Available metabolic strategy of ALA production by overexpression of hemA, hemL and eamA
We constructed an E. coli strain from BW25113 which achieved high efficiency of protein expression through T7 expression system in our earlier work18. This mutant E. coli BW25113-T7 strain was rationally engineered to produce ALA by over-expression of key genes. The biosynthetic pathways of ALA in E. coli and the strategies for constructing ALA production strain are shown in Figure 1. The key genes of ALA synthesis via the C5 pathway in this strategy are hemA and hemL. The hemA gene encodes a NADPH-dependent glutamyl-tRNA reductase which catalyzes the reduction of glutamyl-tRNA to glutamate-1-semialdehyde (GSA)17. The hemL gene encodes glutamate-1-semialdehyde aminotransferase which quickly converts GSA to ALA. The eamA gene encodes an O-acetylserine or cysteine exporter which is capable of translocating dipeptides and amino acid analogs from the cytosol to the periplasm.
Plasmid pET-ALA-LAA was constructed to over-express these three key genes. In this plasmid, these three genes can be tightly controlled and efficiently expressed by the T7-Lac promoter. Cells that harbored pET-ALA-LAA were cultured in special M9YE medium. After 24 h of induced fermentation, an ALA titer (662.3 mg/L) was determined.
Overexpression of gltB, gltD and gltX genes involved in the C5 pathway in E. coli
Glutamate and L-glutamyl-tRNA are both precursors of ALA synthesis via the C5 pathway. To increase glutamate and L-glutamyl-tRNA amounts for ALA synthesis, we overexpressed gltB, gltD and gltX genes alone or in combination.
Glutamate synthase is a tetramer of dimers, with each dimer having one large and one small subunit (gltB and gltD, respectively)19. Glutamate synthase catalyzes the single-step conversion of L-glutamine and alpha-ketoglutarate into two molecules of L-glutamate (Figure 2). In doing so, glutamate synthase simultaneously operates as the major source of L-glutamate for the cell and as a key step in ammonia assimilation during nitrogen-limited growth19-21. The ammonia-dependent activity can be catalyzed very slowly by just the small subunit in the absence of the full complex22. Glutamate-tRNA ligase (GluRS) is a member of the family of aminoacyl-tRNA synthetases, which is encoded by gltX gene23, 24. GluRS charges GlutRNA for both protein and ALA synthesis25.
In this study, we constructed four extra plasmids to overexpress these three genes (Table 1). These four plasmids were electroporated into cells that harbored pET-ALA-LAA. The resulting strains were cultivated in modified minimal medium (see ‘‘Section Materials and Methods’’) supplied with 10 g/L glucose for ALA accumulation analysis. After 24 h of fermentation, ALA concentration was determined (Table 2). Strain BW25113-T7 which only contained pET-ALA-LAA was set as the control.
Beyond our expectation, we found all strains that harbored extra plasmids exhibited decreased ALA accumulation with E. coli BDX having the greatest decrease in 5-ALA accumulation relative to the control (Table 2). We found that all strains harbored extra plasmids grew much more slowly than control strains (Table S1). These results indicate that overexpression of gltB, gltD and gltX genes may not have a positive effect on the ALA biosynthesis in E. coli. This finding suggests that the biosynthesis of L-glutamate and conversion of L-glutamate to GlutRNA are not rate-limiting steps of ALA biosynthesis.
Enhanced Production of 5-ALA by metabolic pathway modification
To further improve the BW25113-T7 strain, following targeted genetic modifications were performed (Figure 1).
HemF is a strictly aerobic enzyme that requires molecular oxygen as the electron acceptor and produces hydrogen peroxide. The knock-out of hemF gene would mightily repress the biosynthesis of protoporphyrinogen IX (PPG) resulting in reduced endogenous loss of 5-ALA. After knocking out the hemF gene in BW25113-T7 (named D1:F), yield of 5-ALA increased 2.06-fold compared to the original strain (named as D0). This result indicated that knock-out of the hemF gene has a positive effect on ALA biosynthesis.
L-glutamate is a precursor of ALA synthesis via the C5 pathway. Therefore, we attempted to increase ALA accumulation by reducing endogenous loss of L-glutamate. GdhA, which encodes glutamate dehydrogenase, catalyzes the NADPH-dependent amination of α-ketoglutarate to yield L-glutamate26. This reaction is reversible (Figure 3A). YbdK catalyzes ATP-dependent ligation of glutamate with cysteine at a low catalytic rate27 (Figure 3B). GadB, a glutamate decarboxylase enzyme, catalyzes cleavage of L-glutamate into carbon dioxide and 4-aminobutanoate28 (Figure 3C). These three genes in the ALA biosynthetic pathway were knocked out (Figure 1) individually or jointly using the E. coli D1:F strain. ALA accumulations of those mutant strains ranged from a reduction to a 2.30-fold increase in ALA accumulation (Table 3). Among the mutant strains, E. coli D3:FYB significantly increased ALA accumulation (2.30-fold) compared with E. coli D0.
The periplasmic binding proteins—mppA, the L-alanyl-g-D-glutamyl-meso-diaminopimelate binding protein, or dppA, the dipeptide binding protein—actively import ALA through an interaction with the dipeptide inner membrane ATP-binding cassette transporter, DppBCDF, in E. coli29. We theorized that inactivation of dppA and/or mppA genes would reduce ALA assimilation and therefore increase ALA accumulation in the medium. As expected, inactivation of dppA improved ALA production but mppA did not (Table 3). To further improve ALA production, we used the E. coli D4:FYAB strain to perform dppA and mppA gene knockouts individually or jointly. We found that mutant strain, E. coli D5:FYABD, showed the highest production of ALA which was 2.42 fold greater than D0 (Table 3).
Growth characteristicS of mutant strains and selection of optimal mutant E. coli strain
After knocking out these genes, growth of mutant strains in different media were examined to assess whether CRISPR/Cas9-mediated gene knock-out affected metabolic characteristics of the bacteria. These strains all harbored pET-ALA-LAA for producing ALA.
These mutant strains were cultured in special M9YE medium (standard M9 medium with additional 2 g/L Yeast Extract and 10 g/L Glucose) or standard LB medium (Figure 4). Growth rate among these strains in LB medium ranged from 0.25 to 0.3 (Table 4). When cultured in special M9YE medium, growth rate of these strains varied from 0.3 to 0.5. These results reveal that knockouts of some genes may lead to growth retardation of bacteria.
We easily selected D5:FYABD as the optimal mutant strain for ALA production based on its growth rate and ALA production (Figure 5). The ALA production was increased 2.42-fold with similar growth rate when compared with the original-type (D0).
Increasing ALA accumulation via C4 pathway
In E. coli, primary ALA biosynthesis is through the C5 pathway. To further increase accumulation of ALA, we tried to synergistically produce ALA via the C4 pathway. The photosynthetic bacterium, Rhodobacter sphaeroides, can accumulate ALA under certain conditions or after mutagenesis30-32. Through metabolic engineering, recombinant E. coli was also able to produce ALA from the C4 pathway through biotransformation. In this aspect, the gene encoding for ALA synthase from R. sphaeroides was introduced into E. coli through genetic engineering33. Biosynthesis of glycine and succinyl-CoA are regulated in E. coli. Consequently, glycine and succinate (the precursor of succinyl-CoA) must be added to the culture medium artificially to provide sufficient substrates for enhanced ALA biosynthesis. In the current study, we overexpressed a modified heterologous hemA from R. sphaeroides using the T7 Expression System (Figure 6). The amino acid sequence of hemARS was modified for better expression in E. coli. Glycine (2g/L) was added to the M9YE medium as a substrate for the C4 pathway. These modifications increased ALA accumulation from 1602.72 mg/L with E. coli D5:FYABD to 2099.7 mg/L for the mutant strain containing the hemARS gene (Table 2). After 24 h cultivation, a yield of 0.210 g ALA per g glucose was achieved.