Predictions

With reference to the aims, it might be expected that, yeasts in batch culture are largely selected to increase their exponential growth rate. If there is a trade-off between rate and yield, and the Crabtree effect indeed represents a high rate/low yield strategy, the yield is expected to decrease. We therefore test if changes in rate and yield are observed in lineages of Crabtree-negative yeasts after 1500 generations of evolution in a high sugar environment. For the Crabtree-positive reference yeast, S. cerevisiae, it would be expected that the changes in rate and yield are small compared to the other species used in the experiment.

An important question to consider is whether the switch from Crabtree-negative to Crabtree- positive is plausible. Most yeasts can ferment in anaerobic conditions; therefore, it is reasonable to assume that they possess the enzymes to ferment. In addition to respiration only two additional enzymes are required to ferment, Pdc and Adh (see Figure 1.1). The main hurdle therefore is regulatory. Firstly, the regulation of which enzyme converts pyruvate, the down regulation of Pdh and upregulation of Pdc must occur to increase the fermentation rate, while the upregulation of Adh must occur to prevent accumulation of toxic acetaldehyde (Pfeiffer &

25

Morley 2014). Secondly, the regulation of the expression of glucose transporters would be a key factor in the move towards Crabtree-positive metabolism. Engineered Saccharomyces cerevisiae

that had all glucose transporters save one removed, exhibited fully respiratory metabolism at high glucose levels and only switches to fermentation in limited oxygen environments (Otterstedt et al. 2004; Maclean & Gudelj 2006). This highlights that the regulation of glucose transporter expression is a key element to yeast exhibiting Crabtree-positive metabolism.

M

Material and Methods

2.5.1

Starting Isolates

Prior to discussing the results of the experimental evolution study, a brief review of the isolate- derived yeast populations (from here these will simply be referred to as populations) is given to highlight the existing metabolic and physiological traits of each yeast. The yeast isolates were sourced originally from Mat Goddard’s collection of natural yeast isolates at Auckland University. The populations are not laboratory yeasts propagated for many generations in isolation but were only recently sourced from natural populations and have had very few generations in a laboratory environment. Those that were isolated from ferment in wine-making were sampled 75% of the way through fermentation and would have originated from the skin of the grapes used not introduced separately (Gayevskiy & Goddard 2012). The cultures obtained from the Goddard lab were single clonal isolates. The yeast species were identified through sequencing the ITS and/or the 26S rDNA region after collection from a number of areas around New Zealand (Goddard 2008b). This section will review the current knowledge of each yeast species, their natural habitat outside of the laboratory, their growth requirements, fermentative ability and appearance. Finally, at the end of this section a compiled table of all known metabolic tests for each yeast species is presented.

2.5.1.1Kodamaea sp.

Kodamaea ohmeri also known as Yamadazyma ohmeri or Pichia ohmeri was assigned to

26

partial sequences of 18s and 26s Ribosomal RNAs (Yamada et al. 1995). It was first isolated from cucumber brine and has been associated with honey bee colonies, and as an emerging pathogen usually in immunocompromised individuals (Biswal et al. 2015; Graham et al. 2011; Piredda & Gaillardin 1994; Etchells & Bell 1950). It has the ability to form pseudohyphae but cannot assimilate nitrate. Appendix A.1 covers more detail on its metabolic capabilities. Cells are roughly 3-6μm in size and generally globose, ellipsoid, ovoid to cylindrical in shape. The species exhibits fermentative abilities, requires biotin for growth and can grow above 30°C (Fiol & Claisse 1991). Our Kodamaea are of unknown species however due to their association to bees (see Table 2.2)

it is assumed they are most likely Kodamaea ohmeri and due to the Biolog results (discussed later) the metabolic similarities would suggest they are most likely the same species but have become vicariant.

2

2.5.1.2Issatchenkia sp.

Issatchenkia orientalis Kudryavtsev was isolated in fruit juice or berries in Russia by Kudravtsev in 1960 (Kurtzman et al. 2011). This yeast is also referred to by the names Candida krusei and Pichia kudriavzevii but has a large number of synonyms which makes collecting a comprehensive record of information on this yeast difficult (Kurtzman et al. 2011). Candida krusei has been isolated from bronchomycosis specimens and human excrement suggesting a pathogenic or opportunistic nature. It has the ability to produce both pseudohyphae but lacks the ability to assimilate potassium nitrate. Appendix A.1 summarises the metabolic traits of this yeast. Cells are roughly 5μm, spheroidal, ellipsoidal or elongate and pseudohyphae present. I. orientalis exhibits fermentative ability and can grow in a vitamin free medium above 30°C (Kurtzman et al. 2011). One of our strains is definitely identified as I. orientalis being sourced from ferment (see Table

2.2 species naming conventions), while our second Issatchenkia strain is of unknown species and origin, so therefore could be slightly different in its metabolic capabilities and indeed its fermentative ones as there is no record for this either.

27

2

2.5.1.3Candida railenensis

Candida railenensis was isolated in decaying wood in Chile (Ramirez & Gonzalez 1984). It was also

found to be abundant on Nothofagus New Zealand (Serjeant et al. 2008) and in acorns of Quercus robur (Isaeva et al. 2009). It was assigned to the Candida genus due to its ability to produce both pseudo and true mycelium and assimilate potassium nitrate, and its lack of ability to produce ascospores (Ramirez & Gonzalez 1984). Appendix A.1 summarises the metabolic traits of this yeast. Cells are roughly 5μm, generally globose, ellipsoidal or elongate, occasionally ogival, triangular or lunate and the cell wall is ascomycetous and two layered. C. railenensis exhibits very limited fermentative ability and cannot grow in vitamin free medium or above 30°C (Kurtzman et al. 2011; Ramirez & Gonzalez 1984). Our strain was sourced from ferment (see Table 2.2).

2.5.1.4Pichia kluyveri

Pichia kluyveri was isolated from rotting cacti (Lachance et al. 1988; Starmer et al. 1987). However it has been found to span cactophilic and non-cactophilic habitats and has been isolated from olives, fruit and coffee beans (Kurtzman et al. 2011; Ganter et al. 2000). It was split into three varieties due to differences in physiological abilities, infertile crosses and a lower degree of genetic similarity than expected (Ganter et al. 2000; Kurtzman et al. 2011; Phaff et al. 1987). Pseudohyphae are formed but true hyphae are not while assimilation of nitrate is negative. Appendix A.1 summarises the metabolic traits of this yeast. Cells are roughly 4-11μm, generally spheroidal, ellipsoidal or elongate and occasionally may be tapered but they are not ogival in shape. P. kluyveri exhibits varying fermentative ability depending on the variety of P.kluyveri it is, for example P.kluyveri var. kluyveri ferments glucose strongly while P. kluyveri var.

cephalocereana is comparatively slow at fermenting. Some shared characteristics include the lack of ability to grow in vitamin-free medium but they can grow above 30°C. The strain we used was most likely P. kluyveri var. kluyveri as the isolate information provided by the Goddard lab records it with fermentative abilities in an anaerobic environment (Anfang et al. 2009).

28

2

2.5.1.5Kluyveromyces nonfermentans

Kluyveromyces nonfermentans was isolated from deep sea mud in Suruga Bay and Sagami Bay,

Japan (Nagahama et al. 1999). It is hypothesised that K. nonfermentans is derived from a

Kluyveromyces aestuarii that evolved in deeper regions of the marine environment (Nagahama et al. 1999). Pseudohyphae and true hyphae are not formed and nitrate is not assimilated (Nagahama et al. 1999; Kurtzman et al. 2011). Cells are around 2-7.5μm in size and spheroidal to ellipsoidal in shape. K. nonfermentans lacks the strong fermentative abilities of the other

Kluyveromyces and has the differentiating lack of ability to assimilate sucrose, lactic acid and succinic acid like other Kluyveromyces. Growth requires vitamins biotin, niacin and thiamin, while growth above 30°C is variable. For full metabolic capabilities see Appendix A.1. Our strain was imported from the Netherlands to the Goddard Laboratory, NZ (under permit 105, July 2006).

2.5.1.6Saccharomyces cerevisiae

Saccharomyces cerevisiae has been used for decades in the brewing of beer and the making of bread. It is one of the most widely used model organisms in biology, with yeast research having resulted in several Nobel Prizes (Suh et al. 2006) including, the Nobel Prize in Physiology or Medicine 2016 by Yoshinori Ohsumi, and the Nobel Prize in Physiology or Medicine 2001 by Leland Hartwell.

Pseudohyphae are formed but septate true hyphae are not and assimilation of nitrate is negative. Appendix A.1 summarises the metabolic traits of this yeast. Cells are roughly 3-10μm, generally globose, ellipsoidal or cylindroidal in shape. S. cerevisiae exhibits very strong fermentative ability,

lacks the ability to grow in vitamin-free medium while growth above 30°C can be variable (Kurtzman et al. 2011). The strain we used was isolated from ferment. It is hypothesised that the

Saccharomyces fermenting ability is adaptive as it modifies the environment to its advantage even though it is energetically inefficient to do so, not just by the production of ethanol but by heat also (Goddard 2008b). It is also theorised that due to lack of evidence of a specific adaptation

29

to fruit, S. cerevisiae may have evolved a more general ability to inhabit and persevere in numerous different environments (Goddard & Greig 2015).

2

2.5.1.7Summary

From the compiled research on the yeast population it can be supposed that each have metabolically different traits, that all the populations save Kluyveromyces nonfermentans have the ability to utilise the fermentative pathway, albeit in an anaerobic setting, and all species have the ability to assimilate glucose and survive the environmental conditions employed for the experimental evolution study. We expect S. cerevisiae to display the most pronounced, and K. nonfermentans the least pronounced Crabtree effect, with the other species being placed between these two extremes; and as shown in Chapter Three, this is confirmed by ethanol assays. It is also apparent that there is a large amount of information left to be discovered about these yeasts, which highlights potential areas for future study. Table 2.2 presents the names of all the yeasts used in this study.

Abbreviated Name Species Goddard Lab Origins

NSC A1 Kodamaea sp. Wax isolate of beehive

in Mangere Bridge

NSC A8 Kodamaea sp. Beesonline isolate

NSC A11 Issatchenkia sp. Chardonnay juice,

Kumeu River, Auckland

NSC B3 Candida railenensis Chardonnay ferment,

Kumeu River, Auckland

NSC B5 Pichia kluyveri Chardonnay juice,

Kumeu River, Auckland

NSC C10 Issatchenkia orientalis

Kudryavtsev

Sauvignon blanc juice, Marlborough

NSC F8 Kluyveromyces nonfermentans Netherlands

SC E2 Saccharomyces cerevisiae Sauvignon blanc

ferment, Hawkes Bay Table 2.2: Species Abbreviated Names and Origins. The table provides the species names of all yeast populations, their allocated abbreviations and the origin of the strains from around New Zealand, save Kluyveromyces nonfermentans which was sourced from the Netherlands.

30

2