Improved production of the non-native cofactor F420 in Escherichia coli

The effect of different carbon sources on cofactor F₄₂₀ yield and growth of E. coli

To investigate the effects of different carbon sources on the production of F₄₂₀, we tested acetate, fumarate, glucose, glycerol, pyruvate, and succinate as carbon sources, as these carbon sources enter central metabolism at different points, have varied uptake mechanisms and therefore distinct bioenergetic consequences for the cell³³. Pyruvate and fumarate, (followed closely by acetate and succinate) supported the greatest F₄₂₀ production per gram of dry cell weight (DCW; Fig. 2A, B; Table 1). However, it should be noted that these carbon sources did not support high levels of biomass formation (Fig. 2C). Indeed, the cell density (measured as OD₆₀₀) varied significantly by carbon source (Fig. 2C). With respect to overall productivity of F₄₂₀ production (expressed as µmol F₄₂₀/L/h), glycerol was the most productive carbon source (Fig. 2B); F₄₂₀ yield with pyruvate was 0.90 µmol/g DCW which is close to the yield of the cofactor NADPH in E. coli of 1.3 µmol/g DCW³⁴ (Fig. 2A). High F₄₂₀ yield and productivity with pyruvate implicitly indicated the impact of this intracellular metabolite as well as its precursor, PEP, on F₄₂₀ biosynthesis.

Phospho-enol pyruvate (PEP) is a key metabolite for F₄₂₀ biosynthesis

To systematically understand the effect of F₄₂₀ biosynthesis on the distribution of flux through the entire metabolic network of the engineered E. coli grown with different carbon sources, we created and utilized the iEco-F420 metabolic model (see Methods) to compare flux profiles. Figure 3 summarises Flux Balance Analysis (FBA) results for two main pathways; glycolysis and the TCA cycle, for in silico growth with glucose, glycerol, and succinate as sole carbon sources, which were selected because of their different F₄₂₀ productivity profiles. FBA predicted assimilation of 72% of glucose, as the sole carbon source, via the phosphoenolpyruvate (PEP): phosphotransferase system (PTS); all enzymes involved in glycolysis were active. Given the defined criteria, FBA predicted no flux through PEP synthase (PPS) or PEP carboxykinase (PPCK) indicating tight control over the pool of PEP during in silico growth with glucose (Fig. 3). These simulation results suggest a key role for PEP during F₄₂₀ biosynthesis.

The metabolic model indicated that growth on succinate results in activation of the gluconeogenesis pathway and PPCK. With glycerol as the sole carbon source the upper glycolytic pathway was turned off (Fig. 3B), resulting in up to 27% higher overall ATP generation. On the other hand, fumarate was predominantly metabolised through aspartase since the glyoxylate shunt was highly active when glycerol was the carbon source, which leads to a reduction in total flux through TCA cycle. These modelling results explain the higher growth (Fig. 2C) and higher capacity for F₄₂₀ production when engineered E. coli cells expressing the F₄₂₀ biosynthetic pathway are grown with glycerol compared with glucose or succinate. Interestingly, with succinate as the carbon source, the iEco-F420 model predicted that pyruvate was produced mainly through malate dehydrogenase (decarboxylating) (Fig. 3A), leaving the PEP pool more accessible for incorporation into F₄₂₀ production, consistent with the experimental yields. These results are consistent with the empirical growth experiments and also indicate a key role for PEP in controlling flux through the F₄₂₀ biosynthesis pathway.

The iEco-F420 model contains 35 reactions consuming PEP: 19 are PEP-dependent phosphotransferases, 10 reactions participate in central carbon metabolism, two occur in cell envelope metabolism, two in tyrosine metabolism, and one in F₄₂₀ biosynthesis (Supplementary File 1; Table S1). In an effort to increase the PEP pool, we used the model to test whether any of these competing reactions were dispensable in silico. However, single gene deletion in silico predictions suggested that removing the reactions involved in cell envelope and tyrosine metabolism would result in cell death.

We next performed flux variability analysis (FVA) for all reactions in the metabolic network, including the PEP-consuming reactions (Supplementary File 1; Tables S2–S13) to specifically explore flux variations in PEP-consuming/producing reactions as a result of maximization of flux through biosynthesis of F₄₂₀. PEP hydratase (enolase) was chosen to interpret flux variations with respect to PEP availability for cellular growth versus F₄₂₀ production. Figure 4 shows the flux profile of PEP hydratase using all six carbon sources. When glucose is the sole carbon source, PEP must be produced through glycolysis to meet cellular objective (i.e., maximizing growth). At maximum biomass (where the blue and red lines showing minimum and maximum fluxes meet in Fig. 4), PEP hydratase flux is positive, meaning that 2-phospho glycerate (2-PG) is fully metabolized to PEP. One engineering objective for increasing the heterologous production of F₄₂₀ requires more carbon to be diverted into the target product rather than biomass, up to the point where the growth of the host is so negatively affected that it becomes uneconomical. When biomass yield drops to 80% of its maximum, for example, the minimum and maximum fluxes through PEP hydratase are still both positive, meaning that essential cellular processes take priority. As a result, 2-PG needs to be metabolized to provide stoichiometric requirements of PEP. However, at 50% of maximum biomass yield, the minimum flux (Fig. 4) through PEP hydratase becomes negative, meaning that the system is more relaxed to divert a portion of PEP for other processes including F₄₂₀ production.

Unlike the flux predictions for PEP hydratase using glucose, PEP is significantly more available for processes other than cellular growth when the carbon source is succinate, fumarate or pyruvate, even at maximum biomass yields (Fig. 4). This is consistent with reports that glucose uptake in E. coli occurs primarily via the PTS, consuming up to 50% of the available PEP in cell^35,36, thereby reducing its availability for F₄₂₀ biosynthesis. Gluconeogenic carbon sources such as pyruvate, succinate, and fumarate increase intracellular PEP levels compared to glucose³³ as their uptake is PEP-independent³⁶. PEP hydratase flux variation with glycerol is the highest among other carbon sources, meaning that glycerol assimilation could potentially lead to greater flexibility in utilising PEP for F₄₂₀ biosynthesis. However, glycerol uptake occurs through the glycolysis pathway and although its uptake requires half the energy (in form of ATP) of glucose, most of the PEP is still required for cellular activities rather than biosynthesis of F₄₂₀. Nonetheless, glycerol remains a candidate carbon source for large-scale F₄₂₀ production compared with glucose when maintaining high cell masses is essential because it allows for higher cellular mass yields while bypassing PTS-dependent PEP depletion. In the case of acetate, ATP-dependent acetate assimilation is the only route for producing acetyl-CoA, which is an essential precursor for the biosynthesis of most amino acids and fatty acids and therefore biomass yield drops significantly (Fig. 4). However, FVA for PEP hydratase indicates the feasibility of utilising PEP for non-cellular activities.

We measured intracellular PEP for engineered E. coli grown with glucose and glycerol to validate the model predictions. When glycerol was used as the sole carbon source, PEP and F₄₂₀ levels were 1.43 and 1.82-fold higher, respectively, compared with when glucose was used as the carbon source. This difference was also borne out in the simulation data (Supplementary Table S1). These results, collectively, demonstrate that the choice of carbon source directly affects intracellular availability of PEP, which, in turn, influences F₄₂₀ levels.

Using 3PG as an alternative to PEP

As PEP is likely to be a flux-limiting metabolite, we explored the possibility of using an alternative metabolite in its place. Three different metabolites have been proposed to be incorporated in the sidechain of F₄₂₀: PEP, 2-phospho-l-lactate and 3-phospho-d-glycerate^18,27,37. While 2-phospho-l-lactate has not been observed in E. coli¹⁸, 3-phosphoglycerate (3PG) is a glycolytic pathway intermediate present in E. coli at 10 times the concentration of PEP³⁸. Moreover, in the context of the iEco-F420 model, PEP-dependent F₄₂₀ biosynthesis requires an additional FMN-dependent reduction step (the FbiB-dependent conversion of dehydro-F₄₂₀-0 into F₄₂₀-0) indicating that additional carbon would need to be diverted into FMN biosynthesis^28,39. Preliminary evidence suggests that 3PG-F₄₂₀, unlike F_O and F_OP, is accepted as a cofactor by F₄₂₀-dependent enzymes with similar kinetics to standard F₄₂₀³⁰. Given the relative abundance of 3PG, we investigated it as an alternative to PEP by substitution of M. smegmatis FbiD with that of P. rhizoxinica.

Although 3PG is present at a higher intracellular concentration than PEP (1.5 mM cf. 0.18 mM)³⁸ and is predicted to provide relatively similar maximum theoretical F₄₂₀ yields (Supplementary Fig. S1), the experimentally determined yield of F₄₂₀-3PG was found to be lower than for F₄₂₀-PEP (Fig. 5). Moreover, no F₄₂₀-3PG formation was observed with either succinate or fumarate as carbon source. This contrasts with the model predictions of feasible theoretical yields for F₄₂₀-3PG with all carbon sources tested (Supplementary Fig. S1). It is possible that the P. rhizoxinica FbiD product, glyceryl-2-diphospho-5ʹ-guanosine, 3PG-F₄₂₀-0 and/or its polyglutamated derivatives are poor substrates for the enzymes catalysing subsequent steps in F₄₂₀ biosynthesis, which had been sourced from Mycobacteria and may have low specificity for 3PG containing F₄₂₀ metabolites (Fig. 5).

Over-expression of PEP synthase increases the yield of F₄₂₀

Given that PEP is a limiting metabolite in F₄₂₀ biosynthesis, we investigated whether production of PEP could be increased. Growth on fumarate and succinate is known to increase the expression of PEP-producing enzymes PPS and PPCK⁴⁰ (Fig. 3A). Indeed, overexpression of PPS has been used to increase PEP concentrations in vivo^41,42 to improve the yield of shikimic acid⁴³, aromatic amino acids^42,44 and lycopene⁴⁵ biosynthesis. However, overexpressing PPS has been reported to negatively affect cell growth due to the excretion of pyruvate and acetate⁴².

In an attempt to increase intracellular PEP concentrations, we overexpressed PPS and PPCK from an IPTG-inducible expression plasmid and studied the effect on F₄₂₀ yield. Consistent with previous reports⁴¹, overexpression of PPS resulted in growth inhibition. Therefore, to improve final biomass concentration, PPS was only induced once cell density (OD₆₀₀) was greater than 1.0, which resulted in significant improvement in F₄₂₀ yield. We tested overexpression of PPS when grown on different carbon sources, as shown in Fig. 6. Overexpression of PPS improved the yield of F₄₂₀ from 0.27 to 0.54 µmol/g DCW using glucose and from 0.53 to 0.80 µmol/g DCW using glycerol. When grown on pyruvate, an F₄₂₀ yield of 1.60 µmol/g DCW was observed without the addition of IPTG. With the addition of IPTG, the yield of F₄₂₀ yield decreased to 0.90 µmol/g DCW. The yield of F₄₂₀ also decreased after PPS induction when grown on succinate or fumarate (Fig. 6A). The pyruvate:PEP node of E. coli metabolism is highly regulated at both the transcriptional and metabolic levels⁴⁶, it is possible that PPS is metabolically regulated during gluconeogenesis or that the reversable flux through PPS is being driven thermodynamically towards pyruvate formation when grown on gluconeogenic carbon sources. With glucose and glycerol, induction of PPS with IPTG resulted in significant improvement in the yield of F₄₂₀ as compared to non-induced PPS. On the contrary, with pyruvate, non-induced PPS resulted in significantly higher yield and productivity of F₄₂₀ compared to IPTG induced PPS. It may be that optimal PPS expression levels differ with different carbon sources. The highest yield of F₄₂₀ obtained was 1.60 µmol/g DCW, with a productivity of 0.17 µmol/h, using pyruvate as carbon source with leaky expression of PPS.

The impact of PPCK overexpression on F₄₂₀ yield was also studied (Fig. 7). Unlike the expression of PPS, no improvement in F₄₂₀ production was observed during PPCK overexpression. We confirmed the protein was expressed in soluble form (Supplementary Fig. S6). It is quite likely that we saw no difference in F₄₂₀ concentration when PPCK was over-expressed because E. coli PPCK activity is metabolite controlled, either by the cellular PEP concentration or PEP:pyruvate ratio⁴⁶. We therefore investigated the potential of uncontrolled PPCK overexpression using the iEco-F₄₂₀ model. We explored the overall capability of the metabolic network to improve flux through FbiB (i.e., production of mature F₄₂₀) by simulating over-expression of PPS or PPCK. The results, shown in Supplementary Fig. S2, indicate that by forcing a higher flux through PPS or PPCK, the maximum FbiB flux (shown by black arrows) drops unless it occurs at a non-zero flux through PPS or PPCK. These results indicate the maximum stoichiometric capacity for F₄₂₀ biosynthesis as a result of over-expressing PPS or PPCK; however, the overall kinetics of the system and regulatory mechanisms for growth with different carbon sources would significantly influence F₄₂₀ yields, in vivo. The experimental results (Fig. 6) confirmed improved F₄₂₀ biosynthesis when using glucose and pyruvate as a result of PPS overexpression, in agreement with the simulation results shown in Supplementary Fig. S2 for these carbon sources. It should be noted that the simulation results of Supplementary Fig. S2 also demonstrate the potential impact of the type of transporter on the flux through CofE when over-expressing PPS or PPCK.

The effect of time and carbon source on polyglutamate chain length

The final step in F₄₂₀ biosynthesis is the addition of between one and nine glutamate residues to the F₄₂₀-0 intermediate to yield F₄₂₀-n (n: number of glutamate residues)^47,48. What influences the tail length of F₄₂₀ is still not clear, although in vitro analysis of F₄₂₀-0:g-glutamate ligases from different organisms has revealed that they typically produce F₄₂₀ species with polyglutamate chain lengths consistent with F₄₂₀ obtained from the native organisms^48,49. The number of glutamate residues influences the cofactor affinity of some F₄₂₀-dependent enzymes; for example, the F₄₂₀-dependent oxidoreductases MSMEG_2027, MSMEG_0777 and MSMEG_3380 from M. smegmatis reportedly having a high affinity for long chain F₄₂₀ rather than shorter-chain F₄₂₀⁵⁰. Similar effects are seen with polyglutamylated folates and folate mimics^51,52,53. Interestingly, F₄₂₀-n composition changes with different growth phases of Methanosarcina barkeri and M. mazei⁵⁴. We therefore investigated the composition of F₄₂₀ during different growth phases of E. coli. The composition of F₄₂₀-n at various time points is shown in Fig. 8. When grown with glucose or glycerol as the carbon source, E. coli initially produced short chain F₄₂₀-(1–4) in higher proportions, which shifted over time to predominantly longer chain F₄₂₀-(5–8) (Fig. 8A, B). CofE from M. smegmatis (the enzyme used in this system) has been shown to produce predominantly longer F₄₂₀ species (5–8) in stationary phase⁵⁰.

Interestingly, we found that the tail length distribution at the end of the exponential phase was influenced by the carbon source used (Fig. 8C, D). Growth on succinate yielded the highest proportion of long chain F₄₂₀, with F₄₂₀-(5–8) comprising > 90%. Glycerol has the next highest proportion of F₄₂₀-(5–8) at > 80%, with glucose and acetate with the lowest levels of F₄₂₀-(5–8) produces (< 30% and < 25%, respectively) (Fig. 8C). The iEco-F420 model was used to guide interpretation of carbon source-dependent tail length distribution. According to the cofactor biosynthesis pathway shown in Fig. 1, two molecules of GTP per molecule of glutamate are required to metabolise an F₄₂₀ molecule with only one glutamate residue. Likewise, in an ideal case where all incoming carbon to the E. coli has to end up in F₄₂₀ with only one glutamate residue, the iEco-F420 model predicted that the ratio of sum of fluxes through all glutamate-producing reactions (({v}_{glu}^{t})) to sum of fluxes through all GTP-producing reactions (({v}_{gtp}^{t})) has to be equal to two regardless of the type of carbon source. However, when the model was used to simulate F₄₂₀ biosynthesis with chain length compositions observed experimentally, flux predictions suggested deviations in the ratio of ({v}_{glu}^{t}) to ({v}_{gtp}^{t}), which depends on the type of carbon source. Interestingly, the ratio of ({v}_{glu}^{t}) to ({v}_{gtp}^{t}) was predicted to be 1.731 and 1.772 using succinate and glycerol, respectively, showing the largest deviation for a ratio of two. On the other hand, the ratio of ({v}_{glu}^{t}) to ({v}_{gtp}^{t}) was predicted by the model to be 1.994, 1.960, and 1.873 using glucose, acetate, and pyruvate, respectively, explaining why the lowest proportion of long chain F₄₂₀ was observed with these carbon sources.

3PG-F₄₂₀ yielded significantly higher fraction of short chain F₄₂₀-(1–4) > 70% (Fig. 8D) compared to PEP derived F₄₂₀ irrespective of the carbon source used. This could be due to the difference in the kinetics of the enzymes for 3PG-F₄₂₀ and PEP-F₄₂₀.

The iEco-F420 metabolic model additionally provided some insights into the energetic differences in F₄₂₀ biosynthesis with only one glutamate tail as well as with varying number of glutamate tails. For all carbon sources examined, yields were higher for F₄₂₀ with only one glutamate than those for a mixture of F₄₂₀ molecules with different chain-lengths. This is because at a fixed growth rate (i.e., constant cell mass yield), total energy production (in the form of ATP) is higher for biosynthesis of F₄₂₀ with one glutamate than that for biosynthesis of a mixture of F₄₂₀ molecules (Fig. 9). Based on the results illustrated in Fig. 9, glucose maintains the highest cell mass per mole of ATP produced by cells, which explains the low F₄₂₀ yield from glucose compared to other carbon sources as shown in Fig. 2A. Assimilation of acetate as the sole carbon source requires the activation of ATP-dependent acetate kinase. Therefore, cells have to produce ATP in order to uptake carbon source for survival, which results in low growth rates (Fig. 2C) and maintaining the lowest cell mass yield per mole ATP produced among other carbon sources (Fig. 9) but, relatively high F₄₂₀ yields (Fig. 2A; Supplementary Fig. S1). According to the modelling predictions, acetate might provide benefits from industrial perspective because, cells would be forced to produce ATP for fueling F₄₂₀ production rather than for their growth.