Exploring putative L-serine exporters in C. glutamicum
In previous studies, homologs of E. coli exporters have been shown to have similar functions in C. glutamicum [17, 19, 20]. Therefore, we hypothesized that the C. glutamicum homolog to EamA(L-serine exporter in E. coli) [2] might be involved in L-serine export in C. glutamicum. According to the NCBI database, EamA belongs to the RhaT superfamily, and 15 records of related proteins associated with RhaT superfamily in C. glutamicum ATCC13032 were obtained. After eliminating duplicate records, three related genes, NCgl2050, NCgl2065, and NCgl0580 genes, were obtained, which might be involved in L-serine export in C. glutamicum.
To verify the function of these putative proteins in C. glutamicum SSAAI (SSAAI), NCgl2050, NCgl2065, and NCgl0580 were deleted in this strain respectively. The results showed that the deletion of NCgl2050 and NCgl2065 did not produce any changes in cell growth and L-serine titer (Fig. 1a and 1b). Strikingly, deletion of NCgl0580 significantly reduced the L-serine titer in SSAAI, but did not affect the growth of the strain (Fig. 1c). SSAAI ΔNCgl0580 produced 11.31 g/L L-serine, which was 56.5% lower than that noted in SSAAI (Fig. 1c P<0.001). However, plasmid-borne overexpression of NCgl0580 compensated for the lack of NCgl0580 with respect to L-serine titer, resulting in 26.76 g/L L-serine titer, similar to that generated by the parent strain SSAAI (Fig. 1d). As shown in Fig. 1d and Fig. 1e, when compared with SSAAI, the strain harboring the plasmid grew slowly to some extent in the logarithmic growth phase, finally reaching cell growth similar to that of SSAAI. This finding suggested that NCgl0580 might act as the L-serine exporter in C. glutamicum, and was named as SerE and its function was further investigated.
Fig. 1 Effect of NCgl2050, NCgl2065, and NCgl0580 deletion and complemented strain on SSAAI. (a) NCgl2050 deletion strain SSAAIΔNCgl2050 (open symbols) and SSAAI (solid symbols). (b) NCgl2065 deletion strain SSAAIΔNCgl2065 (open symbols) and SSAAI (solid symbols). (c) NCgl0580 deletion strain SSAAIΔNCgl0580 (open symbols) and SSAAI (solid symbols). (d) Complemented strain SSAAI∆NCgl0580-NCgl0580 (open symbols) and SSAAI (solid symbols). Squares and circles indicate cell growth OD562 and L-serine titer, respectively. (e) Growth rates of the complemented strain SSAAI∆N0580-NCgl0580 (red) and SSAAI (black).
Localization and function of SerE
According to the NCBI, SerE was presumed to be a hypothetical membrane protein of 301 amino acids, similar to permease of the drug/metabolite transporter (DMT) superfamily. The transmembrane helices of SerE were predicted by TMHMM Server v. 2.0, and SerE exhibited ten transmembrane-spanning helices with both amino- and carboxy-terminal ends in the cytoplasm.
To confirm the localization of SerE, SerE-EGFP fusion protein was expressed in SSAAI. Confocal microscopic observations of SSAAI-egfp and SSAAI-serE-egfp confirmed that EGFP and SerE-EGFP fusion proteins were successfully expressed, respectively (Fig. S1). To further verify the localization of SerE, membrane and cytoplasmic proteins from these two strains were extracted by ultrasonication, and the fluorescence of these proteins was determined using a fluorescence spectrophotometer. The fluorescence of the cytoplasmic proteins of SSAAI-egfp and membrane proteins of SSAAI-serE-egfp (Fig. 2a) affirmed that SerE was localized at the plasma membrane in SSAAI.
To substantiate the function of SerE, a peptide feeding approach was employed by incubating SSAAI and SerE deletion strain, SSAAI ΔserE, with 2 mM of the dipeptide Ser-Ser, respectively, and measuring the concentration of extracellular L-serine. As shown in Fig. 2b, a higher L-serine concentration was detected in SSAAI, when compared with that in SSAAI ΔserE, confirming that SerE is a novel exporter of L-serine in C. glutamicum.
Fig. 2 Fluorescence of cytoplasmic proteins and membrane proteins, and the result of amino acid export of SerE by using peptide feeding approach in SSAAI. (a) Fluorescence of cytoplasmic proteins and membrane proteins of SSAAI-10 (SSAAI harboring plasmid pDXW-10 only, gray bar with slash), SSAAI-egfp (SSAAI overexpressing EGFP protein with pDXW-10, gray bar), and SSAAI-serE-egfp (SSAAI overexpressing SerE-EGFP fusion protein with pDXW-10, white bar). (b) Extracellular concentration of L-serine in SSAAI (solid squares) and serE deletion strain SSAAI ΔserE (solid circles) with 2 mM of the dipeptide Ser-Ser. Extracellular concentration of L-serine in SSAAI (empty squares) without the dipeptide Ser-Ser. (c) Extracellular concentration of L-threonine in SSAAI (solid squares) and serE deletion strain SSAAI ΔserE (solid circles) with 2 mM of the dipeptide Thr-Thr. Extracellular concentration of L-threonine in SSAAI (empty squares) without the dipeptide Thr-Thr.
It is known that L-cysteine export system in E. coli (encoded by eamA) also catalyzes L-serine export [2], and that L-threonine exporter in C. glutamicum (encoded by thrE) also transports L-serine [15]. We therefore analyzed whether the novel exporter SerE could export L-cysteine or L-threonine. The export experiments with dipeptides (Thr-Thr, Cys-Cys) were performed using SSAAI and SSAAI ΔserE. The dipeptides were added at a concentration of 2 mM to the medium, and the extracellular amino acid concentrations at different time intervals were determined by HPLC. The results revealed that the concentration of L-cysteine was comparable in both strains and did not significantly change (data not shown), indicating that SerE might not export L-cysteine. Interestingly, the concentrations of L-threonine in SSAAI ΔserE were lower than those in SSAAI (Fig. 2c), indicating that SerE might be also an exporter of L-threonine in C. glutamicum.
Interaction of a known exporter ThrE and a novel exporter SerE
It is well known that thrE encodes ThrE that can export L-threonine and L-serine in C. glutamicum ATCC13032 [15]. To understand the interaction between ThrE and SerE on L-serine export, thrE was deleted in SSAAI (SSAAI ΔthrE), which did not produce any significant change in L-serine titer in the deletion mutant (Fig. 3a and 3b). In contrast, deletion of SerE significantly reduced the L-serine titer in SSAAI, and resulted in little change in cell growth (Fig. 1c). The SSAAI ΔserE produced 11.31 g/L L-serine, which was 56.5% lower than that produced by SSAAI (Fig. 1c). Subsequently, thrE and serE double deletion mutant was constructed, which exhibited cell growth comparable to that of SSAAI, and produced 10.34 g/L L-serine, which was 60% lower than that observed in SSAAI (Fig. 3a and Fig. 3b).
Furthermore, thrE and serE were overexpressed alone or in combination in SSAAI to obtain SSAAI-thrE, SSAAI-serE, and SSAAI-serE-thrE. While L-serine accumulation in SSAAI-thrE was similar to that in SSAAI, the production of L-serine in SSAAI-serE reached 28.67 g/L, which was 10.5% higher than that noted in SSAAI (Fig. 3a and 3c). However, a decrease in cell growth was observed in SSAAI-serE before 72 h of fermentation, when compared with that found in SSAAI (Fig. 3d). Furthermore, no significant difference in L-serine titer was found in the time courses of both SSAAI-serE and SSAAI-serE-thrE, and SSAAI-serE-thrE exhibited lower cell growth than SSAAI-thrE before 96 h of fermentation (Fig. 3c and 3d). These observations might be due to the inhibition of cell growth resulting from L-serine over-efflux, metabolic burden of overexpression of two membrane-binding proteins, or inhibition of cell growth by L-threonine over-efflux. Taken together, these findings suggested that SerE plays a more important role than ThrE for L-serine export in SSAAI.
Fig. 3 Effect of the exporters thrE and serE deletion or overexpression on SSAAI. (a) Cell growth (gray bar with slash) and L-serine titer (white bar) of SSAAI, thrE deletion strain SSAAI ∆thrE, serE deletion strain SSAAI ∆serE, thrE and serE deletion strain SSAAI ∆serE ∆thrE, thrE overexpression strain SSAAI-thrE, serE overexpression strain SSAAI-serE, and thrE and serE double overexpression strain SSAAI-serE -thrE. (b) Cell growth (open symbols) and L-serine titer (solid symbols) of SSAAI (squares), serE deletion strain SSAAI ∆serE (circles), thrE deletion strain SSAAI ∆thrE (triangles), and thrE and serE deletion strain SSAAI ∆serE ∆thrE (rhombus). (c) Cell growth (open symbols) and L-serine titer (solid symbols) of SSAAI (squares), serE overexpression strain SSAAI-serE (circles), thrE overexpression strain SSAAI-thrE (triangles), and thrE and serE double overexpression strain SSAAI-serE-thrE (rhombus). (d) The growth rates of SSAAI-serE-thrE (red) and SSAAI (black).
Transcriptional regulator of the novel exporter SerE
The gene NEWCgl0581, located upstream of serE and divergently transcribed from serE (Fig. S2), and its product (consisting of 303 amino acids) was found to be a member of the LysR-type transcriptional regulators (LTTRs) family. It must be noted that LTTRs were initially described as regulators of divergently transcribed genes [21]. In a previous study on C. glutamicum, LysG, located upstream of L-lysine exporter gene lysE, was observed to encode a LysR-type transcriptional regulator, confirming that LysG is a positive transcriptional regulator of lysE [22]. Accordingly, we speculated that NCgl0581 might be involved in the control of serE transcription.
To determine the function of NCgl0581, a mutant strain with NCgl0581 deletion was constructed. As shown in Fig. 4a, the growth of SSAAI ΔNCgl0581 was similar to that of the parent strain SSAAI. However, the L-serine titer of SSAAI ΔNCgl0581 was 11.08 g/L, which was 57.4% lower than that of the parent strain (P<0.001), indicating that NCgl0581 played an important role in L-serine production. Subsequently, the effect of NCgl0581 on serE expression was further investigated by using the probe plasmid pDXW-11. Two recombinant strains, SSAAI ΔNCgl0581-1 (harboring the plasmid pDXW-11-1, Fig. 4b) and SSAAI ΔNCgl0581-0 (harboring the plasmid pDXW-11-0, Fig. 4c) were constructed, and their fluorescence during fermentation was measured. The fluorescence of SSAAI ΔNCgl0581-1 was stronger than that of SSAAI ΔNCgl0581-0 during the fermentation process (Fig. 4d), revealing that NCgl0581 functioned as a positive regulator of serE expression. To verify whether the regulatory protein NCgl0581 binds to the upstream region of SerE, EMSA was performed by using the DNA probe labeled with biotin, and the result clearly indicated that NCgl0581 binds to this region (Fig. 4e).
Fig. 4 Verification of the function of NCgl0581. (a) The cell growth (squares) and L-serine titer (circles) of SSAAI (solid symbols) and NCgl0581 deletion strain SSAAIΔNCgl0581 (open symbols), respectively. (b) Plasmid pDXW-11-1 containing fragments of NCgl0581 (gray), intergenic region between NCgl0581 and NCgl0580 (black), and EGFP (green). (c)Plasmid pDXW-11-0 containing fragments of the intergenic region between NCgl0581 and NCgl0580 (black) and EGFP (green). (d) Fluorescence of the two strains, SSAAI ΔNCgl0581-1 (gray bar with slash) and SSAAIΔNCgl0581-0 (white bar). (e) Verification of NCgl0581 binding to the upstream region of SerE by using EMSA. Lane 1: the nuclear extracts with activated specific TF (positive control), Lane 2: the nuclear extracts without activated TF (negative control), Lane 3: Sample.
To confirm whether NCgl0581 is a specific regulator of SerE, transcriptome sequencing was performed using SSAAI and NCgl0581 deletion strain. The findings showed that the transcription levels of 115 genes were altered, including 56 upregulated genes and 59 downregulated genes, in response to NCgl0581 deletion, indicating that NCgl0581 is a global transcriptional regulator in C. glutamicum. The genes with significant transcriptional change (≥4-fold) are shown in Tables 1 and 2.
Table 1 Genes significantly upregulated by NCgl0581 deletion
Gene id
|
SSAAI Δ0581
|
SSAAI
|
Fold change
|
Protein function
|
NCgl2897
|
701.56
|
71.07
|
9.87
|
Starvation-inducible DNA-binding protein
|
NCgl0546
|
17.78
|
2.75
|
6.45
|
Hypothetical protein
|
NCgl1405
|
15.94
|
2.71
|
5.88
|
ABC transporter periplasmic component
|
NCgl1302
|
10.05
|
1.96
|
5.13
|
Aldo/keto reductase
|
NCgl1344
|
286.87
|
55.96
|
5.12
|
Ornithine carbamoyltransferase
|
NCgl1343
|
280.65
|
57.24
|
4.9
|
Acetylornithine aminotransferase
|
NCgl0746
|
43.30
|
9.04
|
4.7
|
Hypothetical protein
|
NCgl1342
|
134.70
|
29.07
|
4.63
|
Acetylglutamate kinase
|
NCgl2946
|
672.93
|
155.87
|
4.31
|
Hypothetical protein
|
NCgl1022
|
89.53
|
21.28
|
4.20
|
Cysteine sulfinate desulfinase
|
NCgl1023
|
368.88
|
88.67
|
4.15
|
Nicotinate-nucleotide pyrophosphorylase
|
NCgl1341
|
108.49
|
27.09
|
4.00
|
Bifunctional ornithine acetyltransferase/N-acetylglutamate synthase
|
Table 2 Genes significantly downregulated by NCgl0581 deletion
Gene id
|
SSAAI Δ0581
|
SSAAI
|
Fold change
|
Protein function
|
NCgl0580
|
18.40
|
5152.54
|
280.02
|
Hypothetical protein
|
NCgl0638
|
1.71
|
20.97
|
12.22
|
ABC transporter permease
|
NCgl0639
|
11.00
|
82.47
|
7.49
|
ABC transporter periplasmic component
|
NCgl2943
|
207.03
|
1355.55
|
6.54
|
Hypothetical protein
|
NCgl0943
|
16.19
|
103.52
|
6.39
|
AraC family transcriptional regulator
|
NCgl0484
|
2.32
|
14.57
|
6.28
|
ABC transporter permease
|
NCgl2942
|
283.52
|
1776.15
|
6.26
|
NADH:flavin oxidoreductase
|
NCgl0166
|
13.41
|
79.70
|
5.94
|
Hypothetical protein
|
NCgl0324
|
2.11
|
11.87
|
5.61
|
Zn-dependent alcohol dehydrogenase
|
NCgl0282
|
5.19
|
28.25
|
5.44
|
4-Hydroxyphenyl-beta-ketoacyl-CoA hydrolase
|
NCgl1975
|
102.94
|
503.75
|
4.89
|
Hypothetical protein
|
NCgl2893
|
1.25
|
6.08
|
4.84
|
Efflux system protein
|
NCgl0155
|
9.11
|
43.69
|
4.79
|
5-Dehydro-2-deoxygluconokinase
|
NCgl0014
|
10.02
|
47.76
|
4.76
|
Hypothetical protein
|
NCgl2953
|
7.68
|
35.80
|
4.66
|
Sugar permease
|
NCgl2744
|
12.26
|
55.19
|
4.50
|
Hypothetical protein
|
NCgl2970
|
15.22
|
67.51
|
4.43
|
ABC transporter periplasmic component
|
NCgl0608
|
23.06
|
100.35
|
4.35
|
ABC transporter permease
|
NCgl0258
|
4.51
|
19.50
|
4.32
|
Arsenite efflux pump ACR3
|
NCgl0281
|
16.83
|
67.69
|
4.02
|
Dehydrogenase
|
The transcriptional level of serE was significantly decreased by 280-fold following NCgl0581 deletion, revealing that NCgl0581 is a positive regulator of serE. Furthermore, NCgl0581 deletion downregulated the two ABC transporter permeases (NCgl0638 and NCgl0484) and ABC transporter periplasmic component (NCgl0639) by 12-, 6.3-, and 7.5-fold, respectively, and upregulated ABC transporter periplasmic component (NCgl1405) by 5.88-fold, suggesting that NCgl0581 is involved in the synthesis of substances transported through ABC transporter.
Overexpression of SerE and NCgl0581
As NCgl0581 could activate the expression of SerE in SSAAI, the overexpression of NCgl0581, serE, or their co-expression was studied, and strains SSAAI-NCgl0581 and SSAAI-NCgl0581-serE were constructed respectively. As shown in Fig. 5a and Fig. 5b, a decrease in cell growth was observed in SSAAI-NCgl0581-serE and SSAAI-NCgl0581 before 96 h of fermentation, and SSAAI-NCgl0581-serE showed the lowest growth rate, the time courses for L-serine production were similar in all the strains. Furthermore, the yield of L-serine to biomass (Yp/x) increased in both SSAAI-NCgl0581-serE and SSAAI-NCgl0581 (Fig. 5c and 5d), suggesting that overexpression of a novel exporter SerE and its transcriptional regulator NCgl0581 was beneficial for L-serine efflux, but not for cell growth. Besides, SSAAI-NCgl0581-serE and SSAAI-NCgl0581 exhibited 9.67% (p <0.05) and 19.17% higher Yp/x in 96 h (p<0.01), respectively, when compared with SSAAI. A similar decrease in cell growth was observed in SSAAI-serE (Fig. 3c); however, the L-serine titer was 28.67 g/L, which was 10.5% higher than that noted in SSAAI. This decrease in cell growth in the recombinant strain could be due to the transportation of the synthesized L-serine out of the cell, resulting in inadequate intracellular L-serine for cell growth. Therefore, our subsequent investigation involved replenishment of L-serine by overexpressing L-serine synthetic pathway key enzyme.
Fig. 5 Effect of serE and NCgl0581 deletion or overexpression on SSAAI. (a) Cell growth (open symbols) and L-serine titer (solid symbols) of SSAAI (squares), NCgl0581 overexpression strain SSAAI-NCgl0581 (circles), and NCgl0581 and serE double overexpression strain SSAAI-NCgl0581-serE (triangles). (b) The growth rates of SSAAI-NCgl0581 (red), SSAAI-NCgl0581-serE (blue) and SSAAI (black). (c) Yp/x of SSAAI (gray bar with slash) and NCgl0581 overexpression strain SSAAI-NCgl0581 (white bar). (d) Yp/x of SSAAI (gray bar with slash) and NCgl0581 and serE double overexpression strain SSAAI-NCgl0581-serE (white bar).
High yield production of L-serine through SerE combined with synthetic pathway
To direct more flux to L-serine synthesis, L-serine exporter SerE and L-serine synthetic pathway key enzyme (containing a feedback insensitive serAΔ197, serC, and serB encoding the deregulated 3-phosphoglycerate dehydrogenase, phosphoserine phosphatase, and phosphoserine aminotransferase, respectively) were co-overexpressed in SSAAI to obtain SSAAI-serE-serAΔ197-serC-serB. The recombinant strain shared similar typical growth curves as the parent strain SSAAI, and achieved a final L-serine titer of 32.8 g/L, which was 22.1% higher than that noted in SSAAI (p<0.001). Subsequently, L-serine exporter serE, serAΔ197, serC and serB were overexpressed in strain A36 to obtainA36-serE-serAΔ197-serC-serB. A36 was stemmed from SSAAI by using ARTP mutation, which produced 34.78 g/L L-serine [23], As shown in Fig. 6, the tandem expression strain A36-serE-serAΔ197-serC-serB shared similar typical growth curves as the parent strain A36 in the overall process. Furthermore, the sucrose level decreased with time in a similar pattern in both the strains. Interestingly, when the incubation time of batch cultivations exceeded 72 h, the cell growth and L-serine titer of A36-serE-serAΔ197-serC-serB were higher than those of the parent strain A36. After 120 h of cultivation, A36-serE-serAΔ197-serC-serB consumed all of the sucrose and achieved a final L-serine titer of 43.9 g/L, with a conversion rate of 0.44 g/g. These results demonstrated that overexpression of L-serine exporter in combination with L-serine synthetic pathway could facilitate L-serine production in C. glutamicum.
Fig. 6 Fermentation process of strain A36 and strain A36-serE-serAΔ197-serC-serB. The cell growth (open symbols), L-serine titer (solid symbols), and residual sucrose (gray symbols) of strain A36 (squares) and A36-serE-serAΔ197-serC-serB (circles) are presented. Three parallel experiments were performed. Error bars indicate standard deviations of the results from three parallel experiments.