The first step for D-xylose metabolism is the uptake of the sugar through the plasma membrane. A lack of transport system may be one of the major reasons for microalgae without the capability to utilize D-xylose as the sole carbon source. There are two kinetically distinct D-xylose transport systems - the high-affinity system (specific for D-xylose) and the low-affinity system (shared with D-glucose) for native D-xylose-metabolizing microbes. Meanwhile, D-xylose also can be taken up by the D-glucose transporters for non-native D-xylose utilizing species, such as the ethanol-producing yeast Saccharomyces cerevisiae[21].
Our results showed that C. sorokiniana could take up D-xylose through a single-carrier system. The maximum D-xylose transport rate of 3.8 nmol min-1 mg-1 DCW with K
m
value of 6.8 mM was obtained in D-glucose induced C. sorokiniana. It has been reported that Chlorella cells possess an inducible active hexose/H+ symport system responsible for uptake of D-glucose from the environment [12]. Similar with the D-xylose-metabolizing microbes, the green microalga C. sorokiniana might share the inducible hexose symporter for D-xylose uptake (Figure 4). However, D-glucose strongly inhibits D-xylose transportation due to the different affinities for these two sugars [21]. Our results demonstrated that D-xylose uptake was suppressed in the presence of D-glucose, D-galactose and D-fructose, but the degrees of inhibition were different due to the different affinities to these hexoses. C. sorokiniana might not be able to transport the pentose sugars (L-arabinose and D-ribose) by using the hexose symporter, because no significant inhibitory effect on D-xylose assimilation was observed for these sugars.
Interestingly, the non-induced C. sorokiniana were also able to take up D-xylose and had similar affinity (K
m
) to D-xylose compared with the induced cells. Thus, the non-induced cells might still utilize the hexose symporter for D-xylose assimilation, which was also evidenced by the severe inhibition of D-xylose uptake in the presence of D-glucose and D-galactose. However, the capacity (V
max
) was much lower than that of the induced algae, indicating a limited quantity of transporters present in the non-induced cells. Tanner [12] suggested that a small amount of transport protein was constitutively present in the algal membrane. Haass and Tanner [13] reported that non-induced Chlorella strains had the sugar uptake capability and the uptake rate increased from 2 to 412-fold after induction by D-glucose (D-glucose induced cells). This was in accordance with our results of D-xylose uptake for the non-induced cells, and the data also demonstrated that D-glucose induced C. sorokiniana exhibited a remarkably increased D-xylose uptake rate.
After D-xylose is transported into the cells, it will be reduced to xylitol by XR and then converted to D-xylulose by XDH in most eukaryotic microorganisms. Subsequently, D-xylulose is converted to D-xylulose 5-phosphate (D-xylulose 5-P) by xylulokinase before entering the PPP pathway [22]. In this study, NAD(P)H-linked XR and NADP+-linked XDH activities were detected in crude cell-free extract obtained from the induced C. sorokiniana after incubation with D-xylose. In fact, XDH from reported wild-type microorganisms exclusively uses NAD+ as the cofactor, while XR prefers NADPH [22]-[24]. The imbalance of redox cofactors will occur due to the different coenzyme usage, resulting in high amounts of xylitol accumulation. This study discovered a unique XDH from the microalga C. sorokiniana, which oxidized xylitol to form xylulose using only NADP+ as a cosubstrate.
Our results still showed that a notable quantity of xylitol was produced during the growth of C. sorokiniana on D-xylose. The imbalance of cofactors is not the reason for this because the XDH of C. sorokiniana utilizes NADP+ as the cofactor, while the XR prefers NADPH. The formation of xylitol is not solely a consequence of coenzyme imbalance and some metabolic factors may also cause its production, such as the activity ratios of XR and XDH. It has been reported that a high ratio of 10:1 of XDH and XR activities were essential to improve the conversion of D-xylose to D-xylulose [25]. However, our data showed that the activity ratio of XDH and XR was only 1.8:1 at pH 8.0 (optimal pH of XDH). Actually, Chlorella tends to maintain an intracellular pH relative constant around 7.0 [26],[27]. Under this condition, the activity of XR was even higher than XDH (Figure 2A) and the equilibrium of the reaction favored xylitol formation.
Unlike yeast and fungi, microalgae are photosynthetic organisms and have the capability to capture light energy through photochemical reactions. During the first stage of photosynthesis, light energy is converted into chemical energy such as NADPH, which can potentially serve as the coenzyme for D-xylose metabolism (Figure 4). Our results showed that the D-xylose consumption was 2-fold higher under light compared with the cultures in the dark. However, in the presence of DCMU, an herbicide specifically blocking electron flow from photosystem II and inhibiting NADPH production, the consumption of D-xylose was almost identical with that of the cultures in the dark. This result also reflected that the improvement of D-xylose utilization by light might be attributed to the extra chemical energy from the light-dependent reaction.
Although C. sorokiniana could take up D-xylose through the inducible hexose symporter and express XR and XDH for D-xylose catabolism, the growth efficiency was very low since the cell number and DCW did not obviously increase. There are several possible reasons for this phenomenon. The synthesis of the hexose symporter could be induced by D-glucose, D-galactose and D-fructose, but the induction was not achievable by pentoses [12]. Although D-glucose induced algae can take up D-xylose efficiently, the reproduced algal cells may not possess such a transport system (or only a very few amount of constitutive transporters), which subsequently hinders the expression of XR and XDH and eventually results in the death of the reproduced cells. Hahn-Hägerdal et al. [22] declared that the inability of S. cerevisiae to utilize D-xylose was due to the low expression of XR and XDH although the genes encoding these enzymes were present in its genome. Additionally, the low activity ratio of XDH and XR limits D-xylose toward the central metabolism. Our data showed that more than half of the D-xylose carbon was secreted out in the form of xylitol, which would lead to inefficient ATP generation [28]. Microalgae require a significant amount of ATP just for maintenance [6]. As a result, the energy and carbon are inadequate to support the cell reproduction and biomass accumulation.
As natural photosynthetic microorganisms, most microalgae can only grow photoautotrophically, but some of them can grow heterotrophically and/or mixotrophically using organic substrates, which shows great potential in producing biofuels and valuable chemicals due to the rapid growth [2]. Generally, D-glucose is the most preferable organic carbon for these species. D-xylose is hardly utilized by many microbes as the sole carbon source due to a lack of efficient uptake system and/or the catalytic enzymes. Many reports have stated that microalgae could not grow on D-xylose [16]-[18]. However, our present work shows different results. The microalga C. sorokiniana could not only assimilate D-xylose from the environment but also had the capability to catalyze it. This is the first time that D-xylose metabolic enzymes in microalgae have been identified. It is meaningful for future genetic engineering since the introduction of an entire D-xylose metabolic pathway from other microbes into the microalga C. sorokiniana is not necessary. Additionally, C. sorokiniana expresses a unique XDH using NADP+ as the cofactor, which has not been reported in any wild-type microorganisms [22]-[24]. The utilization of NADP+ as cofactor is quite significant since XR prefers NADPH, resulting in balanced redox cofactors. This unique XDH has potential to be applied in the genetic modification of some typical strains, such as the ethanol producing yeast S. cerevisiae, to overcome the imbalance of redox cofactors during fermentation of D-xylose.
In conclusion, the presented results demonstrated that the oleaginous microalga C. sorokiniana (UTEX 1602) had the capability to utilize D-xylose. The analysis of sugar uptake kinetics suggested that the inducible hexose symporter might be responsible for the transport of D-xylose across the cell membrane. The enzymatic activities of NAD(P)H-linked XR and NADP+-linked XDH were detected in C. sorokiniana after the assimilation of D-xylose. Culturing C. sorokiniana under light could improve D-xylose utilization due to extra NADPH from the light-dependent reaction of photosynthesis. This study provided a new insight into D-xylose metabolism in microalgae. Future work will continue to identify the key enzymes and related genes of D-xylose metabolic pathway in C. sorokiniana, and genetic engineering will be adopted to improve D-xylose utilization, for example by introducing a D-xylose specific transporter and overexpressing XDH and XR in a reasonable ratio.