Engineering sucrose metabolism in Pseudomonas putida highlights the importance of porins

Abstract

Using agricultural wastes as a substrate for biotechnological processes is of great interest in industrial biotechnology. A prerequisite for using these wastes is the ability of the industrially relevant microorganisms to metabolize the sugars present therein. Therefore, many metabolic engineering approaches are directed towards widening the substrate spectrum of the workhorses of industrial biotechnology like Escherichia coli, yeast or Pseudomonas putida. For instance, neither xylose or arabinose from cellulosic residues, nor sucrose, the main sugar in waste molasses, can be metabolized by most E. coli and P. putida wild types. We evaluated a new, so far uncharacterized gene cluster for sucrose metabolism from Pseudomonas protegens Pf-5 and showed that it enables P. putida to grow on sucrose as the sole carbon and energy source. Even when integrated into the genome of P. putida, the resulting strain grew on sucrose at rates similar to the rate of the wild type on glucose - making it the fastest growing, plasmid-free P. putida strain known so far using sucrose as substrate. Next, we elucidated the role of the porin, an orthologue of the sucrose porin ScrY, in the gene cluster and found that in P. putida, a porin is needed for sucrose transport across the outer membrane. Consequently, native porins were not sufficient to allow unlimited growth on sucrose. Therefore, we concluded that the outer membrane can be a considerable barrier for substrate transport, depending on strain, genotype and culture conditions, all of which should be taken into account in metabolic engineering approaches. We additionally showed the potential of the engineered P. putida strains by growing them on molasses with efficiencies twice as high as obtained with the wild-type P. putida. This can be seen as a further step towards the production of low-value chemicals and biofuels with P. putida from alternative and more affordable substrates in the future.

Effect of Photocaged Isopropyl β‐d‐1‐thiogalactopyranoside Solubility on the Light Responsiveness of LacI‐controlled Expression Systems in Different Bacteria

“…Furthermore, in pseudomonads including P. putida , the outer membrane exhibits a reduced permeability as compared to E. coli . The uptake of small water‐soluble molecules is mainly mediated by a defined set of specific porins such as OprF, which is characterised by a significantly slower diffusion rate compared to the more unspecific E. coli porins OmpF and OmpC [25–26] . As a consequence, the water‐soluble compound 10 b could be transported over the outer membrane in a slower process.…”

Biotransformation of 2,4‐dinitrotoluene in a phototrophic co‐culture of engineered Synechococcus elongatus and Pseudomonas putida

“…R34, transferred into the P . putida chromosome via Tn 7 transposition ( top panel ), and plasmid pSEVA221· cscRABY (Löwe et al , ), encoding the functions needed for sucrose utilization from P . protegens Pf‐5 ( bottom panel ).…”

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

“…The soil bacterium and growingly used robust platform strain P. putida KT2440 can naturally assimilate a spectrum of aromatic compounds and organic acids but only a few plant biomass‐derived carbohydrates: glucose, mannose and fructose (Linger et al , 2014; Belda et al , 2016; Nikel and de Lorenzo, 2018; Jayakody et al , 2018). Its metabolism was engineered to reach out to other sugars, including carbohydrates typically produced upon (hemi)cellulose hydrolysis or pyrolysis (Meijnen et al , 2008; Linger et al , 2016; Löwe et al , 2018). In a recent work, P. putida EM42, a P. putida KT2440‐derived strain with streamlined genome and better physiological properties (Martínez‐García et al , 2014), was empowered with a xylose transporter and isomerase pathway from Escherichia coli along with a cytoplasmic β‐glucosidase BglC from Thermobifida fusca (Dvořák and de Lorenzo, 2018).…”