A mixed-substrate feeding strategy was devised to improve aromatics production by engineered P. putida S12. The approach was based on the assumption that the precursors E4P and PEP were limiting factors for aromatic biosynthesis and that their availability could be improved by stimulating the PP pathway fluxes through pentose co-feeding. A p-hydroxybenzoate producing strain, P. putida S12palB2, was selected as an aromatics-producing model system. As this strain does not have the natural ability to utilize pentoses, a xylose isomerase pathway was introduced, and the initially low growth rate on xylose was improved via an evolutionary selection procedure.
Surprisingly, tenfold less transfers were required to achieve growth characteristics similar to those of the previously evolved xylose-utilizing strain P. putida
S12xylAB2 (Meijnen et al. 2008
). In part, this can be attributed to the targeted disruption of gcd
, since more than ten transfers had been required for strain S12xylAB2 to acquire a gcd
negative phenotype. In addition to the xylose utilization efficiency, also the p
-hydroxybenzoate yield was improved after the evolutionary selection. This phenomenon may be explained by the increased PP pathway activity associated with the improved xylose utilization phenotype, leading to an intrinsically improved E4P and PEP availability, independently from xylose co-feeding.
As anticipated, xylose co-feeding considerably improved the p
-hydroxybenzoate yield. The increased product yield was observed with both glucose and glycerol as the primary substrate and was shown not to be caused by xylose consumption per se. Remarkably, both the product and biomass yield on glycerol were consistently higher compared to glucose, either with or without xylose co-feeding. This may be attributed to regulatory effects (e.g., carbon catabolite repression) but could also indicate that PEP availability is more critical for efficient p
-hydroxybenzoate production than E4P availability. If it is assumed that pyruvate dikinase (PEP synthase) is active only under gluconeogenic conditions (Sauer and Eikmanns 2005
), twice the amount of GAP (and, thus, PEP) can be obtained from glycerol compared to glucose, which is metabolized via the Entner–Doudoroff pathway in pseudomonads (del Castillo et al. 2007
; Fuhrer et al. 2005
). In addition, the glycerol-associated yield improvement appears to be connected to the evolutionary selection, since no such effect has been observed with the parent strains of P. putida
S12pal_xylB7 (Verhoef et al. 2010
). Presumably, the increased PP pathway activity associated with the efficient xylose-utilizing phenotype may allow for a more efficient equilibration of PEP and E4P levels, resulting in more efficient p
-hydroxybenzoate production. It should be noted that the applied proportion of xylose in the feed showed little effect within the range tested, whether the primary substrate was glucose or glycerol. Apparently, the p
-hydroxybenzoate production is not very sensitive to variations in relative xylose concentrations above a certain threshold value.
Unexpectedly, the capacity to transport and/or utilize xylose appeared to be dependent on the primary carbon source. With glycerol, a low concentration of residual xylose was observed that is presumably close to the Km
of the—yet unidentified—xylose transporter in P. putida
S12pal_xylB7. With glucose as the primary substrate, however, the residual xylose concentrations were higher and furthermore increased more than proportionally with increasing amounts of xylose in the feed. Although this phenomenon is still subject to further study, it may be hypothesized that xylose transport in P. putida
S12pal_xylB7 is PEP dependent. This would be consistent with the observed increase in residual xylose concentrations with decreasing glucose feed (an already relatively inefficient source of PEP), the relative independency between residual xylose concentration and glycerol feed (a good source of PEP), and the decreased growth rate on xylose when Pal/Tal was introduced (drain on PEP for p
-hydroxybenzoate production). In that case, replacing any PEP-dependent transport systems would be an obvious target for further strain improvement. The GAP/PEP availability may furthermore be improved by constructing an ED-negative, glycolytic P. putida
S12 strain. The contribution of the (ATP-driven) E. coli
xylose transporter XylFGH to xylose import was presumably limited as observed previously (Meijnen et al. 2008
We have demonstrated that p
-hydroxybenzoate production in P. putida
can be considerably improved by co-feeding different carbon sources that are metabolized via different, (partly artificial) pathways. Thus, the availability of the key aromatics precursors, PEP and E4P, is improved. In addition to p
-hydroxybenzoate, the production of other aromatic compounds derived from aromatic amino acids may be stimulated via this strategy. Moreover, lignocellulosic hydrolysate, the expected major feedstock for future production of biobased fuels and chemicals (Himmel and Bayer 2009
; Kumar et al. 2008
; Lange 2007
), seems to be ideally suited for aromatics production since glucose and xylose are the predominant constituents. Also the improved production on glycerol presents an additional possibility to deploy a cheap and abundant waste substrate for biocatalytic production of (fine) chemicals.