Ag43-mediated display of a thermostable β-glucosidase in Escherichia coli and its use for simultaneous saccharification and fermentation at high temperatures

Muñoz-Gutiérrez, Iván; Moss-Acosta, Cessna L.; Trujillo-Martínez, Berenice; Gosset, Guillermo; Martı́nez, Alfredo

doi:10.1186/s12934-014-0106-3

Cited by 22 publications

(26 citation statements)

References 31 publications

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“…As we detected no β-glucosidase (EC 3.2.1.21) activity in culture supernatants or lysates prepared from cells incubated with cellobiose, we focused initially on the latter bottleneck. Previous attempts to engineer bacteria for cellobiose utilization and valorization included periplasmic or extracellular β-glucosidase expression (Chen et al, 2011; Rutter et al, 2013), or its surface display (Muñoz-Gutiérrez et al, 2014). This last approach was also recently used for P. putida KT2440, but surface display of three cellulases, including β-glucosidase BglA from Clostridium thermocellum , resulted in only trace conversion of cellulosic substrate into glucose (Tozakidis et al, 2016).…”

Section: Resultsmentioning

confidence: 99%

Refactoring the upper sugar metabolism ofPseudomonas putidafor co-utilization of disaccharides, pentoses, and hexoses

Dvořák

Lorenzo

2018

Preprint

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6Running title: Engineering cellobiose and xylose metabolism in P. putida 7 Abstract 1 Given its capacity to tolerate stress, NAD(P)H/ NAD(P) balance, and increased ATP levels, 2 the platform strain Pseudomonas putida EM42, a genome-edited derivative of the soil 3 bacterium P. putida KT2440, can efficiently host a suite of harsh reactions of biotechnological 4 interest. Because of the lifestyle of the original isolate, however, the nutritional repertoire of P. 5 putida EM42 is centered largely on organic acids, aromatic compounds and some hexoses 6 (glucose and fructose). To enlarge the biochemical network of P. putida EM42 to include 7 disaccharides and pentoses, we implanted heterologous genetic modules for D-cellobiose and 8 D-xylose metabolism into the enzymatic complement of this strain. Cellobiose was actively 9 transported into the cells through the ABC complex formed by native proteins PP1015-PP1018. 10The knocked-in -glucosidase BglC from Thermobifida fusca catalyzed intracellular cleavage 11 of the disaccharide to D-glucose, which was then channelled to the default central metabolism. 12Xylose oxidation to the dead end product D-xylonate was prevented by by deleting the gcd 13 gene that encodes the broad substrate range quinone-dependent glucose dehydrogenase. 14 Intracellular intake was then engineered by expressing the Escherichia coli proton-coupled 15 symporter XylE. The sugar was further metabolized by the products of E. coli xylA (xylose 16 isomerase) and xylB (xylulokinase) towards the pentose phosphate pathway. The resulting P. 17 putida strain co-utilized xylose with glucose or cellobiose to complete depletion of the sugars. 18 These results not only show the broadening of the metabolic capacity of a soil bacterium 19 towards new substrates, but also promote P. putida EM42 as a platform for plug-in of new 20 biochemical pathways for utilization and valorization of carbohydrate mixtures from 21 lignocellulose processing.22 23 24recruitment of one heterologous -glucosidase for intracellular cellobiose hydrolysis, and the 1 implementation of three enterobacterial genes (xylose transporter, isomerase and kinase) 2 sufficed to cause disaccharide and the pentose co-utilization by P. putida EM42 with 3 inactivated glucose dehydrogenase while maintaining its ability to use glucose. We also 4 demonstrate that P. putida metabolism generates more ATP when cells are grown on cellobiose 5 instead of glucose. This study expands the catalytic scope of P. putida towards utilization of 6 major components of all three lignocellulose-derived fractions. Moreover, given that the 7 cellobiose, as the bulk by-product of standard cellulose saccharification, frequently remains 8 untouched in the sugar mix (due to the inability of most microorganisms to assimilate it), our 9 results demonstrate rational tailoring of an industrially relevant microbial host to achieve a 10 specific step in such a biotechnological value chain. 11 12 13 14 6 1 Figure 1. Engineering Pseudomonas putida EM42 for co-utilization of D-cellobios...

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Section: Resultsmentioning

confidence: 99%

Refactoring the upper sugar metabolism ofPseudomonas putidafor co-utilization of disaccharides, pentoses, and hexoses

Dvořák

Lorenzo

2018

Preprint

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“…Escherichia coli offers a good platform for the CBP because there are many genetic tools available to facilitate its genetic modification, can grow in mineral salt media without complex supplements, and can ferment a wide variety of fermentable sugars, such as hexoses, pentoses, and uronic acids (Muñoz-Gutiérrez and Martínez 2013;Orencio-Trejo et al 2010). Several strategies involving expression of heterologous cellulases in fermentative ethanologenic strains are currently being pursued (Kojima et al 2012;Linger et al 2010;Muñoz-Gutiérrez et al 2014;Zheng et al 2012) including the development of ethanologenic E. coli strains engineered to heterologously express saccharolytic enzymes to produce ethanol directly from lignocellulosic biomass (Edwards et al 2011;Ryu and Karim 2011).…”

Section: Introductionmentioning

confidence: 99%

Improved ethanol production from biomass by a rumen metagenomic DNA fragment expressed in Escherichia coli MS04 during fermentation

Loaces

Amarelle

Muñoz-Gutiérrez

et al. 2015

Appl Microbiol Biotechnol

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With the aim of improving current ethanologenic Escherichia coli strains, we screened a metagenomic library from bovine ruminal fluid for cellulolytic enzymes. We isolated one fosmid, termed Csd4, which was able to confer to E. coli the ability to grow on complex cellulosic material as the sole carbon source such as avicel, carboxymethyl cellulose, filter paper, pretreated sugarcane bagasse, and xylan. Glucanolytic activity obtained from E. coli transformed with Csd4 was maximal at 24 h of incubation and was inhibited when glucose or xylose were present in the media. The 34,406-bp DNA fragment of Csd4 was completely sequenced, and a putative endoglucanase, a xylosidase/arabinosidase, and a laccase gene were identified. Comparison analysis revealed that Csd4 derived from an organism closely related to Prevotella ruminicola, but no homologies were found with any of the genomes already sequenced. Csd4 was introduced into the ethanologenic E. coli MS04 strain and ethanol production from CMC, avicel, sugarcane bagasse, or filter paper was observed. Exogenously expressed β-glucosidase had a positie effect on cell growth in agreement with the fact that no putative β-glucosidase was found in Csd4. Ethanol production from sugarcane bagasse was improved threefold by Csd4 after saccharification by commercial Trichoderma reesei cellulases underlining the ability of Csd4 to act as a saccharification enhancer to reduce the enzymatic load and time required for cellulose deconstruction.

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“…For example, the IgE Fcε3 domain was integrated into Ag43 and displayed on the surface of E. coli Tan109, with regard to a potential model for asthma and other allergic diseases treatment (Huang et al 2014). The β-glucosidase (BglC) was also displayed on the E. coli MS04 by Ag43 system, and successfully fermented cellulosic materials in the simultaneous saccharification and fermentation (SSF) process (Muñoz-Gutiérrez et al 2014). AT systems are considered to have some striking advantages compared to other surface display systems: a higher number of recombinant proteins can be displayed at the cell surface without reducing cell viability; the β-barrel is freely mobile while serving as an anchor for the recombinant protein within the outer membrane; and an inorganic prosthetic group can be incorporated in an expressed apo-protein (Jose 2006).…”

Section: Introductionmentioning

confidence: 99%

Antigen-43-mediated surface display revealed in Escherichia coli by different fusion sites and proteins

Jing

Guo

2019

Bioresour. Bioprocess.

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Background: Cell surface display system allows for endowing functional proteins expressed on bacterial surface by fusing different anchor proteins. Among PgsA, Blc, and Omp anchor, the antigen 43 (Ag43)-mediated surface display is a novel system in Escherichia coli. Here, we have demonstrated the red fluorescent protein (RFP) and cellulase (EC 3.2.1.4) on the cell surfaces at two different fusion sites in Ag43. Results: We introduced two fusion sites which are unstructured domain (52-138 aa) and autochaperone domain (600-700 aa) at N-terminal for passenger proteins. As a result, the surface-displayed RFP expressed in plasmid pET28a, but the intracellular RFP expressed more than the surface-displayed RFP. Improved display efficiency of Ag43 was present when fusing at the site of the 138th amino acid (aa) compared to fusing at the site of the 700th aa. For endoglucanase, whole-cell surface-displayed Ag43-138-BsCel5 showed the highest specific activity which was 4.65-fold of BsCel5. Cell-displayed cellulase preserved residual activity ranging from 78% to 38% at temperatures from 55 °C to 80 °C, respectively. Conclusions: This study is to demonstrate the novel surface display system of Ag43 in E. coli by targeting two different proteins RFP and BsCel5 that were successfully displayed on the cell surfaces at two different fusion sites. The Ag43 system displays surface heterologous proteins and is a potential whole-cell catalyst in the bioconversion of cellulose.

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Ag43-mediated display of a thermostable β-glucosidase in Escherichia coli and its use for simultaneous saccharification and fermentation at high temperatures

Cited by 22 publications

References 31 publications

Refactoring the upper sugar metabolism ofPseudomonas putidafor co-utilization of disaccharides, pentoses, and hexoses

Refactoring the upper sugar metabolism ofPseudomonas putidafor co-utilization of disaccharides, pentoses, and hexoses

Improved ethanol production from biomass by a rumen metagenomic DNA fragment expressed in Escherichia coli MS04 during fermentation

Antigen-43-mediated surface display revealed in Escherichia coli by different fusion sites and proteins

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