Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
Volume 23
Issue 3
DOI:10.30825/5.ejpau.188.2020.23.3, EJPAU 23(3), #01.
Available Online: http://www.ejpau.media.pl/volume23/issue3/art-01.html


Małgorzata Robak
Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Poland



Despite many studies, the “check points” of metabolic regulation of citric acid (CA) secretion by the yeasts Y.lipolytica still remain unknown. In this manuscript, some possible aspects of strain dependent secretion as well as CA metabolism regulation were discussed. Keys enzymes’ activities, substrate concentration, affinity of the uptake systems, intracellular CA concentration and strains abilities were the main points taken into consideration. The direction for the future studies emerged from this review, mainly connected to cellular and mitochondrial citrate transport systems and cellular substrates transporters (glucose, fructose, glycerol, ethanol and acetate), give promising starting point for future efficient strain development.

Key words: Yarrowia lipolytica, citrate synthase, intracellular CA, glucose consumption, RAPD.


The nonconventional and ubiquitous yeast Yarrowia lipolytica can be used for many biotechnological processes [5, 80].Skotan Ltd., the polish biotechnological company, produces fodder yeast based on Y. lipolytica biomass grown on glycerol [http: //www.skotansa.pl]. Other example of industrial process using this yeast is erythritol production developed by a chinese company Baolinbao Biology Co. Ltd located in Shandong [http://www.importgenius.com/shipments/baolinbao-biology.html]. In the past, a factory producing single cell proteins (SCP) and citric acid (CA) using Y. lipolytica operated in Europe [21].

Citrate is one of the most widely produced compounds using biotechnological processes. Annual global market reaches over 2 million tons, growing at 3.7% a year [68]. Citrate production by filamentous fungus Aspergillus niger meets the biggest part of this demand [42]. However, many years ago, the ability of Y. lipolytica yeast to secrete CA has been shown [6, 18, 47]. Many efforts have been done to find the best strain and elucidate its secreting ability. It was demonstrated, that CA secretion begins under nitrogen, sulfur or phosphorous limitation [7, 8, 78]. To improve the titer of CA secreted by Y.lipolytica, many studies were focused on medium and process optimization [8, 36, 60–64, 82]. Furthermore, searching for unusual substrates utilization [4, 17, 19, 30, 39, 45, 47, 51, 52, 64, 65, 79] and enzyme activities determination [20, 38, 46, 55] were also performed. Additionally, studies focused on searching for the best CA producer based on wild strains screening [37, 69], mutagenisation [66, 78] and genetic engineering [15, 16, 22, 23, 34, 35, 72] were carried out.

Currently, the best strain of Y. lipolytica secrete up to 200 g/L of CA with a productivity rate of 0.1 g/g/h and a yield of 0.65–0.9 g/g of substrate [4, 8, 27, 36, 43, 45, 50, 63]. The highest concentration can be obtained on glucose and on glycerol as carbon sources [50,65]. However, genetically engineered strains, harboring invertase gene, secreted high titer of CA on sucrose, nearly 140 g/L, with a very high yield: 0.75–0.82 g/g [16, 36]. Using hydrophobic substrates (oil, fatty acid, hexadecane) the production of CA is accompanied by isocitric acid secretion (ICA), which constitutes up to 60% of the sum of citric acids [16, 27, 56]. When glycerol was used, less ICA was accumulated - from 8.1 to 37% [27]. The ICA secretion is also strain dependent, and using glucose or glycerol as substrate, some strains produce very low amounts of ICA, even below 1% [63]. The substrate for CA production was chosen based on the availability of the “wastes” from food and biodiesel production. In a recent study, the corn wet milling products were used as low-cost fermentation media [9]. At the moment, glycerol turned out to be the cheapest substrate for CA biosynthesis and the repeated-batch culture – the best mode to conduct the process. Laboratory bioreactors used in yeasts growth were shown on fig. 1.

Fig. 1. Laboratory bioreactors used for yeasts growth and CA production: 5 L (a), 90 L and 30 L (b) in the Department of Biotechnology and Food Microbiology of Wrocław University of Environmental and Life Sciences

Despite many studies, the “check points” of metabolic regulation of CA secretion by Y.lipolytica strains still remain unknown. In this manuscript, some possible aspects of strain dependent CA secretion abilities as well as CA metabolism regulation are discussed. The main points, which have to be taken into consideration are: enzymes’ activities, substrate concentration and the affinity of the uptake systems, intracellular CA concentration and strains abilities.


The activity of keys enzymes of CA biosynthesis (citrate synthase-CS, isocitrate deshydrogenase – ICDH, aconitase- AH and isocitrate lyase- ICL was first measured by Akiyama et al. [1], Glazunova and Finogenova [20], Behrens et al. [6], Matsuoka et al. [41], Franke-Rinker et al. [18], Katajeva and Finogenova [29]. All of the authors found comparable enzyme activities, not connected to the applied substrate [Table 1]. Robak [55] has shown, that CS and AH have no impact on the CA productivity. Activity of these enzymes is similar for both, strains known as producers as well as for these not secreting CA. Furthermore, similar CS and AH activities were measured during growth and CA biosynthesis phase. However, to keep constant CS activity during continuous processes, the amount of ammonium ions have to be dosed at about 1 mmol/L/h (0.054 g/L/h as ammonium chloride). A similar ammonium ions supplementation was suggested by Anastassiadis and Rhem [2] for Candida oleophila CA biosynthesis. CS is the only enzyme in the cell responsible for CA synthesis using acetyl-CoA and oxalate as substrates. Glucose, fructose, glycerol and fatty acids can be used as carbon sources for this process. In turn, in the yeast Saccharomyces cerevisiae three isoformes of CS are present: Cit1, Cit2, Cit3, found in mitochondria, cytoplasm and peroxisomes, respectively [69]. In Y. lipolytica, based on the optimal pH, at least 3 isoforms of CS were suspected [54]. Blasting the S. cerevisiae Cit1, Cit2, Cit3 against the Y.lipolytica proteome, the presence of protein sequence with 59% of identity to Cit3 was revealed on the chromosome E [SGD based blast analysis].

Table 1. Summary of enzyme activities (CS: citrate synthase, AH: aconitase, ICDH: isocitrate deshydrogenase, ICL: iosocitrate lyase) in biomass of Y.lipolytica growing on different substrates. All enzyme activities are expressed as µM/min/mg of protein
Substrate CS   AH ICDH ICL Source
Glucose 0.59-1.34 0.28-0.42 0.26-0.291 0.015-0.033 [20]
and fructose
0.76-1.31 nd 0.015-0.059 nd [55]
Fructose 0.43-6.37 nd 0.033-0.135 nd [unpublished,
Janik & Robak, 2002]
n-paraffins 0.33-1.0 0.067-0.11 0.095 nd [19]
Ethanol 0.72-3.25 nd 0.06-0.22 0.06-0.25 [24]
Glycerol 0.36-5.,87 nd 0-0.0048 0.007-0.126  (unpublished,
Janik & Robak, 2007)
and glycerol
3.17-15.49 nd 0.03-0.26 nd (unpubliszed,
Bąk & Robak, 2008]
Pure glycerol 0.8-0.82 0.04-0.08 0.12-0.14 0.008-0.009 [44]
derived glycer
1.6-1.74 0.02-0.12 0.08-0.09 0.089-0.1 [44]

In the unpublished study, the activity of CS was very high 5.87 mM/min/mg of protein and a slight positive correlation with CA secretion has been observed at the end of the process, for the 2nd and 3rd cycle during repeated-batch culture (RBC). Beside so many studies, no clear correlation between enzyme activities and CA secretion by Y. lipolytica has been established [8;80]. Based on these results, one may conclude, that the regulation of the key enzymes of CA biosynthesis is not the key factor for CA secretion improvement. It is worth to point out, that the lack of correlation between enzymes activities and CA biosynthesis was demonstrated also for A. niger, still the main CA producer on industrial scale [32]. In these fungi, according to Torres et al. [75], seven enzymes were involved in CA biosynthesis. Furthermore, disappointing results were obtained when single (citrate synthase) and double (pyruvate kinase and phosphofructokinase) over-expression were performed to improve CA biosynthesis from glucose. Ruijter et al. [59] demonstrated, that introduction of eleven CS copies into A. niger genome did not enhance CA biosynthesis. To conclude, citrate synthase plays only a minor role in controlling the flux of carbon in the pathway involved in CA overproduction. Similarly, the overexpression of AH in Y. lipolytica did not improve CA secretion [22]. In the case of the biosynthesis of erythritol by Y.lipolytica it was easier. Rapidly it was demonstrated that the key enzyme responsible for erythritol overproduction from glycerol was erythrose reductase [25,73].

A. niger is the main CA producer since over 100 years and despite many decades of intensive studies, only a small part of the CA production process is clearly determined. Until 2019 there is no consensus regarding the mechanism responsible for CA biosynthesis and its secretion [48]. It seem, that over secretion of CA may be controlled by the transport process itself [26, 71].

Currently, the biochemical studies on key enzyme activities are slowed down due to the ever-increasing genomic studies. To understand the CA secretion abilities of A. niger and Y. lipolytica will most likely be obtained through very intensive exploration of genome sequencing data followed by genome scale metabolic models. The first successful attempt, based on gene engineering to enhance citrate production in filamentous fungi, was the disruption of trehalose-6-phosphate synthase A (ggsA), which led to decreased inhibition of hexokinase by trehalose-6-phosphate [3]. Furthermore, expression of Brsa-25 (one of  filament-associated genes from the SSH library generated with the RsaI-digested cDNA) in anti-sense orientation allowed not only for pelleted growth, but also for the production of CA in the presence of normally inhibitory concentration of Mn2+ [10]. In a recent review, the impact of multi-omics data of A.niger on citrate production was discussed [74].

The inactivation of methyl-isocitrate forming enzyme was the first approach based on the gene knock-out strategy in Y.lipolytica [50], followed by rapid evolution of research based on genetic engineering of CA improvement in this yeast and recently published results on mitochondrial protein expression profiles in response to citric acid biosynthesis [68].


The concentration of the applied substrate is the second most important aspect during repeated batch cultures, shoving high and variable impact on metabolic pathways. The concentration of glucose was studied by Kim and Roberts [31] as well as Rane and Sims [53], however, no clearly demonstrated conclusions were observed. Robak [55] demonstrated, that glucose consumption differs among producers and non producers of CA. CA producing strains consumed 81–98% of glucose in the medium whereas non-producers consumed only 40–50% [Table 2]. However, the ability of glucose uptake remained similar. The affinity (Km) of cells toward glucose is similar for all Y. lipolytica strains and most likely is the result of recently identified glucose transporters [33].

Table 2. Glucose uptake and the affinity of its assimilation by Y. lipolytica [55].
 Y. lipolytica   CA secretion Glucose consumption during growth in medium * [%] KM
24 h
B* Low affinity system High affinity system
24 h 32 h 48 h
ATCC 32338A non 50.4 12.0 33.0 40.0 20 1.4
A-101 yes 98.7 39.0 56.0 75.0 22 1.2
A-101.1.22 yes 97.7 46.0 54.0 73.0 17.5 0.87
A-101.1.31 yes 98.7 36.0 46.0 67.0 22 0.64
A-101.1.31.K1 yes 98.4 nd nd nd 16.6 1.9
* A: medium with 10 g/L of glucose as substrate;
  B: medium with 100 g/L of glucose.

Based on the results of CA biosynthesis processes studies in different Y.lipolytica strains (A-101 and its derives) the following equation (1), describing the connection between the rate of substrate consumption and the rate of citrate secretion by 1 g of dry biomass was determined [55]:

Y= 1,5087 X + 2,3377        (1)

Y – g of consumed glucose by 1 g of dry biomass;
X – g CA secreted by 1 g of dry biomass

Interestingly, application of a second, additional substrate enhance CA secretion by Y. lipolytica. Addition of acetate to the production medium contributed to a higher CA biosynthesis and secretion [57, 76]. Using glucose and glycerol or glucose and fructose the concentration CA secreted into the medium was also important [34, 35].

Based on the results described above, it seems to be clear, that the ability of Y. lipolytica to consume and metabolize glucose plays very important role in CA secretion. The question still is, why some strains consume all the available substrate whereas the other use only half of its initial concentration? One of the possible reasons may be the 6-phosphofructokinase (PFK) and its regulation, which in CA secreting Y.lipolytica strains may not be inhibited by CA. It was already shown for Rhodosporium toruloidesPFK during CA secretion [13]. In Y. lipolytica PFK enzyme and gene were characterized by Flores et al. [14]. There is only one gene encoding PFK in Y.lipolytica and the corresponding enzyme exhibit different kinetic characteristics when compared to S. cerevisaie PFK. It is inhibited by 5 mM citrate, due to that, the final activity could be the result of intracellular CA concentration leading to restricted glucose consumption in non CA secreting strains.

Accordingly, there is an ubiquitous need to improve microbial CA production in industrially relevant microorganisms. Furthermore, it is obvious, that overexpression of genes involved in the central carbon metabolism has no significant effect on improvement of CA production. However, the current development of proteomic, transcriptomic and fluxomic techniques will contribute significantly in the future to develop industrially efficient CA producers [67].


It is well documented, that CA is involved in anabolic and catabolic pathways regulation in the cells. CA activates cytosolic acetyl-CoA carboxylase and inhibits glycolytic fluxes on the level of PFK and pyruvate kinase, it also inhibits CS activity. Flores et al. [14] demonstrated, that in Y.lipolytica CJM244 cells PFK is inhibited in 80% by 5 mM citrate. This inhibition probably was not present in some Y. lipolytica strains, especially those secreted CA (A-101 and its derivatives). According to the equation (1) no inhibition of glucose catabolism caused by accumulated CA was observed. It could be explained by the fact, that the ratio of intracellular to secreted CA was different in cells producing and non-producing this metabolite [Table 3].

Table 3. Intracellular and extracellular CA concentration in yeast cells and culture broth at 48 h of the biosynthesis process [55].
Strain Producers of CA CA [mM] Ratio of intra/extra CA
 in culture broth in cells
ATCC 20 320 Yes 113 239  2,12
ATCC 20 324 Yes 173 367  2,12
ATCC 32 338A Non  14,5  77  5,31
A-101 Yes 165  30  0,18
A-101.1.22 Yes 201 101  0,50
A-101.1.31 Yes 149  87  0,58

It was observed, that low ratio of intra to extracellular citrate concentration (<0,6) correlates with high amount of secreted CA. The Y.lipolytica A-101 and its derivatives (acetate negative mutants) must have an “active” CA secretion system which prevents enzymes from inactivation, mainly the PFK present in the cytoplasm and CS occurring in the mitochondrion. Existence of a cellular membrane citrate and glucose anti-porter could be predicted based on this results. Furthermore, the presence of a proton symporter may be also predicted on that basis. However, for Y. lipolytica ATCC 20 320 and ATCC 20 324 strains, another explanation could be considered, mainly a facilitated diffusion transport. Such diffusion was proposed by Marchal et al. [40] as an explanation of CA secretion abilities. It has long been suspected, that the principal mode of CA export from the mitochondria in A. niger involves import of cytosolic malate by the mitochondrial citrate transporter [58]. For the CA secretion from the cells, Papagiani [49] has presented two different transport mechanisms based on simple diffusion and pH gradient. Despite decades of studies on CA biosynthesis by A. niger, only a small part of the production process was understood in details [28,49]. Recently 2 genes encoding mitochondrial citrate transporters were described in Aspergillus luchuensis [26] and 1 in Y. lipolytica [80].


CA secretion very often is a strain trait [Table 3]. Cavallo et al. [8] pointed out strains as being the main factor of CA biosynthesis. In the era of molecular techniques used for strain identification and differentiation, differences in genome sequences among Y. lipolytica strains have been detected. Fig. 2 presents a similarity tree of Y. lipolytica strains based on 3 independent RAPD analysis performed with 4 microsatellite starters: (GTG)5, (GACA)4, (GCA)5, M13 (Barszczeski et Robak, KBN grant report 2008, unpublished data).

Fig. 2. RAPD* similarity of six Y. lipolytica strainsof different origin: A-101 isolated in Poland, 3 mutants of that strains (A-101.1.31, A-101.122, A-101 1.31. K1), W29 isolated in France and ATCC32 338A deposited in USA collection.
*RAPD analyze was performed by method described for Candida sake by Walczak et al. [77].

The Y. lipolytica strains not producing CA (ATCC 32328A) showed only 53% of similarity with CA producers. A similarity of 72% was detected among the producers, and 82-87% among the bests producers. Nowadays, this observation could be verified due to the fact, that full genome sequences of the CA producing strains are available [11,12].

Y.lipolytica strains differ also in acetate utilization (Fig. 3). An improvement in CA to ICA ratio during citrate biosynthesis was described [54, 62, 78].

Fig. 3. Growth of selected Y.lipolytica strains on  MMT medium with glucose (a) and with sodium acetate + uracile (b). Media composition was described in [54], strains: A-101 isolated in Poland, 2 mutants: A-101.1.31 (acetate  negative), A-101 1.31. K1 (smooth revertant  of acetate mutant) and 3 transformants:  A-18 (suc+ura-), Klon 13 (suc+ura+) and B-56-5 (suc+ura+, with 2 copies of Suc2).


Despite many studies and well documented CA secretion by some Y.lipolytica strains, the check points of that biosynthesis still remain unknown. Additionally, some directions for the future studies emerged from the data available in the literature and are mainly connected to cellular and mitochondrial citrate transport systems as well as substrates transporters (glucose, fructose, glycerol, ethanol and acetate transporters). The preliminary data, which can be found in the literature, give promising starting point for future efficient strain development.


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Received: 7.07.2020
Reviewed: 27.07.2020
Accepted: 5.08.2020

Małgorzata Robak
Department of Biotechnology and Food Microbiology,
Wrocław University of Environmental and Life Sciences, Poland
Chełmońskiego 37/41
51-630 Wrocław
email: malgorzata.robak@up.wroc.pl

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