Investigating whether l-serine could be reimported by E. coli
To determine whether l-serine was reimported, E. coli ES was inoculated into Luria-Bertani (LB) medium with an additional 2 g/L or 4 g/L l-serine. An HPLC chromatogram of the l-serine standard solution is shown in Additional file 1: Fig. S1. As shown in Fig. 2, the l-serine concentration decreased to 1.05 g/L and 1.17 g/L at 4 h. Then, the l-serine concentrations were further reduced to less than 0.15 g/L at 6 h. It was observed that the maximum specific growth rates of the strains with an additional 2 g/L and 4 g/L l-serine were 1.47 h−1 and 1.65 h−1, respectively, which were 1.12- and 1.26-fold that of the corresponding control strain, respectively. The final optical densities at 600 nm (OD600) of the strains in LB medium with an additional 2 g/L and 4 g/L l-serine increased by 56% and 67%, respectively, compared to that of the control (OD600 ~ 5.3). This demonstrated that l-serine could be imported and metabolized and provided easily assimilable carbon and nitrogen sources for cell growth. This raises the question of how the uptake takes place and whether it has any effect on l-serine accumulation.
Relevance of transporters for l-serine uptake
The four genes sdaC, cycA, sstT and tdcC, which were reported to be related to l-serine uptake, were overexpressed in E. coli ES [16,17,18,19,20,21,22,23]. The l-serine uptake activity of these strains was evaluated. As shown in Fig. 3, the l-serine uptake activity (30 min) of ES/pSC-12, ES/pSC-11, ES/pSC-14 and ES/pSC-13 was 2.983, 2.79, 2.3 and 2.166 nmol min−1 (mg dry weight)−1, respectively, increasing by 186%, 168%, 121% and 108% compared to that of the parent strain of ES (1.04 nmol min−1 (mg dry weight)−1). The time courses of l-serine uptake are shown in Additional file 2: Fig. S2. This result indicated that cycA and sdaC played critical roles and that sstT and tdcC were also important in l-serine uptake activity.
Effect of single-gene deletions on l-serine uptake
The four genes, sdaC, cycA, sstT and tdcC, were knocked out, resulting in strains ES-1, ES-2, ES-3 and ES-4. As shown in Fig. 4 and Additional file 2: Fig. S3, the l-serine uptake activity (30 min) of ES-1 was 0.79 nmol min−1 (mg dry weight)−1, decreasing by 23% compared to that of the parent strain of ES (1.04 nmol min−1 (mg dry weight)−1). It was surprising that the l-serine uptake activity of ES-2, ES-4 and ES-3 increased by 77%, 48% and 33% compared to that of the control. This abnormal phenomenon was explored through a series of real-time quantitative reverse‐transcription PCR (RT-qPCR) experiments. As shown in Fig. 5, deletion of the cycA and tdcC genes led to a 1.76- and 1.15-fold increase in the relative expression of sdaC, respectively, which was consistent with the increase in l-serine uptake activity in the two mutants. However, the sstT mutants showed similar expression of sdaC with increased l-serine uptake activity compared to ES. These results illustrated that certain regulatory mechanisms of the l-serine uptake system remain unknown.
Effect of multigene deletions on l-serine uptake
The above results demonstrated that sdaC played a significant role in the l-serine uptake system, but the roles of cycA, sstT and tdcC in the uptake system remained unclear. Therefore, the three genes were knocked out combinatorially in the single-gene-deletion strain ES-1, resulting in seven multigene-deletion strains. The mutant strains were named ES-12 (ES ΔsdaCΔcycA), ES-13 (ES ΔsdaCΔsstT), ES-14 (ES ΔsdaCΔtdcC), ES-123 (ES ΔsdaCΔcycAΔsstT), ES-124 (ESΔsdaCΔcycAΔtdcC), ES-134 (ES ΔsdaCΔsstTΔtdcC) and ES-1234 (ES ΔsdaCΔcycAΔsstTΔtdcC). As shown in Fig. 6 and Additional file 2: Fig. S4, among the double-gene-deletion strains, both strains ES-12 and ES-13 showed a low l-serine uptake activity of nearly 0.50 nmol min−1 (mg dry weight)−1, decreasing by 52% compared to that of ES. However, another double-gene deletion strain, ES-14, showed 0.846 nmol min−1 (mg dry weight)−1l-serine uptake activity, which was similar to that of ES-1. Among the triple-gene deletion mutants, the l-serine uptake activity of ES-123 was 0.347 nmol min−1 (mg dry weight)−1. ES-124 and ES-134 showed an l-serine uptake activity of nearly 0.24 nmol min−1 (mg dry weight)−1, decreasing by 76% compared to that of ES. Finally, when all four genes were deleted (ES-1234), the l-serine uptake activity decreased to near zero, which implied that the strain could not import extracellular l-serine effectively.
The impact of the deletion of l-serine uptake genes on growth and l-serine production in shake flask fermentation
To evaluate the l-serine production capability of the l-serine uptake system mutants, the mutant strains were transformed with the plasmid pSC-08 containing the l-serine synthesis genes serAfr, serB and serC and the 3-phosphoglycerate kinase gene pgk and shake flask fermentation was performed. Among single-gene deletion mutants, deletion of sdaC, sstT and tdcC had a slight effect on the density of the cell cultures (Fig. 7), including that all strains in which cycA was deleted showed poor growth, and ES-2/pSC-08 showed a low final OD600 of 2.19, which was 30% less than that of ES/pSC-08 (OD600 ~ 2.9). The strains exhibited poor growth when more genes were knocked out. For example, the final OD600 values of ES-1/pSC-08, ES-13/pSC-08 and ES-134/pSC-08 were 3.2-, 2.43- and 2.23, respectively, which were 1.1-, 0.82- and 0.77-fold the value for ES/pSC-08. respectively.
In addition, as shown in Fig. 7, the strains with lower l-serine uptake activity exhibited higher l-serine production. For instance, ES-1/pSC-08, ES-13/pSC-08 and ES-134/pSC-08 showed 23%, 48% and 77% lower l-serine uptake activity and 69%, 196% and 242% higher l-serine production, respectively, than the control strain ES/pSC-08 (1.04 nmol min−1 (mg dry weight)−1, 130 mg/L). ES-134/pSC-08 achieved the highest l-serine production (445 mg/L) in shake flask fermentation. However, ES-1234/pSC-08, which had nearly no l-serine uptake activity, produced only 384 mg/L l-serine and showed the poorest cell growth.
Fed-batch fermentation in a 5-L fermenter
The four high-yield strains in shake flask fermentation, namely, ES-1/pSC-08, ES-13/pSC-08, ES-134/pSC-08 and ES-1234/pSC-08, were selected for a 36-h fed-batch fermentation in a 5-L fermenter. As shown in Fig. 8 and Additional file 3: Table S1, ES-1/pSC-08 produced 23.8 g/L l-serine with a yield of 0.25 g l-serine/g glucose. The production of ES-13/pSC-08 further increased to 29.6 g/L with a yield of 0.31 g l-serine/g glucose. As expected, the triple-deletion mutant strain ES-134/pSC-08 showed the highest production of 34.8 g/L with a yield of 0.32 g l-serine/g glucose, increasing by 46% compared to that of ES-1/pSC-08. The concentration of l-serine detected at the end of the ES-1234/pSC-08 fed-batch culture was 26.3 g/L, which was similar to that of ES-1/pSC-08, due to the lowest biomass (OD600 ~ 30) of ES-1234/pSC-08.
During the effective l-serine production time (12–36 h), the productivity of ES-1/pSC-08, ES-13/pSC-08, ES-134/pSC-08 and ES-134/pSC-08 was in the range of 23–36, 31–47, 40–56 and 46–57 mg l-serine/g CDW h−1, respectively (Fig. 8). Additionally, ES-134/pSC-08 achieved the highest l-serine titer. However, the l-serine production of ES-1234/pSC-08 was affected by its lowest OD600 although it showed the highest l-serine productivity.