Synthesis of active Olisthodiscus luteus ribulose-1,5-bisphosphate carboxylase in Escherichia coli

1988 ◽  
Vol 11 (6) ◽  
pp. 821-831 ◽  
Author(s):  
Scott Newman ◽  
Rose Ann Cattolico
Keyword(s):  
Escherichia Coli ◽  
Bisphosphate Carboxylase ◽  
Olisthodiscus Luteus

scholarly journals The Physiological Responses of Escherichia coli Triggered by Phosphoribulokinase (PrkA) and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco)

2020 ◽  
Vol 8 (8) ◽  
pp. 1187
Author(s):  
En-Jung Liu ◽  
I-Ting Tseng ◽  
Yi-Ling Chen ◽  
Ju-Jiun Pang ◽  
Zhi-Xuan Shen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Growth ◽  
Physiological Responses ◽  
Lactate Production ◽  
Reducing Power ◽  
Glucose Consumption ◽  
Redox Balance ◽  
Bisphosphate Carboxylase ◽  
E Coli ◽  
Co2 Recycling

Phosphoribulokinase (PrkA) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been proposed to create a heterologous Rubisco-based engineered pathway in Escherichia coli for in situ CO2 recycling. While the feasibility of a Rubisco-based engineered pathway has been shown, heterologous expressions of PrkA and Rubisco also induced physiological responses in E. coli that may compete with CO2 recycling. In this study, the metabolic shifts caused by PrkA and Rubisco were investigated in recombinant strains where ppc and pta genes (encodes phosphoenolpyruvate carboxylase and phosphate acetyltransferase, respectively) were deleted from E. coli MZLF (E. coli BL21(DE3) Δzwf, ΔldhA, Δfrd). It has been shown that the demand for ATP created by the expression of PrkA significantly enhanced the glucose consumptions of E. coli CC (MZLF Δppc) and E. coli CA (MZLF Δppc, Δpta). The accompanying metabolic shift is suggested to be the mgsA route (the methylglyoxal pathway) which results in the lactate production for reaching the redox balance. The overexpression of Rubisco not only enhanced glucose consumption but also bacterial growth. Instead of the mgsA route, the overproduction of the reducing power was balanced by the ethanol production. It is suggested that Rubisco induces a high demand for acetyl-CoA which is subsequently used by the glyoxylate shunt. Therefore, Rubisco can enhance bacterial growth. This study suggests that responses induced by the expression of PrkA and Rubisco will reach a new energy balance profile inside the cell. The new profile results in a new distribution of the carbon flow and thus carbons cannot be majorly directed to the Rubisco-based engineered pathway.

scholarly journals The rbcX Gene Product Promotes the Production and Assembly of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase of Synechococcus sp. PCC7002 in Escherichia coli

2004 ◽  
Vol 45 (10) ◽  
pp. 1390-1395 ◽  
Author(s):  
Takuo Onizuka ◽  
Sumiyo Endo ◽  
Hideo Akiyama ◽  
Shozo Kanai ◽  
Masahiko Hirano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Bisphosphate Carboxylase ◽  
Synechococcus Sp

Evolving improved Synechococcus Rubisco functional expression in Escherichia coli

2008 ◽  
Vol 414 (2) ◽  
pp. 205-214 ◽  
Author(s):  
Oliver Mueller-Cajar ◽  
Spencer M. Whitney
Keyword(s):  
Escherichia Coli ◽  
Large Subunit ◽  
Fold Increase ◽  
Functional Expression ◽  
Kinetic Properties ◽  
Selection System ◽  
Wild Type ◽  
Bisphosphate Carboxylase ◽  
E Coli ◽  
Enhanced Expression

The photosynthetic CO2-fixing enzyme Rubisco [ribulose-P2 (D-ribulose-1,5-bisphosphate) carboxylase/oxygenase] has long been a target for engineering kinetic improvements. Towards this goal we used an RDE (Rubisco-dependent Escherichia coli) selection system to evolve Synechococcus PCC6301 Form I Rubisco under different selection pressures. In the fastest growing colonies, the Rubisco L (large) subunit substitutions I174V, Q212L, M262T, F345L or F345I were repeatedly selected and shown to increase functional Rubisco expression 4- to 7-fold in the RDE and 5- to 17-fold when expressed in XL1-Blue E. coli. Introducing the F345I L-subunit substitution into Synechococcus PCC7002 Rubisco improved its functional expression 11-fold in XL1-Blue cells but could not elicit functional Arabidopsis Rubisco expression in the bacterium. The L subunit substitutions L161M and M169L were complementary in improving Rubisco yield 11-fold, whereas individually they improved yield ∼5-fold. In XL1-Blue cells, additional GroE chaperonin enhanced expression of the I174V, Q212L and M262T mutant Rubiscos but engendered little change in the yield of the more assembly-competent F345I or F345L mutants. In contrast, the Rubisco chaperone RbcX stimulated functional assembly of wild-type and mutant Rubiscos. The kinetic properties of the mutated Rubiscos varied with noticeable reductions in carboxylation and oxygenation efficiency accompanying the Q212L mutation and a 2-fold increase in Kribulose-P2 (KM for the substrate ribulose-P2) for the F345L mutant, which was contrary to the ∼30% reductions in Kribulose-P2 for the other mutants. These results confirm the RDE systems versatility for identifying mutations that improve functional Rubisco expression in E. coli and provide an impetus for developing the system to screen for kinetic improvements.

Ultrastructural and Cytochemical Analyses of the Expression of the Thiobacillus Carboxysome Operon in Escherichia Coli

2001 ◽  
Vol 7 (S2) ◽  
pp. 740-741
Author(s):  
H.C. Aldrich ◽  
S. Elvington ◽  
HE. Machines ◽  
R. Szabady ◽  
K. Feder ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Expression Vector ◽  
Immunogold Labeling ◽  
Bisphosphate Carboxylase ◽  
Functional Entity ◽  
Ultrathin Sections ◽  
Thiobacillus Neapolitanus

The cytoplasm of the bacterium Thiobacillus neapolitanus contains 117 nm diameter polyhedral inclusions, “carboxysomes” (Fig. 1) that contain ribulose-1,5- bisphosphate carboxylase/oxygenase (RuBisCO). Surrounding the polyhedron are nonmembranous proteinaceous plates devoid of lipid. The carboxysomes are composed of at least 8 major peptides, all coded within the same operon. Six (CsoSIA, CsoSIB, CsoSIC, CsoS2A, CsoS2B, and CsoS3) make up the shell, and two are the large (CbbL) and small subunits (CbbS) of RuBisCO. Using immunogold labeling on ultrathin sections, peptides CsoS2A, CsoS2B, and CsoS3 have been localized to the shell. Since the original characterization of the csoSl gene, we have also immunolocalized the CsoSl peptide to the shell.As part of our initial efforts to understand how these components are assembled into the symmetrical, functional entity, the carboxysome operon from T. neapolitanus was cloned into the pET-21a(+) plasmid, an expression vector that codes for resistance to ampicillin.

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