The yeast Kluyveromyces marxianus and its ... - It works!

Appl Microbiol Biotechnol (2008) 79:339–354 

DOI 10.1007/s00253-008-1458-6 

MINI-REVIEW 

The yeast Kluyveromyces marxianus and its 

biotechnological potential 

Gustavo Graciano Fonseca & Elmar Heinzle & 

Christoph Wittmann & Andreas K. Gombert 

Received: 28 January 2008 /Revised: 12 March 2008 /Accepted: 13 March 2008 /Published online: 22 April 2008 

# Springer-Verlag 2008 

Abstract Strains belonging to the yeast species Kluyveromyces 

marxianus have been isolated from a great variety of habitats, 

which results in a high metabolic diversity and a substantial 

degree of intraspecific polymorphism. As a consequence, 

several different biotechnological applications have been 

investigated with this yeast: production of enzymes (βgalactosidase, 

β-glucosidase, inulinase, and polygalacturonases, 

among others), of single-cell protein, of aroma 

compounds, and of ethanol (including high-temperature and 

simultaneous saccharification-fermentation processes); reduction 

of lactose content in food products; production of 

bioingredients from cheese-whey; bioremediation; as an 

anticholesterolemic agent; and as a host for heterologous 

protein production. Compared to its congener and model 

organism, Kluyveromyces lactis, the accumulated knowledge 

on K. marxianus is much smaller and spread over a number of 

different strains. Although there is no publicly available 

genome sequence for this species, 20% of the CBS 712 strain 

genome was randomly sequenced (Llorente et al. in FEBS Lett 

487:71–75, 2000). In spite of these facts, K. marxianus can 

envisage a great biotechnological future because of some of its 

qualities, such as a broad substrate spectrum, thermotolerance, 

high growth rates, and less tendency to ferment when exposed 

G. G. Fonseca : A. K. Gombert 

Department of Chemical Engineering, University of São Paulo, 

São Paulo, São Paulo, Brazil 

G. G. Fonseca : E. Heinzle : C. Wittmann 

Biochemical Engineering Institute, Saarland University, 

Saarbrücken, Germany 

A. K. Gombert (*) 

Department of Biotechnology, Delft University of Technology, 

Julianalaan 67, 

2628BC Delft, The Netherlands 

e-mail: andreas.gombert@poli.usp.br 

to sugar excess, when compared to K. lactis. To increase our 

knowledge on the biology of this species and to enable the 

potential applications to be converted into industrial practice, a 

more systematic approach, including the careful choice of (a) 

reference strain(s) by the scientific community, would certainly 

be of great value. 

Keywords Kluyveromyces marxianus . 

Yeast biotechnology . Yeast physiology . Yeast taxonomy 

Taxonomic history of the present species Kluyveromyces 

marxianus 

Kluyveromyces marxianus was first described in 1888 by E. 

C. Hansen, which at that time was named Saccharomyces 

marxianus after Marx, the person who originally isolated 

this yeast from grapes (Lodder and Kreger-van Rij 1952). 

In their monograph, Lodder and Kreger-van Rij (1952) 

describe ten strains of S. marxianus, among which a 

particular strain labeled Zygosaccharomyces marxianus, 

which had been deposited at the Centraalbureau voor 

Schimmelcultures (CBS) in 1922 by H. Schnegg, was 

arbitrarily chosen as the type strain. This corresponds to the 

present CBS 712 strain. Some differences among the ten 

mentioned strains were already pointed out at that time, 

regarding the formation of pseudomycelium, and the 

capacities of assimilating and fermenting lactose. Rotting 

leaves of sisal, sewage of a sugar factory, and “Lufthefe” 

(aerated yeast) are other habitats from which the strains of 

S. marxianus had been isolated. Already in 1939, Sacchetti 

had observed that inulin is fermented by S. marxianus 

(Lodder and Kreger-van Rij 1952). Although it was already 

recognized at that time that S. marxianus and Saccharomyces 

fragilis, which had been isolated from kefir in 1909 by

340 Appl Microbiol Biotechnol (2008) 79:339–354 

Jörgensen, were very closely related, they were considered 

distinct species (Lodder and Kreger-van Rij 1952). In 1951, 

Luh and Phaff affirmed that S. fragilis “is the only yeast 

species capable of attacking pectin” (Lodder and Kreger-van Rij 

1952). Yoghurt, soft cheese, a lung with tuberculosis, Koumiss 

(a beverage made of fermented mare’s milk), a human lesion 

of tonsils and the pharynx, and feces are other habitats from 

which the 11 strains belonging to the S. fragilis species had 

been obtained from (Lodder and Kreger-van Rij 1952). 

Mainly due to differences in spore and ascus morphology, 

in the capacity of fermenting and oxidizing different 

sugars, and in the occurrence of hybridization between 

strains, when compared to true Saccharomyces yeasts, there 

was a need to reclassify the former species S. fragilis and S. 

marxianus, besides Saccharomyces lactis, into a new taxon 

(van der Walt 1970). In 1956, van der Walt described the 

new genus Kluyveromyces, the type species of which was 

Kluyveromyces polysporus (van der Walt 1956). Later, it 

was found that the latter yeast had very similar properties to 

the three above-mentioned species, and consequently, they 

were all reclassified into the genus Kluyveromyces, which 

encompassed 18 species in the second edition of The 

Yeasts, a taxonomic study (Lodder 1970). Additional 

habitats from which strains had been isolated include Bantu 

Beer, milk of a mastitic cow, asthmatic expectoration, and 

maize meal (van der Walt 1970). Again, Kluyveromyces 

fragilis was considered closely related but still separate 

from K. marxianus, mainly due to the former’s high 

capacity of fermenting lactose. Dairy products and human 

and animal lesions were the prevalent origin of strains in 

the K. fragilis taxon (van der Walt 1970). 

In the third edition of The Yeasts, a taxonomic study 

(Kreger-van Rij 1984), the genus Kluyveromyces was 

divided into 11 species. On the basis of interfertility, the 

taxon K. marxianus was organized into seven varieties, 

which are able to readily hybridize (van der Walt and 

Johannsen 1984). Concomitantly, the former species K. 

fragilis and K. lactis disappeared. 

In the most recent edition of The Yeasts, a taxonomic study 

(Kurtzman and Fell 1998), the chapter on the Kluyveromyces 

genus includes 15 species. The seven varieties within the K. 

marxianus species, proposed in the previous edition of the 

monograph, were eliminated by considering them as either 

independent species (e.g., K. lactis and K. dobzhanskii) or 

synonyms of K. lactis or K. marxianus (Lachance 1998).This 

is due to the examination of the genetic structure of 

populations, in combination with hybridization ability, as 

criteria for classification. Consequently, the former species or 

varieties Kluyveromyces bulgaricus, K. cicerisporus, K. 

fragilis, andK. wikenii could not be considered distinct from 

K. marxianus (Lachance 1998). 

Since the biological concept of species cannot be applied 

to homothallic organisms, such as the majority of yeasts in 

the Kluyveromyces taxon, any classification is always based 

on arbitrary criteria, which have changed along time, as 

discussed above. Since the development of rapid and 

efficient gene sequencing tools, it became natural to utilize 

gene sequences as the criterion for the comparison and 

classification of microorganisms into the different taxa. 

Rather than performing single gene comparisons, the most 

recent reports on the taxonomy of Kluyveromyces yeasts 

employ multigene sequence analyses for elucidating the 

phylogeny of the different strains. Using this strategy, 

Kurtzman and Robnett (2003) showed that the species 

described by Lachance (1998) in the Kluyveromyces genus 

are actually distributed into six clades, indicating the 

polyphyly of this group of yeasts. This is mainly due to 

the previous criteria employed in classification, such as 

ascus morphology (in this particular case, ascus deliquescence), 

which are inadequate as phylogenetic descriptors 

(Kurtzman 2003; Kurtzman and Robnett 2003). It has been 

proposed that genera should be circumscribed according to 

the phylogenetically defined clades, rather than on phenotypic 

analyses (Kurtzman and Robnett 2003). As a result of 

this, the number of species in the Kluyveromyces genus 

decreased to six and the species K. marxianus is proposed 

as the conserved type species (Kurtzman 2003; Lachance 

2007). The type species of the originally described 

Kluyveromyces genus (van der Walt 1956), namely, K. 

polysporus, has been reclassified into the newly proposed 

Vanderwaltozyma genus (Kurtzman 2003; Lachance 2007). 

Biochemistry, metabolism, and physiology 

It should be noted that the great majority of studies 

published on K. marxianus have not aimed at looking into 

its biochemistry, metabolism, or physiology. Most of the 

works that are publicly available explored potential applications 

of this organism (see “Biotechnological applications”), 

without investigating what takes place at the 

intracellular level. Typically, the yeast cells have been 

cultivated on a specific substrate, and measurements have 

been carried out in such a way that only the concentrations 

of a substrate and of a product, besides the cell concentration, 

are determined. In what concerns physiology, carbon 

balances are very rarely looked at, meaning that it is only 

possible to have a rough macroscopic picture of the cellular 

reactions and, hence, of the organism’s physiology. 

Since the 1970’s, a number of studies has been published 

on biochemical and metabolic aspects of different K. 

marxianus strains (a summary is presented in Table 1). 

Some studies were actually aimed at identifying suitable 

classification methods for K. marxianus, which has always 

been a challenging task. These include the observation that, 

in contrast to S. cerevisiae, ergosterol is the only sterol

Appl Microbiol Biotechnol (2008) 79:339–354 341 

Table 1 Compilation of biochemical and metabolic studies performed with the yeast K. marxianus 

Target of the study Strain employed Reference 

Sterol composition K. fragilis NCYC 100 Penman and Duffus (1974) 

Subcellular localization of the enzyme 

alcohol dehydrogenase (ADH) 

K. fragilis H32 Künkel and May (1976) 

Measurement of heat evolution by 

microcalorimetry 

K. fragilis NCYC 100 Beezer et al. (1979) 

Characterization of the enzyme fructose- 

1,6-bisphosphatase 

K. fragilis ATCC 10022 Toyoda and Sy (1984) 

Lactose symporter K. marxianus CBS 397; K. 

Van den Broek et al. (1987); 

marxianus IGC 2902, CBS 712, 

Carvalho-Silva and Spencer- 

IGC 2587, IGC 2671, IGC 3014, 

NRRL Y-1122, CBS 397 

Martins (1990) 

High- and low-affinity glucose transporters K. marxianus IGC 2587 Gasnier (1987) 

Regulation of four transport systems 

identified during the exponential and 

the stationary phases of batch growth 

on glucose 

K. marxianus CBS 397 De Bruijne et al. (1988) 

High- and low-affinity symporters of 

glucose and fructose 

K. marxianus CBS 6556 Postma and Van den Broek (1990) 

Proton-motive force-driven transport 

of galactose 

K. marxianus CBS 397 van Leeuwen et al. (1991) 

Transport of lactic acid K. marxianus IGC 3014 Fonseca et al. (1991) 

Mechanism of the enzyme UDP 

K. fragilis ATCC 10022 (presumed, 

Mukherji and Bhaduri (1992); 

glucose 4-epimerase 

from one of the articles) 

Bhattacharjee and Bhaduri (1992); 

Ray et al. (1995); Majumdar 

et al. (1998) 

Absence of complex I NADH 

ubiquinone oxidoreductase 

K. marxianus K5 (own collection) Büschges et al. (1994) 

Regulation of adenylate cyclase 

by Ras proteins 

K. marxianus CBS 5795W Verzotti et al. (1994) 

Coenzyme Q system and the 

monosaccharide pattern of cell wall 

K. marxianus (various strains) Molnár et al. (1996) 

Behaviour of K. marxianus during 

autolysis 

K. marxianus CBS 397 Amrane and Prigent (1996) 

Presence of killer activity K. marxianus (isolated as a 

result of the work) 

Abranches et al. (1997) 

Glucose repression via Mig1p K. marxianus SGE11 (Montpellier 

University) 

Cassart et al. (1997) 

composition of the cell wall K. marxianus R157, 1586 

(University of New South Wales) 

Nguyen et al. (1998) 

Presence of active efflux pumps 

involved in drug resistance 

K. marxianus IGC 2671 Prudêncio et al. (2000) 

Transport of malic acid via a 

K. marxianus ATCC 10022, KMS3 

Queirós et al. (1998) 

symport mechanism 

(derivative of CBS 6556) 

Identification and characterization 

K. marxianus Y-610 (identical to 

Yoda et al. (2000) 

of a cell-wall acid phosphatase 

ATCC 12424) 

Capacity of using xenobiotic 

K. fragilis UU1; K. marxianus IMB3 Ternan and McMullan (2000); 

compounds as nitrogen source 

Ternan and McMullan (2002) 

Response of the NADP+-dependent 

glutamate dehydrogenase to 

nitrogen repression 

K. marxianus CBS 6556 de Morais (2003) 

Characterization of an amine oxidase K. marxianus CBS 5795 Corpillo et al. 2003 

The transport mechanism of xylose K. marxianus ATCC 52486 Stambuk et al. 2003 

This list does not include enzymes of industrial interest, which are separately listed in Table 2.


present in K. marxianus; the use of microcalorimetry for 

identification purposes; and the characterization of coenzyme 

Q and monosaccharide patterns of cell walls (Table 1). 

In other cases, K. marxianus wassimplyusedasthe 

source of specific compounds, which were the actual focus of 

research (mainly enzymes). Examples of this kind of study 

include those on fructose-1,6-bisphosphatase, uridine diphosphate 

(UDP) glucose-4-epimerase, an acid phosphatase, an 

amine oxidase, protein phosphatases, carboxypeptidases, and 

aminopeptidases (Tables 1 and 2). A number of transport 

studies were also carried out, namely, on the transport of 

sugars (lactose, glucose, fructose, galactose, and xylose) and 

organic acids (lactic and malic acids) (Table 1). Finally, a few 

metabolic studies were carried out with the aim of 

characterizing some non-transport aspects of K. marxianus, 

such as the absence of complex I in the respiratory chain, the 

regulation of adenilate cyclase by Ras proteins, the functional 

characterization of Mig1p (involved in glucose repression), 

analysis of cell-wall composition, presence of efflux pumps 

and their role in drug resistance, and the regulation of the 

nicotinamide adenine dinucleotide phosphate (oxidized form) 

(NADP + )-dependent glutamate dehydrogenase (Table 1). In 

many cases, these studies were performed in parallel with 

other yeasts, mainly with S. cerevisiae. 

In terms of biochemical studies on enzymes that have 

industrial interest, K. marxianus has been used as a source 

of inulinase, β-galactosidase, β-glucosidase, and endopolygalacturonases 

(Table 2). Besides these, some less widespread 

enzymes with potential industrial application, such as 

protein phosphatases, carboxypeptidases, and aminopeptidases 

have also been investigated more recently (Table 2). 

In the 1970s, physiological studies focusing on the 

influence of some common environmental factors on the 

growth of K. marxianus started to appear in the literature, 

as a reflection of the eventual interest in using this yeast for 

industrial applications. Chassang-Douillet et al. (1973) 

presented the first clear physiological comparison of K. 

marxianus and S. cerevisiae, carried out using synthetic 

media and demonstrating that the so-called glucose effect 

was absent in K. marxianus, as opposed to S. cerevisiae. 

Later studies reported on the effects of pH (de Sánchez and 

Castillo 1980), ethanol concentration (Bajpai and Margaritis 

1982), and sugar concentration (Margaritis and Bajpai 1983) 

on the growth kinetics of K. marxianus. Importantly, K. 

marxianus started to be included in comparative biochemical 

and physiological studies on yeast in general, such as those 

related to catabolite repression (Eraso and Gancedo 1984), 

sensitivity toward toxins (Sukroongreung et al. 1984), and 

growth inhibition by fatty acids (Viegas et al. 1989). 

The use of defined synthetic media combined with chemostat 

cultivations for quantitative physiological studies started 

around the 1990s, with works focusing on the regulation of 

respiration and fermentation and on the so-called Crabtreeeffect 

in yeasts (van Urk et al. 1990; Verduyn et al. 1992). It 

was then quantitatively shown that K. marxianus presents a 

strong Crabtree-negative character, since no ethanol production 

was observed after a glucose pulse applied to respiring 

cells, in contrast to what is commonly observed with S. 

Table 2 Studies on the biochemistry of enzymes of industrial interest performed with the yeast K. marxianus 

Enzyme Application Strain Reference 

Inulinase Production of fructose 

syrup from inulin-containing 

feed-stocks 

β-galactosidase Reduction of lactose 

content in foods 

K. fragilis ATCC 12424; 

K. marxianus CBS 6397, 

CBS 6556 

K. fragilis (several strains); 

K. marxianus NCYC 111; 

K. marxianus ATCC 10022; 

K. marxianus IMB3; K. 

marxianus CBS 6556 

Workman and Day (1984); 

Rouwenhorst et al. (1988, 1990a,b) 

Mahoney et al. (1975); Gonçalves and 

Castillo (1982); Bacci Júnior et al. 

(1996); Brady et al. (1995); Martins 

et al. (2002) 

β-glucosidase Hydrolysis of cellulosic materials K. fragilis ATCC 12424 Raynal and Guerineau (1984); 

Leclerc et al. (1987) 

Endopolygalacturonases Reduce of viscosity in fruit K. marxianus CCT 3172; CCT Jia and Wheals (2000) 

processing products 

3172 (overproducing mutant); 

an unidentified NCYC isolate 

Protein phosphatases modification of cheese-making 

qualities of caseins 

K. marxianus (strain not indicated) Jolivet et al. (2001) 

Carboxypeptidases reduction of bitter taste in proteincontaining 

foods 

K. marxianus (own isolate) Ramírez-Zavala et al. (2004b) 

Aminopeptidases direct processing or aging of dairy 

and meat products 

K. marxianus (own isolate) Ramírez-Zavala et al. (2004a) 

Only studies which focused on the biochemistry of enzymes are indicated here. For studies aiming at enzyme production, please refer to the 

section on “Biotechnologial applications”


cerevisiae andevenwithK. lactis, to a lesser extent (Kiers et 

al. 1998). This was later confirmed by Bellaver et al. (2004). 

Castrillo and Ugalde (1993) showed that when oxidoreductive 

metabolism sets in in K. marxianus, as a function of 

increasing glycolytic flux, the maximum respiratory capacity 

of the cells has not yet been achieved, which is in contrast 

with the situation in S. cerevisiae, in which the onset of 

respirofermentative metabolism coincides with the achievement 

of its maximum respiratory capacity. From these studies, 

K. marxianus was classified as facultatively fermentative and 

Crabtree-negative (van Dijken et al. 1993). It is important to 

note that it cannot grow under strictly anaerobic conditions 

and that the occurrence of ethanol formation is almost 

exclusively linked to oxygen limitation (Visser et al. 1990; 

van Dijken et al. 1993; Bellaver et al. 2004). More recently, 

Blank et al. (2005) showedthatK. marxianus presents the 

highest tricarboxylic acid cycle flux during batch growth on 

glucose among the 14 hemiascomycetous yeasts studied 

within the Génolevures consortium (Souciet et al. 2000). 

Other physiological studies report on various issues, 

such as flocculation (Fernandes et al. 1992, 1993), the 

Table 3 Biotechnologically relevant genes sequenced in K. marxianus 

influence of CO2 on the survival of K. marxianus 

(Isenschmid et al. 1995), the influence of the specific 

growth rate on the morphology of the NRRLy2415 strain, 

which displays significant growth in pseudo-hyphal form 

(O’Shea and Walsh 2000), the effects of increased air 

pressure on the biomass yield of K. marxianus (Pinheiro et 

al. 2000), the response of K. marxianus to oxidative agents 

such as hydrogen peroxide (Pinheiro et al. 2002), and the 

macromolecular composition of K. marxianus cells as a 

function of the specific growth rate (Fonseca et al. 2007). 

The later data can be particularly useful for metabolic flux 

analysis studies. 

One important aspect on the physiology of K. marxianus 

is the fact that significantly different growth parameters, 

such as μmax and Yx/s, have been reported not only for 

different strains within the species but also for the same 

strain when investigated in different laboratories (Fonseca 

et al. 2007). 

From the data in Tables 1 and 2 (and also in Table 3), it 

can be observed that the number of strains that have been 

investigated is quite large, and many of them were not 

Gene/function Strain Reference 

β-glucosidase K. fragilis ATCC 12424 Raynal et al. (1987) 

INU1/inulinase K. marxianus ATCC 12424 Laloux et al. (1991) 

LEU2/β-isopropylmalate dehydrogenase K. marxianus CBS 6556 Bergkamp et al. (1991) 

URA3/orotidine-5′-phosphate decarboxylase K. marxianus CBS 6556 Bergkamp et al. (1993b) 

PDC/pyruvate decarboxylase K. marxianus ATCC 10606 Holloway and Subden (1993) 

ADH1/alcohol dehydrogenase K. marxianus ATCC 12424 Ladrière et al. (1993) 

ABF1/a DNA binding protein K. marxianus (strain not indicated) Oberyé et al. (1993) 

the GAP family/glyceraldehyde-3-phosphate 

dehydrogenases 

K. marxianus ATCC 10022 Fernandes et al. (1995) 

LAC4/β-galactosidase K. fragilis (strains not indicated) Huo and Li (1995) 

MIG1/DNA-binding protein involved in 

glucose repression 

K. marxianus SGE11 (Montpellier University) Cassart et al. (1997) 

EPG1/endopolygalacturonase K. marxianus BKM Y-719 Šiekštelė et al. (1999) 

PCPL3/purine-cytosine permease K. marxianus ATCC 12424 Ball et al. (1999) 

ADH2/alcohol dehydrogenase K. marxianus ATCC 12424 Ladrière et al. (2000) 

17% of the genome (1,300 genes by a 

partial random strategy) 

K. marxianus CBS 712 Llorente et al. (2000) 

Inulinase K. marxianus (strain not indicated) GenBank AF178979 

URA3/orotidine-5′-phosphate decarboxylase K. cicerisporus CBS 4857 Zhang et al. (2003) 

QOR/NADPH quinone oxidoreductase K. marxianus KCTC 7155 Kim et al. (2003) 

URA 9/dihydroorotate dehydrogenase 2 K. marxianus NRRL Y-8281 GenBank AY444339 

HIS3/imidazoleglycerol-phosphate dehydratase K. cicerisporus CBS 4857 GenBank AY303539 

OYE/old yellow enzyme C. macedoniensis AKU 4588 Kataoka et al. (2004) 

EPG1–2/endopolygalacturonase K. marxianus CECT 1043 GenBank AY426825 

FPS1/plasma membrane glycerol channel K. marxianus IGC 3886 Neves et al. (2004) 

β-galactosidase K. marxianus (strain not indicated) GenBank AY526090 

Exoinulinase K. marxianus IW 9801 GenBank AY649443 

TPI1/triosephosphate isomerase K. marxianus (strain not indicated) GenBank AJ577476 

Partial gene sequences deposited in public databases and sequences coding for RNA were not included. GenBank accession numbers were only 

given when there is no available article to be cited.


obtained directly from the main culture collections worldwide. 

If, on the one hand, this leads to an interesting 

metabolic diversity and to several potential applications, as 

described below in this review, it makes it difficult, on the 

other hand, to gain fundamental knowledge on the 

metabolism and physiology of this yeast. In this sense, it 

would be necessary that researchers started using a reduced 

number of strains (chosen from key culture collections), 

similarly to the way in which the K. lactis community has 

been using the CBS 2359 strain (Lachance 1998; Fukuhara 

2006). This would allow the development of efficient 

molecular genetic tools for K. marxianus (probably starting 

with genome sequencing), which are the basis for 

performing systematic studies that will finally lead to a 

better understanding of the biology of this species. A 

possibility would be to choose one or two strains with 

characteristics that have given K. marxianus a clear 

advantage over other yeasts: thermotolerance, high growth 

rate, absence of fermentative metabolism upon sugar 

excess, and a broad substrate spectrum. For making this 

choice, an approach as the one reported by van Dijken et al. 

(2000) could be followed. 

Recombinant DNA technology 

As with any (potential) industrial organism, rational genetic 

manipulation is one of the most efficient ways of 

optimizing process yield and/or productivity. In some cases, 

the application of recombinant DNA (rDNA) technology 

may even become a prerequisite for a successful industrial 

process, either to increase product titer and/or purity to 

levels at which the process becomes economically feasible 

or to render the producing host capable of synthesizing a 

heterologous compound. This kind of activity has been well 

known as metabolic engineering, which is now a consolidated 

discipline (Stephanopoulos et al. 1998). rDNA 

technology is also an invaluable technique for genetic and 

physiological studies, which in turn are essential for 

increasing our understanding of K. marxianus. 

Already more than two decades ago, transformation 

methods for inserting foreign DNA into K. marxianus have 

been developed. Das et al. (1984) constructed a plasmid 

called pGL2, containing the kanamycin resistance gene as a 

dominant selectable marker, and the KARS2 autonomously 

replicating sequence of K. lactis. They showed that the 

transformation method of intact cells with alkali cations, 

originally developed for Saccharomyces cerevisiae by Ito et 

al. (1983), also worked in the strain K. fragilis C21. 

However, the transformation efficiency was rather low. 

A breakthrough in molecular biology research of 

Kluyveromyces yeasts was the discovery of the pKD1 

plasmid in the species Kluyveromyces drosophilarum 

(Falcone et al. 1986). The 4.8-kb, 1.65 μm pKD1 plasmid 

proved to have a similar organization but different 

sequences and host specificities, when compared to other 

already known plasmids, such as the 2 μ plasmid of 

Saccharomyces yeasts (Chen et al. 1986). In contrast to the 

latter, pKD1 can be maintained stable in K. lactis, but not in 

S. cerevisiae, in the absence of selective pressure (Bianchi 

et al. 1987). Later, it was shown that the insertion of the 

kanamycin resistance gene, the URA3 gene of S. cerevisiae, 

a replication origin for E. coli, and the ampicillin resistance 

gene into pKD1 rendered a shuttle plasmid that could be 

transformed and maintained in K. marxianus strains CBS 

6556 and CBS 712, though still with low-transformation 

efficiencies (Chen et al. 1989). This is in accordance with 

the fact that ARS and centromere sequences of K. lactis 

work in K. marxianus and vice versa (Das et al. 1984; 

Iborra and Ball 1994). Thus, pKD1-based plasmids have 

become the most common choice for inserting foreign 

DNA sequences into K. marxianus (Bergkamp et al. 1993b; 

Bartkevičiute et al. 2000; Zhang et al. 2003). 

Iborra (1993) reported for the first time transformation 

efficiencies in the order of hundreds to thousands of 

transformants per microgram of DNA with K. marxianus, 

either with the lithium method (Ito et al. 1983) or using 

electroporation (Meilhoc et al. 1990). Similar results were 

obtained more recently by Zhang et al. (2003). 

Besides requiring efficient vectors and transformation 

protocols, foreign gene expression also depends on the 

promoter and eventually a signal sequence for directing the 

synthesized protein into the extracellular environment, 

which usually facilitates downstream operations. For this 

purpose, Bergkamp et al. (1993a) used the promoter and 

prepro-signal sequence of the INU1 (inulinase) gene to 

successfully direct heterologous expression and secretion of 

α-galactosidase in K. marxianus, with dramatically higher 

efficiencies when compared to the use of classical S. 

cerevisiae promoters, such as PGK. 

With the INU1 promoter, heterologous gene expression 

can be fine-tuned by choosing the appropriate carbon 

source. Another regulated promoter that was successfully 

used in K. marxianus is the tetracycline repressible 

promoter (Pecota and da Silva 2005). 

Strong, constitutive promoters for driving heterologous 

gene expression have also been described, such as that of a 

purine-cytosine permease gene (Ball et al. 1999). Instead of 

fine-tuning foreign gene expression according to promoter 

strength or induction properties, Pecota et al. (2007) 

developed an insertion cassette that enables multicopy 

integration of a precise number of gene copies into K. 

marxianus with recycling of the selection marker. 

Auxotrophic mutants of K. marxianus, for their use in 

transformation experiments, have been reported for leucine, 

uracil, histidine, or triptophane requirement (Bergkamp et


al. 1991, 1993b; Basabe et al. 1996; Hong et al. 2007). 

Dominant markers applicable for the selection of K. 

marxianus transformants include at least the kanamycin, 

the aureobasidin A, and the nurseothricin resistance genes 

(Das et al. 1984; Hashida-Okado et al. 1998; Goldstein and 

McCusker 1999; Steensma and Ter Linde 2001; Ribeiro et 

al. 2007). Recycling of the marker gene for multiple gene 

disruptions can be performed in K. marxianus in the same 

way as in S. cerevisiae and K. lactis with the Cre-loxP 

system (Güldener et al. 1996; Ribeiro et al. 2007). 

A number of genes have been cloned and sequenced in 

different K. marxianus strains, and the most relevant ones 

are indicated in Table 3. 

Biotechnological applications 

When evaluating the yeast K. marxianus for biotechnological 

applications, it is impossible not to consider other most 

popular species, mainly S. cerevisiae and K. lactis. The 

former is probably the most employed biocatalyst in the 

biotechnological industry and a model organism in biological 

studies, whereas the latter has been chosen as a model 

Crabtree-negative, lactose-utilizing organism (Lachance 

1998; Fukuhara 2006). The fact that several K. marxianus 

strains have obtained the generally-regarded-as-safe 

(GRAS) status, similarly to S. cerevisiae and K. lactis 

(Hensing et al. 1995) indicates that this aspect does not 

impose any disadvantage for the former, when compared to 

the latter yeasts, in terms of process approval by regulatory 

agencies. The fact that K. lactis was chosen by the scientific 

community as the model organism in the Kluyveromyces 

genus, and not K. marxianus, led not only to a much better 

understanding of its physiology (for reviews, see e.g., 

Schaffrath and Breunig 2000; Wolf et al. 2003; Breunig and 

Steensma 2003 and the whole issue no. 3, vol. 6 of FEMS 

Yeast Research), and to the full sequencing of its genome 

(Dujon et al. 2004) but also to the development of several 

applications, including the expression of more than 40 

heterologous proteins (van Ooyen et al. 2006). This is due, 

to a great extent, to the fact that researchers have used, from 

the beginning, a very small number of K. lactis isolates 

(Fukuhara 2006), which has not been the case in the K. 

marxianus species. 

The development of biotechnological applications with 

K. marxianus has been motivated by a number of 

advantages it has when compared to K. lactis. These 

include at least the fact that it can grow on a broader 

variety of substrates and at higher temperatures, its higher 

specific growth rates, and the lesser tendency to produce 

ethanol it has when exposed to sugar excess (Rouwenhorst 

et al. 1988; Steensma et al. 1988; Bellaver et al. 2004; see 

also “Biochemistry, metabolism, and physiology”). 

One very important aspect of the ecology of K. marxianus 

should be taken into account when considering its biotechnological 

utilization: Individuals have been isolated from an 

enormous variety of habitats (see “Taxonomic history of the 

present species Kluyveromyces marxianus”). 

The obvious consequence is that the metabolic diversity 

is broad, and hence, potential biotechnological applications 

of K. marxianus strains are manifold. A summary of the 

most explored applications with this yeast follows. 

Although several yeasts have been reported for the 

production of aroma compounds, only a few of these can 

find industrial application due to their GRAS status 

(Medeiros et al. 2000, 2001). Kluyveromyces sp. produce 

aroma compounds such as fruit esters, carboxylic acids, 

ketones, furans, alcohols, monoterpene alcohols, and 

isoamyl acetate in liquid fermentation (Scharpf et al. 

1986; Fabre et al. 1995). Of all these compounds, 2-phenyl 

ethanol (2-PE), with rose petals aroma, is the most 

important commercially (Welsh et al. 1989; Leclercq-Perlat 

et al. 2004). Natural 2-PE has a high-value (approximately 

US $1,000 kg −1 ) serving a current world market of 

approximately 7,000 tons per annum (data from 1990; 

Etschmann et al. 2002). This alcohol presents sensorial 

characteristics that influence the quality of the wine, 

distilled drinks, or fermented foods. It is also found in 

fresh beer and is added to various industrial food products 

such as ice creams, bullets, non-alcoholic drinks, gelatines, 

puddings, and bubble gums (Wittmann et al. 2002). The 

influences of the carbon source (Fabre et al. 1998; 

Medeiros et al. 2000), aeration rate (Medeiros et al. 

2001), media composition (Etschmann et al. 2004), and 

cultivation conditions (Etschmann and Schrader 2006) on 

the aroma production using K. marxianus were studied. 

K. marxianus possesses the natural ability to excrete 

enzymes. This is a desired property for cost-efficient 

downstream processing of low- and medium-value enzymes 

(Hensing et al. 1994). The enzymes that hydrolyze pectic 

substances are known as pectic enzymes, pectinases, or 

pectinolytic enzymes (Wimborne and Rickard 1978). 

Pectinases are industrially used in the extraction and 

clarification of fruit juices (i.e., grape and apple; Schwan 

et al. 1997; Blanco et al. 1999). Other interesting 

applications are related to the maceration of vegetables, 

oil extraction, and formulation of animal feed using 

complex mixtures with cellulases to make the nutritional 

assimilation easier (Blanco et al. 1999). K. marxianus has 

considerable economic advantages over Aspergillus as an 

endo-PG source, even without genetic improvement of the 

strains (Harsa et al. 1993). Thus, the use of PGs from K. 

marxianus has attracted considerable interest (Garcia- 

Garibay et al. 1987b; Harsa et al. 1993; Donaghy and 

McKay 1994). Moreover, no other pectinolytic enzyme, 

besides PG, was reported to be secreted by K. marxianus


CCT 3172 in the culture media, which should facilitate the 

process to produce pure enzyme (Schwan et al. 1997). 

In K. marxianus, the pectinolytic enzymes are only 

produced during exponential growth, but almost all PG is 

secreted in the start of the stationary phase (Schwan and 

Rose 1994; Schwan et al. 1997; Serrat et al. 2004). Among 

the cultivation parameters, dissolved oxygen was reported 

to be the key in the production of both biomass and endo- 

PG (Wimborne and Rickard 1978; Garcia-Garibay et al. 

1987a). High rates and yields of biomass production require 

high oxygenation levels that, however, repress endo-PG 

induction (Wimborne and Rickard 1978). It was reported 

that K. marxianus exhibited pectolytic ability when it was 

grown without shaking and under anaerobic conditions, 

and no activity was found at high aeration rates (Barnby 

et al. 1990; Schwan and Rose 1994; García-Garibay 

et al. 1987a). 

The effect of temperature was assessed on both growth 

and endo-PG production in combination with the effect of 

dissolved oxygen (Schwan and Rose 1994). Temperature 

was also reported to have no direct effect on the synthesis 

of this enzyme but influenced the growth rate and had an 

indirect effect due to changes in oxygen solubility (Cruz- 

Guerrero et al. 1999). Addition of pectin in an aerobic 

culture in a fermenter was reported to derepress the 

production of the enzyme (Garcia-Garibay et al. 1987a). 

However, whereas some authors did not find any effect of 

pectin addition to the medium in an endo-PG producing 

strain (Schwan and Rose 1994; Schwan et al. 1997), others 

reported the enhancement of pectinase production by this 

yeast when pectin was added (Wimborne and Rickard 

1978; Lim et al. 1980; Cruz-Guerrero et al. 1999). K. 

marxianus CCT 3172 was able to break down pectin but 

required a usable source of carbon and energy to elaborate 

pectinolytic activity (Schwan and Rose 1994). It had a 

strong endo-PG activity between pH 4–6 with pH 5 as 

optimum (Schwan et al. 1997). Furthermore, the type of 

pectinase excreted by this strain was pointed as a feasible 

alternative to fungal production due to the lower broth 

viscosity, which can make downstream operations easier 

(Almeida et al. 2003a). 

Lactose-intolerance can be circumvented by removing 

lactose from the diet or by converting this sugar into 

glucose and galactose with β-D-galactosidase (Rajoka et al. 

2003). It was reported that only approximately 2% of the 

recognized yeast species are capable of fermenting lactose 

(Barnett et al. 1983), among which strains within the 

Kluyveromyces genus can be found. In a screening 

performed with yeast strains belonging to different genera, 

only two cultures of K. fragilis and Ferrissia fragilis 

showed β-galactosidase activity (Fiedurek and Szczodrak 

1994). Lactose is considered the primary inducer of β-Dgalactosidase 

synthesis (Furlan et al. 2000) and production 

of lactase by K. marxianus using cheese whey as a nutrient 

source has been investigated by several authors (Sonawat et 

al. 1981; Nunes et al. 1993). 

During the stationary phase of growth, the β-D-galactosidase 

activity of K. marxianus CBS 712 and CBS 6556 remained 

approximately constant (Rech et al. 1999). In contrast, a 

reduction of β-D-galactosidase activity was reported by other 

authors in the stationary phase (Mahoney et al. 1975). The 

highest β-D-galactosidase activity of K. marxianus CBS 712 

and CBS 6556 was reported to be at 37°C, decreasing quickly 

at temperatures above 40°C (Rech et al. 1999), while in K. 

marxianus IMB3, the enzyme is optimally active at 50°C 

(Barron et al. 1995a). On the other hand, the β-galactosidase 

of K. marxianus CBS 6556 is more stable than the 

corresponding enzymes of other strains, when stored at low 

temperatures, e.g., 4°C (Rech et al. 1999; Itohetal.1982; 

Brady et al. 1995). When different substrates were investigated 

for β-galactosidase production by K. marxianus, lactose 

supported the highest enzyme activities (Rajoka et al. 2003; 

Rajoka et al. 2004). 

Inulinase is an enzyme that cleaves fructose molecules 

from inulin. Its expression is induced by inulin or sucrose, 

and the enzyme can be excreted to the culture medium or 

remain associated to the cell wall (Rouwenhorst et al. 1988; 

Barranco-Florido et al. 2001). K. marxianus has been 

widely studied for inulinase production, aiming at the 

production of fructose syrup from inulin (Cruz-Guerrero et 

al. 1995). 

Pessoa and Vitolo (1999) obtained the highest inulinase 

activities with the K. marxianus DSM 70106 strain, using 

inulin as the carbon source. Growth of K. marxianus on 

sucrose also proceeds via the action of an extracellular 

inulinase (Hensing et al. 1994), which is repressed when 

growth is not sucrose-limited (Rouwenhorst et al. 1988; 

Parekh and Margaritis 1985; Grootwassink and Hewitt 1983). 

K. marxianus has also been proposed as a source of: (1) 

oligonucleotides, used as flavour enhancers in food products; 

(2) oligosaccharides, used as prebiotics; and (3) 

oligopeptides, immuno stimulators added to dairy products 

that are released in the wort after whey protein proteolysis 

(Belem et al. 1997; Belem and Lee 1998, 1999). Recent 

studies have shown the potential of K. marxianus FII 

510700 biomass as an alternative source to S. cerevisiae for 

yeast autolysates (Lukondeh et al. 2003a), alkali-insoluble 

glucans (Lukondeh et al. 2003c), and a natural bioemulsifier 

(Lukondeh et al. 2003b). 

In the field of bioremediation, processes using K. 

marxianus were developed for the removal of copper ions 

(II) with molasses as a nutrients source (Aksu and Dönmez 

2000). Lead (II) uptake by K. marxianus from contaminated 

molasses had negative effects on cell growth. Nevertheless, 

the decrease in biomass formation did not lead to decreased 

lead (II) uptake; on the contrary, the biosorption ability was


higher at higher initial lead (II) concentrations (Skountzou 

et al. 2003). 

Ethanol production at elevated temperatures has received 

much attention because of the potential cost savings, which 

could be obtained by continuous evaporation of ethanol 

from the broth under reduced pressure (Hacking et al. 1984; 

Gough et al. 1996, 1997, 1998; Banat et al. 1998). This 

topic was recently reviewed for yeasts in general, including 

K. marxianus. The advantages described, besides the 

energy savings due to reduced cooling costs, were higher 

saccharification and fermentation rates, continuous ethanol 

removal, and reduced contamination (Banat et al. 1998). 

However, the temperature increase has a negative effect on 

ethanol yield and also reduces the cell viability (Anderson 

et al. 1986; Ballesteros et al. 1991). K. marxianus was 

reported to produce alcohol at temperatures above 40°C and 

to have a maximum growth temperature of 47°C (Anderson 

et al. 1986), 49°C (Hughes et al. 1984), or even 52°C 

(Banat et al. 1992). Lower ethanol tolerance was observed 

when K. marxianus was compared to S. cerevisiae, and this 

was correlated with the activity of the plasma membrane 

ATPase (Rosa and Sa-Correia 1992; Fernanda and Sa- 

Correia 1992). Hacking et al. (1984) screened yeast strains 

for their ability to ferment glucose to ethanol at high 

temperatures. The tolerance of all species seemed to 

decrease with temperature, but in general, Kluyveromyces 

strains were more thermotolerant than Saccharomyces, 

which in turn can produce higher ethanol yields. Anderson 

et al. (1986) compared K. marxianus strains isolated from 

sugar mills and CBS strains for ethanol production at high 

temperatures. The CBS strains produced the same ethanol 

amounts as the new isolates but with lower cell viability 

and higher cultivation time. Sakanaka et al. (1996) reported 

the fusion of a thermotolerant strain of K. marxianus with a 

high ethanol producing strain of S. cerevisiae; however, 

their fermentative capacity was severely impaired and the 

fusants’ thermostability was lower than for either of the 

parental cells. 

While Schwan and Rose (1994) reported that ethanol 

production in galactose-containing medium was not as high 

as when glucose was the carbon source, Duvnjak et al. 

1987 found that galactose was a better carbon source for 

ethanol production than glucose; however, the strains 

employed in both works were different. The conversion of 

xylose into ethanol by K. marxianus was already reported 

some time ago (Margaritis and Bajpai 1982). 

Different process strategies have been used for ethanol 

production with K. marxianus: batch cultures with elevated 

substrate concentrations (Grubb and Mawson 1993; Barron 

et al. 1996), fed-batch production (Ferrari et al. 1994; 

Gough et al. 1998; Love et al. 1996), continuous system 

(Love et al. 1998), membrane recycle bioreactors (Tin and 

Mawson 1993), two-stage fermentation (Hack et al. 1994; 

Banat et al. 1996), immobilization with β-galactosidase 

(Hahn-Hägerdal 1985), calcium-alginate-immobilized cells 

(Bajpai and Margaritis 1987a,b; Marwaha et al. 1988; 

Nolan et al. 1994; Riordan et al. 1996; Barron et al. 1996; 

Brady et al. 1996, 1997a,b, 1998; Ferguson et al. 1998; 

Gough and Mchale 1998), cells immobilized in poly(vinyl 

alcohol) cryogel beads (Gough et al. 1998), or in Kissiris, a 

mineral glass foam derived from lava (Nigam et al. 1997; 

Love et al. 1996, 1998), extractive fed batch cultures (Jones 

et al. 1993), simultaneous saccharification and fermentation 

processes with added enzymes (Barron et al. 1995b, 1996, 

1997; Boyle et al. 1997; Nilsson et al. 1995; Ballesteros et 

al. 2002a,b, 2004; Kádár et al. 2004) or by cloning of 

heterologous cellulase genes (Hong et al. 2007), and the use 

of mixed cultures (Ward et al. 1995). 

Cheese whey contains lactose and a protein fraction 

sufficiently rich in essential amino acids. Cheese whey 

cultivations with K. marxianus have been proposed, with 

promising results, as a means of reducing the pollution 

caused by this industrial waste stream (Ghaly and Singh 

1989; Giec and Kosikowski 1992; Harden 1996; Aktas et 

al. 2005) and/or to produce single-cell protein (Giec and 

Kosikowski 1992; Ben-Hassan et al. 1992, Ben-Hassan and 

Ghaly 1995; Belem and Lee 1999; Schultz et al. 2006; 

Ghaly and Kamal 2004). Aerobic cultures of microorganisms 

in cheese whey can reduce up to 90–95% of its BOD 

(Grubb and Mawson 1993), resulting in bioingredients of 

high added value for the food industry (Belem et al. 1997). 

Other potential applications of the yeast K. marxianus, 

which can be found in the literature include its use as baker’s 

yeast (Caballero et al. 1995) and as an anticholesterolemic 

agent (Yoshida et al. 2004). The cellular components 

involved in the hypocholesterolemic activity of K. marxianus 

were further examined (Yoshida et al. 2005). 

Last but not the least, K. marxianus has been investigated 

as a host for the production of heterologous proteins. 

In general, yeasts are capable of performing some posttranslational 

modifications of proteins, such as glycosylation 

and/or other modifications required for optimal 

biological activity and stability (Hensing et al. 1995). S. 

cerevisiae has been the most commonly used yeast host for 

the production of heterologous proteins (Romanos et al. 

1992; Gellissen and Hollenberg 1997; Porro et al. 2005). 

Nevertheless, this yeast has some drawbacks, such as its 

strong aerobic fermentation behavior and a tendency to 

hyperglycosylate secreted glycoproteins (Hensing et al. 

1994). K. lactis has also been used for the production of 

heterologous proteins (van den Berg et al. 1990; Panuwatsuk 

and da Silva 2002; Bartkevičiute and Sasnauskas 2003; van 

Ooyen et al. 2006). K. marxianus, which is phylogenetically 

close to K. lactis, is supposed to have a similar 

capacity for synthesis and secretion of high molecular 

weight proteins (Wésolowski-Louvel et al. 1996). Some


examples showing that heterologous protein production in 

this yeast is possible have been reported in the literature 

(Bergkamp et al. 1993a; Bartkevičiute et al. 2000; Zhang et 

al. 2003). More recently, Pecota et al. (2007) successfully 

expressed lactate dehydrogenase activity in K. marxianus, 

using an integrative multi-copy system, resulting in lactate 

production by this yeast. Hong et al. (2007) expressed 

thermostable endo-β-1,4-glucanase, cellobiohydrolase, and 

β-glucosidase, also making use of an integrative system, 

generating a strain capable of converting cellulosic materials 

into ethanol. Although these studies demonstrate that 

the heterologous proteins expressed in K. marxianus were 

functional, the capacity of K. marxianus to perform posttranslational 

modifications of heterologous proteins still 

remains to be investigated. 

Acknowledgements Grants from Fundação de Amparo à Pesquisa 

do Estado de São Paulo (FAPESP) (Brazil), Deutscher Akademischer 

Austausch Dienst (DAAD) (Germany), and Fundação Coordenação de 

Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Brazil) are 

acknowledged. 

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