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-cellobiosetwo important lignocellulosic carbohydratesby 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 b-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. Coproduction of extracellular xylose-born xylonate and intracellular cellobiose-born medium-chain-length polyhydroxyalkanoates was established in proof-ofconcept 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.

Metabolic engineering of Pseudomonas putida for production of the natural sweetener 5‐ketofructose from fructose or sucrose by periplasmic oxidation with a heterologous fructose dehydrogenase

“…Remarkably, the 5‐KF production rate from sucrose was comparable to the one obtained directly with fructose, confirming that Inv1417 is a highly active invertase. Furthermore, our results indicate that P. putida has a high potential to serve as host for periplasmic oxidations not only with native enzymes, as recently published (Dvořák et al ., 2020 ), but also with complex heterologous enzymes like G . japonicus Fdh.…”

Rational engineering of natural polyhydroxyalkanoates producing microorganisms for improved synthesis and recovery

“…By knocking out the gcd gene, coding for the glucose dehydrogenase, the cells no longer synthesized xylonate. The research team then sought to produce mcl ‐PHA on the pentose as feedstock using resting cells but failed to mitigate the synthesis of the coproduct xylonate achieving only traces (0.2 g L −1 ) of the polyester (Dvořák et al, 2020 ) (Table 1 ). Similarly, P. putida KT2440 also exhibits impaired sucrose metabolism.…”

Industrial biotechnology of Pseudomonas putida: advances and prospects

“…The structural composition of PHAs can be adjusted by feeding of precursors (Wang et al 2010), cultivation conditions, and strain engineering (Tripathi et al 2013). The companies Kaneka (Japan), Telles (USA), Jiangsu Nantian (China), Tianjin GreenBio (China), Tepha (USA), DSM (The Netherlands), Biomer Biotechnology (Germany), Bioon (Italy), Polyferm (Canada), and Biomatera (Canada) report to produce PHA polymers on an industrial scale using P. putida (Poltronieri and Kumar 2017 (Dvořák et al 2020b). Such co-production of valuable bioproducts from low-cost substrates along with PHA represents a good opportunity to mitigate the overall PHA production costs (Li et al 2017).…”