Establishing indigoidine production in Saccharomyces cerevisiae
In S. lavendulae, the native pathway to convert l-glutamine into the blue pigment indigoidine consists of the NRPS BpsA and a 4′-phosphopantetheinyl transferase (PPTase), needed to activate the apo-NRPS into its holo-form via the addition of a coenzyme A-derived phosphopantetheine moiety (Fig. 1b) [19, 21]. To establish the indigoidine pathway in S. cerevisiae, we genomically integrated the Bacillus subtilis PPtase sfp, previously shown to successfully activate apo-BpsA [22], and the 3.8 kbp NRPS gene bpsA into S. cerevisiae BJ5465, a protease deficient strain reported to functionally express Sfp [23].
Blue pigment production was successfully observed in the resulting strain 3 days after visible colony formation (Fig. 1c). The pigmentation appeared first in the central colony region and was limited to subpopulations on the colony surface and extended outwards of the colony over the course of 10 days (Additional file 1: Figure S1). This observation indicates that the localization of a given cell within a colony has an effect on the production. This effect could originate from enhanced oxygen availability at the surface of the central colony region as compared to the outer limits or lower layers of a colony [24], as oxygenation is a necessary step in the formation of the pigment [25].
To determine the localization of the pigment within the cell, we performed brightfield microscopy of the transformants. As expected from the phenotype of the colony, the population shows heterogeneity regarding pigment production (Fig. 1d). In cells that produce the blue pigment, it accumulates in foci and forms aggregates.
Carbon source determines efficiency of indigoidine production
While glucose is its preferred carbon source, S. cerevisiae can utilize other sugars such as sucrose, galactose and a variety of non-fermentable substrates including glycerol by adjusting its energy metabolism from fermentation to respiration. The flux through the TCA cycle is significantly increased during respiration compared to that during fermentation (Fig. 1a) [10]. The TCA cycle intermediate alpha-ketoglutarate serves as an indirect precursor pool for indigoidine formation via the amino acids glutamate and glutamine.
Thus, we hypothesized that efficient formation of indigoidine as a product of the TCA cycle takes place predominantly during the respiratory metabolic state and not during fermentative growth. To test this hypothesis, BJ5465.sfp.bpsA was grown on solid rich media containing either 2% glucose or 2% glycerol as sole carbon sources and pigment formation was monitored. When grown on medium containing glycerol, visible blue pigmentation coincided with visible colony formation after 3 days of incubation at 37 °C and increased in intensity to reach maximum pigmentation after additional 4 days (Fig. 2a). Using glucose as a carbon source caused a delay in visible pigmentation but increased growth rate of the colonies as compared to glycerol. Because glycerol is a non-fermentable carbon source, cells are required to shift into respiratory metabolic state, which leads to a decrease in growth rate but an increased flux through the TCA cycle. Furthermore, blue pigment production was absent in spontaneous petite mutants grown on medium containing 2% glucose (Additional file 1: Figure S1), indicating the requirement of functional mitochondria for indigoidine formation. Petite mutants form small colonies on fermentable carbon sources and are unable to grow on non-fermentable carbon sources due to absent or dysfunctional mitochondria and thus TCA cycle deficiency [26]. Therefore, these observations are consistent with our hypothesis that efficient production of indigoidine occurs during respiratory growth.
To determine if the increase in pigmentation for cells grown on rich medium containing glycerol as sole carbon source originates from an increase of number of cells producing the blue pigment or from an enhanced production per cell, we performed light microscopy of cells grown on glycerol. We found the cause for increased colorization of the colonies predominantly to be a result of an increase in the number of pigment-producing cells (Fig. 2b).
In addition to the type of sugar used as a carbon source, the concentration of sugar in the medium has a strong impact on the energy metabolism of S. cerevisiae. In aerobic conditions, S. cerevisiae metabolizes different carbon sources via different metabolic pathways, namely fermentative, mixed respiro-fermentative or purely respiratory for non-fermentable carbon sources [8, 14, 27]. To study the effect of increasing sugar concentration of differentially metabolized carbon sources on the production of indigoidine, we cultivated BJ5465.sfp.bpsA in liquid media containing either glucose, sucrose, galactose or glycerol ranging in concentrations from 1 to 5%.
We expected production of indigoidine to occur when cells enter respiratory growth upon activation of the TCA cycle. Thus, we hypothesized that pigment production would start upon entering growth phase independent of sugar concentration for the non-fermentable carbon source glycerol and galactose. Indeed, we observed pigment formation in all concentrations of glycerol or galactose as carbon source (Fig. 3). While the quantified concentration of indigoidine via a colorimetric assay did not show a strong dependence on initial concentration of sugar present in the cultures grown in glycerol, a slight trend towards more intense coloration of the culture with increasing sugar concentration was observed (Fig. 3a). Even though S. cerevisiae is able to metabolize glycerol, growth remains slow when utilizing this non-fermentable sugar as sole carbon source [28], resulting in slow growth and thus low titers of indigoidine in these cultures. Further, for these cultures only negligible amounts of by-products were detected. In contrast, cultures grown in galactose showed the highest indigoidine production after 3 days with a starting concentration of 3% galactose, decreasing with increasing deviation from this concentration (Fig. 3b). Even though most of the sugar was already consumed after 3 days as quantified by HPLC analysis, indigoidine production increased further with starting concentrations of 2–5% galactose but remained stable for 1% after 5 days (Additional file 1: Figure S3). By-product formation was solely detected in cultures with higher starting galactose concentrations of 4% and 5%, indicating the occurrence of carbon catabolite repression as previously described by Gancedo et al. [7]. The results obtained for cultures grown in glycerol and galactose are consistent with 13C flux studies, showing increased flux to the precursor alpha-ketoglutarate under purely respiratory or respiro-fermentative growth [10].
For sucrose and glucose, pigment production was observed at high titers for low starting sugar concentrations of 1% and 2% but was absent at 4% and 5% initial sugar concentrations, while the by-products ethanol and acetate were detected in increasing amounts with increasing starting sugar concentrations (Fig. 3c; sucrose in Additional file 1: Figure S3).
We hypothesized that the lack of pigment production at higher starting concentrations of these fermentable carbon sources after 3 days could be caused by remaining sugar. Unconsumed sugar present in the medium at sufficient concentrations could cause the cells to remain in the fermentative state, inhibiting flux through the TCA cycle and thus preventing indigoidine production. To test this hypothesis, we performed sugar and by-product quantification using HPLC. This analysis revealed that 99% of the sugar is consumed independent of starting glucose concentrations (Fig. 3c, middle), rendering excess sugar as cause for the absence of pigment production unlikely. This conclusion is supported by the observation that pigment production remained absent in cultures of 4% or 5% glucose or sucrose concentration even after additional 48 h of cultivation (Additional file 1: Figure S3). The absence of pigment production in cultures with high initial sugar concentrations could originate from nitrogen limitations of these cultures at later stages of their growth. This conclusion is in agreement with results from observations made by Brown and Johnson [27] when analyzing the effect of sugar concentrations on cell yield and metabolites of S. cerevisiae cultures.
To gain a detailed understanding of the production profile, we captured the dynamics of metabolite abundances (carbon source, ethanol, acetic acid) and quantified pigment production over the course of 4 days. As expected, the carbon consumption profile of BJ5465.sfp.bpsA grown in medium containing glucose resembles a typical profile for aerobic diauxic growth by S. cerevisiae [29]. In the first 24 h of cultivation, glucose was fully consumed by fermentative metabolism resulting in the production of 6.96 ∓ 0.85 g/L ethanol, 0.07 ∓ 0.01 g/L acetate and biomass accumulation of 5.13 ∓ 0.78 g/L (Fig. 4a). In a subsequent respiratory metabolic growth phase, the non-fermentable carbon source ethanol was consumed leading to slower biomass formation. The shift from glucose consumption to ethanol consumption, marks the onset of indigoidine production after 24 h. These results indicate that indigoidine production coincided with the shift from fermentative to respiratory metabolism for cells grown on glucose containing medium. Thus, we expected that growth on a non-fermentable carbon source would eliminate the delay in indigoidine production caused by an initial fermentative growth phase. Indeed, growth on glycerol resulted in prompt production of blue pigment (Fig. 4b), even though glycerol was consumed at very slow rates throughout the experiment. Ethanol and acetate were produced at negligible amounts during the entire growth phase as expected for respiratory growth. In contrast to growth on glucose, indigoidine production profile during growth on glycerol correlated with the biomass profile.
Altering metabolic states through controlled carbon availability in bioreactors can enhance indigoidine production
Advanced process control available in bioreactors can be used to influence microbial growth and product generation through controlled culture environments. Our previous experiments were performed in tubes and shake flasks in batch-mode, where no additional substrate was added after the start of the cultivations. In these batch fermentations, substrate depletion affected the metabolic state of the cultures. To maintain a specific metabolic state over an extended period of time, fed-batch cultivations with two different substrate feeding strategies, i.e. carbon depletion and carbon excess, were performed in 2 L bioreactors.
Carbon depletion conditions were implemented using a dissolved oxygen (DO) signal-based pulse feeding strategy. The metabolic activity of cells stalls upon depletion of total carbon in the culture. First, glucose is fully consumed, followed by the consumption of other sources of carbon such as fermentative by-products ethanol and acetate. The stall in metabolic activity leads to a reduction in oxygen demand, resulting in a sudden increase (“spike”) of dissolved oxygen levels in the culture. A “pulsed” feed of glucose was triggered upon carbon depletion events detected by a DO spike. Excess availability of carbon was achieved through semi-continuous feeding of glucose with fixed delivery of 4 g/L/h.
We hypothesized that excess carbon conditions would promote fermentative metabolism while carbon depletion would enable respiratory metabolism. Indeed, excess conditions resulted in accumulation of the by-products ethanol and acetate reaching final concentrations of 55.3 g/L and 3.1 g/L, respectively (Fig. 5a). No significant production of the pigment was observed. These observations agree with our hypothesis that fermentative metabolic state and thus the inactivity of the TCA cycle impedes efficient pigment formation.
In contrast, depletion conditions resulted in a high production titer of indigoidine, reaching 980 mg/L at the end of the fermentation while accumulating only negligible amounts of ethanol and acetate throughout (Fig. 5b). Interestingly, excess availability of glucose did not have a significant effect on biomass formation as determined by dry cell weight. These results indicate that the growth parameters selected for glucose starvation conditions imposed a predominantly respiratory metabolic state leading to the activation of the TCA cycle and production of indigoidine.