Screening L-Trp hyperproducing strains using an intracellular L-Trp biosensor
The biosensor is a small-molecule inducible system that produces a fluorescent readout proportional to the amount of product inside of the cell. Here, we used a previously characterized L-trp biosensor (Fig. 1a and Fig. S1). To test the applicability of this biosensor, we introduced it into E. coli DH5α and induced GFPmut2 expression by adding different concentrations of the Ala-Trp dipeptide in vitro. The results showed that there was a good linear relationship between the Ala-Trp concentration and the fluorescence intensity when the dipeptide concentration was less than 2 mM (Fig. S2). In addition, a clear difference in fluorescence intensity was observed in DH5α carrying the L-trp biosensor with and without the addition of Ala-Trp (Fig. S3). These results indicated that the sensor system was capable of reflecting the difference in L-trp concentration by altered fluorescence intensity. Furthermore, to confirm the feasibility of using the L-trp biosensor response to measure intracellular L-trp, we next investigated whether the biosensor could distinguish strains with different L-trp production capacities via different fluorescence signals. The L-trp sensor plasmid was introduced into the strains TPD1, TPD2, TPD3 and TPD4 with increased L-trp productivity (Table S1). As shown in Fig. S4, with the increased titer of L-trp from TPD1 to TPD4, there was an obvious increase in specific GFP fluorescence in the same order. Therefore, the L-trp biosensor showed a good correlation between L-trp concentration and fluorescence intensity, both when L-trp was added at different concentrations in vitro and in strains with different L-trp production capacities, suggesting that this device could be used as a screening platform for selecting L-trp hyperproducing strains.
To establish a high-throughput screening technique, we introduced the L-trp sensor into E. coli TPD4, which was obtained by deleting trpR and overexpressing the trpEDCBA operon, along with the aroG and ppsA genes in E. coli KW. To obtain an L-trp hyperproducing strain, a random mutagenesis library of the TPD4 strain was constructed using exposure to atmospheric and room-temperature plasma (ARTP) for 16 s (Fig. S5). In the control group, colonies without ARTP treatment were cultured under the same conditions for comparison. After ARTP mutagenesis and FACS screening, two mutant strains were finally selected from a library of 2×105 clones. The isolated strains were cultured in fermentation medium and the L-trp concentrations were determined by HPLC. Strains TPD5 and TPD6 showed similar growth characteristics (Fig. 1b), but respectively produced 3.08 and 1.46 times more L-trp than the parental strain TPD4 (Fig. 1c). In addition, FACS spectrograms of TPD4 and TPD5 showing effective separation of the strains confirmed the functionality of the L-trp biosensor (Fig. 1d), which was consistent with their L-trp production. Using this approach, we successfully increased L-trp biosynthesis to a titer of 2.01 g/L and a yield of 0.124 g/g.
Identification of genetic changes in strain TPD5 by whole-genome sequencing and identification of mutations beneficial for L-Trp overproduction
Whole-genome re-sequencing technology enables the complete characterization of mutant genomes and facilitates the identification of relevant mutations for different phenotypes. Compared with the parental strain TPD4, the screened E. coli TPD5 exhibited a 3.08-fold increase of L-trp titer, which greatly improved the L-trp productivity. To determine the genetic basis of the strain’s L-trp overproduction phenotype, the whole genome of stain TPD5 was sequenced and re-annotated (see Supplementary Table S3). Combined with mutations found in gaps, a total of 38 mutations were identified across the genome of TPD5, encompassing 3 insertions, 5 deletions, and 23 substitutions. A total of 29 mutations were located within ORFs and 9 in non-ORF regions (Supplementary Tables S4 and S5). Among these mutations, only a small number affected loci with annotated functions related to the L-trp biosynthesis pathway. These include mlc (deletion) related to the phosphotransferase transport system (PTS), AcnA (S522G) in the TCA cycle, as well as AroG (S211F), TrpE (A63V) and tnaA (deletion) in the L-trp biosynthesis pathway. Some mutations were thought to occupy sites of interest that may be correlated with the strain’s phenotype. For example, FlhD (V84F), encoding a flagellar transcriptional regulator, and AcrR (D139N), encoding a HTH-type transcriptional regulator, both of which are involved in the transcriptional regulation of several flagellar operons and modulate swimming motility, and therefore might be responsible for the physiological changes in the cell.
In order to identify genome changes responsible for L-trp overproduction, 28 mutations located in ORFs that alter amino acid sequences were selected as genetic manipulation targets for the introduction of individual back mutations into the chromosome of the parent strain TPD5 to evaluate their effects on L-trp overproduction. Shake-flask fermentations indicated that a number of these mutations are actually neutral or even harmful (Fig. 2). For instance, AcrR (D139N), ArsB (E303K), GlpQ (R101C), DinD (P7S), OppF (S325A), EntE (A407T), GarL (A190S), DicA (E123K), PolA (G763S), Imp (E288K), LacI (deletion), YdeI (deletion), YgiQ (deletion) and YlbE (insertion) were neutral mutations, with no observable phenotypic changes in the corresponding mutants. PuuP (Y110C) behaved as a harmful mutation, and L-trp production increased to 2.4 g/L after this gene was restored, which may be related to its role as an important growth factor in the synthesis of proteins and nucleic acids. It is likely responsible for the low efficiency in transforming putrescine. In addition to the confirmed neutral and harmful mutations, at least 13 specific mutations were identified as beneficial for L-trp overproduction, with varying contributions.
Among the 13 beneficial mutations, four changes led to a significant decrease of L-trp overproduction after back mutation, including AroG (S211F), RpoS (nonsense mutation Q33*), TrpE (A63V) and TnaA (deletion). Strain TPD5-RM2, in which the AroG (S211F) mutation was restored, reintroducing the wild-type 3-deoxy-D-arabino-heptulosonate-7-phospphate (DAHP) synthase, showed a decrease of L-trp production from 2.01 g/L to 0.86 g/L. The L-trp production of strain TPD5-RM10 was even reduced to 0.09 g/L after restoring the wild-type anthranilate synthase TrpE. Strain TPD5-RM27, in which the degradation pathway tryptophanase TnaA was restored, showed a decrease of L-trp production to 0.9 g/L. In addition, restoring the Q33* nonsense mutation of RpoS, which is a global regulator, reduced the L-trp titer almost 3.3-fold to 0.6 g/L. Our results confirmed that only four mutations among the selected ones were significantly beneficial for L-trp production, accounting for a small part of the total mutations.
Insights into the AroG S180F mutation that leads to L-Trp overproduction
AroG is a DAHP synthetase isozyme that catalyzes the first committed step in the biosynthesis of aromatic amino acids and vitamins. It condenses the two important precursors phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) to form DAHP. However, feedback inhibition by L-phenylalanine (L-phe) results in insufficient DAHP synthase activity to efficiently channel PEP and E4P into the L-trp biosynthesis pathway[24, 25]. In order to remove the feedback inhibition of AroG enzyme, Ger et al. isolated the mutant S180F through screening of phe-resistant colonies. We also found that AroG in the screened strain TPD5 was mutated, but not at the residue S180. Instead, the residue S211 was mutated to F.
As can be seen in Fig. 2, AroG (S211F) had a significant impact on the production of L-trp. In order to explore whether S211F relieved the L-phe feedback inhibition of AroG in the same way as S180F, we analyzed the effect of the mutations at residue 118 and residue 211 on the binding of L-phe. Fig. 3a shows that under normal conditions, the inhibitor L-phe interacts directly with residue 180 (S) and residue 6 (Q) of wild-type AroG. When S180 was mutated to F, the phenyl ring of F blocked the binding of the inhibitor L-phe (Fig. 3b), thus releasing L-phe inhibition. Similarly, although there is no direct interaction between S211 and the inhibitor L-phe, replacement of S211 with F also blocks the binding of the inhibitor L-phe via steric hindrance from the phenyl ring (Fig. 3c). Therefore, AroG (S211F) in strain TPD5 relieved the feedback inhibition of L-phe in the same way as the previously reported S180F mutant, increasing the conversion of PEP and E4P into DAHP, which increased the metabolic flux into the shikimate pathway. The feedback-resistant AroG (S211F) may be a good choice for the modification of other strains that produce aromatic amino acids or vitamins.
Insights into the TrpE A63V mutation that leads to L-Trp overproduction
Anthranilate synthetase (TrpE), which produces anthranilate from chorismate and glutamine in the first step of the terminal branch of the L-trp pathway in E. coli, is subject to feedback inhibition by the end-product L-trp[27, 28]. However, considering that TrpE is a key enzyme in the L-trp branch of the pathway, releasing its feedback inhibition is crucial for increasing L-trp production. In order to explore whether TrpE (A63V) is resistant to L-trp feedback-inhibition, we carried out molecular dynamics (MD) simulations of wild-type TrpE and mutant TrpE (A63V). The wild-type TrpE protein structure for MD simulations was constructed using the network server I-TASSER, on which Schrödinger was used to construct the initial structure of the mutant TrpE (A63V). The binding sites of the substrate chorismate and the inhibitor L-trp after docking are shown in Fig. 4a.
In the absence of L-trp, the wild-type TrpE protein was in its normal, closed conformation (as shown in Fig. 4b, blue). Accordingly, L-trp bound to a specific position of the TrpE protein, resulting in structural changes causing its active-site pocket to shift from a closed conformation to an open conformation (as shown in Fig. 4b, yellow). In the open conformation, TrpE could not bind the substrate chorismate, thus preventing its normal catalytic function. By contrast, the structure of the mutant TrpE (A63V) did not change and remained in a closed conformation regardless of the presence (Fig. 4c, green) or absence (Fig. 4c, pink) of L-trp. This conformation was the same as that of wild-type TrpE performing the normal catalytic function (Fig. 4c, blue). Thus, the feedback-resistance of TrpE was achieved by replacing the residue A63 with V, explaining the reason for the great change in L-trp production caused by this mutation. Here, the mechanism of TrpE inhibition by L-trp was analyzed, which enables us to clearly understand the mechanism of feedback inhibition, laying a foundation for further improvement of TrpE protein in the future.
Effects of the RpoS nonsense mutation Q33* on L-Trp production
RpoS encodes the alternative sigma factor σs, a subunit of RNA polymerase that acts as the master regulator of many stationary-phase genes for adaptation to nutrient deprivation and other stresses in E. coli. Genome-wide analyses of RpoS-dependent gene expression showed that up to 10% of genes in E. coli are under direct or indirect control of RpoS, among which over 130 genes are positively controlled and a surprisingly large number are negatively regulated by this sigma factor. Although the RpoS regulon is a large, conserved system that is critical for adaptation to a variety of stresses and metabolic conditions, its effect on specific metabolic pathways, such as L-trp biosynthesis pathway, remains incompletely characterized. In order to determine the effects of the nonsense mutation RpoS (Q33*) on the increase of L-trp production, we fermented strains TPD5 and TPD5-RM3 and observed the differences in the fermentation process.
As can be seen from the fermentation results, when RpoS was restored, the color of the fermentation broth and cell pellet changed from light brown to pink (Fig. 5a and Fig. S6). In addition, the cell morphology at different fermentation stages was observed by optical microscopy, and it was found that the cells of TPD5-RM3 deteriorated and became abnormal and irregular from 12 h (Fig. S7). Furthermore, cells of TPD5 and TPD5-RM3 fermented for 12 h and 38 h were observed by scanning electron microscopy. As can be seen in Fig. 5b and c, strain TPD5-RM3 exhibited obvious cell surface shrinkage after fermentation for 12 h, and obvious holes appeared on the surface of a small number of cells, which may lead to the leakage of intracellular lysates, leading to the adhesion of cells. This phenomenon was more obvious at 38 h of fermentation, and almost all the cells of strain TPD5-RM3 collapsed, presenting a withered state. Compared with strain TPD5, in which only individual cells showed surface shrinkage, strain TPD5-RM3 showed dramatically reduced vitality. The normal expression of RpoS resulted in an observable phenotypic alteration of the L-trp producing strain, which was specifically visible in the color of the fermentation broth, cell morphology, cell size, etc. This change may be due to the direct or indirect regulation of some cell membrane proteins by RpoS. Moreover, the premature termination of RpoS translation due to the nonsense mutation Q33* impaired cell growth on glucose (Fig. 5d, left) and strongly altered the distribution of intracellular fluxes. After RpoS was recovered, the production of L-trp decreased significantly to only 0.42 g/L, representing a 79.93% reduction (Fig. 5d, middle), while the concentration of acetic acid as a by-product in strain TPD5-RM3 was significantly increased to 2.2 times that of strain TPD5 (Fig. 5e, right). This difference indicates that the carbon flow of cells was affected by RpoS, redirecting more metabolic flux to by-product biosynthesis. Therefore, it can be inferred that RpoS not only has a direct impact on the overall morphology of cells on the macro level, but also significantly influences the carbon flow of strains on the micro level.
Transcriptomic analysis of the role of the global regulator RpoS in the L-Trp biosynthesis pathway
In order to obtained deeper insights into the genetic link between RpoS and the L-trp biosynthesis pathway, comparative transcriptomic analysis was conducted with cells of E. coli TPD5 and TPD5-RM3 harvested during the stationary growth phase. Fig. 6a shows the heatmap of differentially expressed genes, from which it can be seen that the genes expressed in strain TPD5-RM3 were significantly different from those expressed in strain TPD5. A total of 469 genes showed significant variation at the genome-wide transcriptional level between TPD5 and TPD5-RM3 (163 up- and 306 downregulated genes; Fig. 6b and Supplementary Table S6). The correlation of gene expressing levels between the two strains was 0.846 (Fig. S8). Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were carried out. GO functional annotation classified the differentially expressed genes into three categories: biological process, cellular component, and molecular function, while KEGG pathway analysis classified the differential genes into 20 categories with the most significant difference in biosynthesis of secondary metabolites (Fig. 6c, d and Supplementary Table S7).
To determine the functional classes of the differentially expressed genes in stain TPD5-RM3, we carried out a homology-based annotation specifically for all the genes and GO terms related to the dataset shown in Table S7. Significant differences were found in the expression of genes in the categories biological process and molecular function (Fig. 6c). In particular, the enrichment of terms related to metabolic process were distinct (GO:0008152, GO:0009987, GO:0071704, GO:0044238, GO:0044237), indicating that RpoS has an extensive effect on gene expression in metabolic pathways, thus directly affecting the production of most primary metabolites. Moreover, the set of differentially expressed genes was mapped to the KEGG pathways in E. coli W3110 (Supplementary Table S8), showing that a series of enriched pathways exhibited significant differential expression. The transcripts of genes involved in the biosynthesis of secondary metabolites, amino acid and carbon metabolites were significantly different from those in strain TPD5, and thus may directly or indirectly affect the synthesis of L-trp.
The differentially expressed genes nearly all fell into different functional pathways, including biosynthesis of secondary metabolites (49 genes), carbon metabolism (31 genes), biosynthesis of amino acids (30 genes), purine metabolism (19 genes), glycolysis/gluconeogenesis (11 genes), pentose phosphate pathway (8 genes), as well as glycine, serine and threonine metabolism (8 genes). To further analyze the differential expression information, we first focused on genes in the pathways directly related to L-trp biosynthesis (Fig. 7). In the L-trp biosynthesis pathway, transcription of the trp genes, which are organized in a single operon, was strongly upregulated 1.54- (trpE), 2.34- (trpD), 3.43- (trpC), 2.43- (trpB) and 3.36-fold (trpA) in E. coli TPD5-RM3. In contrast to the higher expression of the trp operon in E. coli TPD5-RM3, most genes involved in precursor supply (PEP, E4P, PRPP (5-phospho-α-D-ribose 1-diphosphate), glutamine, L-serine (L-ser)) presented lower expression levels. These included pgi, pfkA/B, fbaA, gapA, pgk, gpmA/M, and eno in the glycolysis pathway, ppsA and fbp in the gluconeogenesis pathway, as well as zwf, gnd, rpiA/B, tktB, talB, deoB and phnN in the pentose phosphate pathway and almost all genes in the TCA cycle. Thus, the insufficiency of crucial precursors, energy or cofactors derived from the central metabolic pathways may have restricted biomass accumulation and L-trp production. Moreover, pathway enrichment analysis also revealed that most genes involved in the glucose absorption system, including ptsH, ptsI, crr, frsA, hfq, sgrR, mlc, crp, and relA-uspD, showed low expression levels in E. coli TPD5-RM3, leading to inadequate nutritional supply. Overall, RpoS attenuates glucose uptake at the beginning of the entire metabolic pathway by reducing the expression level of genes related to the glucose uptake system. Furthermore, the transcription levels of most genes in the glycolysis pathway and pentose phosphate pathway were reduced as a whole, leading to severely weakened metabolic flux in the central metabolic pathways under the premise of inadequate nutrient uptake, which made the cells unable to produce sufficient precursors, energy, and cofactors for L-trp biosynthesis. Therefore, the production of L-trp was very low even though the gene expression intensity of the terminal branch of the L-trp pathway was strong.
Metabolic engineering of TPD5-RM4 for improved L-Trp production
The terminal branch of the L-trp pathway starts from the condensation of chorismate and glutamine and ends with the condensation of indole and L-ser to L-trp (Fig. 1a). However, after the analysis of strain TPD5-RM4, it was found that the concentration of L-ser and its substrate phosphoserine (P-ser) was low, which may be caused by feedback inhibition of the enzyme D-3-phosphoglycerate dehydrogenase (3-PGDH, encoded by serA), leading to insufficient supply of the precursors. In addition, considering that no mutations or manipulations have been made in the L-ser biosynthesis pathway in strain TPD5, the H344A/N364A double mutant of the key enzyme SerA was expressed from the chromosome of strain TPD5-RM4, resulting in strain TPD5-RM4S, which is resistant to feedback inhibition by L-ser. After the overexpression of SerA (H344A/N364A), the concentration of P-ser and L-ser increased significantly (Fig. 8a). As expected, the production of the final product L-trp was further increased to 2.94 g/L (Fig. 8b).
Although the production of L-trp was improved by overexpressing SerA (H344A/N364A), we found that the concentration of glutamate in strain TPD5-RM4S greatly increased to 8.86 mM, which was 368-fold higher than that of strain TPD5-RM4 (Fig. 8a). Glutamate can be converted by glutamine synthetase GlnA to produce glutamine, which is a crucial precursor involved in the first step of the terminal branch of L-trp biosynthesis. Therefore, in order to further expand the metabolic flux of the L-trp pathway, we attempted to convert the large amount of glutamate of strain TPD5-RM4S into glutamine, thus further promoting the synthesis of L-trp. Accordingly, we introduced GlnA (Y405F) from Corynebacterium glutamate into TPD5-RM4S to construct stain TPD5-RM4G. Compared with strain TPD5-RM4S, the concentration of glutamate in strain TPD5-RM4G was reduced to 0.24 mM, successfully transforming the accumulated glutamate into glutamine. The final TPD5-RM4G strain produced L-trp at a titer of 3.18 g/L in shake flask fermentations. Thus, we accomplished a further 57.83% increase of the L-trp titer compared with strain TPD5 by altering the expression levels of genes in the L-ser pathway and boosting the conversion of glutamate to glutamine.