Overexpression of seven different genes restores growth of a ΔpdxB strain of E. coli, which cannot make pyridoxal phosphate (PLP), on M9/glucose.None of the enzymes encoded by these genes has a promiscuous 4-phosphoerythronate dehydrogenase activity that can replace the activity of PdxB.Overexpression of these genes restores PLP synthesis by three different serendipitous pathways that feed into the normal PLP synthesis pathway downstream of the blocked step.Reactions in one of these pathways are catalyzed by low-level activities of enzymes of unknown function and a promiscuous activity of an enzyme that normally has a role in another pathway; one reaction appears to be non-enzymatic.

Evolution or engineering of novel metabolic pathways can endow microbes with new abilities to degrade anthropogenic pollutants or synthesize valuable chemicals. Most studies of the evolution of new pathways have focused on the origins and quality of function of the enzymes involved. However, there is an additional layer of complexity that has received less attention. Introduction of a novel pathway into an existing metabolic network can result in inhibitory cross-talk due to adventitious interactions between metabolites and macromolecules that have not previously encountered one another. Here, we report a thorough examination of inhibitory cross-talk between a novel metabolic pathway for synthesis of pyridoxal 5′-phosphate and the existing metabolic network of Escherichia coli. We demonstrate multiple problematic interactions, including (i) interference by metabolites in the novel pathway with metabolic processes in the existing network, (ii) interference by metabolites in the existing network with the function of the novel pathway, and (iii) diversion of metabolites from the novel pathway by promiscuous activities of enzymes in the existing metabolic network. Identification of the mechanisms of inhibitory cross-talk can reveal the types of adaptations that must occur to enhance the performance of a novel metabolic pathway as well as the fitness of the microbial host. These findings have important implications for evolutionary studies of the emergence of novel pathways in nature as well as genetic engineering of microbes for "green" manufacturing processes. molecular evolution | metabolic engineering | serendipitous pathway | synthetic biology | biodegradation

The genes encoding metabolic enzymes involved in glucose metabolism, the TCA cycle, and biosynthesis of amino acids, purines, pyrimidines, and cofactors would be expected to be essential for growth of Escherichia coli on glucose because the cells must synthesize all of the building blocks for cellular macromolecules. Surprisingly, 80 of 227 of these genes are not essential. Analysis of why these genes are not essential provides insights into the metabolic sophistication of E. coli and into the evolutionary pressures that have shaped its physiology. Alternative routes enabled by interconnecting pathways can allow a defective step to be bypassed. Isozymes, alternative enzymes, broad-specificity enzymes, and multifunctional enzymes can often substitute for a missing enzyme. We expect that the apparent redundancy in these metabolic pathways has arisen due to the need for E. coli to survive in a variety of habitats and therefore to have a metabolism that allows optimal exploitation of varying environmental resources and synthesis of small molecules when they cannot be obtained from the environment.

BackgroundRecently developed methods for genome editing in bacteria take advantage of the introduction of double-strand breaks by I-SceI in a mutation cassette to select for cells in which homologous recombination has healed the break and introduced a desired mutation. This elegantly designed method did not work well in our hands for most genes.ResultsWe corrected a mutation in the gene encoding I-SceI that compromised the function of a previously used Red helper plasmid. Further, we found that transcription extending into the mutation cassette interferes with cleavage by I-SceI. Addition of two transcription terminators upstream of the cleavage site dramatically increases the efficiency of genome editing. We also developed an improved method for modification of essential genes. Inclusion of a segment of the essential gene consisting of synonymous codons restores an open reading frame when the mutation cassette is integrated into the genome and decreases the frequency of recombination events that fail to incorporate the desired mutation. The optimized protocol takes only 5 days and has been 100% successful for over 100 genomic modifications in our hands.ConclusionsThe method we describe here is reliable and versatile, enabling various types of genome editing in Escherichia coli and Salmonella enterica by straightforward modifications of the mutation cassette. We provide detailed descriptions of the methods as well as designs for insertions, deletions, and introduction of point mutations.

PdxB catalyzes the second step in the biosynthesis of pyridoxal phosphate by oxidizing 4-phospho-D-erythronate (4PE) to 2-oxo-3-hydroxy-4-phospho-butanoate (OHPB) with concomitant reduction of NAD+ to NADH. PdxB is a nicotino-enzyme wherein the NAD(H) cofactor remains tightly bound to PdxB. It has been a mystery how PdxB performs multiple turnovers since addition of free NAD+ does not re-oxidize the enzyme-bound NADH following conversion of 4PE to OHPB. We have solved this mystery by demonstrating that a variety of physiologically available α-ketoacids serve as oxidants of PdxB to sustain multiple turnovers. In a coupled assay using the next two enzymes of the biosynthetic pathway for pyridoxal phosphate (SerC and PdxA), we have found that α-ketoglutarate, oxaloacetic acid, and pyruvate are equally good substrates for PdxB (kcat/Km values ~ 1 × 104 M-1s-1). The kinetic parameters for the substrate 4PE include a kcat of 1.4 s-1, a Km of 2.9 μM, and a kcat/Km of 6.7 × 106 M-1s-1. Additionally, we have characterized the stereochemistry of α-ketoglutarate reduction by showing that D-2-HGA, but not L-2-HGA, is a competitive inhibitor vs. 4PE and a noncompetitive inhibitor vs. α-ketoglutarate.

Facile DNA sequencing became possible decades after many enzymes had been purified and characterized. Consequently, there are still “orphan” enyzmes whose activity is known but the genes that encode them have not been identified. Identification of the genes encoding orphan enzymes is important because it allows correct annotation of genes of unknown function or with mis-assigned function. Bis-γ-glutamylcystine reductase (GCR) is an orphan protein that was purified in 1988. This enzyme catalyzes the reduction of bis-γ-glutamylcystine. γ-Glutamylcysteine (γ-Glu-Cys) is the major low molecular weight thiol in halobacteria. We purified GCR from Halobacterium sp. NRC-1 and identified the sequence of 23 tryptic peptides by NanoLC electrospray ionization tandem mass spectrometry. These peptides cover 62% of the protein predicted to be encoded by a gene in Halobacterium sp. NRC-1 that is annotated as mercuric reductase. GCR and mercuric reductase activities were assayed using enzyme that was expressed in E. coli and re-folded from inclusion bodies. The enzyme had robust GCR activity, but no mercuric reductase activity. The genomes of most, but not all, halobacteria for which whole genome sequences are available have close homologs of GCR, suggesting that there is more to be learned about the low molecular weight thiols used in halobacteria.