A P. putida
S12 strain was constructed that efficiently utilizes d
-xylose, as well as l
-arabinose. The expression of xylose isomerase and xylulokinase is essential for the utilization of both pentoses, but the subsequent laboratory evolution is key to the efficiency with which these pentoses are metabolized. The improved yield on xylose attained by the evolved strain could largely be attributed to Gcd having become inactive in the evolved strain, preventing xylose “loss” as a result of oxidation to xylonate. Although targeted disruption of the gcd
gene in wild-type P. putida
S12 resulted in an improved biomass yield on xylose, this strategy did not result in an improved growth rate, indicating that other changes occurred in the evolved strain. It may be speculated that mutations occurred that affected the metabolic fluxes through the PP pathway, which is the expected route by which xylose is metabolized. In P. putida
, a complete PP pathway is present, but metabolic flux analyses on Pseudomonas fluorescens
have shown that this pathway mainly serves to replenish biosynthetic intermediates (4
). Similarly, modest flux distributions have been demonstrated for P. putida
S12 (unpublished data). Therefore, the function of the PP pathway may have changed from anabolic to catabolic in the evolved P. putida
The observation that d
-xylose and l
-arabinose are consumed with equal efficiency, both in terms of biomass yield and specific growth rate, suggests that both pentoses are consumed via the PP pathway. In addition, the evolved strain appears to have acquired an efficient l
-arabinose uptake system, as wild-type P. putida
S12 required the expression of both XylAB and the AraFGH transporter for arabinose utilization. Although coexpressing a high-affinity xylose transporter (XylFGH from E. coli
) did not have a significant effect on xylose metabolism in the nonevolved strain (Table ), the possibility that improved xylose uptake has contributed to more-efficient xylose utilization in strain S12xylAB2 cannot be excluded. Since pentose transporters have been shown to be promiscuous (24
), it may be hypothesized that efficient l
-arabinose uptake has coevolved with improved d
-xylose uptake in strain S12xylAB2.
At this point it is unclear how arabinose is converted into a PP pathway intermediate (Fig. ). The results of enzyme assays showed that l-arabinose is a substrate for XylAB, and growth on arabinose did not occur without the expression of XylAB. The expected product, l-ribulose-5-phosphate, is not a central pathway intermediate, and a C4 epimerization would be required to form d-xylulose-5-phosphate. Indeed, the formation of d-xylulose-5-phosphate from l-arabinose was suggested by the results of the transketolase assay, but no indications that this strain contains an AraD homologue were found in the P. putida S12 genome sequence (unpublished data). Therefore, it is proposed that the endogenous ribulose-5-phosphate-3-epimerase shows nonspecific epimerization activity on l-ribulose-5-phosphate, converting the molecule into d-xylulose-5-phosphate. Further research is ongoing to confirm this hypothesis.
FIG. 4. Utilization of d-xylose and l-arabinose may proceed via (partly) shared pathways. Indications were found that l-arabinose can be converted into d-xylulose-5-phosphate by the combined action of XylAB and a yet-unidentified C4 epimerase (indicated by the (more ...)
Despite the loss of Gcd activity, glucose was still efficiently used as the sole carbon source by the evolved P. putida
S12xylAB2. The glucose catabolism in P. putida
operates through the action of three simultaneous pathways that converge at 6-phosphogluconate. Glucose is preferentially oxidized in the periplasm by Gcd to (2-keto-)gluconate and subsequently phosphorylated in the cytoplasm to yield 6-phosphogluconate (2
). Alternatively, glucose is imported by an ABC-transport system, phosphorylated by glucokinase, and oxidized to 6-phosphogluconate (2
). Apparently, the evolved xylose-utilizing strain can readily switch to this alternative pathway for glucose oxidation without affecting the yield or growth rate.
The absence of active Gcd may itself provide an explanation for the increased gcd
transcription levels observed in both the evolved strain and the gcd
knockout strain. Glucose induces the expression of gcd
), and with active Gcd present, glucose is rapidly oxidized to gluconate and 2-ketogluconate, resulting in a swift downregulation of gcd
. However, without active Gcd, glucose persists in the medium, resulting in increased levels of gcd
mRNA. So, with an intact gcd
gene and associated RBS present in the evolved strain, and apparently even increased transcription levels, the absence of active Gcd must be attributed to some posttranslational effect. The amino acid sequence of Gcd shows that the protein is excreted and that it contains four transmembrane regions (not shown), which is consistent with the periplasmic oxidation of sugars. It raises the possibility that malfunctions appear in the Gcd translocation machinery, leading to faulty localization of the enzyme, improper folding, or inadequate anchoring to the inner membrane. The exact cause of the inactivity of Gcd remains to be investigated.
In conclusion, a P. putida S12 strain was obtained that efficiently utilizes the three most-abundant sugars in lignocellulose, glucose, xylose, and arabinose, as sole carbon sources. The applied evolutionary approach proved to be a powerful method to optimize the initial inefficient xylose-utilizing strain. Transcriptome and proteome analyses, as well as metabolic flux analysis, are currently being performed to identify the changes in the metabolism of the evolved xylose-utilizing strain. The insight gained into the molecular background of the efficient pentose utilization will be employed to incorporate this property into optimized substitute-aromate-producing P. putida S12-derived strains, thereby contributing to the economical feasibility of the production of such biochemicals from renewable feedstock.