Insights into an alternative pathway for glycerol metabolism in a glycerol kinase deficientPseudomonas putidaKT2440

Abstract

16Pseudomonas putida KT2440 is known to metabolise glycerol via glycerol-3-phosphate using 17 glycerol kinase an enzyme previously described as critical for glycerol metabolism (1). 18 However, when glycerol kinase was knocked out in P. putida KT2440 it retained the ability to 19 use glycerol as the sole carbon source, albeit with a much-extended lag period and 2 fold lower 20 final biomass compared to the wild type strain. A metabolomic study identified glycerate as a 21 major and the most abundant intermediate in glycerol metabolism in this mutated strain with 22 levels 21-fold higher than wild type. Erythrose-4-phosphate was detected in the mutant strain, 23 but not in the wild type strain. Glyceraldehyde and glycraldehyde-3-phosphate were detected 24 at similar levels in the mutant strain and the wild type. Transcriptomic studies identified 191 25 genes that were more than 2-fold upregulated in the mutant compared to the wild type and 175 26 that were down regulated. The genes involved in short chain length fatty acid metabolism were 27 highly upregulated in the mutant strain. The genes encoding 3-hydroxybutyrate dehydrogenase 28 were 5.8-fold upregulated and thus the gene was cloned, expressed and purified to reveal it can 29 act on glyceraldehyde but not glycerol as a substrate. 30 31 33Glycerol is a major by-product of plant oil processing. It is also the major by-product of 34 biodiesel production (10 weight %). An increase in production of biodiesel (2) has led to an 35 increase in the availability of glycerol (3). Glycerol could be an interesting substrate for 36 fermentative production of value added products such as 1,3 propanediol, dihydroxyacetone 37 and polyhydroxyalkanoates (4,5). Polyhydroxyalkanoates (PHA) are polymers that are 38 naturally synthesized and stored in a range of microorganisms including Pseudomonads (6). 39Pseudomonas putida KT2440 is a model organism for biocatalysis and synthetic biology. 40While P. putida KT2440 can use glycerol as the sole carbon and energy source for growth, 41 glycerol metabolism in this strain has not been fully characterized. However, the pathway for 42 glycerol metabolism has been extensively studied in the opportunistic pathogen Pseudomonas 43 aeruginosa (7-9). As there is high sequence identity in the proposed glycerol metabolic 44 pathway genes in P. putida KT2440 with genes in P. aeruginosa, some information about 45 glycerol metabolism in P. putida KT2440 can be inferred from the research into P. aeruginosa 46 (10). In P. aeruginosa, the first step in glycerol metabolism is the facilitated diffusion across 47 the cytoplasmic membrane by a glycerol diffusion facilitator (GlpF). The glycerol is then 48 retained intracellularly through phosphorylation by a glycerol kinase (GlpK) to form glycerol-49 3-phosphate, which cannot diffuse back through the cytoplasmic membrane. In P. aeruginosa, 50 the genes for GlpF and GlpK are organised together on one operon (8). The glycerol-3-51 phosphate is converted to dihydroxyacetone-3-phosphate by a cytoplasmic...

The Cellular Response to Lanthanum Is Substrate Specific and Reveals a Novel Route for Glycerol Metabolism in Pseudomonas putida KT2440

“…Upon phosphorylation by GarK, glycerate eventually enters the central metabolism, presumably as glycerate-2-phosphate. This conclusion is supported by the lack of growth of the ΔglpFKRD ΔgarK mutant and the results of a companion study (48), which used a comparative metabolome analysis of glycerol-growing cells of P. putida KT2440 and a ΔglpK deletion strain. The study found a dramatically increased glycerate concentration for the ΔglpK strain, whereas the concentrations of glyceraldehyde and glyceraldehyde-3-phosphate were in similar ranges for the two strains.…”

The cellular response towards lanthanum is substrate specific and reveals a novel route for glycerol metabolism inPseudomonas putidaKT2440

“…Given that the route further proceeds via glycerate and glycerate-2-phosphate, different cellular concentrations of these metabolites would be expected during growth on glycerol in a mutant that is not able to use the GlpFKRD pathway. Interestingly, a companion study to this work employed a metabolome analysis using glycerol-growing cells of P. putida KT2440 and a Δ glpK deletion strain, which can only use the proposed novel route via PedE and PedH (59). In their experiments, the authors observed that the glycerate concentration measured for the Δ glpK strain was dramatically increased compared to the wild type, whereas concentrations of glyceraldehyde and glyceraldehyde-3-phosphate were in the same range for both strains.…”