Refactoring the Embden–Meyerhof–Parnas Pathway as a Whole of

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Refactoring the Embden-Meyerhof-Parnas pathway as a whole of portable GlucoBricks for implantation of glycolytic modules in Gram-negative bacteria Alberto Sánchez-Pascuala, Victor de Lorenzo, and Pablo Ivan Nikel ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00230 • Publication Date (Web): 25 Jan 2017 Downloaded from http://pubs.acs.org on January 26, 2017

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Refactoring the Embden-Meyerhof-Parnas pathway as a whole of portable

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GlucoBricks for implantation of glycolytic modules in Gram-negative bacteria

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Alberto Sánchez-Pascuala, Víctor de Lorenzo*, and Pablo I. Nikel†*

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Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC),

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Campus de Cantoblanco, 28049 Madrid, Spain

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Keywords:

Escherichia coli — Pseudomonas putida — Standardization— Glycolysis — Metabolic Engineering — PHB

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Portable glycolysis for Gram-negative bacteria

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† Current address:

The Novo Nordisk Foundation Center for Biosustainability, Technical

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University of Denmark, 2800 Kongens Lyngby, Denmark

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* Correspondence to: Víctor de Lorenzo ([email protected])

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Centro Nacional de Biotecnología (CNB-CSIC)

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Madrid, Spain

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Tel: (+34 91) 585 45 73

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Pablo I. Nikel ([email protected])

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The Novo Nordisk Foundation Center for Biosustainability,

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Technical University of Denmark

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Lyngby, Denmark

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Tel: (+45 93) 51 19 18

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ABSTRACT

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The Embden-Meyerhof-Parnas (EMP) pathway is generally considered to be the biochemical

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standard for glucose catabolism. Alas, its native genomic organization and the control of gene

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expression in Escherichia coli are both very intricate, which limits the portability of the EMP

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pathway to other biotechnologically-important bacterial hosts that lack the route. In this work, the

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genes encoding all the enzymes of the linear EMP route have been individually recruited from the

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genome of E. coli K-12, edited in silico to remove their endogenous regulatory signals, and

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synthesized de novo following a standard (GlucoBrick) that enables their grouping in the form of

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functional modules at the user's will. After verifying their activity in several glycolytic mutants of E.

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coli, the versatility of these GlucoBricks was demonstrated in quantitative physiology tests and

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biochemical assays carried out in Pseudomonas putida KT2440 and P. aeruginosa PAO1 as the

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heterologous hosts. Specific configurations of GlucoBricks were also adopted to streamline the

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downward circulation of carbon from hexoses to pyruvate in E. coli recombinants, thereby

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resulting in a 3-fold increase of poly(3-hydroxybutyrate) synthesis from glucose. Refactoring

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whole metabolic blocks in the fashion described in this work thus eases the engineering of

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biochemical processes where the optimization of carbon traffic is facilitated by the operation of

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the EMP pathway –which yields more ATP than other glycolytic routes such as the Entner-

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Doudoroff pathway.

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Keywords: Escherichia coli — Pseudomonas putida — Standardization — Glycolysis — Metabolic

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Engineering — PHB

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____________________________________________________________________________

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INTRODUCTION

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The last few years have witnessed an increase in the number of microorganisms that can be

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metabolically engineered within the conceptual frame of Systems Biology and the molecular tools

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of contemporary Synthetic Biology1-5. Longstanding platforms (e.g., Escherichia coli, Bacillus,

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Corynebacterium, and yeast) are increasingly accompanied by others (e.g., Pseudomonas

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species) that, due to their indigenous endurance to environmental stresses, have an improved

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ability to execute harsh biochemical transformations6. Whether well-established or emerging,

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such platforms are empowered by a complex central carbon metabolic network where

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heterologous pathways must nest in any given host. Despite this fact, the majority of ongoing

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Metabolic Engineering efforts are preoccupied with what one could call peripheral aspects7-10,

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e.g., the elimination of competing endogenous pathways, the fine-tuning of gene expression and

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substrate transport, and the re-routing of small molecules at given nodes of the network. In

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contrast, the biochemical core that fuels the microbial cell factory is generally taken for granted

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and hardly ever touched, i.e., few efforts have tried to reshape central enzymatic processes in its

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entirety. Relevant examples of this sort include auxotrophic CO2 fixation11 and CH3OH

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assimilation12 by engineered strains of E. coli. Such efforts suffer from the difficulties of re-

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engineering some of the most extremely interconnected and fine-tuned components of any

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biological system as central metabolism is13,14. Against this background, we wondered whether

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the most archetypal metabolic pathway (i.e., a linear glycolysis) could be reshaped in a way that,

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while maintaining its biochemical role and identity, could be freed of its native regulatory

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complexity and made portable either as a whole or as a functional sub-set of functional elements

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for specific Metabolic Engineering needs.

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In its simplest definition, glycolysis is the metabolic breakdown of glucose into pyruvate (Pyr).

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Thought to have originated from simple chemical constraints in a pre-biotic environment,

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glycolysis is one of the most widespread and conserved metabolic blocks in Nature15. Several

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biochemical sequences lead to the formation of Pyr from hexoses. The most studied example is

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the Embden-Meyerhof-Parnas (EMP) pathway, composed by the sequential activity of ten

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individual enzymes. The first five enzymes are involved in the so-called preparatory phase, which

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uses ATP to convert hexoses into trioses phosphate [i.e., glucose → glyceraldehyde-3-P 3 ACS Paragon Plus Environment

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(GA3P)]. The pay-off phase comprises the second half of the EMP enzymes, and it yields 2

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NADH molecules and 2 ATP molecules per each processed glucose molecule by converting

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trioses phosphate into Pyr [i.e., GA3P → Pyr]. Therefore, the overall stoichiometry of the EMP

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pathway is: glucose + 2NAD+ + 2ADP + 2Pi → 2Pyr + 2NADH + 2H+ + 2ATP. Another glycolytic

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route is the Entner-Doudoroff (ED) pathway, widely distributed in prokaryotes (even more so than

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the EMP route). The overall stoichiometry of the ED pathway is: glucose + NAD+ + NADP+ + ADP

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+ Pi → 2Pyr + NADH + NADPH + 2H+ + ATP. Although this biochemical sequence yields half the

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amount of ATP as compared to that of the EMP pathway16, the ED route has been demonstrated

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to be a key source of reducing power in many environmental microorganisms17,18. Because of a

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lifestyle of constant exposure to physicochemical insults often found in natural scenarios19, such

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bacteria have evolutionarily favored the formation of reducing power20 to counteract oxidative

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stress21. This is in contrast with the generation of ATP and the sheer buildup of biomass that

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appears to be the main metabolic driving force in Enterobacteria. Besides these highly conserved

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biochemical sequences, variants of carbohydrate breakdown processes are also present on other

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prokaryotes. Archaea, for instance, are characterized by the presence of unique, modified

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versions of the EMP and ED pathways which often include non-phosphorylating enzymes22,23.

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The research below was prompted by our ongoing efforts to establish the soil-dweller

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microorganism Pseudomonas putida as a platform for hosting strong redox reactions24-27. This

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bacterium lacks a 6-phosphofructo-1-kinase (Pfk) activity, and therefore the EMP pathway is non-

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functional. The mere addition of Pfk to the biochemical network of P. putida was not only

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insufficient to activate an EMP-based hexose catabolism, but it also resulted in deleterious effects

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on growth and limited resistance to oxidative stress17. Thus, the channeling of glucose towards

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Pyr in P. putida, while delivering enough NAD(P)H and yielding the highest possible amount of

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ATP, demands a multi-tiered approach involving more glycolytic genes or specific combinations

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thereof. To this end, in our present contribution we describe the re-formatting of the entire EMP of

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E. coli in a layout that we have called GlucoBricks, that allows complete or partial implantation of

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the glycolytic route in a variety of Gram-negative bacteria including (but not limited to) P. putida.

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This work finds inspiration in the attempts to decompress the regulatory density of the E. coli T7

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bacteriophage28 and the deconstruction/reconstruction of the whole N2-fixation system of

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Klebsiella oxytoca29 in an easy-to-manipulate setup. This time, however, the objective was the

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implantation of central metabolic functions in different Gram-negative hosts by means of portable,

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standardized modules assembled in promiscuous plasmids belonging to the SEVA (Standard

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European Vector Architecture) collection30. The data discussed below not only demonstrates the

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versatility of the thereby reported device in various bacterial hosts, but it also expands the toolbox

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for deep engineering of central biochemical tasks in a microbial cell factory.

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RESULTS AND DISCUSSION

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Functional elements, layout, and design of glycolytic Modules I and II. The starting point of

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this research was to design a streamlined set of genes to aid implanting (or increasing) glycolytic

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capacities in the microbial cell. To this end, we first pinpointed each of the ten enzymes carrying

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out the complete transformation of glucose into Pyr through the EMP pathway in the extant

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metabolic network of E. coli. In its naturally-occurring configuration the corresponding genes are

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either scattered throughout the genome or arranged in two operons (e.g., fbaA and pgk) under

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the transcriptional control of at least five regulators (Cra, SoxS, Crp, Fur, and FnrS) as well as a

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number of post-transcriptional control devices31. Our first task was to exclusively capture the

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enzymatic complement of the system while releasing the corresponding genes (either separately

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or as a whole) from any host-specific regulatory connection. Against this background, the basic

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organization of what we call a GlucoBrick is sketched in Figure 1. When more than one gene

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encoded a given EMP reaction (i.e., pfkA/pfkB, fbaA/fbaB, gpmA/gpmM, and pykA/pykF), the one

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bringing about the highest activity was selected according to the information available in the

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literature. The structural sequence of each glycolytic gene starts with a leading ATG and ends up

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with a STOP codon. The corresponding DNA was edited to eliminate restriction sites incompatible

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with the assembly standard discussed below, and any internal transcriptional signal was erased.

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Such ORFs are preceded by a standardized Shine-Dalgarno sequence and a DNA spacer, and

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the whole segment is flanked upstream and downstream by restriction sites that match given

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positions of the default multiple cloning site of the SEVA format. SEVA vectors include a choice of

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plasmids with four compatible broad-host range origins of replication (i.e., RK2, pBBR1,

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pRO1600/ColE1, and RSF1010) and six independent antibiotic markers [i.e., ampicillin,

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kanamycin (Km), cloramphenicol, streptomycin, tetracyclin, and gentamicin]. This standard allows

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for the effective propagation and maintenance of up to four plasmids in any given Gram-negative 5 ACS Paragon Plus Environment

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host, and it also enables the simultaneous expression of several genes. Since each glycolytic

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gene was preceded by a synthetic ribosome binding site (RBS), and bracketed by two directional

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SEVA restriction enzymes (i.e., following the standard format Restriction Enzyme 1–RBS–

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glycolytic gene–Restriction Enzyme 2), the assembly standard allows to directly sub-clone or

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swap any gene combination by a simple digestion and ligation step. Moreover, the order of

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insertion of each of the ten GlucoBricks in the SEVA's multiple cloning site reflects the

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biochemical sequence of operation in the EMP pathway, thereby allowing for whatever the

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combination of the parts within the route at stake.

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Building on this concept, we designed two DNA modules that encode all the necessary enzymes

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for the activation of a flawless, linear EMP-based glycolytic route. The EMP pathway was

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purposely split into two catabolic blocks: the upper catabolic block, termed Module I, comprises

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the activities of the preparatory phase; and the lower catabolic block, dubbed Module II, spans

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the activities of the pay-off phase (Figure 1a). Module I thus encodes all the enzymes needed for

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glucose phosphorylation and conversion into trioses phosphate, whereas activities originating

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from Module II use GA3P, the final product of Module I, as the precursor to form Pyr (Figure 1b).

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A detailed map of the DNA architecture of this platform (Figure 2a) indicates that the two DNA

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blocks can be also combined sequentially in a single SEVA vector if desired (and even in

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transposon vectors designed for chromosomal integration of large DNA segments32,33). Note that

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adopting the SEVA standard also enables the user to express any group of genes under the

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control of the large number of constitutive and effector-responsive, broad-host-range

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transcriptional devices available in the database34-36.

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As a first step in the characterization of this tool in E. coli strains, Modules I and II were

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separately cloned in vector pSEVA224 (RK2, KmR), giving rise to pS224—GBI and pS224—GBII,

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respectively (Tables S1 and S2 in the Supporting Information). The expression of the glycolytic

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genes in these low-copy-number plasmids is under control of the LacIQ/Ptrc expression system,

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inducible by addition of isopropyl-β-D-1-thiogalactopyranoside (IPTG) to the culture medium. As

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an additional test of the structural versatility of this plasmid-based GlucoBrick platform, a

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restriction analysis of both pS224—GBI and pS224—GBII was carried out (Figure 2b). All the

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glycolytic genes could be separately recovered upon digestion with the appropriate pair of

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restriction enzymes–thereby facilitating direct sub-cloning into suitable SEVA vectors whenever

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needed as indicated above.

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The activities encoded in the GlucoBricks restore or enhance the growth of glycolytic

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Escherichia coli mutants. The functional characterization of the GlucoBrick system was carried

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out by assessing their phenotypic impact when the corresponding plasmids were introduced in

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glycolytic E. coli mutants lacking either single or several combined activities of the EMP pathway

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(Table 1). E. coli BW25113, a wild-type K-12 strain37, was used as a positive control in all these

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growth experiments, in which the final cell density and the specific growth rate was recorded for

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each strain in cultures containing both Km and IPTG. First, the initial steps in glucose processing

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were targeted at the level of Glk (i.e., glucokinase) and PtsI [i.e., the EI component of the

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phosphoenolpyruvate (PEP):carbohydrate phosphotransferase (PTS) system]. No glucose

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phosphorylation is possible in such mutant as Glk and the PTS-dependent transport of the

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hexose coupled to phosphorylation are both blocked38. Note, however, that glucose transport

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should not be significantly affected, since alternative transporters (e.g., the low-affinity

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galactose:H+ GalP symporter and the ATP-dependent MglABC system) internalize the non-

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phosphorylated hexose when the PTS system is not active39. Accordingly, the ∆glk ∆ptsI strain

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grew in M9GCM semi-synthetic medium (formulated in such a way that all the glycolytic mutants

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tested could grow, albeit some of them did so poorly) but not in M9 minimal medium with glucose

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as the only carbon source. The presence of Module I, however, restored the growth of the double

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mutant on the hexose back to the levels observed in the semi-synthetic medium. When the

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reaction catalyzed by Pfk (which mediates the glycolytic formation of fructose-1,6-P2 from

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fructose-6-P) was eliminated by deleting both pfkA and pfkB, the growth of the resulting strain

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was severely compromised even in M9GCM medium‒and no growth at all was observed in

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minimal medium with glucose. The Pfk activity brought about by Module I was, however, enough

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to restore this growth deficiency in both culture media, and it also lead to a 2.3-fold increase in

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the specific growth rate of the recombinant in M9GCM medium. Another critical step of the EMP

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pathways is TpiA (i.e., triose phosphate isomerase), which plays a central role not only in

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downwards glycolysis but also in gluconeogenesis40. Expectedly, the growth of a ∆tpiA mutant

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was impaired among all the culture conditions tested, probably because of the build-up of

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methylglyoxal as a toxic intermediate41. Expression of tpiA within Module I alleviated this

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metabolic situation, even restoring the growth of the mutant from glucose and significantly

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enhancing it in the semi-synthetic medium.

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The capability of Module II to mediate EMP activities in the pay-off phase was analyzed in

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another set of E. coli glycolytic mutants. The conversion of GA3P into PEP (and, consequently,

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Pyr) was targeted to test Module II. Note that the mere elimination of GA3P dehydrogenase in E.

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coli (represented by GapA, and possibly the GapB isozyme42) does not block Pyr formation, as

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this metabolite could be also produced by the PTS system from PEP. Most of the PEP pool

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comes from the EMP pathway, yet anaplerotic reactions could also generate this intermediate.

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Considering this complex metabolic scenario (and in order to ensure that there is no Pyr

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formation), a triple E. coli mutant was constructed by eliminating gapA (encoding a NAD+-

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dependent GA3P dehydrogenase, and also the major GA3P dehydrogenase prevalent in

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Enterobacteria), epd (also known as gapB, encoding a NAD+‒erythrose-4-P dehydrogenase),

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and also ptsI. Glucose-dependent growth was completely abolished in this mutant, and M9GCM

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medium cultures only attained a modest cell density. Once again, the presence of Module II

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enhanced or even restored the growth of the triple mutant by means of the GapA activity encoded

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therein. Interestingly, the specific growth rate of the recombinant cells in semi-synthetic medium

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increased >6-fold when Module II was transformed in the ∆gapA ∆epd ∆ptsI mutant‒the highest

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among the conditions and strains analyzed in this study. Finally, the ∆pgk and ∆eno mutants had

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a similar qualitative behavior: they grew very poorly in semi-synthetic medium and could not grow

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at all in M9 minimal medium with glucose. The corresponding activities encoded in Module II

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sufficed to restore the growth of the corresponding recombinants on the hexose, while the

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specific growth rate in M9GCM medium increased by ca. 3-fold in either case (Table 1). Note

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that, besides the functional complementation of individual enzyme activities missing in the single

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or multiple mutant strains studied herein, the GlucoBrick blocks can also amplify glycolytic

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activities that are already in place in E. coli, thus boosting the channeling of hexoses through the

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linear EMP pathway. In all the cases studied here, the final cell densities attained by the

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recombinants carrying GlucoBricks in M9 minimal medium with glucose was comparable to that

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of wild-type E. coli BW25113.

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In order to evaluate if these growth phenotypes correlate with higher specific activities of some of

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the enzymes involved in glucose processing, some key steps in the EMP pathway were

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evaluated in cell-free extracts in vitro. The activity of Glk in the glucose-grown ∆glk ∆ptsI mutant

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expressing Module I was 1,370 ± 120 nmol min-1 mg protein-1 upon induction of gene expression

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with IPTG, representing a ca. 23- and 10-fold increase as compared with the same strain carrying

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the empty vector and wild-type BW25113 carrying the empty vector, respectively (note that some

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residual Glk activity was observed in the knock-out strain, possibly arising from some other non-

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specific hexose kinases43). Similarly, introduction and induction of the genes in Module I in the

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∆pfkA ∆pfkB strain resulted in 10-fold increase in the Pfk activity (1,080 ± 70 nmol min-1 mg

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protein-1) as compared with the same strain carrying the empty pSEVA224 vector. In contrast, the

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Pfk activity in wild-type BW25113 was 751 ± 36 nmol min-1 mg protein-1. The residual in vitro

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phosphofructokinase activity observed in the E. coli ∆pfkA ∆pfkB mutant (