2.1 Construction of the L-serine production strain from E. coli W3110
As a prerequisite for L-Ser production, the activity of the branch pathway leading to L-Ser biosynthesis, which involves serA, serC and serB, was enhanced. PGDH, encoded by serA, catalyzes the initial reaction in L-Ser biosynthesis and the catalytic activity of PGDH can be regulated by feedback inhibition by L-Ser in E. coli. The feedback inhibition was overcome by mutation of two residues (344 and 346) to alanine, as previously described, which would remove the hydrogen bonds between L-Ser and the regulatory binding domain. This led to the mutated gene named serAfr. The feedback resistance of the enzyme PGDH, encoded by serAfr, was investigated by overexpressing these genes in BL21(DE3) via the pT7-7 vector. The activity of serAfr could was sustained at 95% with 80 mmol/L L-Ser, whereas the activity of the wild-type enzyme remained at only 10% (Fig. 2A). Then, serAfr, serC, and serB were overexpressed in the low copy number pSC vector containing the PR or PL promoter with resulting in SP-01, SP-02 and SP-03 (Fig. 2B).
To produce L-Ser, the sdaA gene encoding the L-Ser-specific dehydratase was first deleted from E. coli W3110 to construct the SSW-01 strain. Subsequent deletion of glyA, encoding SHMT, which converts L-Ser to glycine, resulted in the double knockout SSW-02 strain. To evaluate the L-Ser production capacity, the resulting plasmids SP-01 (SP-serAfr), SP-02 (SP-serAfrC) and SP-05(SP-serAfrBC) were transformed into SSW-02. As shown in Fig. 2C, strain SSW-02/SP-01 was grown in M9-yeast medium supplemented with 50 mmol glucose, and the final concentration of L-Ser was 155 mg/L after 15 h in a shake flask. An L-Ser concentration of 220 mg/L, 42% higher than that obtained by culturing SSW-02/SP-01, was obtained by culturing SSW-02/SP-02. SSW-02/SP-05 attained the highest L-Ser concentration, 270 mg/L, which exhibited 1.74-fold increase compared to only overexpressing serAfr. The L-Ser accumulation profile shown in Fig. 2C, indicates that the production of L-Ser increased as more biosynthetic genes were overexpressed.
Furthermore, previous studies have shown that only 15% of the carbon assimilated from glucose is directed into the L-Ser biosynthetic pathway in E. coli. Hence, SP-serAfrBCpgk (SP-08) was then constructed to increase the carbon flux from glucose to L-Ser and improve L-Ser productivity via amplification of phosphoglycerate kinase encoded, by pgk (Fig. 2B). Flask culture of the recombinant SSW-02/SP-08 strain produced a final L-Ser concentration of 311 mg/L, 15% higher than that obtained by culturing SSW-02/SP-05 (Fig. 2C). Thus, overexpression of pgk effectively improved the L-Ser production capacity of the strain. To further examine L-Ser production of SSW-02/SP-08, fed-batch fermentation was performed in a 5-L fermenter. The highest L-Ser concentration, 17.7 g/L, was observed at 32 h with a yield of 24% from glucose (Fig. 2D).
2.2 Influence of mutations in glyA on L-serine production and cell growth
A previous study showed that attenuation of glyA transcription resulted in increased L-Ser accumulation, a decrease in the purine pool, poor growth and cell elongation (Fig S1, Additional file 1)[25, 26]. The same phenomenon was observed in this study; SSW-02 cells were elongated and exhibited unstable growth at the early stage of fermentation. We reprogrammed the predominant one-carbon source metabolism with suppressed SHMT activity to increase the stability of the strains. A series of error-prone PCRs were employed to construc a glyA mutation library. Different reductions in SHMT activity were obtained and examined by transforming the recombinant plasmids harboring glyAmut into BL21(DE3). As shown in Table 4, SHMT encoded by glyAmut (K229G) showed an activity of 0.13 U, which decreased by 41% compared to wild type. The mutant K229G was modeled by SWISSMODEL based on the wild type SHMT (PDB ID, 1DFO). As shown in Fig. 3A, close view of the SHMT K229G mutant compared with the wild type SHMT complexed with cofactor LPL (pyridoxal 5′-phosphate) and THFA (PDB ID, 1DFO). The side chain of the K229, which involved the degradation of L-Ser, was removed to obtain the mutant K229G . Sequentially, the glyA gene in SSW-01 was replaced with the appropriate glyAmut(K229G) via CRISPR/Cas9 to generate SSW-03 (△sdaA glyAmut). Then, the L-Ser biosynthesis plasmid SP-08 was transformed into SSW-03, and cell growth and L-Ser production were evaluated. As shown in Fig. 3B, glyAmut introduction resulted in a 24% increase in biomass, and cultured cells maintained stable growth throughout repeated experiments. SSW-03/SP-08 produced 21.6 g/L of L-Ser, an increase of 22% compared to SSW-02/SP-08.
2.3 Influence of sdaB, ilvA, tdcB and tdcG deletion on L-serine production
The L-Ser production capacity of E. coli was significantly increased by overexpression of serAfr, serB, serC and pgk via knockout of the sdaA and mutation of glyA. The four genes other than sdaA and glyA, i.e., sdaB, ilvA, tdcB and tdcG, have been reported to transform L-Ser to pyruvate in E. coli [29, 30]. However, previous studies focused mainly on decreasing the degradation of L-Ser by deleting all of these genes simultaneously, and few studies have systematically investigated the individual contribution of these degradation genes to L-Ser production. To prevent the degradation and improve the production of L-Ser, sdaB, ilvA, tdcB and tdcG were knocked out individually in the SSW-03 background to generate strains SSW-05, SSW-06, SSW-07 and SSW-08 (Fig. 4A). The plasmid SP-08 was transformed into these mutant strains to produce L-Ser. As shown in Fig. 4B, strain SSW-05/SP-08, which had sdaB deletion, showed the highest L-Ser production of 26.5 g/L, an increase of 23% compared to SSW-03/SP-08.This result was expected, because the SSW-05/SP-08 biomass was also increased by 16%, and SSW-05/SP-08 showed an L-Ser productivity of nearly 0.87 g/L/h at 28 h. While the ilvA gene was knocked out, the growth of the strains was severely inhibited, and production could not be induced during fermentation of SSW-07/SP-08 (Fig. 4C). The growth restriction of SSW-06/SP-08 may be due to disruption of branched chain amino acid synthesis by deletion of ilvA. Regarding the tdcB gene, the marginal difference in the L-Ser titer and biomass between the SSW-03/SP-08 and SSW-07/SP-08 strains indicated that deletion of tdcB is insufficient to improve L-Ser production (Fig. 4D). However, fermentation of deletion of tdcG exhibited unexpected results. This tdcG gene knockout strain, SSW-08/SP-08, showed a same biomass and 42% lower L-Ser production than the SSW-03/SP-08 (OD600 ~ 36, 21.6 g/L) (Fig. 4B and Fig. 4E). The complex phenomenon associated with SSW-09/SP-08 may be caused by regulation of the expression of the interrupted operon tdcABCDEFG by deletion of tdcG. These results suggested that only deletion of sdaB improved L-Ser production, increasing the L-Ser titer by 23%; thus, SSW-05 with only deletion of sdaB was selected for the following experiment, which would avoid severely affected in cell growth by knockout all L-Ser dehydratases.
2.4 Effect of engineering L-serine transport system on strain productivity
Moreover, engineering amino acid transport system is also important to further improving strain productivity by blocking reuptake of amino acid and reducing futile cycles [32, 33]. In E. coli, four genes, sstT, cycA, sdaCand tdcC, have been reported to be involved in L-Ser uptake. Notably, sdaC is the only gene described as a highly specific serine transporter, and deletion of sdaC was found to improve L-Ser production in our recent studies. Thus, the highly specific L-Ser uptake gene sdaC was deleted from SSW-05 to reduce the unwanted futile cycles caused by L-Ser reuptake; this deletion resulted in strain SSW-10. As shown in Fig. 5A, the SSW-10/SP-08 produced 30 g/L L-Ser with a yield of 0.37 g/g from glucose, 16% higher than that of SSW-05/SP-08. In addition, the final L-Ser productivity of SSW-10/SP-08 was approximately 0.84 g/L/h, which was almost 1.15-fold that of SSW-05/SP-08.
Efflux pump is an important component of amino acid transport system and it is known to increase strain tolerance by accelerating the export of amino acid from cells. However, no research to date has reported well-characterized L-Ser exporters in E. coli. ThrE has been identified as an L-Ser/L-threonine exporter in C. glutamicum . And thrE family identified as amino acid exporters in select bacteria, archaea and eukaryotes, but no homologues were found in E.coli. Here, heterologous expression of thrE was performed to verify if it works in E.coli. Thus, thrE was cloned into the constructed expression vector SP-08 adjacent to the PR promoter, resulting in the plasmid SP-09 (Fig. 5B). This recombinant plasmid was then transformed into SSW-10. Figure 5B shows the fermentation process of SSW-10/SP-09. Overexpression of thrE resulted in a 9% decrease in the final OD600 value and an 16% increase in L-Ser production compared to those of SSW-10/SP-08. Although the L-Ser production by the final strain SSW-10/SP-09 (35.1 g/L) was lower than L-Ser production by the strains constructed by Maja Rennig (50 g/L), the yield (42%) of L-Ser from glucose of strain SSW-10/SP-09 was higher than that of the strains constructed by Maja Rennig (36%). Moreover, strain SSW-10/SP-09 exhibited highest productivity and yield of L-Ser from glucose observed to date.
2.5 Transcriptomic analysis of of E. coli W3110 and SSW-10/SP-09
To investigate the effect of L-Ser fermentation on intracellular metabolism, transcriptomic analyses of E. coli W3110 and SSW-10/SP-09 were performed in the exponential phase. A total of 1679 transcripts were found to be significantly different under two criteria (p-value < 0.05 and fold change > 2.0). Transcription levels in central carbon metabolism, including glycolysis, tricarboxylic acid(TCA) cycle and amino acid pathways related L-Ser synthesis, were compared.
Expression of the genes related to most reactions in the glycolysis such as pgi, fabAB, tpiA, eno, and pyk was downregulated in SSW-10/SP-09, while that of pgk, encoding phosphoglycerate kinase, was upregulated due to its expression in plasmid SP-09 (Fig. 6A). In the TCA cycle, expression of most genes were also downregulated in SSW-10/SP-09 (Fig. 6B). As a main machinery for adenosine triphosphate(ATP) synthesis, TCA cycle could produce 12.5 ATP molecules per pyruvic acid (PYR) molecule with important intermediates such as oxaloacetate(OAA) and acetyl-CoA(AcCoA). Downregulation of TCA cycle might cause inferior growth with less energy supply. However, the mqo gene encoding malate dehydrogenase that convert malate with quinone to oxaloacetate and reduced quinone was upregulated. Reduced quinone could significantly decrease global DNA methylation level cells, and cause acute oxidative damage. Reduced quinone rise in SSW-10/SP-09 may be another reason for biomass decrease. In this study, the OD600 of SSW-10/SP-09 was 24, a decrease of 35% compared to that of E. coli W3110 (OD600 = 37). Gene sdhC, encoding succinate dehydrogenase (ubiquinone) cytochrome b560 subunit, was related to in oxygen availability and upregulated in SSW-10/SP-09.
Next, we analyzed changes in the expression of genes related to L-Ser production in SSW-10/SP-09 (Fig. 6C). The expression levels of serA, serC and serB increased in varying degrees. Expression of the gene glnA related to conversion from L-glutamic acid (L-Glu) to L-glutamine(L-Gln), which provided NH4+ for L-Ser biosynthesis, was upregulated. It caused damping reaction in L-Glu, L-Gln and 2-oxoglutarate(2-OXO) such as gltB and gltD. Expression of the dsdA encoding D-Ser ammonia-lyase was upregulated. However, expression of cysEKO, ltae and trpAB involved in L-cysteine(L-Cys) and L-tryptophan(L-Trp) biosynthesis were not change. Likewise, SSW-10/SP-09 showed downregulation of glycine cleavage(Gcv) system genes such as gcvT, gcvP and gcvH due to less intracellular glycine(Gly) (Fig. 6D). It could result in a decreased amount of one-carbon units and poor growth . However, metF, encoding 5,10-CH2-THF reductase, involved in one-carbon metabolism drastically increased, which could compensate for one-carbon unit . Expression of the betB encoding the enzymes that converts betaine aldehyde to betaine was upregulated. Betaine could regulate intracellular osmotic pressure and provide methyl . With supplement betaine, production of L-threonine, cobalamin and L-lactate were increased. Expression of genes related to metabolism of L-threonine(L-Thr), a downstream amino acid of L-Ser, was analyzed (Fig. 6E). The expression levels of ilvA, which was involved in both L-Thr and L-Ser dehydration, was decreased.
In addition to the above genes, there were still large number of differentially expressed genes. Among the upregulated genes in “pyrimidine metabolism” of SSW-10/SP-09, five gene sets rutA(expression ratio 25.37), rutB(expression ratio 24.55), rutC(expression ratio 24.09), rutD(expression ratio 22.30) and rutE(expression ratio 22.44). Rut pathway may be proposed to enhance the rate of hydrolysis of aminoacrylate, a toxic side product of L-Ser degradation[48, 49]. Among the downregulated genes, genes in the category “Galactose metabolism” were enriched in SSW-10/SP-09. This category includes gatZ(expression ratio 2− 3.89), gatA(expression ratio 2− 6.60), gatB(expression ratio 2− 8.45), gatC(expression ratio 2− 6.55), gatD(expression ratio 2− 6.87) and gatR’(expression ratio 2− 3.52). Genes gatZABCDR’ related to dihydroxyacetone phosphate synthesis from galactitol and galactosamine were highly involved in biofilms and downregulation of the operon may have connection to cell density decrease .
2.6 Intermediate metabolite analysis of of E. coli W3110 and SSW-10/SP-09
As shown in Fig. 7A, a set of 17 intracellular metabolites, including glycolytic intermediates, intermediate metabolite in TCA cycle and amino acid related L-Ser, were measured. A score plot of the PCA model using 17 intracellular metabolites showed the discrimination of metabolite profiles depending on different strains (Fig. 7B). In the PCA model, the intracellular metabolite profiles of E. coli W3110 and SSW-10/SP-09 were clearly discriminated. Along the axis of PC1 of the score plot, the metabolite profiles of E. coli W3110 were located on the positive side, while the metabolite profiles of SSW-10/SP-09 were located on the negative side. Intracellular glucose-6-phosphate (G6P) concentration of SSW-02/SP-08 increased. It may be caused by downregulated of most downstream genes such as pgi, fabAB and eno in glycolysis (Fig. 8A and Fig. 9A). Intracellular PYR concentration decreased due to weak glycolysis and efficient carbon flux on L-Ser. In the TCA cycle, 2-OXO concentration and malic acid (MAL) concentration showed no significant changes between SSW-10/SP-09 and E. coli W3110. Intracellular L-Ser concentration was 472.5 µg/L/g(DCW), which was 32-fold of control. Consumption of L-Gln, pitched into the second step of L-Ser biosynthesis, caused damage of its precursor L-Glu. High intracellular L-Thr concentration was in favor of maintaining L-Gly concentration [12, 51]. It also was the reason for the lessened concentration of L-valine (L-Val), L-leucine (L-Leu) and L-isoleucine (L-Ile). Higher intracellular L-Thr concentration also caused downregulation of thrABC (encoding homoserine dehydrogenase I, homoserine kinase, and threonine synthetase), which was consistent with the result showed in Fig. 8C, due to its feedback inhibition . Intracellular L-phenylalanine (L-Phe) concentration of SSW-10/SP-09 increased 182% when compared to it of E. coli W3110. While there was no distinct relationship reported between L-Phe and L-Ser production to date.