Engineering Pseudomonas putida S12 for Efficient Utilization of d -Xylose and l -Arabinose

Abstract

The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to utilize xylose as a substrate by expressing xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli. The initial yield on xylose was low (9% [g CDW g substrate ؊1 ], where CDW is cell dry weight), and the growth rate was poor (0.01 h ؊1 ). The main cause of the low yield was the oxidation of xylose into the dead-end product xylonate by endogenous glucose dehydrogenase (Gcd). Subjecting the XylAB-expressing P. putida S12 to laboratory evolution yielded a strain that efficiently utilized xylose (yield, 52% [g CDW g xylose ؊1 ]) at a considerably improved growth rate (0.35 h ؊1 ). The high yield could be attributed in part to Gcd inactivity, whereas the improved growth rate may be connected to alterations in the primary metabolism. Surprisingly, without any further engineering, the evolved D-xylose-utilizing strain metabolized L-arabinose as efficiently as D-xylose. Furthermore, despite the loss of Gcd activity, the ability to utilize glucose was not affected. Thus, a P. putida S12-derived strain was obtained that efficiently utilizes the three main sugars present in lignocellulosic hydrolysate: glucose, xylose, and arabinose. This strain will form the basis for a platform host for the efficient production of biochemicals from renewable feedstock.

Establishment of Oxidative d -Xylose Metabolism in Pseudomonas putida S12

“…Growth experiments were not reproducible, and if any growth was observed in MMX, the growth rate was very low (0.03 h Ϫ1 ). Assuming that D-xylose is transported across the cytoplasmic membrane, an assumption which is supported by previous observations (14), the gcd knockout strain must be able to oxidize D-xylose, but only by using intracellularly expressed XylB. Hence, the location of D-xylose oxidation also differs in this strain.…”

“…An optical density of 1.0 corresponds to 0.49 g (dry weight) of cells liter Ϫ1 . Sugars and organic acids were analyzed by ion chromatography (Dionex ICS3000 system) as described previously (14).…”

“…To determine the interaction between the endogenous and heterologous activities involved in the oxidative D-xylose catabolism in the engineered P. putida S12 strains, D-xylose oxidation and lower-pathway activity assays were performed. As D-xylose can be oxidized by the endogenous periplasmic protein Gcd (14), as well as by the heterologously expressed protein XylB, both activities were determined. The results of the assays are summarized in Table 4.…”

“…In a previous study, the solvent-tolerant bacterium Pseudomonas putida S12, known for its use as a platform host for the production of aromatic compounds (15,16,19,22), was engineered to use D-xylose as a sole carbon source. This was achieved by introducing genes encoding the phosphorylative D-xylose metabolic pathway of Escherichia coli, followed by laboratory evolution (14). Prior to evolutionary improvement, extensive oxidation of D-xylose to D-xylonate occurred, resulting in a very low biomass-for-substrate yield as D-xylonate is a metabolic dead-end product in P. putida.…”

A latent capacity for evolutionary innovation through exaptation in metabolic systems

“…Viability on a new carbon source can be an important adaptation, and anecdotal evidence shows that this ability can originate as a pre-adaptation 13,14 . For example, laboratory evolution of Pseudomonas putida for increased biomass yield on xylose as a carbon source produces strains that utilize arabinose as efficiently as xylose, even though the ancestral strains did not utilize arabinose 14 .…”