Abstract
BackgroundEscherichia coli strains lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS), which is the major bacterial component involved in glucose transport and its phosphorylation, accumulate high amounts of phosphoenolpyruvate that can be diverted to the synthesis of commercially relevant products. However, these strains grow slowly in glucose as sole carbon source due to its inefficient transport and metabolism. Strain PB12, with 400% increased growth rate, was isolated after a 120 hours adaptive laboratory evolution process for the selection of faster growing derivatives in glucose. Analysis of the genetic changes that occurred in the PB12 strain that lacks PTS will allow a better understanding of the basis of its growth adaptation and, therefore, in the design of improved metabolic engineering strategies for enhancing carbon diversion into the aromatic pathways.ResultsWhole genome analyses using two different sequencing methodologies: the Roche NimbleGen Inc. comparative genome sequencing technique, and high throughput sequencing with Illumina Inc. GAIIx, allowed the identification of the genetic changes that occurred in the PB12 strain. Both methods detected 23 non-synonymous and 22 synonymous point mutations. Several non-synonymous mutations mapped in regulatory genes (arcB, barA, rpoD, rna) and in other putative regulatory loci (yjjU, rssA and ypdA). In addition, a chromosomal deletion of 10,328 bp was detected that removed 12 genes, among them, the rppH, mutH and galR genes. Characterization of some of these mutated and deleted genes with their functions and possible functions, are presented.ConclusionsThe deletion of the contiguous rppH, mutH and galR genes that occurred simultaneously, is apparently the main reason for the faster growth of the evolved PB12 strain. In support of this interpretation is the fact that inactivation of the rppH gene in the parental PB11 strain substantially increased its growth rate, very likely by increasing glycolytic mRNA genes stability. Furthermore, galR inactivation allowed glucose transport by GalP into the cell. The deletion of mutH in an already stressed strain that lacks PTS is apparently responsible for the very high mutation rate observed.
Highlights
Escherichia coli strains lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS), which is the major bacterial component involved in glucose transport and its phosphorylation, accumulate high amounts of phosphoenolpyruvate that can be diverted to the synthesis of commercially relevant products
It has been shown that when both types of methodologies were utilized simultaneously for whole genome resequencing of E. coli strains in which growth adaptations by evolution occurred, both techniques reported false positive mutations [4,5]
Since the mutH gene deletion in this strain is responsible of increasing the mutation rate in E. coli [35], it could be possible that the two nonsynonymous mutations detected only by Winter Genomics Inc. (WG), and confirmed by Sanger, are due to de novo changes that occurred in the overnight culture utilized to obtain DNA for genome analysis by WG and not in the fermentation process started with the PB11 strain (Figure 1)
Summary
Escherichia coli strains lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS), which is the major bacterial component involved in glucose transport and its phosphorylation, accumulate high amounts of phosphoenolpyruvate that can be diverted to the synthesis of commercially relevant products. We have constructed and characterized Escherichia coli strains that lack the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS), by deletion of the ptsH, ptsI, and crr genes, which is the major bacterial component involved in glucose transport and its phosphorylation One of these strains, PB11, in spite of growing very slow in glucose (with a specific growth rate (μ) = 0.1 vs 0.7 h-1 as compared to the parental strain JM101), accumulates high amounts of phosphoenolpyruvate, which can be diverted to the synthesis of aromatic compounds. As a metabolic engineering strategy, an adaptive laboratory evolution process for the selection of faster growing derivatives of the PB11 strain was carried out in a fermentor in minimal medium with glucose as the sole carbon source In this process, after entering the stationary phase this carbohydrate was fed by progressively increasing the dilution rate. The evolved PB12 strain that in the absence of PTS uses the galactose permease (GalP), as the parental PB11 strain for glucose transport, has been utilized for overproduction of aromatic compounds [7,9,12,14,15,16,17]
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