Shake flask cultivation of Y. lipolytica PSA02004
SA is an intermediate of TCA cycle and produced through oxidative/reductive TCA cycle [20]. The reductive pathway is not favorable thermodynamically and responsible for glucose repression. Y. lipolytica prefers to use oxidative TCA cycle for SA production [21,22]. Succinate dehydrogenase is one of the enzymes of oxidative TCA cycle, which catalyzes the oxidation of succinate to fumarate and it has five subunits. Gao et al. inactivated sdh5 encoding succinate dehydrogenase assembly factor 2 (YALI0F11957g) in Po1f strain (derived from W29 strain) and obtained a mutant PGC01003 [23], and this strain showed impaired growth on glucose. The PGC01003 strain was subjected to adaptive evolution using glucose-based medium for 21 days and the evolved strain was designated as Y. lipolytica PSA02004 [18]. This strain was cultured on glucose, glycerol, xylose, glucose/xylose and glycerol/xylose (Figure 2). Glucose and glycerol are the preferred carbon sources for Y. lipolytica, and these carbon sources were completely depleted within 72 h concomitant with the cell growth, which also coincided with SA production (5.0-6.0 g/L) (Figure 2A & 2C). However, the strain was unable to grow on xylose as the sole carbon source (data not shown). The co-fermentation of xylose with glucose or glycerol resulted in xylitol accumulation along with SA synthesis (6.5-8.0 g/L) (Figure 2B & 2D) indicating that Y. lipolytica cannot metabolize xylose to grow on it, but it can transform into xylitol with a high conversion yield (~70%). This was also supported by similar cell growth (OD600: 20-22) observed on glucose/glycerol, as well as during co-fermentation with xylose, where xylose is mainly utilized for xylitol synthesis and not contributing for biomass/product manufacturing. The xylose was subjected to carbon catabolite repression in the presence of glucose/glycerol, and rapid consumption of xylose along with xylitol accumulation started after 48 h when large fraction of these co-substrates was utilized. Acetic acid (AA) was obtained as a main byproduct, which was evident in the late log phase of the cell growth, it can be correlated with subsequent drop in pH below 4.5 in all the fermentations. Another important observation was that the amount of SA and AA achieved during co-fermentation was marginally higher in comparison to fermentation on a single carbon source hinting at cryptic xylose metabolism in Y. lipolytica.
Introduction of xylose metabolic pathway in Y. lipolytica PSA02004
As shown in previous section that Y. lipolytica PSA02004 strain showed no growth on minimal medium supplemented with 20 g/L xylose as a sole carbon source. The inability of Y. lipolytica to assimilate xylose for cellular growth impedes its application for lignocellulosic biorefineries. To enable growth on xylose, xylose metabolic pathway was introduced in Y. lipolytica PSA02004. In this study, the engineered strain was constructed by overexpressing the homologous gene of xylose reductase (XR), xylitol dehydrogenase (XDH) and of xylulose kinase (XK) from Y. lipolytica (Po1d strain) cloned under transcription elongation factor (TEF) promoter (Figure 3A). The resulting strain was designated as Y. lipolytica PSA02004PP. With the overexpression of XR, XDH and XK, the strain was able to grow in the medium containing xylose as a sole carbon source. The time course profiles of substrate assimilation, cell growth, product formation and pH were similar to those obtained on glucose or glycerol. There was no xylitol accumulation and probably, all the formed xylitol was funneled towards central carbon metabolism. The maximum OD600 obtained was 14.1 at 72 h. The recombinant strain PSA02004PP was able to produce 3.8 g/L SA from xylose with 0.19 g/g yield. Interestingly, substantial amount of AA (4.1 g/L) was accumulated. The combined production of two organic acids resulted in drop in pH with time (Figure 3B). In addition to cultivation on xylose, the activity of two key enzymes, i.e. XR and XDH, involved in xylose metabolism was monitored throughout fermentation (Figure 3C). The activity profiles revealed that high activities of XR and XDH were maintained during exponential growth and stationary phase. The maximum XR and XDH activity of 0.85 and 0.98 U/mg, respectively, were obtained at 72 h. The slightly high XDH activity than XR allows better synchronization between two enzymes, and results in efficient conversion xylose to xylulose without accumulation of xylitol as by-product.
Co-fermentation of xylose with glucose/glycerol by Y. lipolytica PSA02004PP in shake flasks
The recombinant Y. lipolytica strain carrying a copy of XR, XDH and XK gene showed a superior growth characteristic along with SA synthesis in xylose containing medium under shake flask cultivation. Furthermore, the phenotypic profile on different carbon sources such as glycerol and glucose, and the effect of these substrates on the uptake of xylose were investigated. The recombinant strain produced SA titer of 5.7 and 5.0 g/L with glycerol and glucose as carbon source, respectively (Figure 4A & 4C). While in case of co-fermentation with glucose or glycerol, the consumption of xylose was slowed down, indicating some signs of catabolite repression effect. The co-fermentation of glucose and xylose resulted in the maximum OD600 value of 22.7 with SA titer of 9.9 g/L at 96 h (Figure 4B). While OD600 of 30.1 was achieved with similar resultant SA concentration (10.0 g/L) at a faster rate in 72 h using a mixture of glycerol and xylose (Figure 4D). Additional accumulation of AA was observed both in individual sugars as well as with mixed substrates, which also reduced the pH of the fermentation broth besides reducing the SA yield. After the introduction of xylose metabolic pathway, no xylitol accumulation was observed with co-fermentations, and significant improvement in SA synthesis was noticed in comparison to control where xylose was transformed into xylitol in presence of glucose/glycerol. Thus, there was clear shift in metabolism with entry of xylose into central carbon metabolism.
Substrate inhibition studies on recombinant Y. lipolytica PSA02004PP
The effects of initial xylose concentration on the substrate uptake rate, cell growth, product and byproduct formation ability of Y. lipolytica PSA02004PP were investigated by growing the strain at different initial concentration of xylose ranging from 20-120 g/L. The aim of the experiment was to determine optimal level of xylose for cell growth and SA production. Figure 5 (A-E) shows the time course profiles for xylose uptake, cell growth (OD600), SA, AA and xylitol production. Xylose was completely consumed in 72 h for fermentation media with an initial level of 20 and 40 g/L. Beyond 40 g/L, residual xylose was noticed even at 120 h. The uptake of xylose was reduced after 48 h at 60, 80, 100 and 120 g/L. The amount of unutilized xylose at 60, 80, 100 and 120 g/L was 8.6, 36.1, 51.9 and 77.3 g/L, respectively (Figure 5A). There was a linear increase in cell growth (i.e. OD600) from 11.7 to 17.2, as the initial xylose concentration was enhanced from 20 g/L to 60 g/L (Figure 5B). Above 60 g/L, there was gradual decline in the biomass formation indicating the substrate inhibition. Similar trend was obtained with SA; producing a maximum of 3.8, 6.6 and 10.0 g/L at 20, 40 and 60 g/L initial xylose concentration, respectively (Figure 5C). Further increase in initial xylose concentration retarded the yield and productivity of SA. AA was identified as the main byproduct and accumulation enhanced at higher substrate concentration. The AA level reached 10-13 g/L at initial xylose concentration 80-120 g/L (Figure 5D). Interestingly, xylitol formation was observed at xylose level above 40 g/L and significantly increased from 1.3 g/L to 10.5 g/L as initial xylose concentration was raised from 60 g/L to 120 g/L (Figure 5E). The continuous increment in AA and xylitol production with increase in xylose levels can be due to overflow metabolism at higher substrate concentrations. The initial xylose concentration of 60 g/L was selected for further experiments to achieve an optimal balance between SA titer, yield and productivity.
Batch cultivation of Y. lipolytica PSA02004PP in bench-top scale bioreactor
The batch cultivation of recombinant Y. lipolytica PSA02004PP was conducted in bench-top bioreactor in order to understand the phenotypic characteristic of strain. The initial concentration of pure xylose was 60 g/L. The strain was able to produce maximum biomass concentration of 7.3 g/L (OD600: 34.9) with pure xylose substrate (Figure 6A). The xylose was almost completely consumed (>99%) in 84 h, which was reflected in concomitant termination of biomass, SA and AA formation. The highest SA level of 11.2 g/L with the yield of 0.19 g/g was obtained in and 8.5 g/L of AA was generated in the same duration. The experiment was repeated with crude xylose-rich hydrolysate derived from SCB (Figure 6B). Hydrolysate after pre-treatment often contains inhibitors which can negatively impact the performance of microorganisms. The comparison was made to evaluate the robustness of strain in presence of fermentation inhibitor such as furfural and AA. The cell growth (OD600: 25.3; 5.3 g/L) was unaffected as biomass yield was almost the same in both cases. The strain accumulated 5.6 g/L SA with a yield of 0.14 g/g. In both fermentation, accumulation of substantial amount of AA (~8.5 g/L) along with SA resulted in significant reduction in pH. Furthermore, no accumulation of xylitol was observed during fermentation, indicating active pentose phosphate pathway resulted in enhanced biomass formation.
Fed-batch fermentation for SA production
Based on the batch fermentation study, where the strain displayed excellent xylose uptake capability with simultaneous biosynthesis of SA, fed-batch fermentation was conducted to further improve SA production. The strain was evaluated in fed-batch fermentation with minimal medium without controlling pH. The batch phase was completed in 72 h of fermentation, where the initial xylose concentration was reduced to 10.3 g/L, and the strain was in exponential phase with a maximum OD600 of 32.0, which is equivalent to 6.7 g/L biomass concentration. The SA and AA concentration at 72 h were 10.8 g/L and 11.6 g/L, respectively. The cell metabolism coupled with accumulation of these organic acids caused reduction in pH level to 3.9 and thereafter, pH was stable till the end of fermentation. The feeding was started after 72 h to maintain a xylose level above 10 g/L (Figure 7). The cell growth was continued till 108 h, thereafter, cell reached stationary phase and remained stable (OD600: 50-56). Despite a low pH, synthesis of biomass and SA was continued with a smooth rate. The highest biomass concentration of 11.8 g/L was observed at 156 h of fermentation. The maximum SA concentration was 22.3 g/L, which was coincided with cell growth. The fermentation resulted in the buildup of 25.0 g/L AA, a major by-product which was obtained in higher amount than the desired product SA.