemical Industries, Ltd., Osaka,
Japan) was added to the synthetic dextrose (SD) medium (0.67% yeast nitrogen basewithout amino acids (Sigma-Aldrich, St. Louis, MO, USA) and 2% glucose). To
obtain better mutant growth, 0.03% uracil was added to the YPD medium. Liquid yeast
shake-flask cultures were incubated at 30°C with shaking at 120 rpm. E. coli was grownin Luria–Bertani (LB) medium (1% tryptone, 0.5% yeast extract, and 0.5% NaCl)
supplemented with 0.01% sodium ampicillin if necessary. Liquid E. coli cultures wereincubated at 37°C with shaking at 120 rpm. S. bombicola was grown on a sophorolipid
production medium (10% glucose, 10% methyl palmitate, 0.5% yeast extract, and 1%
urea; pH adjusted to 5.0 using HCl), or on an APG production medium comprising 15%
37

glucose, 0.5% sodium citrate·3H2O, 0.04% yeast extract, 0.0154% NH4Cl, 0.1%
KH2PO4, 0.07% MgSO4·7H2O, 0.05% NaCl, 0.027% CaCl2·2H2O, and 0.016%
K2HPO4·3H2O. The seed culture (5 mL of YPD in a 100-mL test tube) was incubatedfor 48 h at 30°C with shaking at 250 rpm using a rotary shaker. An 1-mL aliquot of thisculture was added to 100 mL of production medium in a 500-mL Sakaguchi flask. After
48 h, 10 g L1 of secondary alcohol was added to the microbial culture in the case of
APG production medium. The pH was not adjusted and the total incubation time was
120 h.

Washing cells from cultures
Culture samples (20 mL) were centrifuged at approximately 5,000 ×g for 10 min at 4°C
to obtain cell pellets. The pellets were washed twice by resuspending in 0.08% NaCl (10mL) and centrifuging at 5,000 ×g and 4°C for 1 min.

Preparation of cell extracts and microsomes
The washed cell pellet was resuspended in 1/15 M potassium buffer (10 mL, pH 7.4).
Phenylmethylsulfonyl fluoride was added to a final concentration of 1 mM prior to
breaking the cells by shaking vigorously with 0.5-mm glass beads three times at 2,700rpm for 1 min using a Multi-Beads Shocker (Yasui Kikai, Osaka, Japan). The broken
cell suspension was centrifuged at approximately 15,000 ×g for 60 min at 4°C. The
supernatant consisted of soluble cell extract. Microsomes were prepared from the cell
extract by centrifugation at 100,000 ×g for 1.5 h at 4°C. The supernatant was removed
and the microsomal pellet resuspended in 1/15 M potassium buffer (200 μL, pH 7.4)
containing 1 mM phenylmethylsulfonyl fluoride. This mixture was assayed for FAO
38

activity. Cell extracts from E. coli were prepared in the same manner.

Nucleic acid extraction
Yeast genomic DNA was isolated using the Mr. Gentle High Recovery Kit (TaKaRa
Bio). Plasmid DNA was isolated using a plasmid purification kit (Roche, Basel,
Switzerland).

Transformation
S. bombicola cells were transformed via electroporation (Wang et al., 2001) using a
Gene Pulser Xcell (Bio-Rad, Carlsbad, CA, USA) set to 25 μF, 350 Ω, and 2.5 kV.
Transformants were selected on SD plates. E. coli cells were transformed as described
previously (Sambrook and Russell 2001).

Polymerase chain reaction (PCR) and sequencing analysis
The primers used in this study are shown in Table 2. PCR was performed using KOD
FX Neo (TOYOBO, Osaka, Japan). PCR amplification was performed with initialdenaturation at 94°C for 2 min, followed by 30 cycles of 98°C for 30 s, 55°C for 30 s,
and 72°C for 2 min, followed by a final 2-min elongation at 72°C. All DNA sequenceswere determined using a Big Dye ver. 3.1 kit (ABI) and 3500 Genetic Analyzer(Applied Biosystems, Foster City, CA, USA).

FAO activity assay
The assay procedure followed the protocol of Kemp et al. (1988) with modifications.
The assay was a two-enzyme coupled reaction. 1-Dodecanol was used as the standard in39

the general FAO assay. The final reaction mixture for the general AO assay comprised
100mM

sodiumphosphatebuffer

(100

μL,

pH

7.4),

2,2’-azino-di[3-ethylbenzothiazoline-(6)-sulfonic acid] solution in deionized water (50 μL, 2.8 mg
mL1), horseradish peroxidase solution (30 μL, 47 units mL1; Wako Pure Chemical
Industries, Ltd., Osaka, Japan), microsomal protein or cell extract solution (10 μL), and5 mM substrate solution (10 μL). FAO activity was measured at 30°C and 405 nm, andwas reported as the specific activity units per milligram of protein (1 U = 1 μmol ofoxidized substrate min1).

Protein determination
The protein concentration in the extracts was determined using a Bradford protein assay
kit (Bio-Rad). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–
PAGE) was performed according to the method of Laemmli (1970).

Creation of knockout fragments
The strategy for knocking out the UGTA1 and FAO1 genes is shown in Fig. 1. The
1,000-bp upstream and 1,000-bp downstream fragments of the UGTA1 gene were
amplified using the primers UGTAUS-in F and UGTAUS-ura R, and Ura-UGTADS F
and UGTADSin R, respectively. Primers Pura3F and ura3R were prepared according to
manufacturer guidelines and used to integrate the functional S. bombicola URA3
sequence into UGTA1. Primers pUC F and pUC R were used to amplify a linear DNA
fragment of pUC118 (TaKaRa Bio). pUC-ΔUGTA1 was constructed such that thesefragments were ligated using the In-Fusion HD Kit (TaKaRa Bio). Primer pairUGTAUS-in F and UGTADSin R was used to amplify a 3,124-bp fragment, which
40

contained the URA3 sequence and 1,020-bp of the UGTA1 upstream and 1081-bp
downstream flanking sequences from pUC-ΔUGTA1. The fragment was used totransform the ura3-auxotrophic KSM-ura3Δ strain. Primers Pura3 R and FAO1DS F
were prepared and used to integrate the functional hygromycin resistance gene HPT
sequence from pPREP4 (Thermo Fisher Scientific) into FAO1. Primers Pura3 R andFAO1DS F were used to amplify a linear DNA fragment of pUC-ΔFAO1 (Takahashi etal., 2016). pUC-ΔFAO1::HPT was constructed such that these fragments were ligatedusing the In-Fusion HD Kit. The primer pair FAO1USinF and FAO1DSinR was used toamplify a 3,400-bp fragment, which contained the HPT sequence and 1,230-bp of the
FAO1 upstream and promoter sequence of URA3, and 1,027-bp downstream flanking
sequences from pUC-ΔFAO1::HPT. The fragment was used to transform theKSM-ugtaΔ strain.

High-pressure liquid chromatography (HPLC)–mass spectroscopy (MS) analysis
A Prominence UFLC HPLC System (Shimadzu, Kyoto, Japan) and liquid
chromatography (LC)-MS 2020 system (Shimadzu) were used for HPLC-MS analysis.
Glycolipid samples were analyzed using an Acclaim Surfactant column (4.6 u 150 mm,
5 μm; Dionex, Sunnyvale, CA, USA) at 40°C. A gradient of two eluents, namely a 0.1%
formic acid (w/w) aqueous solution and acetonitrile, were used to separate thecomponents. The gradient started at 20% acetonitrile for 3 min and then increased
linearly to 95% acetonitrile over 37 min, holding at 95% acetonitrile for 5 min. The
mixture was maintained at this composition for 30 min and then brought back to 20%
acetonitrile over 5 min. A flow rate of 0.5 mL min1 was used. Scan analysis was
performed for molecules with masses between 30 and 1,500 Da.
41

Sampling and analytical methods
Analytical glycolipid samples were prepared as follows. 1-Butanol (500 μL) containing
0.05% 1-octadecanol as an internal standard was added to culture broth (500 μL) andshaken vigorously for 5 min. After centrifugation at 3,000 ×g for 5 min, the upper
solvent layer was removed, placed in fresh glass culture tubes (ST-13M, Tokyo
Rikakikai Co., Ltd., Tokyo, Japan), and dried using a centrifugal evaporator. For
alkaline hydrolysis of the ester linkages, the solid sample was refluxed in 1 M NaOH
solution (500 μL) for 4 h. After adjusting the mixture to pH 4 with 1 M HCl, 1-butanol(1 mL) was added and shaken vigorously for 5 min. After centrifugation at 3,000 ×g for
5 min, the upper solvent layer was removed, placed in fresh glass culture tubes(ST-13M), and dried using a centrifugal evaporator. A 1-(trimethylsilyl)imidazole
solution (500 μL; GL Sciences Inc., Tokyo, Japan) was added and incubated at 60°C for
10 min. Equal volumes (500 μL) of hexane and saturated saline were added and shaken
vigorously for 5 min. After centrifugation at 3,000 × g for 5 min, the upper solvent layer
was analyzed by gas chromatography/MS (GC-MS) on Agilent 7890A and 5975C
systems (Agilent Technologies, Santa Clara, CA, USA) using a DB-1ms column (20 m
× 0.10 mm × 0.1 μm) and helium gas as the mobile phase at a flow rate 0.3 mL min 1.
The following time program was used: 100°C for 2 min, then increased to 300°C at 15°C min1 (20 min). n-Dodecyl-E-D-maltoside (Calbiochem, EMD Millipore, Billerica,
MA, USA) was used as a standard. Colony-forming units were determined by plating
serial dilutions on YPD agar plates, which were incubated at 30°C for 3 days.

Results42

Growth of the KSM-fao1Δ strain in secondary alcohol substrates
S. bombicola strains were grown on a basal medium containing 2-tetradecanol as thesole carbon source. We determined the ability of KSM-ura3Δ::URA3 and KSM-fao1Δ
strains to use several carbon sources. As shown in Table 3, both strains grew on glucose
as the sole carbon source. The KSM-ura3Δ::URA3 strain, but not the KSM-fao1Δ
strain, also grew on media containing 2-tetradecanol or 2-hexadecanol.

Expression of FAO1 in E. coli and its AO activity
FAO1 was expressed in E. coli BL21(DE3) according to a previous report (Takahashi etal., 2016) and its substrate specificity was verified (Table 4). FAO1 had oxidase activity
toward primary alcohols, but no activity was observed toward secondary alcohols.

Structural analysis of glycolipids produced by KSM-fao1Δ from secondary
alcohols
Although FAO1 had no secondary alcohol oxidizing activity, the assimilability of
secondary alcohol disappeared in the FAO1 mutant. To estimate the secondary alcohol
metabolic pathway in S. bombicola, we analyzed the products from 2-tetradecanol in
KSM-ura3Δ::URA3 and KSM-fao1Δ. Each strain was inoculated into the APG
production medium, 2-tetradecanol was added at 20 g L1, and the culture wasconducted for 2 days. The main product (peak 1) and minor product (peak 2) weredetected in strains KSM-ura3Δ::URA3 and KSM-fao1Δ using GC (Fig. 2). Based on aprevious study (Brakemeier et al., 1995), we estimated that the main product was a
2-tetradecylsophoroside. In contrast, only the culture of KSM-fao1Δstrain showedbyproducts (peaks 3–6).
43

To estimate the structure of byproduct peaks, the supernatant was analyzed by
HPLC/MS. The molecular weights of monoacetylated and diacetylated 2-tetradecyl
disaccharide were 580 Da and 622 Da, respectively. These ions were observed at 10.5
min (peak A in Fig. 3) and 11 min (peak B in Fig. 3) in the culture of the
KSM-ura3Δ::URA3 strain. The major (peak A) and minor (peak B) product ionsappeared at m/z 579.5 and 621.5, respectively, in negative ion mode. Based on aprevious study (Fleurackers et al., 2010), we estimated that peak A was monoacetylated
2-O-tetradecyl-(2-O-β-D-glucopyranosyl-β-D-glucopyranoside)

and

peak

B

was

diacetylated 2-O-tetradecyl-(2′-O-β-D-glucopyranosyl-β-D-glucopyranoside).
In contrast, another peak was observed at 9 min (peak C) in the supernatant of
KSM-fao1Δ with m/z 637.5 in negative ion mode. The molecular weight of peak C was
16 larger than that of peak B, and peak C was eluted at a higher polarity position thanpeak B. Therefore, peak C was attributed to a hydroxyl group added to any position ofdiacetylated 2-O-tetradecyl-(2′-O-β-D-glucopyranosyl-β-D-glucopyranoside).
Subsequently, the position of the additional hydroxyl group was analyzed by
GC-MS. When GC-MS analysis is performed after trimethylsilylation of an alkyl
compound bearing a hydroxyl group, a fragment ion on the alkyl terminal side
containing a hydroxyl group is obtained. Fragment ions of m/z 73 and 117 wereobserved from peak 3, while fragment ions of m/z 73, 103, and 117 were observed frompeak 4. The fragment ions at m/z 103 and 117 were derived from TMS compounds ofω-terminal and ω–1, respectively. This suggested that peaks 3 and 4 were
2,13-tetradecanediol and 1,13-tetradecanediol, respectively. Subsequently, fragment
ions at m/z 73, 117, and 271.1, m/z 73 and 103, and m/z 73, 117 and 271.1 were detectedfrom peaks 2, 5, and 6, respectively. Therefore, peak 5 was attributed to an alkyl
44

sophoroside in which the sugar was transferred to the hydroxy group at position 13 of
1,13-tetradecanediol (peak 4). In the cell of the KSM-fao1Δ strain, the primary alcoholoxidizing activity was greatly reduced, but the oxidase activity for the secondary
alcohol was hardly changed. Therefore, compared with KSM-ura3Δ::URA3, theKSM-fao1Δ strain was thought to accumulate α-ω-type diols. As peak 6 was observed
only in the KSM-fao1Δ strain, it was attributed to 1,13-tetradecanediol glycosylated on
the hydroxy group at position 1. Peak 2, which is commonly observed in both strains,
was thought to be attributed to glycosylation at either of the hydroxy groups in
2,13-tetradecanediol (Fig. 4).

Construction of an FAO1–UGTA1-double negative S. bombicola strain
The glycosyltransferases involved in APG production have not been reported, but
UGTA 1, known as the ω or ω-1 hydroxy fatty acid glycosyltransferase for sophorolipid
biosynthesis, was considered a candidate. KSM-ugtaΔfao1Δ was inoculated into the
APG production medium, 2-tetradecanol was added at 20 g L1, and the culture wasconducted for 2 days. Peaks 1, 2, 5, and 6 were not detected (Fig. 2(c)). Therefore,
KSM-ugtaΔfao1Δ had lost the ability to glycosylate alcohols or diols. The amount ofboth diols reached 2.5 g L1 with a yield of 11.6% (mol/mol).

Discussion
The results of this study suggested that peroxisomal fatty alcohol oxidase, FAO1, plays
crucial roles in the metabolism of secondary alcohols in S. bombicola.
In wild-type S. bombicola, the production of glycolipids from primary alcohols
is hardly observed, but sophorose lipids are known to be formed from secondary45

alcohols (Takahashi et al., 2016; Brakemeier et al., 1995). In FAO1 deficient strains,
glycosylated products of 1,13-tetradecanediol and 2,13-tetradecanediol were observedin the cultures containing 2-tetradecanol. 2,13-Tetradecanediol was detected in theculture solution of the wild-type, while 1,13-tetradecanediol was not. FAO1 has highoxidizing activity for 1-tetradecanol, but not for 2-tetradecanol. In the wild-type strain,
the primary hydroxyl group of 1,13-tetradecanediol should be rapidly oxidized by FAO1,
while 2,13-tetradecanediol should accumulate in the yeast cells because FAO1 does noteasily oxidize secondary alcohols, resulting in detection of the glycosylated product.
Disruption of FAO1 suppressed the assimilation of primary alcohol and produced aglycosylated product derived from 1,13-tetradecanediol. Therefore, the initial step of thesecondary alcohol utilization pathway in S. bombicola was thought to be hydroxylation
at the ω-terminal.
Subsequently, UGTA1 involved in sophorolipid biosynthesis was deleted against
KSM-fao1Δ strain to clarify the factor for glycosylation. In the culture of
KSM-ugtaΔfao1Δ strain, glycosylated products were not detected and the formation of
1,13-tetradecanediol and 2,13-tetradecanediol was observed from 2-tetradecanol. The
synthesis of alkyl sophorosides in S. bombicola was shown to be due to UGTA1. Tosummarize, the utilization of secondary alcohol in S. bombicola is thought to proceed
via the route shown in Fig. 5.
In addition, productivity improvements of long-chain diols were recognized
after deletion of UGTA1 and FAO1. Long-chain diols are expected to be used as finechemicals. ω-Terminal hydroxylation of fatty alcohols is a difficult reaction to achieve
in organic synthesis, and no examples of preparing (α,ω)-diols in one step from
1-decanol or 1-tetradecanol have been reported. There are also few reports of such46

microbial reactions. Genetically modified E. coli can produce 722 mg L1 of
1,8-octanediol or 15 mg L1 of 1,9-nonanediol (every 4 h) from the corresponding
alcohols (Fujii et al., 2006; Scheps et al., 2011). The diol production system reported
herein shows higher productivity than those in the previous reports.
This study not only elucidates the metabolic pathway of secondary alcohols in S.
bombicola, but also provides a system for producing long-chain diols such as
tetradecanediol.

47

FiguresFig. 1 Gene knockout strategy for UGTA1 and FAO1.
(A, D) S. bombicola wild-type UGTA1 or FAO1 allele, (B, E) knockout cassette, (C, F)
mutant allele. Sites of homologous recombination between the genome and cassette areindicated and all primers used to create the cassette and control the genotype of themutants are indicated.

48

Fig. 2 Chromatogram of GC-FID analysis of glycolipids from S. bombicola grown on
2-tetradecanol: (a) KSM-ura3Δ::URA3, (b) KSM-fao1Δ, (c) KSM-ugtaΔfao1Δ. IS,
internal standard (1-octadecanol).

49

Fig. 3 HPLC-ESI MS analysis of glycolipids from S. bombicola grown on
2-tetradecanol: (a) KSM-ura3Δ::URA3, (b) KSM-fao1Δ. E(–), negative ion mode; E(+),
positive ion mode.

50

Fig. 4 Putative structure of products from the FAO1 knockout strain grown on
2-tetradecanol.

51

Fig. 5 Secondary alcohol metabolic pathway in S. bombicola and the new pathwayobserved after FAO1 deletion.

52

Tables
Table 1 Microbial strains and plasmids used or constructed in this study.

StrainProperties

Source or reference

KSM36
KSM-ura3Δ

S. bombicola KSM36
KSM36ΔURA3

FERM-BP 799
1

KSM-ura3Δ::URA3

KSMΔURA3::URA3
KSM36ΔURA3,

1

KSM-fao1Δ
KSM-ugtaΔ
KSM-ugtaΔfao1Δ
NBRC10243

FAO1::URA3
KSM36ΔURA3,
UGTA1::URA3
KSM36ΔURA3,
UGTA1::URA3, FAO1::HPT
Type strain

Escherichia coli

1
This study
This study
NITE Biological Resource
Center, Japan
TaKaRa BIO

BL21(DE3)
Construction of an
pUC-ΔFAO1

FAO1-negative S.
bombicola strain

53

1

Table 2 Primers used for isolating, cloning, expression, and knocking out the S.
bombicola FAO1 gene.

PrimerSequence (5'-3')Purpose in this study

Pura3 FAGTACATATTTTTCGAAACAGCTCGCAA

Cloning of URA3
Construction

ura3 RCTAAGAAACTCATCTTGACTGAACTTTTCof KSM-fao1Δ
andKSM-ura3Δ::URA3

pUC F

GTACCGAGCTCGAATTCGTpUC R

GCAGGCATGCAAGCTTGGCUGTAUS-in F

AGCTTGCATGCCTGCTATTAACTCCGCAGCATGAC

Construction

UGTAUS-ura R

CGAAAAATATGTACTGAATATTCGTAGGGAGAAGC

of KSM-ugtaΔ

Ura-UGTADS FAGATGAGTTTCTTAGTAGAATCGTACGATCAAATCUGTADSin R

ATTCGAGCTCGGTACTCCTTGCCTCATTCCACCTCFAO1DS FAAGCCTTATATCGAATACAC

Pura3 RATTATTTCTCTACAGTAGTG

Pura-HPT in F

CTGTAGAGAAATAATATGAAAAAGCCTGAACTCAC

HPT-FAO1DS in
R

TTCGATATAAGGCTTTTATGAACAAACGACCCAAC

FAO1USinF

AGCTTGCATGCCTGCTTTAAATCCAGAAAGAACTG

FAO1DSinR

ATTCGAGCTCGGTACGACACTTCTCAGGAACCCTC

pUC118 F

GGCGAAAGGGGGATGTGC

Constructionof KSM-ugtaΔfao1Δ

Checkingfor
illegitimate

pUC118 R

recombinationof

GCACCCCAGGCTTTACAC

pUC118 region

54

Table 3 Growth of KSM36 and mutant strains grown in secondary alcohols.

Substrates/strains

KSM36

KSM-ura3Δ::URA3

KSM-fao1Δ

Glucose+++2-tetradecanol
2-hexadecanol

+
+

+
+




+, Growth; –, no growth.

Table 4 Substrate specificity of FAO1 for primary or secondary alcohols.
Substrates

Relative activity

1-tetradecanol

100%

㻌

2-tetradecanol

n.d.

㻌

n.d., not detected.

References
Brakemeier, A., Lang, S., Wullbrandt, D., Merschel, L., Benninghoven, A., Buschmann,N., Wagner, F. (1995) Novel sophorose lipids from microbial conversion of 2-alkanols.
Biotechnol. Lett. 17: 1183–1188.

Cheng, Q., Sanglard, D., Vanhanen, S., Huan, T.L., Bombelli, P., Smith, A., Slabas, A.R(2005) Candida yeast long chain fatty alcohol oxidase is a c-type haemoprotein andplays an important role in long chain fatty acid metabolism. Biochim. Biophys. Acta
1735: 192–203.

55

Fleurackers, S.J.J., Van Bogaert, I.N.A., Develter, D. (2010) On the production andidentification of medium-chained sophorolipids. Euro. J. Lipid Sci. Technol. 112: 655–
662.

Fujii, T., Narikawa, T., Sumisa, F., Takeda, K., Kato, J. (2006) Production ofα,ω-alkanediols using Escherichia coli expressing a cytochrome P450 fromAcinetobacter sp. OC4. Biosci. Biotech. Biochem. 70: 1379–1385.

Inoue, S., Ito, S. (1982) Sophorolipids from Torulopsis bombicola as microbial
surfactants in alkane fermentations. Biotechnol. Lett. 4: 3–8.

Kemp, G.D., Dickinson, F.M., Ratledge, C. (1988) Inducible long chain alcohol oxidasefrom alkane-grown Candida tropicalis. Appl. Microbiol. Biotechnol. 29: 370–374.

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the headof bacteriophage T4. Nature 227: 680–685.

Sambrook, J., Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed.
New York: Cold Spring Harbor Laboratory Press

Scheps, D., Malca, S., Hoffmann, H., Nestl, B.M., Hauer, B. (2011) Regioselective
ω-hydroxylation of medium-chain n-alkanes and primary alcohols by CYP153 enzymes
from Mycobacterium marinum and Polaromonas sp. strain JS666. Org. Biomol. Chem.
9: 6727–6733.
56

Spencer, J.F.T., Gorin, P.A.J., Tulloch, A.P. (1970) Torulopsis bombicola sp. Antonie
Van Leeuwenhoek 36: 129–133.

Takahashi, F., Igarashi, K., Hagihara, H. (2016) Identification of the fatty alcoholoxidase FAO1 from Starmerella bombicola and improved novel glycolipids production
in an FAO1 knockout mutant. Appl. Microbiol. Biotechnol. 100: 9519–9528.

Van Beilen, J.B., Li, Z., Duetz, W.A., Smits, T.H.M., Witholt, B. (2003) Diversity ofalkane hydroxylase systems in the environment. Oil Gas Sci. Technol. 58: 427–440

Wang, T-T., Choi, Y-J., Lee, B.H. (2001) Transformation systems of non-Saccharomyces
yeasts. Crit. Rev. Biotechnol. 21: 177–218.

57

General Discussion

In the thesis, I aimed to perform the basic molecular biological research and clarify the
fatty alcohol metabolism pathway in S. bombicola to improve the glycolipid production
system of this yeast. In Chapter 1, I described cDNA analysis, construction of arecombinant system, and identification of the primary alcohol oxidase in S. bombicola
were performed. In Chapter 2, I demonstrated that primary alcohol oxidase is also
involved in the secondary alcohol utilization pathway and determined the fatty alcohol
utilization pathway in this yeast.
I performed cDNA and genome analyses of S. bombicola. The FAO1 gene does
not contain intron, similar to the case for other genes in S. bombicola (Van Bogaert, etal., 2009). The physiological significance of non-intron containing genes in this yeast
remains to be elucidated and will be investigated in future research. Regarding therecombination system, the electroporation method achieved higher transformation
efficiency than a previously reported system (Van Bogaert et al., 2008). The results ofdestruction of FAO1 suggested that S. bombicola is a haploid yeast. S. bombicola
appears to be easy to analyze in terms of strain improvement for industrial applicationand fundamental molecular biology.
In Chapter 1, I showed that FAO 1 is a fatty alcohol oxidase by expression
analysis in E. coli, and identified it as a major fatty alcohol oxidase localized inperoxisomes by the FAO1 disruption in S. bombicola. Hommel and Ratledge (1990)
suggested that two alcohol oxidase activities exist in S. bombicola. Although we did notreport the findings, we identified an oxidase FAO2 that shows weak homology to S.

58

bombicola FAO1, but the alcohol utilization was not decreased by deletion of FAO2.
Recently, another group reported a destruction study of FAO2 in S. bombicola (Van
Renterghem et al., 2018). The deletion of the FAO1 improved the productivity ofbolaform sophorolipids, while deletion of the FAO2 did not. These results supported thatthe notion that FAO1 is the main fatty alcohol oxidase in S. bombicola. The function of
FAO2 remains to be elucidated.
Deletion of FAO1 improved the yields of tetradecanol-based sophorolipids, andtetradecanediol-based sophorolipids. Bolaform sophorolipids are considered to be
excellent as hypoallergenic, film-forming ability, degreasing agents, or suppression ofscale formation in comparison with normal APGs (Van Renterghem et al., 2018).
Through functional analyses of FAO1, we were able to construct a production systemfor bolaform glycolipids with excellent physical properties.
As another issue, I tried to elucidate the metabolic utilization pathway of
secondary alcohols in S. bombicola. The FAO1-deficient strain was unable to assimilate
secondary alcohols, suggesting that FAO1 had a role in secondary alcohol metabolism.
However, FAO1 did not show oxidation activity for secondary alcohol. The initialoxidation stage in the alcohol metabolism pathway was not considered to be oxidationof the hydroxyl group on the secondary alcohol. As a result of structural analysis of the
product using secondary alcohols as substrates in the wild-type and FAO1-deficientstrain, a diol-type glycolipid having an ω-terminal hydroxyl group was only detected in
the FAO1-deficient strain. Moreover, when both of UGTA1, a glycosyltransferases forsophorolipid biosynthesis, and FAO1 were deleted, the diol-containing APGs weredisappeared and accumulation of ω or ω-1 diols was observed. From these results,
secondary alcohol metabolism in S.bombicola involves initial hydroxylation at the ω59terminal, then oxidiation by FAO1, and finally assimilation in the beta-oxidation
pathway.
The above results showed that FAO1 makes a large contribution to the fatty
alcohol metabolism pathway in S. bombicola and that fatty alcohols can be efficiently
glycosylated in the FAO1-deficient strain. This study is considered to be useful not only
for the basic results elucidating the alcohol metabolism pathway in S. bombicola but
also for the method for synthesis of novel glycolipids.
Finally, the glycosyltransferase for alcohol and diol was unknown, and this study
revealed that UGTA1 catalyzes the reaction. Multiple hydroxylases in S. bombicola
have been identified and are involved in sophorolipid biosynthesis or alkane
assimilation (Van Bogaert et al., 2009). These enzymes hydroxylate the ω or ω-1
positions of fatty acids, alcohols and alkanes. Unlike the oxidase enzymes possessed byother alkane-utilizing microorganisms, the hydroxylases in S. bombicola have highhydroxylation activity at the ω-1 position, necessarily producing secondary alcohols.
When the glycosyltransferase is deficient, the secondary alcohols remain in the yeast
body without being assimilated. As one hypothesis, it seems that the glucose additionand secretion system developed as a secondary alcohol export pathway, and then further
developed into a sophorolipid biosynthesis system.


References
Hommel, R., Ratledge, C. (1990) Evidence for two fatty alcohol oxidases in thebiosurfactant-producing yeast Candida (Torulopsis) bombicola. FEMS Microbiol. Lett.
70: 183–186.

60

Van Bogaert, I.N.A., De Maeseneire, S.L., Develter, D., Soetaert, W., Vandamme, E.J.
(2008)Developmentofatransformationandselectionsystemfortheglycolipid-producing yeast Candida bombicola. Yeast 25: 273–278.

Van Bogaert, I.N.A., Demey, M., Develter, D., Soetaert, W., Vandamme, E.J. (2009)
Importance of the cytochrome P450 monooxygenase CYP52 family for the
sophorolipid̺producing yeast Candida bombicola. Yeast Res. 9: 87–94.

Van Renterghem, L., Roelants, S.L.K.W., Baccile, N., Uyttersprot, K., Taelman, M.C.,
Everaert, B., Mincke, S., Ledegen, S., Debrouwer, S., Scholtens, K., Stevens, C.,
Soetaert, W. (2018) From lab to market: An integrated bioprocess design approach for
new-to-nature biosurfactants produced by Starmerella bombicola. Biotechnol. Bioeng.
115: 1195–1206.

61

Acknowledgments

I wish to express my sincere gratitude and appreciation to my supervisor, Prof. Takashi
Yamamoto at Hiroshima University for valuable advice, supervision, and crucial
contributions throughout this study, and for the excellent opportunities provided to me.
I am deeply grateful to Yoshinori Takema, Tadashi Hase and Atsushi Ouchi, from
Kao Corporation who provided me with this opportunity, as well as constructive
comments and warm support. I would like to give heartfelt thanks to Tadayuki Imanaka
who was my professor in my Kyoto University era and provided valuable advice.
Special thanks also go to Toshiaki Fukui (Tokyo Institute of Technology) and Takeshi
Tanaka (National Institute of Advanced Industrial Science and Technology) who gaveme invaluable comments and warm encouragement.
I am greatly indebted to Katsuya Ozaki, Hiroshi Hagihara and Yasushi Takimura
from Kao Corporation, who have been responsible for helping me in this research. This
work could not have been completed without their help. My special thanks go to mysenior associate, Kazuaki Igarashi at Kao Corporation, who is a best collaborator forthis work.
I would like to give special thanks to my colleagues at Kao Corporation; Kenji
Manabe, Takuya Morimoto, Atsuko Hayase, Akihito Kawahara, Yuichi Tsuboi, Nozomu
Shibata, Tatsuya Osamura, Jitsurou Kaneda, Kyoushiro Nonaka, Kousuke Ike, Mao
Shaku, and Hikaru Nagai, for their warm encouragement.
Finally, special thanks to my mother, wife, daughter, and late father for theirunflagging encouragement and mental and financial support throughout my study.

62