Refactoring the Embden-Meyerhof-Parnas Pathway as a Whole of Portable GlucoBricks for Implantation of Glycolytic Modules in Gram-Negative Bacteria

Abstract

The Embden-Meyerhof-Parnas (EMP) pathway is generally considered to be the biochemical standard for glucose catabolism. Alas, its native genomic organization and the control of gene expression in Escherichia coli are both very intricate, which limits the portability of the EMP pathway to other biotechnologically important bacterial hosts that lack the route. In this work, the genes encoding all the enzymes of the linear EMP route have been individually recruited from the genome of E. coli K-12, edited in silico to remove their endogenous regulatory signals, and synthesized de novo following a standard (GlucoBrick) that enables their grouping in the form of functional modules at the user's will. After verifying their activity in several glycolytic mutants of E. coli, the versatility of these GlucoBricks was demonstrated in quantitative physiology tests and biochemical assays carried out in Pseudomonas putida KT2440 and P. aeruginosa PAO1 as the heterologous hosts. Specific configurations of GlucoBricks were also adopted to streamline the downward circulation of carbon from hexoses to pyruvate in E. coli recombinants, thereby resulting in a 3-fold increase of poly(3-hydroxybutyrate) synthesis from glucose. Refactoring whole metabolic blocks in the fashion described in this work thus eases the engineering of biochemical processes where the optimization of carbon traffic is facilitated by the operation of the EMP pathway-which yields more ATP than other glycolytic routes such as the Entner-Doudoroff pathway.

Keywords: Escherichia coli; PHB; Pseudomonas putida; glycolysis; metabolic engineering; standardization.

Figure 1

Schematic representation of the GlucoBrick platform layout and the cognate glycolytic reactions. (a) The minimal set of genes from Escherichia coli K-12 needed for the activation of a functional and linear Embden–Meyerhof–Parnas pathway were edited according to the Standard European Architecture Vector rules and assembled into two synthetic operons. The first operon, termed Module I, encodes all the reactions within the upper catabolic block of the pathway (i.e., bioreactions of the preparatory phase of glycolysis). The second operon, termed Module II, spans the reactions of the lower catabolic block of the pathway (i.e., bioreactions of the pay-off phase of glycolysis). All the glycolytic reactions are shown below the gene encoding them. Note that each gene is preceded by a synthetic regulatory element, indicated by a purple circle, composed of a ribosome binding site (sequence underlined) and a short spacer sequence. (b) Linear glycolytic pathway encoded by the GlucoBrick platform, transforming glucose into glyceraldehyde-3-P (GA3P) by means of the activities of Module I; and GA3P into pyruvate (Pyr) by means of the activities of Module II. The two sets of glycolytic transformations are indicated with blue and red arrows, representing the genes within Modules I and II, respectively. Other abbreviations used in this outline are as follows: G6P, glucose-6-P; F6P, fructose-6-P; FBP, fructose-1,6-P₂; DHAP, dihydroxyacetone-P; BPG, glycerate-1,3-P₂; 3PG, glycerate-3-P; 2PG, glycerate-2-P; and PEP, phosphoenolpyruvate.

Figure 2

Genetic architecture of the GlucoBrick platform. (a) Physical map of Modules I and II, indicating restriction enzymes bracketing individual glycolytic genes. The enzyme targets are colored in the sequence of the multiple cloning site of all the plasmids belonging to the Standard European Architecture Vector to identify the DNA block they belong to (i.e., blue, Module I; and red, Module II). Other restriction targets that could be used to add different regulatory or structural elements and thereby expand the usability of this platform are shown in gray. The abbreviations used in this outline are GA3P, glyceraldehyde-3-P; and Pyr, pyruvate. (b) Restriction analysis of Module I and II. Plasmids pS224·GBI (upper panel) and pS224·GBII (lower panel) were digested with the appropriate enzymes as indicated and the products were separated by electrophoresis in a 0.7% (w/v) agarose gel. Plasmid pS224·GBI was digested with AvrII-BamHI (i, releases the whole Module I segment); AvrII-EcoRI (ii, releases glk); EcoRI-SacI (iii, releases pgi); SacI-KpnI (iv, releases pfkA); KpnI-SmaI (v, releases fbaA); and SmaI-BamHI (vi, releases tpiA). Plasmid pS224·GBII was digested with BamHI-HindIII (i, releases the whole Module II segment); BamHI-XbaI (ii, releases gapA); XbaI-SalI (iii, releases pgk); SalI-PstI (iv, releases gpmA); PstI-SphI (v, releases eno); and SphI-HindIII (vi, releases pykF).

Figure 3

Characterization of physiological parameters in recombinant Pseudomonas putida and P. aeruginosa strains carrying Module I. (a) Schematic representation of central carbon metabolism in Pseudomonas species. Glucose catabolism occurs mainly through the activity of the Entner–Doudoroff (ED) pathway, but part of the trioses-P thereby generated are recycled back to hexoses-P by means of the EDEMP cycle, that also encompasses activities from the Embden–Meyerhof–Parnas (EMP) and the pentose phosphate (PP) pathways. Note that a set of peripheral reactions can also oxidize glucose to gluconate and/or 2-ketogluconate (2KG) before any phosphorylation of the intermediates occurs. Each metabolic block is indicated with a different color along with the relevant enzymes catalyzing each step, and the EDEMP cycle is shaded in blue in this diagram. Note that the 6-phosphofructo-1-kinase activity, missing in most Pseudomonas species, is highlighted with a dashed gray arrow. The abbreviations used for the metabolic intermediates are as indicated in the legend to Figure 1; other abbreviations are as follows: 6PG, 6-phosphogluconate; KDPG, 2-keto-3-deoxy-6-phosphogluconate; acetyl-CoA, acetyl-coenzyme A; 2KG, 2-ketogluconate; and 2K6PG, 2-keto-6-phosphogluconate. (b) Glucose consumption profile and (c) growth curves of P. putida KT2440, its Δglk derivative, and P. aeruginosa PAO1, carrying either the control vector (pSEVA224) or pS224·GBI (Module I). Glucose consumption is reported as the mean value ± standard deviation from duplicate measurements in at least three independent experiments. CDW, cell dry weight. Significant differences (P < 0.05, as evaluated by means of the Student’s t test) in the pairwise comparison of a given recombinant to the control strain, carrying the empty pSEVA224 vector, are indicated by an asterisk. In the growth curves, each data point represents the mean value of the optical density measured at 600 nm (OD₆₀₀) of quadruplicate measurements from at least three independent experiments. The specific growth rates were calculated from these data during exponential growth, and the inset shows the mean values ± standard deviations for each strain.

Figure 4

Biochemical characterization of native and implanted enzyme activities in Pseudomonas species. (a) In vitro quantification of the specific (Sp) glucokinase (Glk) activity, which phosphorylates glucose to glucose-6-P (G6P) in wild-type (WT) P. putida KT2440 and its Δglk derivative (left panel), and WT P. aeruginosa PAO1 (right panel) carrying either the empty pSEVA224 vector or Module I. (b) In vitro quantification of the specific (Sp) 6-phosphofructo-1-kinase (PfkA) activity, which converts fructose-6-P (F6P) into fructose-1,6-P₂ (FBP) in WT P. putida KT2440 and its Δglk derivative (left panel), and WT P. aeruginosa PAO1 (right panel) carrying either the empty pSEVA224 vector or Module I. (c) In vitro quantification of the specific (Sp) activities of aldolase, phosphoglucoisomerase, and triose phosphate isomerase in P. putida KT2440 carrying either the empty pSEVA224 vector or Module I. These three activities, combined with Glk and PfkA, constitute the preparatory phase of the Embden–Meyerhof–Parnas pathway (i.e., from glucose to glyceraldehyde-3-P). All the strains tested were grown on M9 minimal medium added with glucose at 20 mM and cells were harvested in midexponential phase for these in vitro enzymatic assays. Each bar represents the mean value of the corresponding enzymatic activity ± standard deviation of quadruplicate measurements from at least two independent experiments. Significant differences (P < 0.05, as evaluated by means of the Student’s t test) in the pairwise comparison of a given recombinant to the control strain, carrying the empty pSEVA224 vector, are indicated by an asterisk.

Figure 5

Enhanced poly(3-hydroxybutyrate) synthesis in recombinant Escherichia coli carrying Modules I and II. (a) Three enzymes are necessary for de novo synthesis of poly(3-hydroxybutyrate) (PHB). In Cupriavidus necator, from which the cognate genes were harnessed, PHB accumulation depends on the sequential activity of a 3-ketoacyl-coenzyme A (CoA) thiolase (PhaA), a NADPH-dependent 3-acetoacetyl-CoA reductase (PhaB1), and a PHB synthase (PhaC1). PhaA and PhaB1 catalyze the condensation of two molecules of acetyl-CoA to 3-acetoacetyl-CoA and the reduction of this intermediate to R-(−)-3-hydroxybutyryl-CoA (3-HB-CoA), respectively. PhaC1 polymerizes 3-HB-CoA monomers to PHB by releasing one CoA-SH molecule per monomer added. Note that acetyl-CoA can also be used in the major fermentation pathway of E. coli, that produces acetate. The main metabolic blocks within the biochemical network are identified with different colors in the outline: the Embden–Meyerhof–Parnas (EMP) pathway, red; the pentose phosphate (PP) pathway, purple; and the tricarboxylic acid (TCA) cycle and gluconeogenesis, green. Abbreviations of metabolic intermediates are as shown in the caption to Figure 1; other abbreviations are as follows: 6PG, 6-phosphogluconate; acetyl-CoA, acetyl-coenzyme A; OAA, oxaloacetate; and 2-OX, 2-oxoglutarate. (b) Glucose consumption profile and (c) PHB accumulation by E. coli BW25113 carrying plasmid pAeT41 (i.e., constitutively expressing the phaC1AB1 gene cluster from C. necator) transformed with plasmids carrying the genes or modules indicated (see Table 2 for further details). Cells were grown aerobically in LB medium added with glucose at 10 g L^–1 for 24 h. Each parameter is reported as the mean value ± standard deviation from duplicate measurements in at least three independent experiments. CDW, cell dry weight. Significant differences (P < 0.05, as evaluated by means of the Student’s t test) in the pairwise comparison of a given recombinant to the control strain, carrying the empty pSEVA224 vector, are indicated by an asterisk.