**4.1 Review of desaturase studies**

An enzyme that removes hydrogen atoms from a fatty acid derivative, thereby creating a double bond, is called a desaturase, and desaturases are key enzymes for the maintenance of cell life cycles. Desaturases are divided into three types based on substrate specificity; acyl-CoA, acyl-ACP, and acyl-lipid types (Fox et al., 2004). Desaturases are further sub-divided into membrane-bound and soluble types. Membrane-bound enzymes are very unstable and are known to be very difficult to purify. However, recently a very interesting report has been published regarding the purification of human stearoyl-CoA desaturase (Gorden & Fox, 2008). In contrast, there have been some reports regarding the purification and clarification of the crystal structure of the soluble types of enzymes **(**Lindqvist et al., 1996).

reduced esterase activity by UV irradiation. Of the colonies which showed lower growth than KSM-MT66 on the minimum agar containing IP-C16:0, one mutant, designated KSM-

Using this mutant strain T64, and optimizing culture conditions, more than 60 g/l of IP-C16:1Δ6 could be produced in a flask (Table 1). Optimization of culture conditions in a 30 l jar fermentor, resulted in production of 50 g/l over 4 days of cultivation (Fig. 8). Furthermore, C16:1Δ6 can be easily obtained by simple hydrolysis of IP-C16:1Δ6 (Araki et

**Reaction condition KSM- MT66 KSM-T64** 

Reaction process Resting Growing

Monosodium glutamate 1.0% 2.0%

MgSO4·7H2O 2 mM 2 mM MnSO4·6H2O -a 2 µM FeSO4·7H2O - 60 µM CuSO4·5H2O - 5 µM ZnSO4·7H2O - - Thiamine 2 mM - Yeast extract (P-21) - 0.8%

IP-C16:0 20% 22%

IP-C16:1 productivity 50 g *l*-1 61 g *l*-1

250 mM 350 mM pH 7 pH 7.3

T64, displayed 40% of the esterase activity of KSM-MT66.

Metal

Phosphate buffer

Table 1. Production of IP-C16:1Δ6 by *Rhodococcus* sp. KSM-MT66 and KSM-T64.

**expression of two novel desaturases from** *Rhodococcu***s sp.** 

**4. The production of C16:1∆6 by genetically modified bacteria: Cloning and** 

An enzyme that removes hydrogen atoms from a fatty acid derivative, thereby creating a double bond, is called a desaturase, and desaturases are key enzymes for the maintenance of cell life cycles. Desaturases are divided into three types based on substrate specificity; acyl-CoA, acyl-ACP, and acyl-lipid types (Fox et al., 2004). Desaturases are further sub-divided into membrane-bound and soluble types. Membrane-bound enzymes are very unstable and are known to be very difficult to purify. However, recently a very interesting report has been published regarding the purification of human stearoyl-CoA desaturase (Gorden & Fox, 2008). In contrast, there have been some reports regarding the purification and clarification of the crystal structure of the soluble types of enzymes **(**Lindqvist et al., 1996).

al., 2007).

Reaction ingredient

a Not added.

**4.1 Review of desaturase studies** 

Delta 6-desaturases have been reported to be obtained from animals, plants, fungi and cyanobacteria (Aki et al., 1999; Cahoon et al., 1994; Inagaki et al., 2003; Okayasu et al., 1981; Reddy & Thomas, 1996; Zhan et al., 2004). Human and rat fatty acid desaturase 2 (FADS2) -encoding delta 6-desaturases recognize a saturated fatty acid as a substrate under some conditions (Ge et al., 2003; Guillou et al., 2003), and a human delta 6 desaturase gene has been reported to be expressed only in the sebaceous gland. Regarding microbial delta 6-desaturases, industrial producers of unsaturated fatty acids have focused mainly on fungi of the *Mortierella* spp., especially *Mortierella alpina* and *Mortierella cincinelloides*. Two types of delta 6-desaturases have been reported to be expressed by *Mortierella alpina.* The corresponding genes were isolated and the sequences and functions of these genes have been analyzed (Huang et al., 1999; Sakuradani et al., 1999; Sakuradani & Shimizu, 2003). However, even though many delta 6-desaturase genes have been obtained, no delta 6-desaturase genes have been purified from *Rhodococcus* sp.

We aimed to obtain delta 6-desaturase genes from the *Rhodococcus* sp. KSM-T64 strain, which shows 40% of the esterase activity of the parent strain, KSM-MT66.

Fig. 8. Production of IP-C16:1Δ6 in a 30 l-jar fermentor under optimized conditions.

IP-C16:1Δ6 (●), monosodium glutamate (MSG) (○). Cultivation was performed at 26 ºC, with agitation at 350 rpm, aeration at 0.3 vvm and pressure at 0.2 kg/cm2. The *Rhodococcus* sp. KSM-T64 was used.

#### **4.2 Cloning of delta 6-desaturase genes from the** *Rhodococcus* **sp. KSM-T64 strain**

The production of IP-C16:1Δ6 by *Rhodococcus* sp. was improved by the addition of metal ions as mentioned above. We therefore targeted the membrane-bound delta 6-desaturase and attempted to clone a gene whose encoded protein could introduce a *cis*-double bond**.**  We cloned the delta 6-desaturase gene on the basis of previously reported conserved sequences. Histidine motifs (Shanklin & Fox, 1994; Shanklin, 2009) that bind to ferric ions and whose sequences are known to be conserved in membrane-bound desaturases in a

Improvement of Atopic Dermatitis by Human Sebaceous Fatty Acids and Related Lipids 319

Fatty acids extracted from *E.coli* transformants that expressed Rdes1 and Rdes2 were analyzed using GC and GC/MS. The level of C16:1Δ6 was increased 2.5-fold in the Rdes1 expressing transformant and 4.8-fold in the Rdes2-expressing transformant, over that of

Although the level of C16:1Δ6 fatty acids in the transformants was higher than that of control, it was unclear if these delta 6-desaturase genes of the *Rhodococcus* sp. produced

**ORF1(mature)** 1:VAITDIKEFSHLTEADVEALGRELDQIRLDIEDSRGIRDARYIRRVIRVQRALELGGRIA 60 **ORF2(mature)** 1:MAIADVKEYAHLTDADIEALGRELDAIRRDIEESRGEKDARYVRNVIRLQRSLEIGGRAV 60

Fig. 10. Sequence homology of the proteins encoded by Rdes1 (ORF1) and Rdes2 (ORF2).

**5. Further investigation of human sebaceous fatty acids combined with chemical synthesis: Structure-activity relationship of oxa-fatty acids** 

The homology search was performed using Genetyx Win ver.6.1. The gap penalty score was set at –10 as an insert and –3 as an extend. The three histidine rich regions (Histidine motifs)

C16:1Δ6 has a double bond and, consequently, its stability is a concern, which raises doubts regarding adequate stability of C16:1Δ6 in products. The stability of C16:1Δ6 can be improved by replacing the unsaturated fatty acids with the corresponding oxa-fatty acids (Alkoxy fatty acids). Such a change would also be expected to lead to an improvement in antimicrobial properties of the fatty acid. We describe the results of our investigations

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

*His box1 box1*

extracellular IP-C16:1Δ6 or not (Araki et al., 2005).

\*

regarding such a replacement in the section below.

that are conserved in membrane binding desaturases are boxed.

*His box3*

*His box2*

control.

**ORF1(mature) ORF2(mature)**

**ORF1(mature) ORF2(mature)**

**ORF1(mature) ORF2(mature)**

**ORF1(mature) ORF2(mature)**

**ORF1(mature) ORF2(mature)**

**ORF1(mature) ORF2(mature)**

**ORF1(mature) ORF2(mature)**

number of species including worms, borages, *Mucor* sp. and *Mortierella* sp., were targeted for primer design for PCR amplification (Fig. 9). These primers were used for degenerated PCR and nested PCR. In addition, inverse PCR was performed using the obtained PCR products. Approximately 5.5 kbp of sequence was obtained using these methods. Within this sequence, there were sequences that corresponded to two tandem desaturase-like proteins; the first ORF (Rdes1) was comprised of 420 amino acids and the second ORF (Rdes2) was comprised of 413 amino acids from inffered start codons individually. The estimated molecular mass of the Rdes1 protein was 47985 Da and that of Rdes2 was 46951 Da. Three histidine motifs that are established consensus sequences in desaturase proteins were found in these ORF sequences (Fig. 10).

Fig. 9. Construction of PCR primers based on the histidine motifs that are conserved among delta 6-desaturases obtained from various organisms.

The sequence of primers 1, 2, 3, and 4 was; 5'-YTGGTGGAAGGRYAABCAYAA-3', 5'- RGGGAAVARRTGGTG-3', 5'-CAYAAYNMDCAYCA-3', and 5'-RGGGAAVARRTGGTG-3' respectively. Y, R, B, N, M, D, V, and W were mixed primers of C/T, AA/G, C/T/G, A/C/G/T, A/C, A/G, A/C/G and A/T respectively.

Among the membrane-bound delta 6-desaturases which were clarified the function except for putative and probable delta 6-desaturases, Rdes1 and Rdes2 had about 30% to *Homo sapiens* as an amino acid sequence. An unknown ORF whose function has not yet been clarified is present upstream of these two delta 6-desaturase genes. A domain search revealed that this ORF had an oxidoreductase NAD-binding domain, an oxidoreductase FAD-binding domain, a 2Fe-2S iron·sulfur cluster domain and a ferric reductase domain (data not shown).

#### **4.3 Expression of delta 6-desaturase genes in** *Escherichia coli*

Plasmids incorporating the delta 6-desaturase genes Rdes1 and Rdes2 were individually introduced into competent *E. coli* BL21 (DE3) cells, and the corresponding proteins were produced by IPTG induction. Production of very high amounts of these proteins was confirmed using SDS-PAGE. The molecular mass of each of these expressed proteins was 45 kDa. These proteins were identified by comparison with a control cell lysate which was prepared from cells transformed with vector alone**.** 

number of species including worms, borages, *Mucor* sp. and *Mortierella* sp., were targeted for primer design for PCR amplification (Fig. 9). These primers were used for degenerated PCR and nested PCR. In addition, inverse PCR was performed using the obtained PCR products. Approximately 5.5 kbp of sequence was obtained using these methods. Within this sequence, there were sequences that corresponded to two tandem desaturase-like proteins; the first ORF (Rdes1) was comprised of 420 amino acids and the second ORF (Rdes2) was comprised of 413 amino acids from inffered start codons individually. The estimated molecular mass of the Rdes1 protein was 47985 Da and that of Rdes2 was 46951 Da. Three histidine motifs that are established consensus sequences in desaturase proteins

> **WWKDKHNTHH WWKWNHNAHH WWKWNHNAHH WWKDKHNTHH WWKDKHNTHH WWNHRHFQHH WWNHRHFQHH**

*His box1 His box1 His box2 box2 His box3 box3*

**WXXHXXXH WXXHXXXH WWXXXHXXHH**

Fig. 9. Construction of PCR primers based on the histidine motifs that are conserved among

Among the membrane-bound delta 6-desaturases which were clarified the function except for putative and probable delta 6-desaturases, Rdes1 and Rdes2 had about 30% to *Homo sapiens* as an amino acid sequence. An unknown ORF whose function has not yet been clarified is present upstream of these two delta 6-desaturase genes. A domain search revealed that this ORF had an oxidoreductase NAD-binding domain, an oxidoreductase FAD-binding domain, a 2Fe-2S iron·sulfur cluster domain and a ferric reductase domain

Plasmids incorporating the delta 6-desaturase genes Rdes1 and Rdes2 were individually introduced into competent *E. coli* BL21 (DE3) cells, and the corresponding proteins were produced by IPTG induction. Production of very high amounts of these proteins was confirmed using SDS-PAGE. The molecular mass of each of these expressed proteins was 45 kDa. These proteins were identified by comparison with a control cell lysate which was

The sequence of primers 1, 2, 3, and 4 was; 5'-YTGGTGGAAGGRYAABCAYAA-3', 5'- RGGGAAVARRTGGTG-3', 5'-CAYAAYNMDCAYCA-3', and 5'-RGGGAAVARRTGGTG-3' respectively. Y, R, B, N, M, D, V, and W were mixed primers of C/T, AA/G, C/T/G,

**WWXXXHXXHH XXQIEHHLFP XXQIEHHLFP**

**1 2 3 4**

**NYQIEHHLFP NYQIEHHLFP QFQIEHHLFP QFQIEHHLFP NVQIEHHLFP NVQIEHHLFP NFQIEHHLFP NFQIEHHLFP NFQIEHHLFP NFQIEHHLFP**

were found in these ORF sequences (Fig. 10).

**WLAHDFLH WLAHDFLH WIGHDAGH WIGHDAGH WLTHEFCH WLTHEFCH WLQHDYGH WLQHDYGH WLQHDYGH WLQHDYGH**

delta 6-desaturases obtained from various organisms.

A/C/G/T, A/C, A/G, A/C/G and A/T respectively.

prepared from cells transformed with vector alone**.** 

**4.3 Expression of delta 6-desaturase genes in** *Escherichia coli*

*Mortierella alpina* **1S-4** *Borago officinalis Caenorhabditis elegans Rattu norvegicus Homo sapiens*

(data not shown).

Fatty acids extracted from *E.coli* transformants that expressed Rdes1 and Rdes2 were analyzed using GC and GC/MS. The level of C16:1Δ6 was increased 2.5-fold in the Rdes1 expressing transformant and 4.8-fold in the Rdes2-expressing transformant, over that of control.

Although the level of C16:1Δ6 fatty acids in the transformants was higher than that of control, it was unclear if these delta 6-desaturase genes of the *Rhodococcus* sp. produced extracellular IP-C16:1Δ6 or not (Araki et al., 2005).


Fig. 10. Sequence homology of the proteins encoded by Rdes1 (ORF1) and Rdes2 (ORF2).

The homology search was performed using Genetyx Win ver.6.1. The gap penalty score was set at –10 as an insert and –3 as an extend. The three histidine rich regions (Histidine motifs) that are conserved in membrane binding desaturases are boxed.
