Refactoring the upper sugar metabolism of Pseudomonas putida for co-utilization of cellobiose, xylose, and glucose

Víctor de Lorenzo,Pavel Dvořák

doi:10.1016/j.ymben.2018.05.019

Abstract

Given its capacity to tolerate stress, NAD(P)H/ NAD(P) balance, and increased ATP levels, the platform strain Pseudomonas putida EM42, a genome-edited derivative of the soil bacterium P. putida KT2440, can efficiently host a suite of harsh reactions of biotechnological interest. Because of the lifestyle of the original isolate, however, the nutritional repertoire of P. putida EM42 is centered largely on organic acids, aromatic compounds and some hexoses (glucose and fructose). To enlarge the biochemical network of P. putida EM42 to include disaccharides and pentoses, we implanted heterologous genetic modules for D-cellobiose and D-xylose metabolism into the enzymatic complement of this strain. Cellobiose was actively transported into the cells through the ABC complex formed by native proteins PP1015-PP1018. The knocked-in β-glucosidase BglC from Thermobifida fusca catalyzed intracellular cleavage of the disaccharide to D-glucose, which was then channelled to the default central metabolism. Xylose oxidation to the dead end product D-xylonate was prevented by deleting the gcd gene that encodes the broad substrate range quinone-dependent glucose dehydrogenase. Intracellular intake was then engineered by expressing the Escherichia coli proton-coupled symporter XylE. The sugar was further metabolized by the products of E. coli xylA (xylose isomerase) and xylB (xylulokinase) towards the pentose phosphate pathway. The resulting P. putida strain co-utilized xylose with glucose or cellobiose to complete depletion of the sugars. These results not only show the broadening of the metabolic capacity of a soil bacterium towards new substrates, but also promote P. putida EM42 as a platform for plug-in of new biochemical pathways for utilization and valorization of carbohydrate mixtures from lignocellulose processing.

Highlights

IntroductionDue to the physicochemical stresses that prevail in the niches in which the soil bacteriumPseudomonas putida thrives (it is typically abundant in sites contaminated by industrial pollutants), this microorganism is endowed with a large number of traits desirable in hosts of harsh biotransformations of industrial interest (Nikel et al, 2014)
Due to the physicochemical stresses that prevail in the niches in which the soil bacteriumPseudomonas putida thrives, this microorganism is endowed with a large number of traits desirable in hosts of harsh biotransformations of industrial interest (Nikel et al, 2014)
Within 24 h of culture in minimal medium, P. putida EM42 expressing the intracellular -glucosidase BglC utilized 5 g L-1 cellobiose as a sole carbon source, outperforming the best cellobiose-utilizing E. coli strain constructed to date

Summary

Introduction

Due to the physicochemical stresses that prevail in the niches in which the soil bacteriumPseudomonas putida thrives (it is typically abundant in sites contaminated by industrial pollutants), this microorganism is endowed with a large number of traits desirable in hosts of harsh biotransformations of industrial interest (Nikel et al, 2014). The P. putida strain KT2440 is a saprophytic, non-pathogenic, GRAS-certified (Generally Recognized as Safe) bacterium; as the most thoroughly characterized laboratory pseudomonad, it has an expanding catalogue of available systems and synthetic biology tools (Aparicio et al, 2017; Elmore et al, 2017; Martínez-García and de Lorenzo, 2017, p.; Nogales et al, 2017). This bacterium is becoming a laboratory workhorse as well as a valued cell factory This limits the options for its use as a platform to process the carbohydrate products of cellulosic and lignocellulosic waste

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Conclusion