The Ehrlich pathway for amino acid catabolism in yeasts

• CA q q

The Ehriich pathway for amino acid

catabohsm in yeasts

Gabriele Romagnoli

2014

The Ehrlich pathway for amino acid catabolism

in yeasts

ter verkrijging van de graad van doctor

aan de Technische Universiteit Delft,

op gezag van de Rector Magnificus

prof ir. K.Ch.A.M. Luyben,

voorzitter van het College voor Promoties,

in het openbaar te verdedigen op

2 juni 2014 om 10:00 uur

Proefschrift

door

G a b r i e l e R O M A G N O L I

Master in Industrial and Environmental Biotechnology,

University of Rome "La Sapienza",

Dit proefschrift is goedgel<:eurd door de promotor: Prof dr. J.T. Pronk Copromotor: Dr. J-M. Daran Samenstelling promodecommissie: Rector Magnificus, Prof dr. J.T. Pronk, Dr. J-M. Daran, Prof dr. A. Querol Prof dr. H.J. Bosch Prof dr. S. de Vries Dr. G . M . de Billerbeck Dr. E.J. Smid voorzitter

Technische Universiteit Delft, promotor Technische Universiteit Delft, copromotor

Instituto de Agroqufmica y Tecnologia de Alimentos

Universiteit Wageningen Technische Universiteit Delft INSA de Toulouse

Universiteit Wageningen

The studies presented in this thesis were performed at the Industrial Microbiology section, Department o f Biotechnology, Delft University o f Technology, The Netherlands. The Industrial Microbiology Section is part o f t h e Kluyver Centre for Genomics o f Industrial Fermentation, which is supported by the Netherlands Genomics Inhiative.

COVER: Photo provided by Arianne Vasquez

Table of Contents

7 C h a p t e r 1

General Introduction

C h a p t e r 2

substrate specificity of thiamme-pyrophosphate-dependent

2-oxo-acid decarboxylases in Saccharomyces cerevisiae

67 C h a p t e r s

Functional analysts and transcriptional regulaüon of two orthologs of

AROlO, encoding broad-substrate-specificity 2-oxo-aad

decarboxylases, in the brewing yeast Sacckaromyces pastonanus

CBS1483

C h a p t e r 4

Beletion ofthe Saccharomyces cerevisiae AROS gene, encoding an

aromatie amino aeid transaminase, enhances phenylethanol

production from glucose

C h a p t e r s

An alternative, arginase-indepeudeut pathway for arginme

metabolism iu Kluyveromyces lacüs involves guanidiuobutyrase as:

a

key enzyme

S u m m a r y S a m e n v a t t i n g C u r r i c u l u m V i t a e l y b L i s t o f P u b l i c a t i o n s A c k n o w l e d g e m e n t s

Chapter1

General Introduction

Chapler 1

Nitrogen in living cells

Considerations on taw life and

biocl,e„,ical

nelworks in living cells

.nigh,

have o r , g , „ a . e d .end .„ focus

on

carbon chenris.^. While .here

,na,

he

g o X a

or d o , „ g so n should be .aken into aecoaa. ,ha. „ih-ogen is/as, as es en . L e as „ e „ „ „ I , - as carbon. For e«.a,p,e. „ i „ - „ , e ' is f l , : « 7

pephd,c bonds .ha. enable .he asscnbly o f pro.eins (Llinas & Klein ,975) a d .n.e,«,ons be.wee„ D N A and RNA s.rands rely „ „ „i.rogen'- o . „

functional groups (Yanson a ai, 1979). containing Currently, the nitrogen in the biosphere is mostly present in its rather inert

tnolecular form m (Walker, 1,77). Nitrogen fixation (reduction o f N to h more teactive N H , ) is essentia, to make molecular nit „gen availa le f , 1 production o f organic nitrogen compounds by iiving ce.ls. To nnde t a l d importance ot such a process one need only consider that 1% o f ,he o n c l generate worldwide each year is nsed by the Habei^Bosch c s m tndnstrial y convert atmospheric nitrogen m ammonia. Early l „ p „ T e s e : on Pt-ebtotic formation o f organic nitrogen compounds assnmed a strongly ncii atmosphere (Miher & Hamid, ,959). in which reduced nitrogen i tl e „rm ammonia was already present. However, according to eunent'thcoriet c r a n d

(Walkei, 1977). Dinitrogen is one of the least reactive compounds found in na ure hecause of the very high strength of its triple bond ( C o l ^ J Z o 1''62). F o i this reason, prebiotic formation of organic nitrogen compounds

• R-N c N i t r o g e n f i x a t i o n NHj "(mmonificatio R-N F i g u r e 1 T h r e e m a i n e v e n t s d u r i n g the e v o l u t i o n o f t h e N i t r o g e n c v c l e R N I rJ- . u

bilrodiictioii

required events that could generate very high temperatures, such as l.ghtnmg stdkes or meteor e n t i x and thereby overcome the extremely high activation energy needed for splitting the dinitrogen bond (Boyd, 1998; Jenmskens &

Stenbaek-Nielsen, 2004). The products generated after breaking open the dinitrogen bond depend on the composition of the primordial attnosphe.-e. Current geochemicai evidence points to a non-reducing atmosphere made primarily o f carbon dioxide, molecular nitrogen and water (Miller & Sdtlesinge,, ,984) Under those conditions, electric discharges would have formed CO and NO(Borucki & Chameides, 1984), which would have been converted to N O 3 , NO.- and fmally N H 3 through a series o f photochemical and aqueous phase

,.eacbons(Mancinelli & McKay, 1988; Summers & Chang, 1993) (F.gure l A ) ^ Currently, the most accepted route for the prebiotic formation o f ammo ac.ds and nucieobases is the reaction o f ammonia and HCN with aldehydes and ketones (Miller & Van Trump, 1981; Summers & Lerner, 1996). The first organisms to evolve probably depended on N H 3 produced in abiotic reactions and, subsequendy, evolved the capability to deaminate organic nitrogen compounds released by (dead) biomass in the process now known as ammonification(Gutschick, 1981) (Figure I B ) . , u- k

Once the concentration o f abiotically formed N H 3 m the biosphere ran out due to the proliferation o f living cells, organisms able to use abiot.cally produced N O 3 - and N O f as nitrogen source would have had a strong selective advantage. The advent o f microbial reduction o f nitrite and nitrate to ammoma riones 1985) (denitrification/assimilato.7 nitrate reducdon) would then have preceded the 'great oxidation event' in which the Earth's atmosphere switched L m reducing to oxidizing due to the activity o f oxygenic phototrophs

(Schidlowski, 1976; Sessions ei al, i m ) . The appearance o f oxygen m h atmosphere was followed by the evolution o f organisms capable o f oxid.zmg the ammonium, denved from biomass degi-adadon, to NO," and NO3 (nitrification)(Schmidt, 1978) (Figure I C ) and provided additional source o l nittate to the biosphere in addition to abiotic nttrate formation.

As during evolution, the amount o f biomass on the planet gi'ew, so did the demand for nitrogen and a further expansion of life required a new biotic pathway f o r fixation o f molecular nhrogen. Biological nitrogen fixation was first descnbed by the Dutch scientist Beijerinck in 1901(Beijennck, 1901) and, when f u l l y coupled, requires 16 molecules o f ATP for every molecule o f N , reduced. Despite the fact it is a very energy-demanding process, uittogen fixation provides a Sttong evolutionary benefit in situations where other more readily assimilable

Chapler I

nitrogen species are in short supply. Another microbial process that plays a key role in the nitrogen cycle is the anaerobic ammonium oxidation (anammox) It has been recently shown that bacteria performing this reaction are responsible for a substantial fraction of N loss in the ocean (Daalsgard, 2003). Evolution o f diese microorganisms is not yet f u l l y understood (Strous, 2006) and is complicated by the fact that they are veiy hard to culture.

The pre-biotic earth environment defined the first organisms and their metabolic pathways by supplying the building blocks and substrates necessary for their origin. Since the origin o f life, biodc nitrogen metabolism evolved and differentiated. Currently, microorganisms are, together, able to metabolize a wide array o f nitrogenous compounds. Ammonium (NH4-) is considered to be the preferred nitrogen source for many bacteria and flingi (Marzluf, 1997; Merrick & Edwards, 1995). In addition to N H , ^ glutamate and glutamine serve as the key nitrogen donors for biosynthetic reactions in all known cells and are also used as preferred mtrogen sources by many microorganisms (Magasanik, 1993- Merrick & Edwards, 1995).

In the absence o f their preferred nitrogen sources, many microorganisms are able to induce padiways for the utilization o f alternative nitrogen-containing compounds, ranging from amino acids to polyamines and purines. In many microorganisms, evolution in environments with scarce or vaiying nitrogen supplies is refiected by the presence in their genomes o f genes for the uptake and catabohsm o f a wide variety o f nitrogenous compounds. The biochemist.^ genetics and regulation o f nitrogen source metabolism have been particularly' well studied in one model micro-organism, the yeast Saccharomyces cerevisiae (Cooper, 1982).

Saccharomyces: habitats a n d nitrogen availability

Saccharomyces cerevisiae is one o f t h e most extensively used microorganisms

for the production o f fermented products like beer wine and bread. S cerevisiae .s used m an increasing number o f industrial processes including pharmaceutical pi-oducts (insulin (Bayne et ai, 1988), antibodies (Gerngross, 2004), artemisinin (Ro el ai, 2006)), but also for the large scale production o f ethanol ( L i n & Tanaka, 2006) and other bulk chemicals (lactate (Ishida et ai, 2005), malic acid (Zelle et ai, 2008) ). Additionally, S cerevisiae has been the model system for molecular genetic research because the mechanisms o f replication recombination, as well as the core metabolism are generally conserved between yeasts and larger eukaryotes, including mammals (Botstein et al 1997)

Introduction

Obtaining the complete sequence o f its genome (Goffeau et al, 1996) increased the potential o f genetic manipulation and has proven to be extremely useful towards the sequencing o f other yeasts and higher eukaryotic genomes. This allowed exploring differences in core metabolism between related yeast species, including the nitrogen metabolism that, as described more in detail further on during this thesis, is extremely important for the formation o f flavours. In 1986 Large et. al (Large, 1986) presented an extensive study on the utilization o f many nitrogen sources from various yeast genera and pointed out an heterogeneity o f metabolic routes for compounds like urea, lysine or polyamines. Although most laboratory studies have been done using one o f these nitrogen compounds as sole nitrogen source, a wide vanety o f nitrogen sources is present in the yeast natural environment.

Despite the extensive studies o f Saccharomyces cerevisiae as a laboratoiy model and as an industrial 'work horse', surprisingly little is known about the natural habitat o f Saccharomyces yeasts. S. paradoxus is traditionally associated with natural environments, while S. cerevisiae was a domesticated organism adapted to man-made fermentations and largely absent f r o m natural habitats. Only recently, it has been shown that both S. paradoxus and S. cerevisiae can be readily isolated from natural habitats by selective enrichment (Naumov et al,

1998; Sampaio & Gon5alves, 2008).

S. cerevisiae has frequently been isolated f r o m the bark o f certain

deciduous trees (Sampaio & Gonyalves, 2008), from fermenting fruits and f r o m other high-sugar, plant-related environments such as nectar and sap fluxes (Nordin el al, 2001). In perennial trees such as oak, nitrogen is stored both as soluble amino acids and proteins. The relative abundance o f these two potendal nitrogen sources for yeast gi-owth is still an argument o f debate and depends on several factors, including the season, the part o f the tree, fertilization and extraction method. Arginine and proline represent the major soluble nitrogen storage compounds in most trees (Kato, 1986; Titus & Kang, 1982) and can be found both in bark, wood and the phloem (Nordin el al, 2001). O f course, when considering the ecology o f 5'. cerevisiae's habitats, the long history and enormous scale o f its domestication by mankind cannot be neglected (Liti el al, 2009). Domestication may also have influenced nitrogen metabolism, by adaptation o f the yeast genome and its regulation to the nitrogen sources in fermentadon processes. Wine fermentation provides a relevant example o f the importance o f nitrogen source utilization for yeast physiology and industrial application. During fermentation o f grape must, a few sugars such as glucose and fructose are present

Chapter 1

in abundance wiiile nid'ogen availability is limited to some free ammonium and small amounts o f amino acids o f which arginine, proline and glutamine are the most abundant. Scarcity o f assimilable nitrogen in its natural habitats may have contributed to the versatility o f S. cerevisiae with respect to nitrogen sources, combined with its narrow range o f carbon substrates. On the other hand, the absence, in S. cerevisiae, o f proteases capable o f degi-ading larger peptides (Strauss et ai, 2001) limits the variety o f molecules that this yeast can use as a source o f nitrogen. The amount o f yeast assimilable nitrogen ( Y A N ) present in grapes depends on many factors, including gi-ape variety (Butzke, 1998), vineyard soil (Spayd etal, 1995), climatic and weather condidons (Stines el ai, 2000). Nitrogen availability affects many sensory characteristics and influences the synthesis o f important aromatic compounds like fusel alcohols, as well as trehalose and glycerol, which both contribute to the mouth feel o f wine (Lubbers

et a/., 2001).

Arginine, proline and glutamine are the most abundant amino acids in grape must, followed by alanine, threonine and serine (Monteiro & Bisson, 1991). Arginine and glutamine are quickly used early on during the fermentation' while proline is not consumed because the catabolism requires molecular oxygen and is repressed by the presence o f ammonium in the must (Ingledew et al 1987).

Nitrogen metabolism in Saccharomyces yeasts is also highly relevant for beer fermentation. The total concentration o f nitrogen and the variety o f amino acids present in wort influences flavour, colour and stability o f beer. The main nitrogen sources for yeast metabolism in wort are single amino acids, small pepddes and ammonium released during the malting and the mashing processes (Lekkas et ai, 2007). During the wort boiling step the formation o f maltol by the

G r o u p A Fast a b s o r p t i o n G r o u p B I n t e r m e d i a t e a b s o r p t i o n G r o u p C G r o u p D S l o w a b s o r p t i o n L i t t l e o r n o G l u t a m a t e V a l i n e _{G l y c i n e P r o l i n e} A s p a r t a t e M e t h i o n i n e _{P h e n y l a l a n i n e} A s p a r a g i n e L e u c i n e T y r o s i n e G l u t a m i n e I s o l e u c i n e T r y p t o p h a n S e r i n e H i s t i d i n e A l a n i n e T h r e o n i n e _{A m m o n i a} L y s i n e A r g i n i n e

T a b l e 1 C l a s s i f i c a t i o n o f a m i n o a c i d s i n w o r t according to their rate o f

D "

Introduction

reaction between proline and maltose contributes to the browning o f the fmal product (Mabrouk, 1979). During the fermentadon, only about 40% o f t h e total nitrogen is metabolized and the rest contributes to haze development and foam stability (Lekkas et al., 2007). Wort amino acids have been classified according to the rate at which they are metabolized during fermentation (Table 1).

During bread making, Saccharomyces cerevisiae has a great impact on the amino acid content o f dough. Amino acids are important precursor for flavours and play an important role during the bread making process, which will be discussed later on in this introduction.

Central nitrogen metabolism

A l l known strains of Saccharomyces cerevisiae are able to use more than 50 compounds as the sole source o f nitrogen for gi'owth (Large, 1986). Their repertoire o f nitrogen sources includes L-amino acids, D-amino acids (LaRue & Spencer, 1967), ammonium, urea, purines and polyamines. In contrast to some other yeasts, S cerevisiae is unable to use nitrate or nitrite as nitrogen sources. The conversion o f all nitrogen sources o f S cerevisiae involves ammonia, glutamate and/or glutamine as 'entry compounds', with the two amino acids serving as amino group donors for further reactions (Cooper, 1982). The central nitrogen metabolism in .S'. cerevisiae can be summarized by four reactions, catalysed by only 5 enzymes (Figure 2). These are two NADP-dependent glutamate dehydrogenases encoded by GDHl (Moye et al, 1985) and GDH3 (Avendano et al, 1997); NAD-dependent glutamate dehydrogenase encoded by

GDH2 (Deluna et al, 2001); glutamine synthase encoded by GLNl (Minehart &

F i g u r e 2 M a i n r e a c t i o n s i n v o l v e d i n c e n t r a l n i t r o g e n m e t a b o l i s m . 1) G d h l a n d G d h 3 , N A D P - d e p e n d e n t g l u t a m a t e d e h y d r o g e n a s e ; 2 ) G d h 2 N A D - d e p e n d e n t g l u t a m a t e d e h y d r o g e n a s e ; 3 ) G l n l , g l u t a m i n e s y n t h a s e ; 4) G I t l , g l u t a m a t e synthase

Chapter 1

Magasanik, 1992) and glutamate synthase encoded by GLTl (Filetici et ai, 1996). The GDH2 gene encodes for the NAD-dependent glutamate deliydrogenase and, during gi-owth on glutamate-yielding nitrogen sources, has the important function o f providing ammonia for the synthesis o f glutamine (Miller & Magasanik, 1990).

Under condidons o f ammonium sufficiency, the major pathway f o r glutamate synthesis is catalysed by the Gdhl and Gdh3 isoforms o f glutamate dehydrogenase and consist o f the reductive amination o f 2-oxoglutarate, with ammonia as the nitrogen donor (Deluna et ai, 2001). The combined action o f glutamine synthetase Glnl and the glutamate synthase GItl provides an alternative pathway to produce glutamate, which is present in S. cerevisiae but absent from many other related yeasts. The activity o f these key enzymes is tightly regulated according to the type and concentration o f nitrogen source present (Valenzuela et ai, 1998). The glutamate dehydrogenases have a fairly low affinity for ammonium (Deluna et ai, 2001). Therefore, when ammonium concentrations are low, the alternative high-affinity pathway via glutamine synthase and glutamate synthase is induced. The fight regulation o f expression and activity o f this high-affinity pathway is probably related to the fact that ammonium assimiladon via G l n l and GItl requires a net investment o f A T P (Tempest e/a/., 1973).

Amino acid assimilation in S. cerevisiae

In many natural and industrial contexts, amino acids represent the most relevant source o f nitrogen for growth o f S. cerevisiae. Amino acids can be grouped into different categories based on their catabolism by S. cerevisiae and the degradation pathway employed (Figure 3). Deamination o f glutamine, asparagine, threonine and serine leads to ammonia production (Messenguy et al., 2006). Tryptophan, tyrosine, phenylalanine, isoleucine, leucine, valine, alanine, methionine, lysine, alanine, G A B A , ornithine and aspartate are initially transaminated to form glutamate (Messenguy el al., 2006), leaving the deaminated carbon skeleton o f the amino acid that acted as amino donor in the form o f 2-oxo acid. T w o subcategories can be identified within this gi'oup, depending on the metabolic fate o f this carbon skeleton. In the case o f lysine, alanine, G A B A , and aspartate, the resulting carbon skeletons can be fed into central carbon metabolism as glutarate, pyruvate, oxaloacetate and succinate respectively. For other amino acids, the remaining 2-oxo acids undergo a decarboxylation followed by either a reduction or an oxidation o f the resulting

Introduction

Deam^iation

Central carbon metabolism

Transamination

Higher Alcohols Asparagine Serine Threonine . Glyeinc A l a n i n e Aspartate Lysine G A B A Urea Leueine Isoleucine Valine M e t h i o n i n e Tryptophan Tyrosine A r g i n i n e Phenylalanine F i g u r e 3 R e p r e s e n t a t i o n o f m a i n r e a c t i o n s i n v o l v e d i n a m i n o a c i d c a t a b o l i s m b y Saccharomyces cerevisiae See the t e x t f o r the d e s c r i p t i o n . (1) denotes a N A D P - d e p e n d e n t g l u t a m a t e d e h y d r o g e n a s e ; (2) N A D - d e p e n d e n t g l u t a m a t e

d e h y d r o g e n a s e . See the t e x t f o r t h e d e s c r i p t i o n

aldehydes. This pathway was initially proposed by Ehrlich and, because it plays a central role in this PhD thesis, w i l l be described in more detail in the following paragraphs.

Arginine represents a special case. Arginine metabolism in 5". cerevisiae is inhiated by a cleavage reaction, catalysed by arginase that yields the non-proteinogenic amino acid ornithine and urea (Sumrada & Cooper, 1982). Further catabolism o f ornithine proceeds via proline (Brandriss & Magasanik, 1980), which is transformed into glutamate in two reaction steps that take place in the mitochondria (Brandriss & Magasanik, 1979).

The Ehrlich pathway

In 5*. cerevisiae, the catabolism o f branched-chain, sulfur-containing and aromatic amino acids proceeds through the Ehrlich pathway (Ehrlich & Herter, 1904), which involves three enzyme-catalysed reacdons. First, hansamination yields a 2-oxo acid, which is subsequently decarboxylated, yielding the corresponding aldehyde. In the third step the aldehyde can be reduced to an

Chapter 1

alcohol or oxidized to an acid in anaerobic or aerobic conditions, respectively. Despite the fact that this pathway was originally described by Ehrlich over one hundred years ago (Ehrlich & Herter, 1904), its key enzymes have not been studied in detail. Identification o f the responsible enzymes is complicated by their redundancy and broad substrate specificity. Both o f these factors are already evident in the first transamination reactions o f t h e Ehrlich pathway.

Transamination o f the branched-chain amino acids leucine, isoleucine and valine is catalysed by the transaminases encoded by the BATI and BAT2 genes (Kispal et ai, 1996). The first enzyme is highly expressed during exponential gi'owth, repressed during stationary phase and is localized in the mitochondria (Eden etal, 1996). Instead, BAT2 is cytosolic and has the opposite expression pattern (Eden et ai, 1996). Although a batl bal2 double mutant is auxotrophic for isoleucine, valine and leucine (Eden et al., 1996; Kispal et ai, 1996), producdon o f the corresponding fusel alcohols amylalcohol, isobutanol and isoamylalcohol is not totally abolished. This observadon indicates the presence o f a BAT-independent transaminase activity (Eden el al, 2001).

The aromatic amino transferase I (Aro8) and the aromatic amino transferase I I (Aro9) are responsible for transamination o f the aromatic amino acids phenylalanine, tyrosine and tiyptophan (Iraqui el al., 1998). Only an aro8

aro9 double mutant is fully auxotrophic for phenylalanine and tyrosine, A m i n o acid Q- ketoglutarate glulaiTiate \ a-Keto acid Oxidation: ALDl. ALD2. ALDi. ALD-1,

AIDS. Aim NADH CO, A Akieliyde Transamination: AROS ARO'J BATI BAT2 Decarboxylation: AROIO PDCI PDC5 PDC6 TUB Reduction: ADHl, ADH2. ADH3, ADH4, ADH5, ADH6, SFAl

Fusel acid _{Fusel alcoiiol}

F i g u r e 4 E h r l i c h p a t h w a y . C a t a b o l i s m o f b r a n c h e d c h a i n , s u l f i j r c o n t a i n i n g a n d a r o m a t i c a m i n o a c i d s r e s u l t s i n t h e f o r m a d o n o f f u s e l a l c o h o l a n d f u s e l acids.

Introduction

indicating tliat both isoenzymes are able to catalyse the last step o f phenylalanine and tyrosine biosynthesis. Aro9 has a predominant role in the first step o f tryptophan degi'adation, since aro9 mutant strains grow veiy poorly when tryptophan is the sole nitrogen source (Urrestarazu et ai, 1998). On the other hand, AR09 deletion has no effect for gi-owth on phenylalanine or tyrosine, indicating that Aro8 or other transaminases can also ensure their degi-adation (Iraqui etal., 1998).

Transcription o f the branched-chain and aromatic aminotransferases is not tightly linked with the presence o f their prefeiTed substrates in gi-owth media. For example, genome-wide transcript profiles o f chemostat cultures containing either phenylalanine, methionine or leucine as sole nitrogen source revealed a consistent upregulation o f both AR09 and BAT2 in comparison with cultures in which ammonia was the nitrogen source (Boer et al, 2007).

The second step i n the Eliiiich pathway is the decarboxylation o f the 2-oxo acid. In the yeast genome five genes share sequence similarity w i t h TPP-dependent 2-oxo acid decarboxylase. In addition to the pyruvate decarboxylase-encoding genes PDCI, PDC5 and PDC6 this set o f genes encompasses THI3 and

AROIO. O f this gene set, AROIO is the only gene whose transcription is strongly

correlated with the nitrogen source present in the medium (Vuralhan et al, 2003). More precisely, transcription o f AROIO is upregulated in the presence o f aromatic or branched chain amino acids, while low transcript levels are found during gi-owth on ammonium or proline. Enzyme activity assays confirmed that ArolO is able to decarboxylate a broad array o f substrates including branched chain (ketomethylvalerate, ketoisocaproate), sulfur-containing (methylthiooxobutanoate) or aromatic (phenylpyruvate) oxo acids (Vuralhan et

al, 2005). However, delefion o f AROIO did not completely abolish

decarboxylation o f phenylpyruvate, thus indicating the existence o f an AROIO-independent activity that required the presence o f a functional THIS allele and one o f t h e pyruvate decarboxylase genes PDCI, PDC5 or PDC6 (Vuralhan et al, 2005). In order to shed light on the substrate specificity o f these putative 2-oxo acids decarboxylases a systematic study o f their substrate specificity was urgently needed (see Chapter 2 o f this thesis).

The last step in the Ehrlich pathway is the reducdon or oxidation o f t h e 'fusel aldehyde' to the corresponding alcohol or acid. Which o f these two products is formed strongly depends on the redox state o f the cells. In aerobic glucose-limited chemostat cultures gi-own with phenylalanine, leucine or methionine as sole nitrogen source, the corresponding fusel acids were the main

Chapter 1

degiadation products (Boer etal., 2007). Conversely, under anaerobic conditions, fusel alcohols were the predominant catabolic products o f amino acid catabolism (Dickinson et al, 2000; Dickinson et al, 1998). A plausible explanation is that higher N A D H / N A D + ratios during anaerobic growth favour the reduction o f t h e fusel aldehyde. This might even have a positive impact on energy metabolism since fusel alcohol production can replace glycerol production from glucose, which requires a net input o f ATP, as an 'electron sink' for excess N A D H generated in biosynthedc reacdons (Hazelwood et al, 2008).

Identification o f t h e oxidoreductases responsible for the last step o f t h e Ehrlich pathway is challenging, due to the large variety o f genes encoding alcohol dehydrogenases ( A D H ) and aldehyde dehydrogenases ( A L D ) harboured by the .S". cerevisiae genome. By testing all possible combination o f deletions o f the five A D H genes it was shown that the final step in the Ehrlich pathway can be catalysed by any o f the ethanol dehydrogenases ( A d h I , Adh2, Adh3, Adh4 and Adh5) or by the formaldehyde dehydrogenase Sfa I (Dickinson et al, 2003). Even less is known on the specificities o f t h e different aldehyde dehydrogenases. Only recendy, it was shown that deletion in the ALD3 gene led to a decrease o f phenylacetate production under aerobic conditions, suggesting that it has a more important role in phenylacetaldehyde oxidation than the highly homologous

ALD2 gene ( K i m et al, 2013). This effect may at least partly be due to the much

higher expression level o f ALD2 compared to ALD2 (Pigeau & Inglis, 2007) and does therefore not necessarily reflect a different substrate specificity o f the encoded enzymes.

Regulation of the E h r l i c h pathway

In S. cerevisiae, amino acid catabolism is under the control o f a transcription regulation mechanism called Nitrogen Catabolite Repression (NCR) (Schure el

al, 2000). This mechanism prevents synthesis o f proteins necessary for

utilization o f non-preferred nitrogen sources when a preferred one is available. NCR involves a complex network o f regulation, mediated by four main transcription factors called G A T A factors (Hofman-Bang, 1999). As long as a preferred nitrogen source is present the TOR kinases phosphorylate the transcriptional activators Gln3 and GatI (Beck & Hall, 1999). The phosphorylated forms interact with Ure2 (Blinder el al, 1996) in the cytosol, thereby preventing their migration to the nucleus and the consequent activation o f nitrogen regulated genes (Cox el al, 2000). Under nitrogen-limited conditions, the kinases are less active and the dephosphorylated forms o f Gln3 and GatI can

Jnlmditclioii

freely migi'ate to the nucleus. Here, they activate transcription o f target genes important for amino acid transport and catabolism, and compete with the negative regulators Dal80 and Gzf3 (Hofman-Bang, 1999). The four transcription factors cooperatively modulate transcription, dependent on the nitrogen sources available.

In addition to G A T A factors, pathway-specific transcription factors regulate the expression o f genes for the utilization o f individual (sets o f ) nitrogen sources. AroSO is a member o f the ZniCyse protein family and activates expression o f the AR09 and the AROIO genes in response to aromatic amino acids (Iraqui et al, 1999). The current hypothesis is that the inducer directly binds to AroSO, thereby causing conformational changes that enable recognition o f specific sequences in the AR09 and AROIO promoters. A recent study showed that AroSO is also required for recruitment o f GatI to its target promoters (Lee et

al, 2013). The two complementary mechanisms (induction by aroinatic amino

acids and nitrogen catabolite repression) have a different weight/importance on the regulation o f AR09 and AROIO. The presence o f tiyptophan induces the

AR09 transcription up to 30 times, while rapamycin treatment (mimicking a

nitrogen starvation state) triggers only a 2 to 3 f o l d increase in expression (Lee & Hahn, 2013). This demonstrates a higher contribution o f the specific inducer than N C R on AR09 expression, a phenomenon previously reported in other N C R -sensitive genes involved in catabolism o f arginine (Smart el al, 1996).

T h e E h r l i c h p a t h w a y in other m i c r o o r g a n i s m s

In 1904, Felix Ehrlich first noted the structural similarities between the branched-chain amino acids leucine, isoleucine and the corresponding higher alcohols isoamylalcohol and amylalcohol (Ehrlich & Herter, 1904). Supplementation o f yeast cultures with branched-chain amino acids resulted in accumulation o f the corresponding higher alcohols showing that, indeed, S. cerevisiae was able to catalyse this conversion. This discovery in S. cerevisiae was an important milestone in the understanding o f amino acid catabolism. Since the early studies, the Ehrlich pathway was found in many other microorganisms, including 'unconventional' yeasts belonging to the genus Lachcmcea. Recently, mixed cultures o f S. cerevisiae and L. Ihermotolerans have been used in traditional fermentation to enhance wine chemical profile and sensory characteristics (Gobbi

et al, 2012). Yarrowia lipolylica is another unconventional yeast able to convert

phenylalanine into phenylethanol showing potential for future applications (Celinska et al, 2013). But the Ehdich pathway is present in many more

Chapter 1

microorganisms otlier tlian yeasts. This broadened the interest from traditional yeast fermentation processes to other areas, including the producdon o f antibiotics and biofuels, as well as the role o f t h e Ehrlich pathway in important human pathogens. For example, higher alcohols produced via the Ehi-lich pathway in the pathogenic yeast Candida albicans are in fact important quorum sensing molecules necessaiy for b i o f i l m formation (Ghosh et ai, 2008). This is a highly relevant observation, since biofilm formation is essential for the colonization o f human tissues by C. albicans (Ramage et al, 2001). Aromatic alcohols such as phenylethanol, tyrosol and tryptophol, are especially important for its switch f r o m planktonic to filamentous form (Chen & Fink, 2006). Synthesis o f these alcohols in C. albicans involves the same set o f enzymes as in

S. cerevisiae, encoded by a group o f genes with high similarity to their S. cerevisiae orthologs (Ghosh et al, 2008). Deletion o f the ScAROSO ortholog

resulted in a 2 to 5 fold reduction in aromatic alcohol production and simultaneous downregulation o f the CaAR09 and CaAROlO genes. Clearly, Ehrlich pathway enzymes and transcriptional regulation are highly similar between these two yeast species.

The filamentous fungus Pemcillium chrysogemim is the major producer o f penicillin antibiotics (Elander, 2003). High-level production o f penicillin G requires the addition o f phenylacetate to the gi'owth media (Moyer & Coghill, 1947). Phenylacetate, which is a side chain precursor for penicillin G, is currently chemically synthesized from petrochemical raw materials. It was shown recently that phenylacetate can actually be produced in vivo by P. chrysogemm via a fungal Ehrlich pathway (Veiga et al, 2012). Among the genes overexpressed during growth with phenylalanine as sole nitrogen source, the P c l 2 g l l 8 6 0 gene was found to share 50% sequence similarity to the S.

cerevisiae AR08 transaminase gene. Based on sequence similarity with S. cerevisiae decarboxylases, the Pcl3g09300 gene was demonstrated to encode a

decarboxylase with affinity for both pyruvate and phenylpyruvate (Veiga et al, 2012). Overexpression o f the Pcl3g09300 gene in a S. cerevisiae strain, in which all five genes with sequence similarity to TPP-dependent 2-oxo acid decarboxylases were deleted, restored its ability to gi'ow on phenylalanine. This functional analysis o f an Ehrlich pathway in P. chiysogemim may open the way towards synthesis o f t h e complete penicillin G molecule, from glucose as the sole carbon source, by engineered strains.

Activity o f Ehilich-type pathways for amino acid catabolism is not confined to eukaiyotic microorganisms. For example, 2-oxo acid decarboxylases

Inlrodiiction

w i t h broad substrate specificity have been identified in industrially important lactic acid bacteria such as Lactococciis lactis (Plaza et ai, 2004). The kivd gene encoding this decarboxylase was successfully used in a metabolic engineering strategy for production o f biofuel in E. coli (Atsumi el al., 2008). The search for other decarboxylases with catalytic and kinetic properties suitable for industrial biofuel production led to exploration o f biodiversity in more extreme enviromnents. Psychrobacter ciyohalolentis was initially isolated from samples o f Siberian permafrost and is able to giow at temperatures between -10 and 30X2. Growth with different amino acids as the nitrogen source resulted in the production o f fusel alcohols (Wei et al., 2013) indicating the presence o f t h e Ehrlich pathway also in this species. The gene encoding for the 2-oxo acid decarboxylase was cloned and characterized in E. coli and the encoded protein was able to decarboxylate the branched- chain and aromatic 2-oxo acids tested. The wide range o f organisms harbouring the Ehrlich pathway, and therefore the larger variety o f kinetic features, represent a valuable resource for future applicadons.

Biotechnological relevance of the E h r l i c h pathway

Higher alcohols are the final products o f the reductive Ehrlich pathway. These compounds and their esters have an important impact on the organoleptic properties o f a wide range o f fermented products, including beer, wine, bread and fermented dairy products (Ferreira el al., 2000; Garcia el ai, 1994; Smit et al., 2005).

For example, the addition o f yeast results in a more aromatic bread due to the accumulation o f higher alcohols such as isobutanol and phenylethanol,which are

C o m p o u n d U p a c n a m e F l a v o u r Typical amount in beer" Typical amount in w i n e ' ' Flavour threshold in water P h e n y l e t h a n o l 2 - p h e n y l e t h a n o l flowery, 15,1 6,1 1 roses I s o a m y l a l c o h o l 3 - m e t h y l b u t a n o l banana 4 9 , 6 1,41 1 M e t h i o n o l 3 - ( m e t h y l t h i o ) p r o p a n o l p o t a t o 0 , 9 9 0 , 0 1 8 0,25 A c t i v e 2 - m e t h y l b u t a n o l a p p l e 14,4

_-

0 , 3 2 a m y l a l c o h o l 0,025 A c e t a l d e h y d e s w e e t . 5,1

_-

0,025 p u n g e n t I s o b u t a n o l 2 - m e t h y l p i o p a n o l m a l t y 15 9,2 0,1 P h e n y l a c e t a t e p h e n y l a c e t i c a c i d 0 , 2 5 7 0 , 0 0 7 1 P r o p a n o l n - p r o p a n o l a l c o h o l i c 10,8 3,6 0,6 T a b l e 2 M a i n f i i s e l a l c o h o l r e l e v a n t f o r f l a v o u r s i n f e r m e n t e d beverages. A l l a m o u n t s are expressed i n m g / L a = ( F r i t s c h & S c h i e b e r l e , 2 0 0 5 ) , b = ( T a o el al., 2 0 0 8 )

Chapter 1

directly derived from the catabolism o f valine and phenylalanine, respectively(Hansen & Schieberle, 2005). Other compounds derived from the yeast amino acid catabolism are aldehydes such as 3-methyl butanal, phenylacetaldehyde and 3-(methylthio)-propanal that contribute especially to the crumb flavour (Schieberle, 1996). Amino acids also react non-enzymatically with free sugars in the Maillard reacdons during the baking process forming a wide variety o f compounds (Martins et al, 2000). Proline and ornithine are responsible for the formation o f 2-acetyl-pyrroline (ACPY) and 2-acetyltetrahydropyridine ( A C T P Y ) , which are the main contributors to the roasted flavors o f baked products (Schieberle, 1990). In beer and wine the flavour o f t h e fmal product is determined by many compounds which include fusel alcohol, esters, vicinal diketones and organic sulfur compounds. These last two, are typically present in green beer, but are significandy reduced during lagering (Renger et al, 1992). The remaining fusel alcohols and esters contribute to the quality o f the finished beer. Isoamylalcohol and phenylethanol are the most important fusel alcohols in beer, with fmal concentrations that are far above their flavour thresholds (Landaud et al, 2001) (Table 2).

Another interesting field o f application o f the Ehrlich pathway is the production o f biofuels. Several fusel alcohols have better fuel characteristics than ethanol as gasoline replacements. Higher alcohols (C4 and C5) have an energy density that is closer to gasoline than that o f ethanol, are not hygi-oscopic (preventing tank and engine corrosion) and, moreover, are less volatile than ethanol. With the exception o f w-butanol and isobutanol, these higher alcohols are only produced in trace amounts by microorganisms. Efforts have been made to divert the 2-oxo acid intermediates o f amino acid metabolism to the formation o f biofuels through engineered Ehrtich pathways. This approach is made extremely attractive by the fact that the relevant 2-oxo acids are natural intermediates o f bacterial amino acid catabolism. Building a functional pathway would then only require the introduction o f two steps: decarboxylation and reduction. Atsumi et al coexpressed the 2-oxo acid decarboxylase f r o m

Lactococciis lactis and the alcohol dehydrogenase ADH2 gene fi-om S. cerevisiae

in E. coli, resulting in the production o f a wide variety o f higher alcohols including /7-propanol, isobutanol and phenylethanol (Atsumi et al, 2008). A main drawback in this strategy was the production o f an alcohol mixture rather than a single alcohol, which is due to the broad substrate specificity o f the heterologous enzymes expressed. Further improvements were achieved by overexpressing the pathway for the synthesis o f the isobutanol precursor

I III 10 dl I cüo II

2-ketoisovalerate and by deleting genes involved in competing pathways. A resulting engineered E. coli strain was able to produce more than 20 g/1 o f isobutanol (Peralta-Yahya & Keasling, 2010). Other hosts such as Bacillus

siibtilis and Coiynebacterium glulamiciim were also used to produce isobutanol

up to 2.6 g/l and 4.9 g/l, respectively (Brat et al, 2012a).

One o f t h e major challenges in microbial production o f isobutanol is the low tolerance o f many microorganisms to the products as well as to inhibitors present in industrial substrates. S. cerevisiae is known for its higher tolerance to ethanol and inhibitors. Moreover, this yeast can withstand low pH, thereby limiting the risk o f bacterial contamination, and is not susceptible to phages. For these reasons, many scientists have sought to explore the potential o f S.

cerevisiae for isobutanol production. Several studies focused on the

overexpression o f valine biosynthetic genes (Chen el al, 2011), all o f which are located in the mitochondria. Synthesis of valine and conversion into isobutanol then occur in two separate compartments. Brat D. el al (Brat el al, 2012b) reported improved yields by relocating the entire pathway to the cytosol, illustrating the relevance o f including metabolic compartmentation into metabolic engineering strategies.

n-Butanol is another higher alcohol with interesting fuel properties that could derive f r o m an Ehrlich-like pathway. Its production in microorganisms was initially reported in Closlridia and relies on a CoA-dependent non-Ehrlich pathway (Weizmann & Rosenfeld, 1937). The C. acetobiitilicitm butanol pathway was cloned into S. cerevisiae but resulted in very low product yields (Steen et al, 2008). Production o f butanol from the non proteogenic amino acid norvaline was previously reported and indicates that the machinery for this bioconversion is present in yeast (Ingraham et al, 1961). A major challenge in exploiting this capacity is to increase the availability o f norvaline, or the transaminated product 2-oxo pentanoate, in S. cerevisiae. A first step was recently made by P. Branduardi et al (Branduardi et al, 2013). They introduced the glycine oxidase goxB gene f r o m B. siibtilis, allowing the conversion o f glycine into glyoxylate. Glyoxylate was further transformed into p-ethylmalate and finally 2-oxo pentanoate by Mis I and Leu2 respectively. Although 2-oxo pentanoate is not a known intermediate o f the yeast metabolism, it was decarboxylated by the pyruvate decarboxylase Pdcl, yielding butyraldehyde that was subsequendy reduced to the final desired product n-butanol. This approach resulted in up to 92 and 58 mg/l o f butanol and isobutanol, respectively. It is important to note that the decarboxylation step plays a key role in all the above

Chapter 1

mentioned strategies. This is not surprising, since it represents the only irreversible reaction in the Ehj-lich pathway. Its substrate specificity and activity therefore have a major impact on the array o f higher alcohols and their concentrations obtained during a fermentation process.

Phenylethanol is another veiy interesting compound due to its rose-like aroma and it is used extensively in the cosmetic and perfume industiy. Yeasts produce phenylethanol as a product o f phenylalanine degradation and many studies have been focused on the screening o f several yeast species in order to identify the best candidate for an industrial process. On the other hand the conversion o f phenylalanine is not optimal and therefore it would be preferable to convert a cheaper substrate such as glucose into phenylethanol. This was achieved by deregulation o f the aromatic amino acids biosynthetic pathway, via deletion o f the AR03 gene and a point mutation in the AR04 gene, which resulted in phenylethanol and tyrosol synthesis directly f r o m glucose (Luttik et

ai, 2008). This approach provided consequently the precursor for the aromatic

alcohols biosynthesis but the catabolic pathway is still repressed by the presence o f ammonium in the medium. Another disadvantage is the accumulation o f a mixture o f alcohols rather than the pure product. Both these aspects are addressed in Chapter 5.

Scope of this thesis

As discussed above, the Ehrlich pathway already plays a key role in many food and beverage fermentation processes and is intensively explored in metabolic engineering strategies for the production o f biofuels and fine chemicals. Currently, S. cerevisiae represents the most readily accessible and relevant microorganism to study and engineer the Ehriich pathway due to the availability o f powerful genetics and genomics tools as well as its immediate connection to the food and biofuels industries. Despite its crucial role in many biotechnological processes, several aspects o f the Ehrlich pathway in S. cerevisiae are not completely understood. The relevance o f t h e different TPP-dependent 2-oxo acid decarboxylase (iso) enzymes for fusel alcohols production in S. cerevisiae has been an argument o f debate for decades (Dickinson et al., 1998; Dickinson et al, 2000; Dickinson el al, 2003). In Chapter 2 we aimed to clarify the contribution o f t h e single enzymes via the systematic study o f their substrate specificity. To prevent the overlapping o f activities belonging to the different isoenzymes often reported in previous works, we constructed a platform strain lacking all five (putative) 2-oxo acid decarboxylase genes. In this 'decarboxylase-negative'

hitrodiictioii

strain, eacli o f tliese five genes was then individually overexpressed. We performed chemostat cultures to ensure the maximum reproducibility and tested the activity o f cell extracts o f the resulting strains with a wide a m y o f aromatic, branched-chain, sulfur-containing and linear-chain 2-oxo acids. The methodologies developed and the results acquired in Chapter 2 were directly applied to get more insight into flavour formation in the brewing yeast

Saccharomycespastorianus. Saccharomyces pastorianus is a hybrid yeast, whose

genome consists o f subgenomes highly similar to those o f S. cerevisiae and S.

eiibayaniis. The impact o f the two genomes on flavour production and its

regulation is poorly understood and in Chapter 3 the two alleles o f AROIO, encoding a broad substrate decarboxylase, derived from the S. cerevisiae and S.

euhayaims were characterized.

Another research line focused on the utilization o f the Ehrlich pathway for the production o f phenylethanol. Phenylethanol is the most used chemical in the perfume and cosmetic industry but extraction from plants is costly and inefficient. In Chapter 4 a screening strategy was applied to identify deletions that would lead to a deregulation o f the Ehrlich pathway. In order to increase the production o f phenylethanol the identified mutation was combined with mutations that were reported to boost the aromatic amino acid biosynthesis. This PhD project on amino acid catabolism was performed in the Kluyver Centre for Genomics o f Industrial Femientation, a Netherlands-based public-private research consortium with an international industrial platform. This project attracted the attention o f one o f the Kluyver Centre's industrial partners, who expressed an active interest in characterizing the arginine degradation pathway in

Kluyveromyces lactis, a non-Saccharomyces yeast relevant for the food and feed

industiy. Since their interest was well aligned with the research in this PhD project, it was decided to take this topic on board. Arginine is one o f the most abundant amino acids in environments where many yeasts belonging to the

Saccharomyces gi'oup can be found (wood bark, wine and beer fermentation). In S. cerevisiae, the arginine catabolism has been extensively studied, but little is

known about arginine metabolism by other yeasts. Chapter 5 describes a genomics, systems-biology and molecular genetics-based analysis o f pathways for arginine metabolism in K. lactis, culminating in the identification o f an Ehrlich-type pathway for arginine metabolism that is both new to and potentially widespread among fungi.

Chapter 1

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