Biotransformation of d-xylose to d-xylonate coupled to medium-chain-length polyhydroxyalkanoate production in cellobiose-grown Pseudomonas putida EM42

Abstract

Co-production of two or more desirable compounds from low-cost substrates by a single microbial catalyst could greatly improve the economic competitiveness of many biotechnological processes. However, reports demonstrating the adoption of such co-production strategy are still scarce. In this study, the ability of genome-edited strain Pseudomonas putida EM42 to simultaneously valorize d-xylose and d-cellobiose - two important lignocellulosic carbohydrates - by converting them into the platform chemical d-xylonate and medium-chain-length polyhydroxyalkanoates, respectively, was investigated. Biotransformation experiments performed with P. putida resting cells showed that promiscuous periplasmic glucose oxidation route can efficiently generate extracellular xylonate with a high yield. Xylose oxidation was subsequently coupled to the growth of P. putida with cytoplasmic β-glucosidase BglC from Thermobifida fusca on d-cellobiose. This disaccharide turned out to be a better co-substrate for xylose-to-xylonate biotransformation than monomeric glucose. This was because unlike glucose, cellobiose did not block oxidation of the pentose by periplasmic glucose dehydrogenase Gcd, but, similarly to glucose, it was a suitable substrate for polyhydroxyalkanoate formation in P. putida. Co-production of extracellular xylose-born xylonate and intracellular cellobiose-born medium-chain-length polyhydroxyalkanoates was established in proof-of-concept experiments with P. putida grown on the disaccharide. This study highlights the potential of P. putida EM42 as a microbial platform for the production of xylonate, identifies cellobiose as a new substrate for mcl-PHA production, and proposes a fresh strategy for the simultaneous valorization of xylose and cellobiose.

Fig. 1

Co‐production of d‐xylonate and medium‐chain‐length polyhydroxyalkanoates from d‐xylose and d‐cellobiose, respectively, in bglC + P. putida EM42. Innate periplasmic oxidative route and introduced cytoplasmic β‐glucosidase BglC from Thermobifida fusca allow simultaneous valorization of d‐xylose and d‐cellobiose in Pseudomonas putida EM42. d‐xylose is oxidized to platform chemical d‐xylonate which is released into the medium. d‐cellobiose, on the other hand, is transported into the cell, cleaved in two d‐glucose molecules by BglC and gives rise to acetyl‐CoA, a precursor molecule for the production of intracellular biopolymers (polyhydroxyalkanoates, PHA) via de novo fatty acid synthesis in nitrogen‐limited conditions. Periplasmic space and cytoplasm are shown in dark and pale grey, respectively. Abbreviations: Gcd, glucose dehydrogenase; Gnl, gluconolactonase; PQQ and PQQH, pyrroloquinoline quinone and its reduced form, respectively; TCA cycle, tricarboxylic acid cycle; mcl‐PHA, medium‐chain‐length polyhydroxyalkanoates.

Fig. 2

Biotransformation of xylose to xylonate by P. putida EM42 resting cell. Incubation of resting cells of (A) Pseudomonas putida EM42 and (B) its deletion mutant P. putida EM42 Δgcd in minimal medium with 5 g l⁻¹ d‐xylose. Experiments were carried out in 25 ml of M9 minimal medium in flasks shaken at 170 r.p.m. and 30°C. (C) Incubation of P. putida EM42 resting cells in 25 ml of minimal medium with 10 g l⁻¹ d‐xylose in flasks shaken at 170 r.p.m. and 30°C. (D) Incubation of P. putida EM42 resting cells in 25 ml of buffered M9 minimal medium (100 mM sodium phosphate buffer) with 10 g l⁻¹ d‐xylose in flasks shaken at 300 r.p.m. and 30°C. In all experiments, minimal medium was inoculated to the initial A ₆₀₀ of 0.5 using cells obtained from an overnight culture in lysogeny broth. d‐xylose, filled squares (■); d‐xylonate, filled circles (●); d‐xylono‐λ‐lactone, filled triangles (▲); cell biomass, open diamonds (◊). Data points shown as mean ± SD of three biological replicates.

Fig. 3

Biotransformation of d‐xylose to d‐xylonate by P. putida EM42 growing on d‐glucose or d‐cellobiose. Two‐day cultures of (A) Pseudomonas putida EM42 in minimal medium with 10 g l⁻¹ d‐xylose and 5 g l⁻¹ d‐glucose used as a sole carbon source for growth. (B,C,D) Cultures of Pseudomonas putida EM42 pSEVA2213_bglC in minimal medium with 10 g l⁻¹ d‐xylose and 5 g l⁻¹ d‐cellobiose used as a sole carbon source. Experiments (A) and (B) were carried out in 25 ml of minimal medium in flasks shaken at 170 r.p.m. and 30°C. Minimal medium was inoculated to the initial A ₆₀₀ of 0.1 using cells obtained from an overnight culture in lysogeny broth. Experiments (C) and (D) were performed in flask with 25 ml of minimal medium buffered with 100 mM sodium phosphate buffer and shaken at 300 r.p.m. (30°C). Cells used for inoculation of the main culture to the initial A ₆₀₀ of 0.1 were pre‐grown overnight in lysogeny broth (C) or in minimal medium with 5 g l⁻¹ d‐cellobiose (D). d‐xylose, filled squares (■); d‐xylonate, filled circles (●); d‐xylono‐λ‐lactone, filled triangles (▲); d‐glucose, filled diamonds (♦); d‐cellobiose, open circles (○); cell biomass, open diamonds (◊). Data points shown as mean ± SD of three biological replicates. Please note that the elevated xylonate concentrations detected after 4 and 8 h in the culture (A) do not reflect the real levels of the xylose oxidation product. Hydroxamate method (Lien, 1959) used here for xylonate quantification was originally designed for the detection of gluconate and its lactone, which temporarily accumulated in the culture medium during glucose utilization in (A). Accumulation of gluconate at the times 4 and 8 h was verified also by the specific d‐Gluconic Acid/ d‐Glucono‐δ‐lactone Assay Kit (Megazyme, data not shown).

Fig. 4

Co‐production of d‐xylonate and PHA from d‐xylose and d‐cellobiose, respectively, by cellobiose‐grown P. putida EM42 pSEVA2213_bglC. (A) Initial culture inoculated from overnight pre‐culture in lysogeny broth was carried out in 25 ml of nitrogen‐limited M9 minimal medium with 100 mM sodium phosphate buffer, 5 g l⁻¹ cellobiose and 10 g l⁻¹ xylose in flasks shaken at 300 r.p.m. and 30°C. (B) Relative fluorescence of bacterial population analysed by flow cytometry every 24 h during the three‐day culture. Cells were stained by Nile Red and processed as described in Supplementary Information. (C) Confocal microscopy of P. putida cells collected at denoted time intervals. Stained bacteria were processed as described in Supplementary Information. White scale bars show 2 μm distance. (D) Culture inoculated from overnight pre‐cultures in M9 minimal medium with 5 g l⁻¹ cellobiose was carried out in the same conditions as were described for (A). (E) Content and monomer composition of medium‐chain‐length polyhydroxyalkanoates in cell dry weight of P. putida EM42 pSEVA2213_bglC cells collected at the end of the two‐day culture (graph D). d‐xylose, filled squares (■); d‐xylonate, filled circles (●); d‐xylono‐λ‐lactone, filled triangles (▲); d‐glucose, filled diamonds (♦); d‐cellobiose, open circles (○); cell biomass, open diamonds (◊). Data points and columns in (A), (B) and (D) show mean ± SD of three biological replicates.