A metabolic and physiological design study of Pseudomonas putida KT2440 capable of anaerobic respiration

Abstract

Background: Pseudomonas putida KT2440 is a metabolically versatile, HV1-certified, genetically accessible, and thus interesting microbial chassis for biotechnological applications. However, its obligate aerobic nature hampers production of oxygen sensitive products and drives up costs in large scale fermentation. The inability to perform anaerobic fermentation has been attributed to insufficient ATP production and an inability to produce pyrimidines under these conditions. Addressing these bottlenecks enabled growth under micro-oxic conditions but does not lead to growth or survival under anoxic conditions.

Results: Here, a data-driven approach was used to develop a rational design for a P. putida KT2440 derivative strain capable of anaerobic respiration. To come to the design, data derived from a genome comparison of 1628 Pseudomonas strains was combined with genome-scale metabolic modelling simulations and a transcriptome dataset of 47 samples representing 14 environmental conditions from the facultative anaerobe Pseudomonas aeruginosa.

Conclusions: The results indicate that the implementation of anaerobic respiration in P. putida KT2440 would require at least 49 additional genes of known function, at least 8 genes encoding proteins of unknown function, and 3 externally added vitamins.

Keywords: Anaerobic fermentation; Anaerobic respiration; Bioinformatics; Computational design; Microbial lifestyle engineering; Pseudomonas.

Fig. 1

Protein domain content of 344 aerobic and 1284 facultative anaerobic Pseudomonas strains. Facultative anaerobic strains capable of respiration are indicated in blue, aerobic strains in red. a 2D Plot of PCA. Position of (P. putida KT2440 is marked with an arrow. Labels on the axes indicate fraction of the total variance explained by each component. b Observed distance tree based on presence/absence of protein domains. c Details of the main branch harbouring P. putida KT2440 (position indicated with an arrow). This branch consists of 138 anaerobic and 87 aerobic Pseudomonas species

Fig. 2

Overview of in silico approaches to identify limitations to anaerobic respiration in P. putida. a Comparative genomics workflow. Genomes of the P. putida group and the anaerobic Pseudomonas group were systematically annotated using SAPP [24, 29], the protein domains were extracted, and both all domains or only the domains common to all anaerobic Pseudomonas species (the core domains) were selected using a 95% persistence threshold. Analysis was performed on the whole set of genomes (left) or a genome cluster of closely related strains (right). Each of these methods resulted in a list of protein domains related to an aerobic lifestyle (purple) or an anaerobic lifestyle (light green). b Transcriptome analysis. c GSM simulations. GSM iJP962 [5] and iJN1411 [35] were expanded with indicated reaction sets and tested for anaerobic growth under anaerobic conditions. Colours indicate final implementation in the design (green). Model and genome base predictions were combined to obtain a final design