Potential glucose transmembrane pathways for R. sphaeroides ATCC 17023
By retrieving the NCBI database, the only integral PTS, fructose-specific PTS (PTSFru), was found in the genome of R. sphaeroides ATCC 17023, which is encoded by the gene cluster fruAB (RSP_1788 and RSP_1786). fruB encodes EI and HPr, the two sugar-nonspecific protein constituents of the PTS, and fruA encodes the sugar-specific transporter. It was reported that PTSFru encoded by fruAB simultaneously had a function of glucose transmembrane in some E. coil strains [4]. Additionally, fruAB in R. sphaeroides may have a similar function as that of the abovementioned E. coil strains. Glucose transported into cells with non-PTS must be phosphorylated before subsequent metabolism. Although the non-PTS-type glucose-specific transporter in R. sphaeroides ATCC 17023 has not been identified, the enzyme glucokinase (glk, RSP_2875), which plays a role in glucose phosphorylation, exists in the genome [3, 18]. Additionally, the glucokinase activity had been determined in some R. sphaeroides strains when cultured with glucose as the sole carbon source [19]. Considering the above information, R. sphaeroides ATCC 17023 should possess the non-PTS. R. sphaeroides ATCC 17023 metabolizes glucose exclusively with the Entner–Doudoroff pathway (ED) under aerobic and anaerobic conditions because of the lack of phosphofructokinase in the Embden–Meyerhof–Parnas pathway (EMP) [16]. According to the abovementioned analysis, metabolic networks that contain the glucose transmembrane and catabolism were constructed and the result is depicted in Fig. 1.
Influence of PTS and non-PTS on bacterial growth and glucose metabolism
To clarify whether PTS (fruAB), non-PTS, or both of R. sphaeroides ATCC 17023 influences cellular glucose metabolism, two mutants (△fruA△fruB and △glk) were constructed using an in-frame markerless deletion method. The △fruA△fruB was a mutant with double knock out of fruA and fruB. Considering that no non-PTS type glucose transporter has been identified in R. sphaeroides presently, glk was knocked out to study the function of non-PTS in glucose metabolism. Subsequently, these mutants’ growth and glucose consumption were studied under aerobic incubation using glucose as the sole carbon source (Fig. 2). All mutant strains showed a lag phase at the beginning of cultivation (between 0 and 12 h), which was similar to that of the R. sphaeroides ATCC 17023 (WT) (Fig. 2a). Evident variations were observed among these strains since then. The WT went into an exponential growth phase and displayed a rapid growth rate than others between 12 and 24 h. Furthermore, the △fruA△fruB went into an exponential growth phase though the growth rate was slower than the WT; however, △glk still showed a slow growth rate. After that, the growth rate of the WT became slower from 36 to 72 h, going into a decline phase at 72 h. △fruA△fruB showed a faster growth rate between 24 and 36 h, and then the rate gradually slowed. The stationary growth phase was observed at approximately 60 h, and the decline phase appeared at 72 h for △fruA△fruB.
In contrast, △glk continually kept a slow growth status between 24 and 36 h and went into a long stationary growth phase till the end of the experiment. Interestingly, the Biomax obtained by △fruA△fruB was 3.22 ± 0.04 g DCW/L, which was much higher than that of the WT (Biomax was 2.23 ± 0.07 g DCW/L) (Table 2). Although the △glk showed a typical bacterial growth process, both the growth rate and Biomax were much weaker than the WT during the whole culture process. Additionally, the Biomax was 0.82 ± 0.01 g DCW/L achieved by the △glk, and glucose concentration was determined simultaneously (Fig. 2b). At the beginning of cultivation (0–12 h), the strains showed slow glucose consumption rates that fit the characteristics of the lag phase. During the culture time between 12 and 24 h, the WT and △fruA△fruB sped up glucose consumption, though △fruA△fruB had a little slower consumption rate than the WT. The result could explain the reason why △fruA△fruB grew slower than the WT. Afterward, the residual glucose concentration in the group with △fruA△fruB was less than the WT. Finally, △fruA△fruB exhausted the glucose within 72 h, which was 12 h earlier than the WT. Compared with the WT, △glk showed a low ability on glucose consumption during the entire process. After incubation for 96 h, there was still 3.73 ± 0.21 g/L residual glucose in the medium. The rglc of △glk was only 0.026 ± 0.001 g/L/h, whereas the rglc of the △fruA△fruB could get to 0.086 ± 0.002 g/L/h, which was approximately 1.18 times that of the WT (Table 2). Besides the rglc, the Yb/glc of the △fruA△fruB was also promoted, approximately 43.4% higher than that of WT. Additionally, we constructed the mutant strains, △fruA and △fruB. The results revealed that the two mutants showed similar growth and glucose metabolism status as those of the △fruA△fruB (unpublished data). Summarily, glk mutation seriously inhibited the growth and glucose metabolism of R. sphaeroides. Considering that glk is a vital gene involved in non-PTS, we speculated that the non-PTS played a major role in transporting glucose for R. sphaeroides ATCC 17023 during the entire process. However, deleting fruAB also influenced bacterial growth and glucose metabolism. The depressing effect of the fruAB mutation on growth appeared at the early incubation phase (12–24 h), whereas it showed a promotion effect on growth at the later phase (24–72 h). The influence of fruAB mutation on growth could be indirectly explained by the status of glucose metabolism. Therefore, we supposed that the PTS and non-PTS had a synergistic influence on glucose metabolism at the early culture phase. The relative expression level of the fruA and glk in WT during cultivation was determined by RT-qPCR to verify the hypothesis further. The result was depicted in Fig. 2c; both fruA and glk showed an increasing tendency in the early culture phase (12–24 h). After that, the expression level of fruA showed a decreased tendency since 36 h, whereas the glk still kept increasing from 36 to 48 h. The results illustrate that non-PTS and PTS have a synergistic function on glucose metabolism during the early phase, and the non-PTS played a major role in glucose metabolism.
Table 2
Growth and glucose consumption assay of the R. sphaeroides mutant strains and WT
Strain | Time (h) | Glucose metabolism (g/L) | Biomax * (g DCW/L) | rglc ** (g/L/h) | Yb/glc *** (g/g) |
WT | 84b | 6.21 ± 0.21a | 2.23 ± 0.07 b (72 h) | 0.074 ± 0.003 b | 0.36 ± 0.02 b |
△glk | 96a | 2.47 ± 0.04b | 0.82 ± 0.01 c (96 h) | 0.026 ± 0.001 c | 0.33 ± 0.01 c |
△fruA△fruB | 72c | 6.19 ± 0.16a | 3.22 ± 0.04 a (60 h) | 0.086 ± 0.002 a | 0.52 ± 0.03 a |
Note:*, Biomax the maximum biomass; **, rglc the average glucose consumption rate; ***, Yb/glc biomass yield to glucose consumption vs. the Biomax. Statistics analysis was performed based on one-way ANOVA method and the data in the same column with the same letters (a-c) meant no significant difference (P ≤ 0.05). |
Enhancing the non-PTS pathway to promote cellular glucose metabolism
According to the above study, blocking the non-PTS inhibited the glucose metabolism of R. sphaeroides ATCC 17023. Whether overexpressing the non-PTS-type glucose transporter helps in improving glucose catabolism. In this section, the galactose:H+ symporter (galP) from E. coil K-12 substr. W3110A was selected for the study. First, three mutants, △fruA△fruB/bp, △fruA△fruB/galPOP, and △fruA△fruB/tac::galPOP, were constructed with the overexpression vector, pBBR1MCS-2. The △fruA△fruB/bp was directly introduced to the blank plasmid in △fruA△fruB. The △fruA△fruB/galPOP was introduced to the plasmid, only harboring the gene, galP. The △fruA△fruB/tac::galPOP is inserted with a strong promoter tac before the gene galP based on the △fruA△fruB/galPOP. Subsequently, these mutant strains were separately cultivated with glucose as the carbon source, and the biomass and glucose concentration was determined every 12 h. The △fruA△fruB/bp showed almost no difference from that of △fruA△fruB in growth and glucose metabolism (Fig. 3). This means that the plasmid introduction did not influence bacterial growth and glucose metabolism. Compared with △fruA△fruB/bp, the growth rate of △fruA△fruB/galPOP was increased at the early phase (12–24 h), but the growth status was the same as that of △fruA△fruB/bp between 24 and 48 h (Fig. 3a). From 48 h, the biomass achieved by △fruA△fruB/galPOP was higher than that of △fruA△fruB/bp though the growth trends were similar. The higher biomass achieved by △fruA△fruB/galPOP could be explained by the faster glucose consumption rate than the △fruA△fruB/bp during this period. The result also suggested that the overexpression of galP could improve cellular glucose metabolism. For △fruA△fruB/tac::galPOP, the growth improved further than △fruA△fruB/galPOP at the early phase (12–24 h), and then, it still kept a fast growth status than others until the time glucose was nearly exhausted. Additionally, the biomass quantity achieved was higher than that of △fruA△fruB/galPOP. The Biomax was 4.01 ± 0.15 g DCW/L achieved by △fruA△fruB/tac::galPOP, which was the highest value among these strains. For glucose metabolism, △fruA△fruB/tac::galPOP exhausted the glucose in the medium within 60 h, and the rglc reached 0.107 ± 0.003 g/L/h (Table 3). Furthermore, glk was overexpressed in △fruA△fruB (△fruA△fruB/tac::glk). However, both the growth and glucose metabolism decreased compared with △fruA△fruB/bp (Fig.S4). Additionally, the result suggested that the original glk expression level was fitting for glucose metabolism. Maybe, overexpression of the glk produced excessive glucose-6P, which is toxic to cells.
Table 3
Growth and glucose metabolism of R. sphaeroides strains
Strain | Time (h) | Glucose metabolism (g/L) | Biomax * (g DCW/L) | rglc ** (g/L/h) | Ybio/glc *** (g/g) |
WT | 84b | 6.21 ± 0.21a | 2.23 ± 0.07d (72 h) | 0.074 ± 0.003c | 0.36 ± 0.02 b |
△fruA△fruB/bp | 72c | 6.18 ± 0.32a | 3.21 ± 0.11c (72 h) | 0.086 ± 0.002b | 0.52 ± 0.02c |
△fruA△fruB/ galPOP | 72c | 6.21 ± 0.12a | 3.43 ± 0.17b (72 h) | 0.086 ± 0.008b | 0.55 ± 0.05b |
△fruA△fruB/tac::galPOP | 60a | 6.20 ± 0.17a | 4.01 ± 0.15a (60 h) | 0.103 ± 0.003a | 0.65 ± 0.07a |
Note:*, Biomax the maximum biomass; **, rglc the average glucose consumption rate; ***, Ybio/glc biomass yield to glucose consumption versus the Biomax. Statistics analysis was performed based on one-way ANOVA method and the data in the same column with the same letters (a-d) meant no significant difference (P ≤ 0.05). |
Improving CoQ 10 productivity of R. sphaeroides
The CoQ10 content of these mutants was determined, and the result is presented in Table 4. Compared with the WT, △glk, △fruA△fruB, and △fruA△fruB/tac::galPOP synthesized a low content of CoQ10 when incubated for 24 h; especially, the CoQ10 content of △glk was 1.12 ± 0.04 mg/g DCW. After that, CoQ10 content of △fruA△fruB and △fruA△fruB/tac::galPOP was increased after 48 h, whereas the CoQ10 content of the WT and △glk showed a slight reduction. As incubation proceeded (48–96 h), the CoQ10 content of the WT and △glk stopped reducing and increased. Simultaneously, △fruA△fruB and △fruA△fruB/tac::galPOP increased in the CoQ10 content. Finally, the CoQ10 content of △fruA△fruB and △fruA△fruB/tac::galPOP reached 5.02 ± 0.18 and 5.11 ± 0.14 mg/g DCW, respectively. The maximum CoQ10 content of △fruA△fruB/tac::galPOP was increased by 29.4% than the WT. It can be proposed that the mutation of fruAB improved biomass yield to glucose and bacterial glucose metabolism rate but also enhanced the CoQ10 synthesis of R. sphaeroides. Moreover, strengthening glucose transportation by overexpressing galP showed little help to strengthen CoQ10 synthesis.
Table 4
The CoQ10 content of WT and mutants cultured in SMM
Culture time (h) | CoQ10 content (mg/g DCW) |
WT | △glk | △fruA△fruB | △fruA△fruB/tac::galPOP |
24 | 3.79 ± 0.11a | 3.12 ± 0.04d | 3.23 ± 0.17c | 3.43 ± 0.16b |
48 | 3.62 ± 0.06b | 2.53 ± 0.33c | 3.65 ± 0.05b | 3.76 ± 0.21a |
72 | 3.83 ± 0.13b | 2.85 ± 0.05c | 4.97 ± 0.15a | 5.01 ± 0.33a |
96 | 3.95 ± 0.21c | 3.02 ± 0.19d | 5.02 ± 0.18b | 5.11 ± 0.14a |
Note: statistical analysis was performed based on one-way ANOVA and the data with the same letters (a-e) means no significant difference (P ≤ 0.05) for each line. |
Although galP overexpression in △fruA△fruB played a role in promoting the CoQ10 synthesis ability of R. sphaeroides, the strategy can promote glucose metabolism rate, which shortens the fermentation time. The inactivation of fruAB improved biomass yield to glucose and the bacterial CoQ10 synthetic ability. Considering the advantages of the two strategies, △fruA△fruB/tac::galPOP was applied to CoQ10 fermentation in a lab-scale tank (10 L), evaluating whether the CoQ10 fermentation is improved. △fruA△fruB/tac::galPOP showed an evident improvement in growth compared with the WT/bp during the fermentation process (12–72 h) (Fig. 4a). The Biomax of △fruA△fruB/tac::galPOP was harvested at 72 h of fermentation, which was 24 h earlier than the WT/bp. Moreover, the value of the Biomax reached 17.24 ± 0.97 g DCW/L, which was promoted by approximately 16% higher than that of the WT/bp (14.85 ± 0.57 g DCW/L). Simultaneously, the glucose concentration in the medium was almost exhausted after 72 h for △fruA△fruB/tac::galPOP (< 5 g/L), whereas there was more than 10-g/L residual glucose residual for the WT/bp. In the aspect of CoQ10 synthesis (Fig. 4b), the yield gradually increased as the fermentation proceeded for both strains. At 48 h incubation, the yield of △fruA△fruB/tac::galPOP showed a higher level than that of the WT/bp, and the phenomenon lasted to the end. The maximum CoQ10 yield of △fruA△fruB/tac::galPOP reached 78.14 ± 2.31 mg/L, which was approximately 49.76% higher than that of the WT/bp. Moreover, △fruA△fruB/tac::galPOP achieved the maximum CoQ10 yield at 72 h, which was 24 h earlier than the WT/bp.