High yield production of L-serine through a novel identified exporter combined with synthetic pathway in Corynebacterium glutamicum [post]

2020 unpublished
L-Serine has wide and expanding applications in industry with a fast-growing market demand. Strategies for achieving and improving L-serine production in C. glutamicum have focused on inhibiting its degradation and enhancing its biosynthetic pathway, however, L-serine yield remains relatively low. Exporters play an essential role in the fermentative production of amino acids. Improvements to L-serine yield should consider the improvement of L-serine export from the cell. In C. glutamicum ,
more » ... . glutamicum , ThrE, which can export L-threonine and L-serine, is the only identified L-serine exporter so far. Results In this study, a novel L-serine exporter NCgl0580 was identified and characterized in C. glutamicum ΔSSAAI (SSAAI), and named as SerE (encoded by serE ). Deletion of serE in SSAAI led to a 56.5% decrease in L-serine titer, whereas overexpression of serE compensated for the lack of serE with respect to L-serine titer. A fusion protein with SerE and enhanced green fluorescent protein (EGFP) was constructed to confirm that SerE localized at the plasma membrane. The function of SerE was studied by peptide feeding approaches, and the results showed that SerE is a novel exporter for L-serine and L-threonine in C. glutamicum. Subsequently, the interaction of a known L-serine exporter ThrE and SerE was studied, and the results suggested that SerE is more important than ThrE in Lserine export in SSAAI. In addition, probe plasmid and electrophoretic mobility shift assays (EMSA) revealed NCgl0581 as the transcriptional regulator of SerE. Comparative transcriptomics between SSAAI and the NCgl0581 deletion strain showed that NCgl0581 is a positive regulator of NCgl0580. Finally, by overexpressing the novel exporter SerE combined with L-serine synthetic pathway key enzyme serA Δ197, serC and serB , the resulting strain lead to the L-serine titer of 43.9 g/L with yield of 0.44 g/g sucrose, which was the highest yield reported so far for any organism. Conclusions This study provides a novel target for L-serine and L-threonine export engineering as well as a novel global transcriptional regulator NCgl0581 in C. glutamicum . Background L-serine has been identified as one of the top 30 most interesting building blocks for a range of chemicals and materials, and is used in cosmetic, pharmaceutical, and food industries [1, 2] . 4 Metabolic engineering of Corynebacterium glutamicum (C.glutamicum) for L-serine production has been focused on its terminal synthesis pathways and degradation pathways, and proven to be very useful for improving L-serine production in this organism [3] [4][5][6]; however, the L-serine productivity is still low for large-scale L-serine production, the highest reported L-serine titer reached 42.62 g/L and 50 g/L with the yield to be 0.21 g/g sucrose and 0.26 g/g glucose in C. glutamicum and E.coli respectively, however, L-serine has the potential to be made from sugar by fermentation with a very high theoretical yield (1.17 g/g glucose,1.22 g/g sucrose) [2] . Improvements to L-serine production should consider the improvement of L-serine export from the cell. Export plays an essential role in metabolic engineering strategies for production of amino acids [7], as it reduces intracellular amino acid concentrations, and thereby alleviates feedback inhibition and circumvents toxicity problems [1, [8][9][10]. In recent decades, a number of export systems have been identified for excreting amino acids, such as L-lysine, L-cysteine, L-glutamate, L-threonine, Larginine, L-methionine, and branched-chain amino acids, in C. glutamicum and E.coli [11] [12] [13] [14] [15] [16] [17] . However, to the best of our knowledge, except for ThrE (L-threonine and L-serine exporter) [15, 18] , no other L-serine exporters have been reported in C. glutamicum so far. In E. coli, Mundhada et al. found that intracellular L-serine accumulation was toxic to the engineered strain modified to produce L-serine, and that following overexpression of eamA (which encodes L-cysteine exporter in E. coli), the engineered strain exhibited increased tolerance toward L-serine with higher L-serine productivity [2] . Therefore, L-serine exporter in C. glutamicum could be a potential target for strain optimization to further improve L-serine production. It has been reported that homologs similar to the exporters in E. coli might fulfil a comparable function in C. glutamicum [17, 19, 20]. Accordingly, we hypothesized that the homolog to eamA (Lserine exporter in E. coli) might be involved in L-serine export in C. glutamicum. In the present study, three homologs to eamA, namely, NCgl2050, NCgl2065, and NCgl0580, were determined, and their functions were identified by targeted gene deletion, respectively. The results showed that one of the genes, NCgl0580, was involved in L-serine export. Subsequently, localization and function of NCgl0580 were investigated, and the interaction of a known L-serine exporter ThrE (encoded by thrE) 5 and the novel exporter NCgl0580 was studied. Furthermore, the transcriptional regulator of NCgl0580 was identified and studied. Finally, the effect of overexpression of L-serine exporter in combination with L-serine synthetic pathway enzyme to L-serine production were evaluated. Results Exploring putative L-serine exporters in C. glutamicum In past 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 (Lserine exporter in E. coli) [2] might be involved in L-serine export in this organism. 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, 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 respectively deleted in this strain. 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) . 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 , when compared with SSAAI, the strain harboring the plasmid grew slowly to some extent in the logarithmic growth phase, finally reaching similar cell growth to 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. 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 glutamicum. Transcriptional regulators and their roles in expression control of target genes are important for metabolic engineering of C. glutamicum for industrial applications [36] , and this study provided a new member of transcriptional regulator family. When L-serine exporter SerE was overexpressed alone, which resulted in increase of 10.8% of Lserine titer, at same time, a decrease in cell growth was observed in recombinant strain, we inferred that L-serine was transported out of the cell, when L-serine was synthesized, and the intracellular Lserine was not enough for the cell growth, to replenish L-serine by overexpressing L-serine synthetic pathway key enzyme, the cell growth was restored, and L-serine titer was increased to 43.9 g/L, and L-serine yield was 0.44 g/g sucrose, which were the highest titer and yield reported so far for any organism. The results indicated that serAΔ197, serC, serB overexpression ensured sufficient L-serine supply avoided the cell growth inhibition. In Mundhada's study, 37 g/L of L-serine was produced with a yield of 0.24 g/g glucose in E. coli [2] , and in their previous study, L-serine titer was 11.7 g/L with highest yield of 0.43 g/g glucose [31] . Interestingly, we found that L-serine titer increased significantly by overexpressing serB in A36, with L-serine titer of 37.9 g/L, which was 24% higher than that of A36, serB encoded phosphoserine phosphatase (PSP, EC 3.1.3.3), catalyzed the last step of Lserine biosynthesis, however, L-serine titer had not significantly change when serAΔ197, serC was overexpressed in A36 respectively (with L-serine titer of 31.1 g/L and 32.78 g/L) (Fig.S5) , and these results were in consistent with the previous report completely, in which overexpression of the mutant allele serAΔ197 in C. glutamicum13032 either alone or in combination with overexpression of serC and serB did not result in significant L-serine accumulation [3],which might be due to the poor accumulation and intracellular conversion of L-serine in C. glutamicum13032. In a recent study, 50 g/L of L-serine was produced with glucose as carbon source in E. coli, with a yield of 0.26-0.30 g/g glucose [37], in which 50 g/L is the highest reported so far for L-serine production, however, the yield was also lower than our present study (0.44 g/g sucrose). It is possible that fine controlling the three enzymes of the L-serine biosynthesis pathway to enhance L-serine production further. Conclusion 15 In the present work, a novel exporter SerE and its positive regulator NCgl0581 were identified in C. glutamicum, with SerE also exhibiting the ability to accept L-threonine and NCgl0581 acting as a novel global transcriptional regulator in C. glutamicum, and by overexpressing novel exporter combined with L-serine synthetic pathway enzyme, increased L-serine yield significantly. These results enrich our understanding of amino acid transport and can provide additional targets for exporter engineering in C. glutamicum. Materials And Methods Strains, plasmids, and growth conditions The strains and plasmids used in this study are listed in Table 3 . E. coli JM109 was used as the cloning host, and was grown in lysogeny broth (LB) medium (containing 5.0 g/L yeast extract, 10.0 g/L tryptone, and 10.0 g/L NaCl) at 37 o C and 220 rpm. The engineered SSAAI (CGMCC No.15170) was constructed in our laboratory by knocking out 591 bp of the C-terminal domain of serA, deleting sdaA, avtA, and alaT, as well as attenuating ilvBN in the genome of C. glutamicum SYPS-062-33a (CGMCC No. 8667). The seed and fermentation media for C. glutamicum were prepared as described previously [5]. The C. glutamicum strains were pre-incubated in the seed medium overnight to an optical density (OD 562 ) of about 25, and then inoculated at an initial concentration of OD 562 =1 into a 250 mL flask containing 25 mL of the fermentation medium at 30 o C and 120 rpm. The antibiotic kanamycin (50 mg/L) was added when necessary. Samples were withdrawn periodically for the measurement of residual sugar, amino acids, and OD 562 as described previously [5]. TABLE 3 Strains and plasmids used in this study. Strain/Plasmid Description E. coli JM109 recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1 C. glutamicum SSAAI C. glutamicum SYPS-33a with deletion of the 591 bp in the C-terminus of serA example, to achieve thrE deletion, the homologous-arm fragments for thrE deletion were amplified from SSAAI chromosome using the primer pairs thrE1/2 for the upstream fragment and thrE3/4 for the downstream fragment. Then, with the two fragments as templates, a crossover PCR was performed using the primer pair thrE1/4. The truncated product of thrE was digested with XbaⅠ and HindⅢ and ligated to the vector pK18mobsacB that was similarly treated. The recombinant plasmid pK18mobsacBΔthrE was transformed into SSAAI competent cells by electroporation, and chromosomal deletion was performed by selecting cells that were kanamycin resistant and sucrose nonresistant, and verified by PCR. The pDXW-10 and pDXW-11 plasmids were used to overexpress genes in C. glutamicum [39, 40]. The recombinant plasmids were constructed as follows: the genes thrE and serE were amplified, digested, and ligated to the pDXW-10 plasmid that was digested with HindⅢ/BglⅡ. The plasmid harboring the fusion protein, SerE-EGFP (enhanced green fluorescent protein) was constructed by using the method reported in a previous study [19] . To confirm the role of NCgl0581 on NCgl0580 expression, the fragment consisting of intergenic region of NCgl0581 and NCgl0580 and EGFP with or without NCgl0581 was ligated to the plasmid pDXW-11 by Clon Express MultiS One Step Cloning Kit (Vazyme, Nanjing, China). The strains were constructed by electroporation with the corresponding plasmids. The genes, serAΔ197, serB, and serC, were PCR amplified from SSAAI using primers shown in Table S2 . To construct plasmid pDXW-10-serAΔ197, the resultant fragment of serAΔ197 was digested with EcoRI and NotI and cloned into pDXW-10. To construct plasmid pDXW-10-serAΔ197-serB-serC, PCR fragments of serB, and serC were digested with the appropriate restriction enzymes and successively cloned into the corresponding plasmids to form plasmid pDXW-10-serAΔ197-serB-serC. The resulting plasmid (pDXW-10-serAΔ197-serB-serC) was then subjected to double digestion by NdeI and PacI for cloning of NCgl0580 to form pDXW-10-serAΔ197-serB-serC-NCgl0580. Confocal microscopic observation The strains SSAAI-10 (SSAAI harboring plasmid pDXW-10), SSAAI-egfp, and SSAAI-serE-egfp were grown in the seed medium and harvested during the exponential phase. The cells were washed twice 19 and maintained in PBS (pH 7.4), mounted on a microscope slide, and observed under a Leica laser scanning confocal microscope (Leica, TCS SP8; Leica, Wetzlar, Germany) equipped with a HC PL Apo 63x/1.40 Oil CS2 oil-Immersion objective, with excitation filter at 488 nm and emission filter at 510-550 nm. The digital images were acquired and analyzed with Lecia Application Suite X 2.0. Membrane and cytoplasmic protein extraction and fluorescence measurements The strains SSAAI-10, SSAAI-egfp, and SSAAI-serE-egfp were used for extracting membrane and cytoplasmic proteins to determine SerE localization. The extraction was performed using Membrane and a Cytoplasmic Protein Extraction Kit according to the manufacturer's protocol (Beyotime, Nanjing, China). The cells were washed twice with PBS (pH 7.4) and disrupted by ultrasonication on ice (pulse, 4 s; interval, 6 s; total duration, 30 min) (Sonics Vibra-Cell ™ , Sonics, Newtown, CT, USA). The supernatant containing cytoplasmic proteins was collected by centrifugation (700 × g, 4 o C for 10 min), and the precipitate was used for extracting membrane proteins. The protein concentration was determined with a Modified BCA Protein Assay Kit (Sangon, China). After extraction, the fluorescence intensity (excitation at 488 nm, emission at 517 nm) of the membrane and cytoplasmic proteins was determined using fluorescence spectrophotometer (Synergy H4; BioTek, Winooski, VT, USA). Amino acid export assay For ascertaining the function of serE, a dipeptide Ser-Ser addition assay was conducted [15] . In brief, pre-incubated cells (in seed medium) were washed once with CGXⅡ minimal medium [41], inoculated into CGXⅡ minimal medium with 2 mM Ser-Ser (other dipeptide), and incubated for 2 h at 30 o C. Then, the cells were harvested, washed once with cold CGXⅡ minimal medium, and resuspended in CGXⅡ minimal medium. Amino acid excretion was initiated by adding 2 mM Ser-Ser (another dipeptide). HPLC was used to determine the concentrations of amino acids [19] . Analytical procedures Cell density (OD 562 ) was measured using an AOE UV-1200S UV/vis spectrophotometer (AOE Instruments Co. Inc., Shanghai, China). Glucose concentration was determined using SBA-40E glucose analyzer (Biology Institute of Shandong Academy of Sciences, China). For measurement of 20 extracellular L-serine concentration in shake-flask fermentation, 500 μL of the culture were centrifuged at 700 ×g for 5 min, and the supernatant was used for detection after appropriate dilution. To ascertain intracellular L-serine concentration, 300 μL of the culture were centrifuged at 700 ×g and 4 o C for 10 min, and 300 μL of water were added to the cells. The cells were disrupted by FastPrep-24 5G instrument (5 m/s, 120 s, MP Biomedicals, Shanghai, China). The cytoplasmic volume was assumed to be 2 μL/mg dry cell weight [27]. The titers of intracellular and extracellular L-serine and other amino acids were analyzed by HPLC using phenyl isothiocyanate as a precolumn derivatization agent, according to our previously study [8].
doi:10.21203/rs.3.rs-16998/v1 fatcat:lj2hgofnw5bbbaaqs35ns7lxdm