Non-conventional yeasts for food and additives production in a circular economy perspective

ABSTRACT

Yeast species have been spontaneously participating in food production for millennia, but the scope of applications was greatly expanded since their key role in beer and wine fermentations was clearly acknowledged. The workhorse for industry and scientific research has always been Saccharomyces cerevisiae. It occupies the largest share of the dynamic yeast market, that could further increase thanks to the better exploitation of other yeast species.

Food-related ‘non-conventional’ yeasts (NCY) represent a treasure trove for bioprospecting, with their huge untapped potential related to a great diversity of metabolic capabilities linked to niche adaptations. They are at the crossroad of bioprocesses and biorefineries, characterized by low biosafety risk and produce food and additives, being also able to contribute to production of building blocks and energy recovered from the generated waste and by-products.

Considering that the usual pattern for bioprocess development focuses on single strains or species, in this review we suggest that bioprospecting at the genus level could be very promising. Candida, Starmerella, Kluyveromyces and Lachancea were briefly reviewed as case studies, showing that a taxonomy- and genome-based rationale could open multiple possibilities to unlock the biotechnological potential of NCY bioresources.

bioprospecting, non-Saccharomyces, food biotechnology, bioprocess, biorefinery, taxonomy

INTRODUCTION

The global yeast market was worth of USD 3.9 billion in 2020 and is expected to reach USD 6.1 billion by 2025, showing a compound annual growth rate of 9.6% during this period (MarketsandMarkets^TM2020). The food industry is responsible for the largest share of this fast-developing market and the well-known ‘conventional’ yeast Saccharomyces cerevisiae remains for sure the most exploited species. This historical workhorse secured its leading position in the industry thanks mainly to the ability to withstand multiple stressful conditions in conversion processes, especially ethanol, osmotic and oxidative stresses, while showing very efficient fermentation performance. Other ‘non-Saccharomyces’ yeasts species have gained increasing attention from scientific researchers, industry leaders and consumers alike. The term ‘non-conventional yeasts’ (NCY) is getting used, as some authors include Schizosaccharomyces pombe among the ‘conventional’ yeasts, but the boundaries between conventional and NCY become fuzzier, as the latter gain more importance and the distinction between traditional and non-traditional applications is also becoming blurred (Sibirny and Scheffers 2002; Kręgiel, Pawlikowska and Antolak 2017).

NCY include several species already well-known and exploited, e.g. Yarrowia lipolytica, Pichia kudriavzevii, Debaryomyces hansenii, Candida utilis (now Cyberlindnera jadinii, see below) and Kluyveromyces marxianus (Navarrete and Martínez 2020). A non-exhaustive list of NCY genera is reported in Table 1 together with the number of papers available in PubMed (https://pubmed.ncbi.nlm.nih.gov, accessed in May 2021) referring to biotechnological applications.

Table 1.

Overview of information available for selected genera of ascomycetous non-conventional yeasts. Mycobank column records the number of valid names for the respective genus names (genus name itself and valid species names). All other columns contain the number of papers retrieved in PubMed (https://pubmed.ncbi.nlm.nih.gov, accessed on May 2021), using the genus as search term, alone or in combination with keywords related with the scope of the present review. Genera selected as case studies are underlined.

Genus name	Mycobank	‘Genus Name’	AND ‘Food’	AND ‘Fermentation’	AND ‘Biotechnology’	AND ‘Collection’	AND ‘Biodiversity’	AND ‘Bioprocess’	AND ‘Waste’	AND ‘Sustainable’	AND ‘Biorefinery’	AND ‘Volatile Fatty Acids’
Saccharomyces	460	139 569	6628	9233	9624	1434	435	500	671	534	140	107
Candida	755	74 146	3084	204	3697	1176	477	233	394	182	26	33
Schizosaccharomyces	35	13 116	188	168	756	119	14	6	7	15	2	0
Pichia	225	11 036	1554	1948	2454	123	147	231	151	82	27	11
Kluyveromyces	39	2985	546	675	440	33	69	79	68	35	18	4
Yarrowia	11	2019	411	327	485	42	29	92	113	80	22	10
Hansenula	82	1343	75	130	261	18	2	15	14	2	4	0
Debaryomyces	103	982	375	202	96	35	72	7	29	8	3	2
Zygosaccharomyces	108	703	406	221	107	21	23	13	11	4	0	1
Metschnikowia	77	535	185	169	65	33	55	0	7	7	1	1
Hanseniaspora	25	486	273	280	53	22	71	3	5	2	0	0
Torulaspora	37	407	234	231	45	16	36	3	8	1	0	1
Brettanomyces	23	296	173	116	39	10	7	2	2	0	0	0
Meyerozyma	9	206	71	62	43	12	31	9	8	4	2	0
Starmerella	43	189	68	91	44	7	19	4	3	12	1	1
Lachancea	11	180	71	94	28	17	15	1	3	0	0	0
Non-conventional yeasts		109	39	43	45	8	7	4	5	4	5	1
Non-Saccharomyces yeasts		276	165	225	54	13	24	2	4	1	0	0

Food-related NCY could be promising candidates for bioprospecting, at the crossroad of bioprocesses (related to food or feed and ingredient productions) and biorefineries. Indeed, yeasts are widely used in the elaboration of food and feed products, commonly found in the production of fermented foods and beverages, functional foods, food additives and ingredients (Arevalo-Villena et al. 2017; Rai, Pandey and Sahoo 2019; Tofalo et al. 2020; Zhou, Semumu and Gamero 2021). Further, food wastes (FWs) and agri-food by-products could be used as substrates for bioconversions and production of biobased molecules such as lactic acid and succinic acid (Pleissner et al. 2016; Li et al. 2019) and Volatile Fatty Acids (VFAs), important building blocks for the chemical industry (Strazzera et al. 2018; Do, Theron and Fickers 2019; Llamas et al. 2020).

Due to their use in food, some NCY species are also included in the Qualified Presumption of Safety (QPS) list by the European Food Safety Authority (EFSA). Besides Saccharomyces species (S. bayanus, S. cerevisiae, and S. pastorianus), the QPS list by EFSA includes 17 species: Candida cylindracea, Cyberlindnera jadinii (anamorph C. utilis), D. hansenii (anamorph Candida famata), Hanseniaspora uvarum (anamorph Kloeckera apiculata), Kluyveromyces lactis (anamorph Candida spherica), K. marxianus (anamorph Candida kefyr), Komagataella pastoris, Komagataella phaffi, Ogatae angusta, S. pombe, Wickerhamomyces anomalus (anamorph Candida pelliculosa), Xanthophyllomyces dendrorhous (anamorph Phaffia rhodozyma), Y. lipolytica and Zygosaccharomyces rouxii (EFSA 2018, 2021a, b; it must be noticed here, that the term ‘anamorph’, still used in EFSA documents, no longer has any status (McNeill and Turland 2011) and the term ‘synonym’ should be used instead). This could be seen as an advantage for bioprocesses, as safe microorganisms could contribute to the sustainability of the process itself, e.g. being less risky for operators. The QPS status granted by EFSA is comparable to the Generally Recognized as Safe (GRAS) status given by the United States Food and Drug Administration (FDA) to safe microorganisms (Deckers et al. 2020). Further, some species are reported in the International Dairy Federation inventory of microbial food cultures with safety demonstration in fermented food products (FIL-IDF 2018).

As for safety, genome sequencing is a powerful tool that can reveal the presence of genes such as virulence factors or genes conferring resistance towards antifungal drugs; this applies to natural biodiversity (EFSA 2021c) and Genetically Modified Microorganisms (GMM; EFSA 2011). GMM can be obtained by multiple molecular biology techniques to produce new ingredients, to improve the yield of existing processes, or to adapt an interesting metabolism to new applications (Hanlon and Sewalt 2021).

Genome sequencing is also the most important tool to provide information on the biotechnological potential of microorganisms, including NCY, and on the taxonomic placement of strains.

The usual pattern for bioprocess development focuses on single strains in certain species. Indeed, expertise built on the model species is precious for biotechnology, as genetic tools, nutrition characteristics and process parameters can be finely tuned to improve yields and product quality at their maximum. Indeed, different strains of the same species could be exploited with relatively low effort, and expertise on a specific group of microorganisms provides an efficient way to maximize the results minimizing the efforts of designing new processes for new products, e.g. by engineering specific strains with appropriate tools.

In the last decade, we have witnessed the description of new species in agri-food and environmental microbiology, revealing an impressive biodiversity ecologically widespread with a possibly unprecedented biotechnological potential. In this oceanic diversity, the existing taxonomic approach, devised to order and name species, could be useful to highlight novel bioprospecting candidates above species level. In fact, strains belonging to different species, but in the same genus (if coherently delineated), could represent a ‘compromise’ between already available tools, which must be adapted to new strains, but a different genomic background, which could have the potential to develop disruptive innovations. As ecology of species also shapes physiology, and genomes contain all the information capacity of the strains, then an updated approach, where accurate taxonomy and genomics combine to reveal the potential for novel metabolic pathways, could give an important contribution to both basic research and biotechnological innovation. Therefore, genera could be promising groups for bioprospecting.

This review covers four genera belonging to phylum Ascomycota, namely Candida, Starmerella, Kluyveromyces and Lachancea, as case studies. In particular, Candida and Kluyveromyces include species among the most widely used, while Starmerella and Lachancea have been described based on the characteristics of species formerly included in Candida and Kluyveromyces, respectively. For each genus, the characteristics and applications of the most used species are reported first, to enlarge then the perspective to the number of species included in the respective genus, that could be targeted in bioprospecting. Data also show that the taxonomical reconsideration of the most numerous genera could lead to the recognition of smaller and more homogeneous groups that could be more easily studied, giving a new boost to biotechnological discovery process.

Genus Candida

The genus Candida represents species for which a sexual cycle has not been documented, spread among distinct phylogenetic clades. The polyphyletic, multifaceted nature of Candida covers a variety of species of diverse origins and provides little information regarding evolutionary relationships. It should be noted that the classification of Candida species was revised in recent years and some of the species were transferred to other genera/species based on analysis of and comparison between different molecular markers (Daniel, Lachance and Kurtzman 2014; Kurtzman et al. 2018; Table 2). However, a general reorganization of the genus needs to be planned and achieved.

Table 2.

Examples of changes in the taxonomy of yeasts previously classified as Candida (Mycobank, https://www.mycobank.org/, checked on 21^st July 2021).

Species (year of effective publication)	Current name (year of new taxonomy publication)
Candida auringiensis (1978)	Groenewaldozyma auringiensis (2016)
Candida batistae (1999)	Starmerella batistae (2018)
Candida cellulosicola (2011)	Spencermartinsiella cellulosicola (2016)
Candida chrysomelidarum (2006)	Metschnikowia chrysomelidarum (2018)
Candida curvata (1952)	Cutaneotrichosporon curvatum (2015)
Candida etchellsii (1978)	Starmerella etchellsii (2018)
Candida fructus (1978)	Clavispora fructus (2018)
Candida galli (2004)	Yarrowia galli (2017)
Candida gelsemii (2007)	Metschnikowia gelsemii (2018)
Candida guilliermondii (Candida guilliermondii var. guilliermondii) (1938)	Meyerozyma guilliermondii (2010)
Candida ishiwadae (1969)	Nakazawaea ishiwadae (2014)
Candida kofuensis (1999)	Metschnikowia kofuensis (2018)
Candida picachoensis (2004)	Metschnikowia picachoensis (2018)
Candida pimensis (2004)	Metschnikowia pimensis (2018)
Candida rancensis (1984)	Metschnikowia rancensis (2018)
Candida shehatae (1967)	Scheffersomyces shehatae (2012)
Candida stellata (1978)	Starmerella stellata (2018)
Candida utilis (1952)	Cyberlindnera jadinii (2009)
Candida zemplinina (2003)	Starmerella bacillaris (2012)

The two most well-known and studied species are C. utilis and C. guilliermondii. However, those two names are not up-to-date, being Cy. jadinii and Meyerozyma guilliermondii the current ones, respectively. For this reason, description of the two species is not included here, and relevant information can be retrieved elsewhere (Sousa-Silva et al. 2021; Yan et al. 2021).

The genus Candida includes important pathogenic species belonging to Candida clade/Lodderomyces clade and CTG clade (the last lineage contains fungal species with non-standard genetic code that switched CUG codon from leucine to serine and occurs in at least 75 Candida species), as well as non-pathogenic yeasts in the order Saccharomycetales with emerging applications in biotechnology with prospective commercial benefits (Fitzpatrick et al. 2010; Santos et al. 2011; Krassowski et al. 2018). The most important non-pathogenic species are indicated in Table 3 and are briefly reviewed below. It must be noted that, usually, at least one genome sequence is present in GenBank for industrially relevant species.

Table 3.

List of industrially relevant Candida species, in alphabetical order (checked on July 27th, 2021); taxonomic information related to former names and type strains were retrieved from Mycobank, https://www.mycobank.org/.

Species	Former name	Sequenced strain	Genome accession numbers
Candida boidinii	Candida alcomigas, Candida koshuensis, Candida methanolica, Candida methylica, Candida olivaria, Candida ooitensis, Candida queretana, Koleckera boidini, Torulopsis enoki, Candida silvicola var. melibiosica	JCM 9604TNRRL Y-2332TUNISS-Cb60NDK27A1Cb18GF002DBVPG6799DBVPG8035TOMC-Y47DBVPG7578TOMC-Y13Y01308	BCGP00000000NHAQ00000000MSRY00000000MSSE00000000MSRX00000000LMZO00000000MSSB00000000MSSD00000000MSSA00000000MSSC00000000MSRZ00000000PKKY00000000
Candida ethanolica	Torulopsis ethanolitolerans var. ethanolitoleran, Torulopsis ethanolitolerans var. minor	M2	ANNA00000000
Candida glabrata	Cryprococcus glabratus, Torulopsis glabrata, Terulopsis stercoralis	CBS 138BG2ATCC 2001DSY562DSY565044040UAB047-W10D4OL1522B1A1B3B3AFFUL887FFUL8872ACCTCC M202019	CR380947-59CP048230-42CP048118-30MVOE00000000MVOF00000000SKBJ00000000SKBK00000000NETP00000000SKBI00000000LLZY00000000LLWO00000000LMAA00000000LMAB00000000LMAY00000000FMSJ00000000FWDN00000000LLZZ00000000AYJS00000000
Candida intermedia	Candida intermedia var. intermedia, Candida intermedia var. ethanophila, Blastodendrion intermedius, Cryptococcus intermedius, Mycotorula intermedia	CBS 141 442PYCC 4715JCM 1607T	LT635756-63LT635764-71BCGD00000000
Candida maltosa	Candida cloacae, Candida novellus, Candida subtropicallis	Xu316	AOGT00000000
Candida oleophila	Candida rignihuensis	/	/
Candida parapsilosis	Monilia parapsilosis, Mycocandida parapsilosis, Candida parapsilosis var. parapsilosis, Mycotorula parapsilosis, Blastodendrion globosum, Schizoblastosporion globosum, Blastodendrion gracile, Schizoblastosporion gracile, Brettanomyces petrophilum, Candida osornensis, Candida montrocheri, Monilia onychophila, Mycocandida onychophila, Mycotorula onychophila, Mycotorula vesica, Pseudomycoderma vesicum, Pseudomycoderma vesica, Blastodendrion intestinale var. epidermicum, Castellania epidermica, Mycotorula parapsilopsis	ATCC 22019TFDAARGOS_653FDAARGOS_652FDAARGOS_650FDAARGOS_65190–137CBS6318CBS1954GA1DE0235USM039KUSM026N3_182_000G1S2_005_002R2a	JADLIH000000000JABWAC00000000JABWAB00000000JABVZZ000000000JABWAA00000000VUYR00000000CBZQ000000000CBZP000000000CBZX000000000VTQU00000000JADCQT000000000JADCQS000000000SCGV00000000SCGQ00000000
Candida sake	Eutorulopsis sake, Torulopsis sake, Candida australis, Candida salmonicola, Candida vanrijiae, Torula lambica, Hansenula lambica, Mycotorula lambica, Candida tropicalis var. lambica, Torulopsis austromarina, Candida austromarina, Candida vanriji	CBA6005H14_1_4C	QELA00000000JADPYB000000000
Candida succiphila	Candida methanolphaga	JCM 9445T	BCGL00000000
Candida tenuis	Yamadazyma tenuis, Mastigomyces philippovii	ATCC 10573T	AEIM00000000
Candida tropicalis(type species)	Oidium tropicale, Monilia tropicalis, Atelosaccharomyces tropicalis, Castellania tropicalis, Endomyces tropicalis, Myceloblastanon tropicale, Procandida tropicalis, Candida albicans var. Tropicalis, Candida tropicalis var. Tropicalis, Mycotorula tropicalis, Blastodendrion irritans, Parasaccharomyces irritans, Candida benhamii, Candida insolita, Candida paratropicalis, Candida vulgaris, Geotrichoides vulgaris, Cryptococcus interdigitalis, Mycotorula interdigitalis, Syringospora interdigitalis, Torulopsis interdigitalis, Cryptococcus mattletii, Endomyces bronchialis, Candida bronchialis, Castellania bronchialis, Monilia bronchialis, Myceloblastanon bronchiale, Endomyces burgesii, Endomyces cruzii, Zymonema cruzii, Endomyces enterica, Endomyces insolitus, Candida insolita, Castellania insolita, Monilia insolita, Myceloblastanon insolitum, Endomyces niveus, Candida nivea, Castellania nivea, Monilia nivea, Myceloblastanon niveum, Endomyces paratropicalis, Atelosaccharomyces paratropicalis, Candida paratropicalis, Castellania paratropicalis, Monilia paratropicalis, Myceloblastanon paratropicale, Mycocandida paratropicalis, Endomyces perryi, Monilia perryi, Parendomyces perryi, Parendomyces perryii, Monilia aegyptiaca, Castellania aegyptiaca, Monilia argentina, Mycotoruloides argentina, Castellania burgesii, Monilia burgesii, Monilia candida, Monilia bonordenii, Myceloblastanon candidum, Monilia candida, Monilia kefyr, Monilia metatropicalis, Castellania metatropicalis, Monilia murmanica, Monilia pseudobronchialis, Candida pseudobronchialis, Mycotorula dimorpha, Syringospora dimorpha, Mycotorula japonica, Mycotorula trimorpha, Mycotoruloides trimorpha, Parasaccharomyces candidus, Parasaccharomyces taticei, Pseudomonilia miso-alpha, Saccharomyces linguae-pilosae, Castellania linguae-pilosae, Cryptococcus linguae-pilosae, Myceloblastanon linguae-pilosae, Torulopsis linguae-pilosae, Saccharomyces pleomorphus, Torulopsis tonsillae, Cryptococcus tonsillae, Candida bimundalis var. Chlamydospora, Torulopsis candida var. Nitratophila, Endomyces entericus, Candida enterica, Castellania enterica, Monilia enterica, Myceloblastanon entericum, Mycoderma issavi, Monilia issavi, Syringospora issavi, Geotrichum issavi, Monilia burgessi, Geotrichum vulgaris, Endomyces burgessii, Monilia burgessii, Castellania burgessii, Candida benhamiae, Parasaccharomyces talicei	IBUN-090–03 567MYA-3404121Y6604MYA-3404MYA-3404MYA-3404	JAHFZN000000000JAFIQD000000000JGYC00000000PKKZ00000000CP047869-75AAFN00000000PQTP00000000
Candida viswanathii	Procandida viswanathii, Candida citrica, Trichosporon lodderae, Candida lodderae, Fermentotrichon lodderae, Candida viswanathii, Fermentotrichon lodderi, Trichosporon lodderi	ATCC 20 962	QLNQ00000000

The biocatalytic and biotransformation properties and biomass of non-pathogenic Candida yeasts are applicable in food and feed, food additives and supplements, beverages, pharmaceuticals and cosmetic production. Papon, Courdavault and Clastre (2014) underlined the biotechnological potential of the Candida species that belong to CTG clade in the era of synthetic biology, thanks to an enhanced protein diversity and expanded capacity of adaptation of these yeasts to environmental conditions. Also, they may metabolize inexpensive substrates and tolerate extreme stresses. The Candida species that belong to CTG clade, like C. tropicalis, C. tenuis, C. maltosa or Candida oleophila have an important potential in production of different industrially valuable metabolites, such as microbial protein, citric acid, xylitol or xylose reductase (Jiang et al. 2016; Golaghaiee, Ardestani and Ghorbani 2017; Hossain et al. 2018; Uthayakumar et al. 2021). The application of ‘omics’ techniques and metabolic engineering may help to maximize the biotechnological potential of these yeasts; more recently, several approaches to genetically modify Candida yeasts using CRISPR-mediated systems were also developed (Uthayakumar et al.2021).

Majority of today's food ingredients, including flavor food additives, need to be so-called natural ingredients to meet consumers demands. Production of natural flavor compounds from agro-wastes by yeasts is current a promising aspect in biotechnology of flavors. The genus Candida includes some strains/species with the ability to synthesize desirable flavor compounds during food fermentation processes. For example, Candida parapsilosis CS2.53 was used among excellent aroma-producing yeasts to enhance the flavor of soy sauce during fermentation of high-salt liquid-state moromi (Jiang et al. 2021). Candida tropicalis was able to produce popular flavorings in food like d-limonene and methyl-butanoate growing on olive mill waste (Güneşer et al. 2017). However, in the last years C. tropicalis and C. parapsilosis were frequently recognized as important non-Candida albicans opportunistic human pathogens that primarily infects the immunocompromised patients (Silva et al. 2012).

Some Candida species can produce alternative, natural and low-calorie sugar substitutes used in the food industry as sweeteners. Biotransformation of L-arabinose by C. parapsilosis DSM 70125 results in arabitol production, a five-carbon sugar alcohol sweetener. The quoted Candida strain was improved by genome shuffling technology for the efficient production of arabitol. Obtained fusants (GSII-3 and GSII-16 strains) were more efficient than the wild-type strain in arabitol synthesis (Kordowska-Wiater, Lisiecka and Kostro 2018). Yeast bioconversion of different fat-rich wastes, like animal fat treatment wastewaters, olive oil mill wastewaters, soap stock of soybean oil refining, soap stock of olive oil pomace refining, waste cooking oil, soap stock wash-water and other, may result in microbial oils synthesis. Some yeast species belonging to the genus Candida are known to be oleaginous microorganisms, including C. tropicalis and C. viswanathii. They were reported as single cell oil producers with the fatty acid composition and degree of unsaturation varied with the growth substrates with possible use in food and feed as nutritional supplement (Ayadi et al. 2016; Bettencourt et al. 2020). Waste substrates rich in VFAs may be utilized as carbon sources during cultivation of oleaginous yeast. Cultivation of Candida sp. on acetic acid, propionic acid and a combination of either acid with glucose as carbon or energy sources resulted in high lipid to biomass ratio (Kolouchová et al. 2015).

Several studies are currently focused on developing novel and effective control methods against pre- and post-harvest decay in different agricultural commodities to avoid the use of large amounts of fungicides. A number of microorganisms, including Candida, showed a bioprotective effect. It was observed that Candida sake may act against major postharvest pathogens of apple including Botrytis cinerea and Rhizopus nigricans (Kasfi et al. 2018). Thanks to the activity of proteinaceous compounds, Candidaethanolica was recently proposed as promising biological control against fungal growth like Aspergillus and Penicillium genera during cocoa fermentation processes (Ruggirello et al. 2019). The inhibitory effect of C. parapsilosis IP1698 on growth of different aflatoxigenic strains of Aspergillus species and aflatoxin production was observed (Niknejad et al. 2012). The species C. oleophila was used to control post-harvest diseases of fruits and vegetables in the first-generation yeast-based commercial biocontrol product (Sui et. al. 2020). The whole genome of C. oleophila was recently sequenced, assembled and annotated by Sui et. al. (2020) to get information about biocontrol-related genes of the species and to understand the molecular mechanism responsible for the activity. Identification of such molecular markers may help to select new effective biocontrol agents.

Candida intermedia and C. tropicalis are extracellular producers of citric acid with important application in food technology (Max et al. 2010).

Pectin-rich agro-industrial wastes, generated in high amounts from the industrial processing of fruits and vegetables, are also sustainable for biorefineries. The main sugars present in pectin-rich agro-industrial hydrolysates, like D-galacturonic acid and L-arabinose, are not commonly used by most yeasts. However, Candida succiphila or Candida sp. (YB-2248) metabolize arabinose to ethanol (Martins et al. 2020). These authors point out that hydrolysates of pectin-rich residues contain acetic acid at higher concentration which may limit the growth of many yeasts. Acetic acid is also the product of acetyl groups hydrolysis present in hemicelluloses (Kolouchová et al. 2015). Utilization of carboxylic acids, like acetate and lactate, is known for opportunistic pathogens belonging to Candida species. This metabolism is used to survive and successfully thrive in unfavorable environmental conditions and nutrient-limited conditions. The Candida glabrata drug:H⁺ antiporter (DHA) CgDtr1 is the only acetate exporter known in Candida species. It is reported as involved in weak acid stress resistance and export of acetate (Alves et al. 2020). Another toxic compound present in pectin-rich hydrolysates is methanol, which toxicity mechanisms are poorly studied. There are several NCY that can efficiently use methanol as the sole carbon and energy source. The most well-known methylotrophic yeasts important for biotechnological applications (production of single cell proteins, pectinase or ethanol) are Candida boidinii, C. parapsilosis and C. glabrata (Martins et al. 2020). The sophorolipids isolated from C. albicans and C. glabrata cultures were suggested as possible food emulsions stabilizers with antibacterial properties against pathogenic bacteria (Kumar Gaura et al. 2019).

Candida yeasts have great advantage in bioprocesses and biorefineries due to robustness with a wide range of physiochemical tolerance and efficient growth on different inexpensive carbon and nitrogen sources. They are able to utilize organic acids, alcohols, pentose sugars, urea, ammonium salts, various amino acids or pyrimidine and are scarcely affected by extremes in pH. Some Candida strains may adapt to high osmotic stress related with elevated concentrations of sugars or salt in culture medium, which are common stresses in biotechnology. Candida species that can grow on 60% glucose and 16% sodium chloride have been described, including C. andamanensis, C. boleticola or C. suratensis (Kurtzman et al. 2015). For those, no genome sequence is available to date. Furfural is one of the typical inhibitors generated in hydrothermal treatment of lignocellulosic biomass. The mechanism of furfural detoxification and metabolic response in C. tropicalis, which shows intrinsic tolerance to inhibitor furfural, was studied by Wang et al. (2016).

Remarkably, considering that the most accessible and abundant renewable raw material on the world is lignocellulosic biomass, mainly consisting of cellulose and hemicelluloses with β(1,4)-xylan as the main component of hemicellulose, the ability of different yeasts to utilize xylose and other pentoses released during hemicellulose hydrolysis is rather a unique feature. There already have been identified strains of the genus Candida being capable of using xylose as a carbon source. They have been mainly found in xylose-rich material and habitats, especially in wood-ingesting insects, insect frass, rotting wood, peat collected from tropical peat swamp forest and moss, but also water environments (Kaewwichian et al. 2019). The discussed yeasts strains can become biocatalysts of different processes based on xylose utilization on industrial scale or may support genetic engineering strategies to modify other microorganisms. The strain Candida akabanensis UFVJM-R131 ferments the hemicellulosic hydrolyzate obtained by acid treatment of the sunflower seed cake and converts xylose into ethanol. The strain is also capable to co-fermenting xylose and glucose, which is a unique yeast ability. New global cellular metabolic engineering tools may allow to develop yeasts co-fermenting hexoses and pentoses (Young, Lee and Alper 2010; Valinhas et al. 2018). The strain C. intermedia PYCC 4715 was the first yeast identified with high xylose-transport capacity via glucose/xylose–H⁺ symporter. Its glucose/xylose transporter gene was used for S. cerevisiae transformation (Leandro, Gonçalves and Spencer-Martins 2006). Another Candida D-xylose-fermenting yeast is Candida kantuleensis (Nitiyon et al. 2018).

As for the genus, in Mycobank there are currently 754 legitimate species names ascribed to the genus Candida, but they include synonyms and species already reclassified. Also, many interesting isolates are unclassified Candida. Thus, the metabolic potential of the genus is still largely undiscovered, and its biodiversity maybe associated with unregistered biotechnological advantages, but the not completely resolved taxonomy makes it difficult to systematically access this biodiversity.

Genus Starmerella

As for Starmerella, among the biotechnologically relevant species (Table 4), the best studied for industrial application is Starmerellabombicola, originally isolated from bumblebee honey in Canada and in concentrated grape juice in South Africa. The interest toward S. bombicola is related to the industrial production of sophorolipids, a class of biosurfactants with antimicrobial activity that are principally used in the cleaning and cosmetic industries in place of traditional agents (i.e. triclosans) which raise environmental and medical concerns. Further, sophorolipids can be employed for the bioremediation of soil pollution by hydrocarbons, as anticancer agents in biomedicine, as antimicrobial additives in lubricants and as taste-modulating agents and emulsifiers in food (van Bogaert et al. 2013; Roelants et al. 2019). Comparative genomics of S. bombicola sequences allowed the identification of sophorolipid biosynthetic cluster genes and their expression levels in related proteomics experiments, thus unravelling the optimal conditions for sophorolipid production (Gonçalves et al. 2020).

Table 4.

List of industrially relevant Starmerella species, in alphabetical order (checked on June 18th, 2021); taxonomic information related to former names and type strains were retrieved from Mycobank, https://www.mycobank.org/.

Species	Former name	Sequenced strain	Genome accession numbers
Starmerella apicola	Torulopsis apicola, Candida apicola	NRRL Y-50 540NRRL Y-2481	LBNK00000000NRDU00000000
Starmerella bacillaris	Candida zemplinina	NPSCBS 9494TPYCC 3044PAS13FRI751	NQLE00000000QLKO00000000PEOB00000000MWPI00000000MWSF00000000
Starmerella batistae	Candida batistae	/	/
Starmerella bombicola(type species)	Candida bombicola	JCM 9596NRRL Y-17069TNBRC 10 243PYCC 5882	BCGO00000000NRDR00000000BBSW00000000PEOC00000000
Starmerella floricola	Candida floricola	/	/
Starmerella kuoi	Candida kuoi	NRRL Y-27208T	NRDS00000000
Starmerella riodocensis	Candida riodocensis	NRRL Y-27 859	NRDT00000000
Starmerella stellata	Saccharomyces stellatus, Torulopsis stellata, Cryptococcus stellatus, Candida stellata	/	/

The first-generation substrates for sophorolipids production by S. bombicola mainly include hydrophobic molecules, such as alkanes, fatty acids and fatty acid esters (Lang et al. 2000; van Bogaert and Soetaert 2011; van Bogaert et al. 2015; Paulino et al. 2016). Other unconventional substrates were investigated, such as petroselinic acid, coconut oil, meadowfoam seed oil, eicosapentaenoic and docosahexaenoic acids (van Bogaert et al. 2010; Li et al. 2013; Delbeke et al. 2016). Also, S. bombicola submerged fermentations were conducted starting from animal fat, waste cooking/frying oil, sugarcane and soy molasses, lignocellulosic biomass and exotic oils (tapis, castor or jatropha oil), while in solid state fermentations mango kernels, sunflower and safflower oil cakes and winterization oil cake with sugar beet molasses were investigated (Parekh, Patravale and Pandit 2012; Rashad et al. 2014; Jiménez-Peñalver et al. 2016; Nooman et al. 2017). Comparable efficiencies with traditional substrates in terms of yield, titer and productivity were not reached so far, but the selection or design of highly productive strains and their combined application based on the use of waste or side-product as substrate represents the most promising strategy towards the development of a more sustainable, bio-based sophorolipids production system (Roelants et al. 2019). In this perspective, other Starmerella native sophorolipid producers (even though with lower production titers and productivity compared to S. bombicola) such as S. apicola, S. riodocensis, S. stellata, S. batistae and S. floricola can be employed in optimized conditions (Kurtzmann et al. Konishi et al. 2008, 2010; Konishi et al. 2017). Among these, S. apicola produces not only sophorolipids, but also membrane fatty acids, and enzymes, such as reductases and proteases, which can be interesting features in the food area, particularly in winemaking (Reid et al. 2012). Further, strains of S. kuoi, isolated from secondary peat swamp forests in Thailand exhibited antifungal activity against Rhizoctonia solani, a rice fungal pathogen that causes the sheath blight disease, the second most important rice disease in the world. The potential of this species to be used as biological control agent needs to be further investigated (Satianpakiranakorn, Khunnamwong and Limtong, 2020).

Within the fermented food biotechnology, Starmerellabacillaris (formerly Candida zemplinina) showed unique biotechnological applicability as co-starter culture in the production of fermented low-alcohol beverages with higher glycerol content (Lemos Junior et al. 2021). The use of S. bacillaris in combination with S. cerevisiae drives the complete fermentation of the major sugars present in musts and releases valuable compounds, such as mannoproteins and glutathione which confer stability and prevent oxidative reactions, besides meeting the increasing consumer demand for alcoholic beverages with reduced levels of alcohol (Lemos Junior et al. 2021; Raymond Eder and Rosa 2021). Strains of S. bacillaris were also explored as biocontrol agents on grapes and apples as alternatives of synthetic fungicides thanks to their antifungal activity towards Botrytis cinerea, Penicillium expansum and Alternaria alternata (Lemos Junior et al. 2016; Nadai et al. 2018; Lemos Junior et al. 2020; Lemos Junior et al. 2021; Raymond Eder and Rosa 2021).

As for safety, S. bacillaris is not included in the QPS list by the EFSA, but safety aspects related to human health were also phenotypically evaluated in 17 strains selected for biocontrol (Lemos Junior et al. 2020); none of the strains raised safety concerns, considering growth at 37°C (the temperature of the human body), formation of pseudo hyphae (a virulence factor in human fungal pathogens) and hydrolysis of the peptide bond of proteins (responsible for the cellular lysis).

Starmerella is related to the genus Wickerhamiella and together they form the Wickerhamiella/Starmerella (W/S) clade, which in the Saccharomycotina species tree branches close to Y. lipolytica, another important species for industrial applications (Gonçalves et al. 2020; Raymond Eder and Rosa 2021). The genus was proposed in 1998 to accommodate the teleomorph of Candida bombicola, isolated from bumblebee honey (Rosa and Lachance 1998). Since then, 43 other species were added to the genus, most of them isolated from flowers and pollinating insects: 26 species as a result of reclassification, mainly from the genus Candida (Santos et al. 2018), while 18 were newly described. Main characteristics of the species are an unusually small cell size, the absence of filaments, the formation of conjugated asci with single and rugose ascospores released terminally from the asci and the fermentative capacity, which could be due to the uptake of an alcohol dehydrogenase gene of bacterial origin (Gonçalves et al. 2020). The strict adaptation to flowers and insects is reflected in the limited nutritional versatility of Starmerella species, due to the loss of several metabolic traits during their evolutionary history; however, this specialization has led to horizontal acquisitions of functional genes encoding fructose transporters, siderophores and thiamine salvage traits (Čadež et al. 2020). Indeed, the horizontal genes acquisition is far more frequent compared to other yeasts, for unknown reasons (De Graeve et al. 2018; Gonçalves et al. 2020).

So far, only 21 Starmerella genomes (size range: 9.275–11.60 Mbp) belonging to 13 species are available, with S. bacillaris and S. bombicola the most represented with five and four genome sequences, respectively (Table 4). Interestingly, genome sequencing of the four strains of S. bombicola allowed the prediction of approximately 4599–4629 protein-coding genes, but only 198 of them were described in detail (Gonçalves et al. 2020); while functions related to carbohydrate polymer synthesis and degradation, such as secretory aspartyl protease (SAP2p), exoinulinases and invertases, detected in the genome of S. apicola NRRL Y-50540 showed high sequence divergence (35% identity) with the same proteins in other yeast species, which could be associated to differences in terms of substrate recognition (Vega-Alvarado et al. 2015). Further, other current questions waiting to be answered are related to the independent evolution of different forms of fructophily, which is the preference for fructose over glucose when they are both available, and the role of the alcohol dehydrogenase-coding gene of bacterial origin in non-fermenting species (Gonçalves et al. 2020).

Genus Kluyveromyces

The most widely used species of Kluyveromyces are K. lactis and K. marxianus (Spohner et al. 2016; Karim, Gerliani and Aïder 2020; Table 5, including all the described species). They are often associated with fermented dairy products, such as artisanal cheese and kefir, but can also be isolated from plants and other habitats (Lane and Morrissey 2010). These two species can utilize xylose, xylitol, cellobiose, lactose and arabinose (Nonklang et al. 2008). Kluyveromyces lactis was the first species after S. cerevisiae to obtain GRAS status (Bonekamp and Oosterom 1994). Kluyveromyces marxianus has also achieved QPS status, due to its long use in dairy products (EFSA 2013).

Table 5.

List of all described species in genus Kluyveromyces, in alphabetical order (checked on July 27th, 2021); taxonomic information related to former names and type strains were retrieved from Mycobank, https://www.mycobank.org/.

Species	Former name	Sequenced strain	Genome accession numbers
Kluyveromyces aestuarii	Saccharomyces aestuarii, Dekkeromyces aestuarii,Zygofabospora aestuarii	NRRL YB-4510TATCC 18862T	PPJO00000000 AEAS00000000
Kluyveromyces dobzhanskii	Saccharomyces dobzhanskii, Dekkeromyces dobzhanskii, Guilliermondella dobzhanskii, Zygofabospora dobzhanskii, Kluyveromyces marxianus var. dobzhanskii, Dekkeromyces dobzhanskii	NRRL Y-1974TCBS 2104T	PPJP00000000CCBQ000000000
Kluyveromyces lactis	Torulaspora lactis, Guilliermondella lactis, Zygofabospora lactis, Kluyveromyces lactis, Kluyveromyces marxianus var. lactis, Dekkeromyces lactis, Saccharomyces lactis, Saccharomyces phaseolospora, Saccharomyces sociasii, Dekkeromyces vanudenii, Kluyveromyces vanudenii, Kluyveromyces marxianus var. vanudenii, Zygofabospora lactis var. vanudenii, Torula sphaerica, Candida sphaerica, Cryptococcus sphaericus, Torulopsis sphaerica, Torulopsis manchurica, Zygofabospora krassilnikovii, Dekkeromyces krassilnikovii, Zygofabospora lactis var. krassilnikovii, Zygosaccharomyces casei, Zygosaccharomyces lactis, Zygosaccharomyces mrakii, Zygosaccharomyces versicolor, Saccharomyces drosophilarum, Dekkeromyces drosophilarum, Guilliermondella drosophilarum, Kluyveromyces drosophilarum, Zygofabospora drosophilarum, Kluyveromyces lactis var. drosophilarum, Kluyveromyces marxianus var. drosophilarum, Kluyveromyces drosophilarum, Dekkeromyces drosophilarum, Zygofabospora lactis var. drosophilarum, Saccharomyces drosophilarum var. drosophilarum, Saccharomyces drosophilarum var. acellobiosus, Saccharomyces sociasi, Saccharomyces phaseolosporus, Kluyveromyces phaseolosporus, Dekkeromyces phaseolosporus, Zygofabospora lactis var. phaseolospora, Cryptococcus spaericus, Candida spherica	NRRL Y-1140CBS 2105GG799	NC_006037–42CP042455-60CP021239-44
Kluyveromyces marxianus(type species)	Saccharomyces marxianus, Dekkeromyces marxianus, Guilliermondella marxiana, Zygofabospora marxiana, Zygorenospora marxiana, Zygosaccharomyces marxianus, Kluyveromyces marxianus var. marxianus, Blastodendrion procerum, Candida macedonienas, Candida mortifera, Monilia mortifera, Mycocandida mortifera, Cryptococcus kartulisii, Castellania kartulisii, Monilia kartulisii, Myceloblastanon kartulisii, Cryptococcus sulphureus, Monilia sulphurea, Mycoderma sulphureum, Mycoderma sulfureum, Endomyces pseudotropicalis, Atelosaccharomyces pseudotropicalis, Castellania pseudotropicalis, Myceloblastanon pseudotropicale, Mycotorula pseudotropicalis, Candida pseudotropicalis, Monilia pseudotropicalis, Mycocandida pseudotropicalis, Candida pseudotropicalis var. Pseudotropicalis, Monilia pseudotropicalis var. Pseudotropicalis, Hansenula pozolis, Kluyveromyces cicerisporus, Dekkeromyces cicerisporus, Kluyveromyces wikenii, Dekkeromyces wikenii, Kluyveromyces marxianus var. Wikenii, Monilia macedoniensoides, Candida macedoniensis var. macedoniensoides, Monilia macedoniensis var. macedoniensoides, Castellania macedoniensoides, Mycotorula lactis, Pseudomycoderma mazzae, Saccharomyces cavernicola, Saccharomyces fragrans, Saccharomyces macedoniensis, Fabospora macedoniensis, Dekkeromyces macedoniensis, Saccharomyces muciparus, Dekkeromyces muciparus, Torula cremoris, Torula lactosa, Mycotorula lactosa, Candida pseudotropicalis var. lactosa, Torulopsis lactica, Zygosaccharomyces ashbyi, Monilia macedoniensis, Mycotorula macedoniensis, Blastodendrion macedoniensis, Castellania macedoniensis, Myceloblastanon macedoniense, Mycotoruloides macedoniensis, Candida macedoniensis, Candida macedoniensis var. macedoniensis, Saccharomyces fragilis, Dekkeromyces fragilis, Fabospora fragilis, Guilliermondella fragilis, Kluyveromyces fragilis, Zygorenospora fragilis, Dekkeromyces fragilis, Kluyveromyces fragilis, Saccharomyces kefyr, Candida kefyr, Cryptococcus kefyr, Geotrichoides kefyr, Mycotorula kefyr, Torulopsis kefyr, Candida kefyr, Monilia macedoniensis var. macedoniensis, Monilia pseudotropicalis var. metapseudotropicalis, Mycocandida pinoyisimilis var. citelliana, Saccharomyces chevalieri var. atypicus, Saccharomyces fragilis var. fragilis, Saccharomyces fragilis var. bulgaricus, Dekkeromyces bulgaricus, Kluyveromyces bulgaricus, Kluyveromyces marxianus var. bulgaricus, Saccharomyces kefyr var. kefyr, Zygosaccharomyces ashbyii, Cryptococcus sulfureus, Kluyveromyces cicerosporus, Monilia sulfurea, Mycocandida pinoysimilis var. citelliana	FIM1NBRC 1777CBS 6556NRRL Y-6860ATCC 8635B0399DMB1L03LHW-OIIPE453UFS-Y2791Olga-1Olga-2100656–19DMKU3-1042KCTC 17 555	CP015054-60AP014599-07/CP067326-33CP067318-24CP023456.1–63JACVOB01000000LXZY01000000BBIL01000000VOSP01000000PYUN01000000LDJA01000000LYPD01000000PUHV01000000PUHT01000000CABJCX010000000 NC_036025–32AKFM02000000
Kluyveromyces nonfermentans	Zygofabospora nonfermentans	NRRL Y-27 343	QYLQ00000000
Kluyveromyces starmeri	-	UFMG-CM-Y3682T	WACN00000000
Kluyveromyces wickerhamii	Saccharomyces wickerhamii, Dekkeromyces wickerhamii, Guilliermondella wickerhamii, Kluyveromyces wickerhamii, Zygofabospora wickerhamii, Dekkeromyces wickerhamii	UCD 54–210T	AEAV00000000

Kluyveromyces lactis and K. marxianus are the only lactose-fermenting species frequently found in milk and dairy products; they also possess weak proteolytic and lipolytic activities. The ability to metabolize milk constituents (lactose, proteins and fat) makes them very important in cheese ripening and fermented milk drink (kefir and kumis) production, as they contribute to maturation and aroma formation.

Kluyveromyces marxianus is very promising to be used as a probiotic due to the capacity of modifications in the cell immunity, adhesion and human gut microbiota with also antioxidative, anti-inflammatory and hypocholesterolemic properties (Xie et al. 2015; Cho et al. 2018); it can survive in the digestive tract, resisting to acid and bile. The latter capacities and a higher ability to adhere to the Caco-2 cells suggested that it might have higher antioxidant activities (Cho et al. 2018). Yoshida et al. (2004) investigated the hypocholesterolemic activities of 81 yeast strains from different species, and the highest potentiality in hypocholesterolemic activity was observed in K. marxianus YIT 8292. Therefore, K. marxianus could be introduced as a potential probiotic yeast and a novel food supplement with the ability to prevent hypercholesterolemia.

Kluyveromyces marxianus and K. lactis can metabolize a variety of low-cost substrates including cheese whey or other dairy wastes which has economic and ecological benefits and makes them the indispensable candidates for commercial industrial applications (Löser et al. 2013; Morrissey et al. 2015). They can produce high value-added bioingredients, such as oligosaccharides, used as prebiotics to increase the growth of Bifidobacterium spp. in the human and animal intestines; oligonucleotides, usually used as enhancers of flavors in food products; and oligopeptides, used as immuno-stimulators (Belem and Lee 1998). When added to foods, these compounds act as immunopotentiators, lower the low-density lipoprotein-cholesterol, risk factor for cardiovascular diseases, promote protection against bacterial infections, enhance food flavors and stabilize food emulsions (Collins and Reid 2016).

Thermostable inulinases obtained from K. marxianus are used for the enzymatic hydrolysis of inulin to produce fructo-oligosaccharides and fructose syrups containing 95% fructose. Fructo-oligosaccharides are used as prebiotic food ingredients, whereas fructose could be an alternative sweetener to sucrose and can increase iron absorption in children. The fructose production through enzymatic hydrolysis of poly- and oligo-saccharides of plant extracts by immobilized inulinases of K. marxianus would be an efficient and advantageous approach for commercial sugar production (Holyavka, Artyukhov and Kovaleva 2016). Furthermore, the inulinases produced by K. marxianus using xylose medium could be another promising option to produce high concentration fructose syrup at industrial level (Hoshida et al. 2018).

Some of the most important applications of K. lactis include the industrial production of β-galactosidase and recombinant chymosin. The β-galactosidase is used to produce lactose-free dairy products and prebiotic galacto-oligosaccharides (Audic, Chaufer and Daufin 2003; Czermak et al. 2004; Guerrero et al. 2015). Recombinant bovine chymosin from K. lactis is an important protein for cheese production and shows a higher specific activity than traditional rennet (Almeida et al. 2015). Other commercially relevant proteins produced using K. lactis include inulinase, phospholipase B, chitinase, xylanase and the sweet-tasting protein brazzein (Jo, Noh and Kong 2013). Kluyveromyces lactis is also used for the manufacture of infant nutrition products, single cell proteins (Magalhães et al. 2011). Several metabolites are produced commercially in K. lactis including lactate, the D-gluconic acid, which is derived from D-xylose (Toivari et al. 2012), and D-arabitol, produced directly from whey (Toyoda and Ohtaguchi 2011).

Recently, the interest in K. marxianus and K. lactis has increased due to their high ability to utilize low-cost substrates and high biomass production, which could ultimately lead to high yields of bioemulsifier, which can be used as emulsifiers, solubilizers, wetting, foaming, antiadhesive and antimicrobial agents (Karim, Gerliani and Aïder 2020). It was found that the emulsification properties of mannoprotein extracted from K. marxianus FII 510700 cell walls were like mannoprotein obtained from the cell walls of S. cerevisiae (Lukondeh, Ashbolt and Rogers 2003; Hajhosseini et al. 2020).

Several Kluyveromyces strains are promising candidates for the synthesis of significant amounts of aromatic compounds; in particular, K. marxianus possesses high potential to produce 2-phenyethanol, alcohols, furanones, fruit esters, ketones, carboxylic acids and aromatic hydrocarbons using, as substrate for cultivation, different agro-industrial wastes, such as pepper and tomato pomaces, grape and acid whey (Morrissey et al. 2015; Güneşer et al. 2016).

Using lactose fermenting yeast could be an attractive approach for bread production. Caballero et al. (1995) carried out an experiment with K. marxianus strains (NRRL-Y-1109 and NRRL-Y-2415) as baker's yeast since this yeast shows high growth in whey without any previous treatment. It was observed that K. marxianus displayed a higher proofing activity in the doughs prepared with whey or lactose than the commercial baker's yeast strains. Furthermore, the use of K. marxianus together with Lactobacillus delbrueckii ssp. bulgaricus or L. helveticus as starter cultures to make sourdough bread could lead to longer shelf-life and better sensory quality of the breads (Plessas et al. 2008).

Whole wheat bread contains relatively high levels of fructans, which are the main source of oligo-, di- and mono-saccharides and polyols (FODMAPs) in our diet. It was found that wheat fructans were more accessible to K. marxianus inulinase compared to S. cerevisiae invertase; and subsequently, a higher degradation of fructans might be obtained by K. marxianus inulinase (Struyf et al. 2017), which is a very important result, because a diet low in FODMAPs could reduce the abdominals symptoms to about 70% of the patients suffering from irritable bowel syndrome (Struyf et al. 2018).

Kluyveromyces marxianus and K. lactis are categorized as Crabtree-negative and as such, do not undergo aerobic alcoholic fermentation (Fonseca et al. 2008; Lane et al. 2011). This can be a beneficial phenotype for industrial production of those compounds which are linked to biomass formation (i.e. biomass-directed applications, protein production) since ethanol formation as a toxic or unintended by-product under aerobic condition could be avoided (Wagner and Alper 2016). Being Crabtree-negative makes them preferable for large-scale fermentation. It should be mentioned that there are some contradictory reports in the literature of the ‘Crabtree status’ of K. marxianus and K. lactis. The results of some experiments show that both species have the genes required for ethanol production under certain conditions. The strength of Crabtree effect can be influenced by extrinsic factors and varies even within species, which explains why some, but not all, strains of K. marxianus and K. lactis are very effective producers of ethanol (Hong et al. 2007; Merico et al. 2009). This apparently conflicting finding is probably due to the strain variability, as most of the studies utilized only one representative strain of each species. It can be concluded that a high degree of intra-species disparity exists for this yeast, not only in terms of its genetics, but also of its physiology (Lane et al. 2011).

Moving to the genus perspective, Kluyveromyces was created by van der Walt (1956) to accommodate K. polysporus, an unusual yeast that formed large numbers of ascospores (sometimes 50 or more). In 1970, the genus comprised 21 species, but the analysis of genomic sequences in 2003 led to its reorganization to only six species: K. marxianus, K aestuarii, K. dobzhanskii, K. lactis, K. wickerhamii and K. nonfermentans (Lachance 2007).

These six Kluyveromyces species vary widely in their ability to metabolize lactose. Three phenotypic groups were described:

1. lactose positive K. lactis var. lactis and dairy strains of K. marxianus (B haplotype);

2. lactose negative K. dobzhanskii and K. lactis var. drosophilarum, unable to utilize this sugar at all;

3. Kluyver effect positive for lactose K. aestuarii, K. nonfermentans, K. wickerhamii and non-dairy isolates of K. marxianus (A and C haplotypes), can respire but not ferment the sugar (Fukuhara 2006).

Recently, the novel species K. starmeri was described, which is phylogenetically closer to K. marxianus, K. dobzhanskii and K. lactis. As other cactophilic yeast species, K. starmeri is nutritionally specialized, able to assimilate only nine of the standard carbon sources, among which lactose is not included (Freitas et al. 2020).

Lactose positive K. marxianus and K. lactis carry two neighboring genes LAC12 and LAC4 that are accountable for lactose fermentation (Fukuhara 2006; Rodicio and Heinisch 2013). LAC12 is a membrane permease that imports lactose into the cell, and LAC4 is an intracellular lactase (β-galactosidase) that hydrolyses lactose into glucose and galactose. The uptake of lactose by LAC12 and its hydrolysis by LAC4 are sufficient to allow fermentative growth of K. lactis and K. marxianus in oxygen-limiting conditions (Ortiz-Merino et al. 2018). The two varieties of K. lactis, var. lactis (domestic and milk-associated) and var. drosophilarum (wild, insect-associated), have different LAC4 and LAC12 genetic make-ups, not functional in the latter (Naumov et al. 2006). Also, not all strains of K. marxianus consume lactose efficiently, due to polymorphisms in LAC12 gene. Three distinct genomic haplotypes of K. marxianus (A, B and C) are known, of which only B haplotype is dairy associated and carries a LAC12_L, the LAC12 variant with efficient lactose-uptake properties among the four LAC12 genes present in K. marxianus (Ortiz-Merino et al. 2018; Varela et al. 2019).

Kluyveromyces and Saccharomyces are part of the family Saccharomycetaceae. Kluyveromyces species are affiliated with the pre-Whole Genome Duplication (WGD) clade, while species of Saccharomyces belong to the post-WGD. Separation of these clades based on the presence of the WGD event explains existed fundamental differences between them (Lane and Morrissey 2010; Lane et al. 2011).

Genus Lachancea

Strains of Lachancea thermotolerans, previously Kluyveromyces thermotolerans, are by far the most studied among the Lachancea species (Table 6), thanks to a significant technological potential: they were among the first non-conventional yeasts to became commercially available as starter cultures for winemaking (Kurtzman 2003; Porter, Divol and Setati 2019b). The ability to produce lactic acid (Binati et al. 2020; Gatto et al. 2020), a very uncommon metabolic activity among yeasts, can be a valuable source for the biological acidification of wines, and other interesting features from the oenological perspective (e.g. low production of volatile acidity, reduction of ethanol content, etc.) were comprehensively discussed elsewhere (e.g. Benito 2020).

Table 6.

List of industrially relevant Lachancea species, in alphabetical order (checked on July 27th, 2021); taxonomic information related to former names and type strains were retrieved from Mycobank, https://www.mycobank.org/

Species	Former name	Sequenced strain	Genome accession numbers
Lachancea fermentati	Debaryomyces manchuricu, Zymodebaryomyces manchuricus, Torulaspora manchurica, Saccharomyces albasitensis, Saccharomyces amurcae, Torulaspora amurcae, Saccharomyces astigiensis, Saccharomyces malacitensis, Saccharomyces montanus, Torulaspora montana, Saccharomyces nilssonii var. malacitensis, Zymodebaryomyces mandshuricus	CBS 6772	FJUO00000000
Lachancea kluyveri	Torulaspora kluyveri, Saccharomyces silvestris, Saccharomyces smittii	NRRL Y-12651T	AACE00000000
Lachancea lanzarotensis	-	CBS 12615T	CDLU00000000
Lachancea thermotolerans(type species)	Kluyveromyces thermotolerans, Saccharomyces thermotolerans, Zygofabospora thermotolerans, Saccharomyces drosophilae, Zygosaccharomyces drosophilae, Saccharomyces veronae, Kluyveromyces veronae	SOL13COLC27CBS 6340T	WVSD01000000WVSE01000000CU928165-71, 80

Recent screening efforts on the probiotic properties of NCY have included representatives from the genus Lachancea, in particular L. thermotolerans stood out as one of the most promising probiotic yeasts (Agarbati et al. 2020; Fernández-Pacheco et al. 2021).

Strains of L. thermotolerans are also promising candidates for fermentation of residues from the soybean processing chain. During the production of soymilk and tofu there are two by-products generated, liquid soy whey and solid soybean pulp (okara), which are currently discarded or used as animal feed. The fermentation of such substrates by different NCY could produce an alcoholic beverage or a food ingredient with more pleasant flavor characteristics, thanks to the positive yeast biotransformation (Vong and Liu 2017; Chua, Lu and Liu 2018). Interestingly, one L. thermotolerans isolated from honeybee gut in Iran was suggested to produce sophorolipids (Mousavi, Beheshti-Maal and Massah 2015).

Recent genotyping analysis of L. thermotolerans strains using microsatellites showed a clear separation of strains from anthropic and natural environments, as well as the formation of clusters based on geographical origin, suggesting that geography and adaptation to grape- and wine-related environments were drivers of genetic evolution. These studies also revealed a striking intra-specific diversity with distinct phenotypic profiles associated with the genotypic groups formed, mainly related to important oenological traits (Banilas, Sgouros and Nisiotou 2016; Hranilovic et al. 2017, 2018).

Strains of L. thermotolerans and Lachancea fermentati were isolated from traditional fermented foods in the Mediterranean area, such as a distillate of fermented honey by-products (Gaglio et al. 2017), and a product from the fermentation of dates (Abekhti et al. 2021). Indeed, L. fermentati was also frequently isolated from a wide variety of niches (Porter, Divol and Setati 2019b). The species L. thermotolerans and L. fermentati are the most promising species in beer brewing too, thanks to the very efficient alcoholic fermentation of sugars into high lactic acid and low ethanol concentrations. Their use is of particular interest, especially for the growing market of low-alcohol beer and sour beer, where the biological acidification and reduced ethanol yield are interesting features, alongside the enhancement of aromatic complexity and organoleptic differentiation (Domizio et al. 2016; Osburn et al. 2018; Bellut et al. 2019; Bellut, Krogerus and Arendt 2020; Zdaniewicz et al. 2020).

Lachancea kluyveri was proposed as a model species, because it diverged from S. cerevisiae prior to the ancestral whole-genome duplication (WGD), and evolved through an introgression event (Brion et al. 2015; Friedrich et al. 2015). A further advancement on the studies with L. kluyveri was the recent integration of ‘omics’ data to reconstruct a genome-scale metabolic model, aimed to understand the metabolism of ethyl acetate and uracil (pyrimidine degradation). Besides the value of L. kluyveri as a model for genomic studies, this interesting yeast is characterized by a weak Crabtree positive metabolism, therefore it is able to produce more biomass than other yeasts and potentially produce valuable biomolecules under industrial conditions (Nanda et al. 2020).

Lachancea lanzarotensis and L. kluyveri are, to a lesser extent than L. thermotolerans and L. fermentati, isolated from grape berries/must (González, Alcoba-Flórez and Laich 2013; Binati et al. 2019; Porter, Divol and Setati 2019a). Enzymatic activities, stress response and monoculture fermentation performance, although to a much more limited coverage than L. thermotolerans, were investigated, with L. kluyveri showing pectinase activity, while L. fermentati and L. lanzarotensis positive for β-glucosidase, but none of the tested strains have protease or esterase activity (Binati et al. 2019; Porter, Divol and Setati 2019a, 2019b). In microvinification trials, it has been shown that lactic acid production is a strain-dependent character in L. thermotolerans, while single strains of L. kluyveri and L. fermentati showed a completely different behavior: the former did not produce detectable amounts of lactic acid, while the latter produced an exceptionally high quantity (Binati 2019; Gatto et al. 2020). As for acetic acid, again there is a considerable intraspecific variability, but L. thermotolerans was consistently reported lower producer compared to the other three Lachancea species.

Whole-genome sequencing was used to investigate differences at the strain level and link genotypes with phenotypes, giving important insights especially in the distinctive lactic acid metabolism associated with some representatives of the genus Lachancea. One study dealt with L. fermentati kombucha isolates used in beer brewing (Bellut, Krogerus and Arendt 2020) and another focused on L. thermotolerans grape isolates promising for multistarter wine fermentations (Gatto et al. 2020).

The above-mentioned species belong to a genus proposed in 2003, after a reorganization of the family Saccharomycetaceae based on a multigene sequence analysis, comprising former members of Kluyveromyces, Saccharomyces and Zygosaccharomyces (Kurtzman 2003). The initial species reclassified to the new genus were Lachancea cidri, L. fermentati, L. kluyveri, L. thermotolerans and Lachancea waltii; from which L. thermotolerans was chosen as type species. Following the description of new species in the last two decades, including Lachancea dasiensis, Lachancea meyersii, Lachancea mirantina, Lachancea nothofagi, Lachancea lanzarotensis and Lachanceaquebecensis, there are currently 11 species assigned to the ascomycetous genus Lachancea. Most of them are ubiquitous, including organisms isolated from soil, plants, insects, but also fermentations of food/beverages and various substrates in both natural and industrial processes (Friedrich et al. 2012; Freel et al. 2015; Porter, Divol and Setati 2019b). To date, the complete genome assemblies of 15 strains from the genus Lachancea are available in public databases, including all legitimate species, but L. cidri. Most of them show a haploid genome containing eight chromosomes. A phylogenetic analysis of the D1/D2 domain of the 26S rRNA gene grouped the 11 species in four clusters (Porter, Divol and Setati 2019b), while the phylogenomic tree obtained with all the available genomes of Lachancea in the NCBI database (10 species) revealed two distinct clades, one containing L. dasiensis, L. nothofagi, L. meyersii and L. lanzarotensis, and the other formed by L. mirantina, L. kluyveri, L. fermentati, L. waltii, L. quebecensis and L. thermotolerans (Gatto et al. 2020). As also observed by Freel et al. (2015), the two species L. thermotolerans and L. quebecensis are very closely related (Gatto et al. 2020).

CONCLUDING REMARKS

A huge biodiversity exists in yeast genera which include model species, and expertise built on model species could leverage the exploration of new strains outside the same species, but within the same genus, thus unlocking the biotechnological potential of NCY bioresources.

Reliable molecular (genetic) approaches to provide a meaningful classification, and genome sequencing for pre-screening are prerequisites for the success of this approach. They should be complemented by physiological and technological testing and will allow effective screening and selection of novel strains from species different from the models to successfully exploit NCY as platform organisms to produce biochemicals.

As a final remark, not only Ascomycota, defined based on the formation of asci and ascospores (Kurtzman 2014; Table 1), could be investigated, but also Basidiomycota (defined by the formation of typical basidia with basidiospores). Many of these yeasts can utilize refractory substrates (i.e. pentoses, sugar alcohols and components in lignocellulose); and, due to their ecology, they produce cold-adapted enzymes and substances, such as carotenoids and mycosporines. Further, by virtue of their strong oxidative metabolism, some species play a central role in environmental remediation, including radionuclide extraction from the environment and metal absorption, and in the degradation of pollutants, aromatic compounds, chemicals, plastics and polymers (Johnson 2013).

Coherent use of updated microbial names is advocated to avoid jeopardized information and difficult interpretation of results in comparison with literature. Also, availability of public data, such as genome sequences and microbial strains deposited in culture collections (CCs) and microbial biological resource centres (mBRCs) could be crucial for NCY exploitation, following what it is happening for prokaryotes (e.g. Shi et al. 2021). Indeed, the H2020 funded project IS MIRRI21 (https://ismirri21.mirri.org/), focused on the implementation of the pan-European Microbial Resource Infrastructure, could contribute to boost knowledge and exploitation of, among others, NCY. Maintainment of an updated database openly available for user communities, including academia, bioindustries, and other stakeholders could support collaborative research via the MIRRI Collaborative Work Environment.

Funding

This publication is based upon work from COST Action CA18229 Yeast4Bio (https://yeast4bio.eu/), supported by COST (European Cooperation in Science and Technology). This work has been funded by the Horizon 2020 Framework Programme of the European Union.

Conflicts of interest

None declared.

Author notes

Renato L. Binati and Elisa Salvetti are authors equally contributed to this work