Role of WEs in nature

In nature, WEs are incredibly widespread substances. They occur in all kind of organisms, e.g.

bacteria (Barney et al., 2012a; Kalscheuer, 2002; Röttig and Steinbüchel, 2013; Santala et al., 2014), insects (Blomquist et al., 1972; Buckner et al., 2009; Rottler et al., 2013), vertebrates (Bagge et al., 2012; Cheng, 2004a; Miklaszewska et al., 2013; Rantamäki et al., 2013; Takagi and Itabashi, 1977), nematodes (Penkov et al., 2014) and plants (Bernard and Joubès, 2013; Duncan et al., 1974; Razeq et al., 2014; Yeats and Rose, 2013). In this respect, functions of WEs are highly diverse as well, ranging from acting as storage compounds, structural components or UV-barriers to functioning as repellents or buoyancy-regulating agents.

In humans, sebaceous glands-derived sebum consists of approximately 25 % WEs (Downing and Strauss, 1974). The actual role of sebum on our skin is not fully understood yet (Smith and Thiboutot, 2008), but it’s anticipated to deliver antioxidants, pheromones and antibiotic lipids to the body surface (Kligman, 1963; Thiele et al., 1999). In vivo experiments furthermore indicate an efficient evaporation-retarding effect of WEs in the tear film of the eye (Craig and Tomlinson, 1997). After initial problems upon reproduction of these findings in vitro (Herok et al., 2009), it’s known today that the evaporation-retarding effects of WEs in tear films seem to be closely associated with their specific melting temperature. WEs adopt a unique physical state at their melting temperature, which makes them able to efficiently cover a liquid’s surface, inducing an anti-evaporative effect (Rantamäki et al., 2013).

A unique case of utilization of WEs in mammals can be found in the sperm whale. Unlike other mammals, sperm whales possess a specialised organ in their forehead which can harbour huge amount of a so called “spermaceti oil” (Perry et al., 1999). The biological function of the spermaceti organ is a matter of discussion until today. Theories span over influence of the spermaceti oil on the whale’s buoyancy (Clarke, 1978) to a function as a weapon (Carrier et al., 2002) or in orientation through the animal’s sonar (Zimmer et al., 2005). Spermaceti oil consists of 70 to 100 % of WEs, while

INTRODUCTION

moiety (Morris, 1973, 1975). In contrast, the Japanese lipid bank website asserts hexadecanoic acid to be the major fatty acid moiety with amounts of over 90 % (Japanese Conference on the Biochemistry of lipids (JCBL), 2014).

The best known insect-derived WEs are produced by honey bees like Apis mellifera. Bees use beeswax as a structural component in order to build honeycombs which can house larvae, honey or pollen. Chemically, beeswax is a blend of an estimated 300 substances. With 35 %, monoesters make up the major part of beeswax. Further constituents comprise hydrocarbons (14 %), diesters (14 %), free acids (12 %) and to a lower extend also triesters, hydroxymonoesters, hydroxypolyesters, acid polyesters and free alcohols (Tulloch, 1970, 1971). In comparison to that, the wax of bumble bees contains approximately twice the amount of hydrocarbons (Tulloch, 1970). Interestingly, bees seem to use their waxes not only for production of honeycombs, but also for orientation. It was shown, that members of different nests also differ in their cuticular wax composition. Traces of these cuticular wax compositions are left behind by the insects at their nests, allowing them to efficiently distinguish between their own and foreign nests (Buckner et al., 2009; Rottler et al., 2013). The use of WEs as structural components is not limited to bees. Larvae of the nematode Pristionchus pacifics uses a very long chain WE in the course of host finding. Coverage of their cuticle with the WEs make the larvae sticky, resulting in a congregation of up to one thousand individuals. The so formed structures are called “dauer towers” and enable the larvae to reach a potential host more easily. The respective WEs which are synthesised by the larvae derive from two C30 chains carrying six double bonds each. With 60 carbon atoms in length, these WEs are one of the longest WEs ever described in a living organism (Penkov et al., 2014).

As WEs consist of highly reduced carbon atoms, possess a high energy density and are osmotically inert, they are perfect storage compounds (Wältermann and Steinbüchel, 2005). Despite of the fact that most bacteria known today synthesise polyhydroxyalkanoate (PHA) or poly(3-hydroxybutyrate) (PHB) as a carbon storage compound, several species which synthesise TAGs or WEs have been described as well (Alvarez and Steinbüchel, 2002; Wältermann et al., 2007). WE synthesising bacteria are mostly derived from the gram-negative species of Acinetobacter, in which WE accumulation was first described 1971 (Gallagher, 1971). Today, wax production and accumulation has also been discovered in other species, such as Alcanivorax, Marinobacter, Thalassolituus, Psychrobacter, Micrococcus, Moraxella, Corynebacterium or Nocardia (Röttig and Steinbüchel, 2013). In all of those species, WEs are accumulated as a supply for times of food deficiency. In contrast, the gram-positive actinobacterium Mycobacterium tuberculosis is reliant on WEs in the process of dormancy induction for persistence of the organism in a host. This induction is impaired in knockout mutants devoid of two FARs responsible for the synthesis of the WEs’ fatty alcohol moiety. As a consequence, the bacterium is not able to seal its cell wall with the help of WEs and prevent nutrient uptake.

Decreased nutrient uptake is necessary for the inhibition of replication (Sirakova et al., 2012).

In contrast to the well described synthesis of WEs as storage compounds in Acinetobacter and several other bacterial species, there is only one known example for a comparable WE utilization in eukaryotes. S. chinensis, also called Jojoba, is a slowly growing desert scrub, which is believed to reach a live span of over one hundred years (Gentry, 1958). Its seeds contain approximately 50 % of fluid WEs instead of TAGs as storage lipids (Greene and Foster, 1933). Since Jojoba WEs are the only natural source of renewable WEs to date (Ash et al., 2005), Jojoba plants are being cultivated in specialised farms in order to harvest the WE containing seeds. However, the costs for Jojoba-derived WEs are rather high due to its slow growth and its shrubby habitus, making the harvest of the seeds more complicated than for normal crop plants. After spermaceti oil became unavailable for lubrication purposes in the 1980s, Jojoba-derived WEs were used in the industry as supplements for

lubricants. Other utilization of Jojoba oil in industry include cosmetics, in which Jojoba oil can be used in shampoos, lotions, crèmes etc. (Green, 2013; McCarthy, 2014). Interestingly, the wax also seems to be suitable for treatment of powdery mildew, a fungal disease affecting a wide range of plants (Hicks and Siemer, 2001). The molecular composition of Jojoba wax comprises mostly of dienoic WEs of 40 and 42 carbon atoms (27 % and 48 % of the total WE load, respectively) (Tada et al., 2005). The major fatty acid moiety within Jojoba wax is eicosenoic acid (20:1-COOH), whereas the most abundant alcohol moieties are eicosenol (20:1-OH) and docosenol (22:1-OH) (Duncan et al., 1974;

Spencer et al., 1977). Jojoba WE molecules are thus different from spermaceti-derived WEs in respect to their higher chain lengths as well as in respect to their higher grade of desaturation.

OBJECTIVES

2 OBJECTIVES OF THIS WORK

Current knowledge about WE biosynthesis indicates low substrate specificities for most described FARs and WSs. This circumstance results in problems concerning application of FARs and WSs for biotechnological approaches, since their low substrate specificities are often not able to realize distinct WE compositions. Instead, WE blends are often highly influenced by the substrate pool compositions of FARs and WSs. Thus, factors acting upstream of WE synthesising enzymes have a prominent role in determination of the final WE composition. As a consequence, the production of defined WE blends in genetically modified systems by the sole expression of FARs and WSs is difficult.

One strategy to overcome this issue is to adjust respective substrate pools by metabolic engineering in order to obtain a desired WE blend. However, respective modifications complicate the approach, as they have to be done in addition to the expression of FARs and WSs. Moreover, they may interfere with the metabolism of the host, resulting in further difficulties. Also, the provision of two different acyl-CoA substrate pools within one cell is a problem, as respective modifications in the hosts metabolism might interfere with each other. Yet, different acyl-CoAs would be necessary in order to produce WEs consisting of two moieties differing in chain length, grade of desaturation or methyl branching.

In order to overcome these difficulties, modified enzymes with high substrate specificities might be an appropriate tool. If a certain specificity for both, FARs and WSs, could be assured regardless of the composition of the substrate pool, no additional metabolic engineering would be necessary in order to produce WEs of defined compositions. Moreover, modified FARs and WSs with high specificities could be used for the production of compounds which are only synthesised to an unsatisfactory extend by currently known wild type enzymes, for instance FAEE and FAME.

The construction of optimised FARs and WSs with distinct substrate specificities requires detailed knowledge about substrate specificity determining structures in respective enzymes. However, to date this knowledge is scarce. Neither structures of FARs nor of WSs are reported until now.

Moreover, there is only one study describing a change of substrate specificity in FARs through protein engineering (Chacon et al., 2013). Likewise, only one report on changing substrate specificity in a WSD-type WS through protein engineering was published (Barney et al., 2012b). Although both studies are important contributions towards a detailed understanding of substrate specificity in FARs and WSs, they also illustrate the infancy of the whole field. Hence, significant efforts are necessary in order to be able to construct WE biosynthetic enzymes with distinct substrate specificities.

In the present study, the main aims were therefore to contribute to the elucidation of structure-function relationships in FARs and WSs. For this purpose, biochemical and biophysical properties of four FARs, two WSs as well as of a soluble DGAT3 have been studied in order to obtain structural insights into the respective enzyme classes.

3 MATERIALS