scholarly journals Engineering of a Highly Efficient Escherichia coli Strain for Mevalonate Fermentation through Chromosomal Integration

2016 ◽  
Vol 82 (24) ◽  
pp. 7176-7184 ◽  
Author(s):  
Jilong Wang ◽  
Suthamat Niyompanich ◽  
Yi-Shu Tai ◽  
Jingyu Wang ◽  
Wenqin Bai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shake Flask ◽  
Mevalonate Pathway ◽  
High Titer ◽  
Scale Up ◽  
Coli Strain ◽  
High Yield ◽  
Glycolytic Flux ◽  
Chromosomal Integration ◽  
Theoretical Yield

ABSTRACTChromosomal integration of heterologous metabolic pathways is optimal for industrially relevant fermentation, as plasmid-based fermentation causes extra metabolic burden and genetic instabilities. In this work, chromosomal integration was adapted for the production of mevalonate, which can be readily converted into β-methyl-δ-valerolactone, a monomer for the production of mechanically tunable polyesters. The mevalonate pathway, driven by a constitutive promoter, was integrated into the chromosome ofEscherichia colito replace the native fermentation geneadhEorldhA. The engineered strains (CMEV-1 and CMEV-2) did not require inducer or antibiotic and showed slightly higher maximal productivities (0.38 to ∼0.43 g/liter/h) and yields (67.8 to ∼71.4% of the maximum theoretical yield) than those of the plasmid-based fermentation. Since the glycolysis pathway is the first module for mevalonate synthesis,atpFHdeletion was employed to improve the glycolytic rate and the production rate of mevalonate. Shake flask fermentation results showed that the deletion ofatpFHin CMEV-1 resulted in a 2.1-fold increase in the maximum productivity. Furthermore, enhancement of the downstream pathway by integrating two copies of the mevalonate pathway genes into the chromosome further improved the mevalonate yield. Finally, our fed-batch fermentation showed that, with deletion of theatpFHandsucAgenes and integration of two copies of the mevalonate pathway genes into the chromosome, the engineered strain CMEV-7 exhibited both high maximal productivity (∼1.01 g/liter/h) and high yield (86.1% of the maximum theoretical yield, 30 g/liter mevalonate from 61 g/liter glucose after 48 h in a shake flask).IMPORTANCEMetabolic engineering has succeeded in producing various chemicals. However, few of these chemicals are commercially competitive with the conventional petroleum-derived materials. In this work, chromosomal integration of the heterologous pathway and subsequent optimization strategies ensure stable and efficient (i.e., high-titer, high-yield, and high-productivity) production of mevalonate, which demonstrates the potential for scale-up fermentation. Among the optimization strategies, we demonstrated that enhancement of the glycolytic flux significantly improved the productivity. This result provides an example of how to tune the carbon flux for the optimal production of exogenous chemicals.

scholarly journals Purification and properties of cystathionine β-lyase from Arabidopsis thaliana overexpressed in Escherichia coli

1996 ◽  
Vol 320 (2) ◽  
pp. 383-392 ◽  
Author(s):  
Stéphane RAVANEL ◽  
Dominique JOB ◽  
Roland DOUCE
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Biochemical Properties ◽  
Coli Strain ◽  
High Yield ◽  
Purification And Properties ◽  
Key Enzyme ◽  
Schiff Base Formation ◽  
Insight Into ◽  
Base Formation

Cystathionine β-lyase is a key enzyme in sulphur metabolism that catalyses the second reaction specific for methionine biosynthesis, the pyridoxal 5´-phosphate-dependent β-cleavage of cystathionine to produce homocysteine. To obtain insight into the biochemical properties of the plant enzyme, the cDNA encoding cystathionine β-lyase from Arabidopsis thaliana was used to construct an overproducing Escherichia coli strain. The recombinant enzyme was isolated at high yield (29 mg of pure protein/litre of cell culture) using an efficient two-step purification procedure. Physicochemical properties of the Arabidopsis cystathionine β-lyase were similar to those previously reported for the bacterial enzymes. In particular, the native recombinant protein is a tetramer composed of four identical subunits of 46 kDa, each being associated with one molecule of pyridoxal 5´-phosphate. Interaction between the apoenzyme and pyridoxal 5´-phosphate is extremely tight, being characterized by a Kd value of 0.5 µM. Purification and sequencing of the phosphopyridoxyl peptide established that Schiff base formation between the cofactor and the holoenzyme occurs at lysine-278. The substrate specificity of the recombinant cystathionine β-lyase resembles that of the enzyme isolated from other sources, cystathionine and djenkolate being the most effective substrates. The cystathionine analogue aminoethoxyvinylglycine irreversibly inactivates the recombinant cystathionine β-lyase. The inactivation is accompanied by dramatic modification of the spectral properties of the enzyme that can be attributed to the attack of the azomethine linkage between pyridoxal 5´-phosphate and lysine-278 of the polypeptide by aminoethoxyvinylglycine.

scholarly journals Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli

2008 ◽  
Vol 51 (3) ◽  
pp. 473-482 ◽  
Author(s):  
Dorismey Vieira Tokano ◽  
Marisa Emiko Kawaichi ◽  
Emerson José Venâncio ◽  
Marilda Carlos Vidotto
Keyword(s):  
Escherichia Coli ◽  
Biological Activity ◽  
Iron Uptake ◽  
Expression Vector ◽  
Iron Acquisition ◽  
Coli Strain ◽  
High Yield ◽  
High Similarity ◽  
E Coli

The aim of this work was to isolate, clone and characterize the iron uptake gene iutA from avian pathogenic E. coli (APEC). The iutA gene was isolated from the strain APEC 9, serotype O2:H9, which was cloned in the expression vector pET101/D-TOPO. The gene of 2.2 Kb was sequenced (AY602767, which showed high similarity to the iutA gene from three plasmids, two from APEC, pAPEC-02-ColV (AY545598.4) and pTJ100 (AY553855.1), and one from a human invasive E. coli strain, the pColV K30. The recombinant protein IutA was over expressed in E. coli BL21(DE-3) and was solubilized with urea and purified by Ni-NTA column. This method produced a relatively high yield of r-IutA of approximately 74kDa, which was used to produce the antibody anti-IutA. This anti-IutA reacted with the protein r-IutA and native IutA of APEC 9, as demonstrated by Western blot, showing that the r-IutA conserved epitopes and its antigenicity was preserved. The anti-IutA IgY was able to inhibit the IutA biological activity, inhibiting the sensitivity to cloacin DF13 of APEC9. However, it did not inhibit the growth of APEC9 in M9 and did not protect the chickens inoculated with the APEC, suggesting that the APEC possessed another iron acquisition mechanism distinct of aerobactin.

scholarly journals High Glycolytic Flux Improves Pyruvate Production by a Metabolically Engineered Escherichia coli Strain

2008 ◽  
Vol 74 (21) ◽  
pp. 6649-6655 ◽  
Author(s):  
Yihui Zhu ◽  
Mark A. Eiteman ◽  
Ronni Altman ◽  
Elliot Altman
Keyword(s):  
Escherichia Coli ◽  
Nadh Oxidase ◽  
Pyruvate Dehydrogenase Complex ◽  
Formation Rate ◽  
Batch Process ◽  
Defined Medium ◽  
Coli Strain ◽  
Glycolytic Flux ◽  
Exponential Feeding ◽  
Phosphoenolpyruvate Synthase

ABSTRACT We report pyruvate formation in Escherichia coli strain ALS929 containing mutations in the aceEF, pfl, poxB, pps, and ldhA genes which encode, respectively, the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase. The glycolytic rate and pyruvate productivity were compared using glucose-, acetate-, nitrogen-, or phosphorus-limited chemostats at a growth rate of 0.15 h−1. Of these four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux (1.60 g/g � h), pyruvate formation rate (1.11 g/g � h), and pyruvate yield (0.70 g/g). Additional mutations in atpFH and arcA (strain ALS1059) further elevated the steady-state glycolytic flux to 2.38 g/g � h in an acetate-limited chemostat, with heterologous NADH oxidase expression causing only modest additional improvement. A fed-batch process with strain ALS1059 using defined medium with 5 mM betaine as osmoprotectant and an exponential feeding rate of 0.15 h−1 achieved 90 g/liter pyruvate, with an overall productivity of 2.1 g/liter � h and yield of 0.68 g/g.

scholarly journals Fermentative Production of Thymidine by a Metabolically Engineered Escherichia coli Strain

2009 ◽  
Vol 75 (8) ◽  
pp. 2423-2432 ◽  
Author(s):  
Hyeon Cheol Lee ◽  
Jin Ha Kim ◽  
Jin Sook Kim ◽  
Wonhee Jang ◽  
Sang Yong Kim
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Excision Repair ◽  
Coli Strain ◽  
High Yield ◽  
Gene Encoding ◽  
E Coli ◽  
Fermentative Production ◽  
Batch Fermenter ◽  
Degradation Activity

ABSTRACT Thymidine is an important precursor in the production of various antiviral drugs, including azidothymidine for the treatment of AIDS. Since thymidine-containing nucleotides are synthesized only by the de novo pathway during DNA synthesis, it is not easy to produce a large amount of thymidine biologically. In order to develop a host strain to produce thymidine, thymidine phosphorylase, thymidine kinase, and uridine phosphorylase genes were deleted from an Escherichia coli BL21 strain to develop BLdtu. Since the genes coding for the enzymes related to the nucleotide salvage pathway were disrupted, BLdtu was unable to utilize thymidine or thymine, and thymidine degradation activity was completely abrogated. We additionally expressed T4 thymidylate synthase, T4 nucleotide diphosphate reductase, bacteriophage PBS2 TMP phosphohydrolase, E. coli dCTP deaminase, and E. coli uridine kinase in the BLdtu strain to develop a thymidine-producing strain (BLdtu24). BLdtu24 produced 649.3 mg liter−1 of thymidine in a 7-liter batch fermenter for 24 h, and neither thymine nor uridine was detected. However, the dUTP/dTTP ratio was increased in BLdtu24, which could lead to increased double-strand breakages and eventually to cell deaths during fermentation. To enhance thymidine production and to prevent cell deaths during fermentation, we disrupted a gene (encoding uracil-DNA N-glycosylase) involved in DNA excision repair to suppress the consumption of dTTP and developed BLdtug24. Compared with the thymidine production in BLdtu24, the thymidine production in BLdtug24 was increased by ∼1.2-fold (740.3 mg liter−1). Here, we show that a thymidine-producing strain with a relatively high yield can be developed using a metabolic engineering approach.

scholarly journals Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli

2011 ◽  
Vol 77 (9) ◽  
pp. 2905-2915 ◽  
Author(s):  
Claire R. Shen ◽  
Ethan I. Lan ◽  
Yasumasa Dekishima ◽  
Antonino Baez ◽  
Kwang Myung Cho ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Driving Forces ◽  
High Titer ◽  
Catalytic Efficiency ◽  
Anaerobic Growth ◽  
High Yield ◽  
Efficient Production ◽  
Acetyl Coa ◽  
Content Type ◽  
Irreversible Reaction

ABSTRACT1-Butanol, an important chemical feedstock and advanced biofuel, is produced byClostridiumspecies. Various efforts have been made to transfer the clostridial 1-butanol pathway into other microorganisms. However, in contrast to similar compounds, only limited titers of 1-butanol were attained. In this work, we constructed a modified clostridial 1-butanol pathway inEscherichia colito provide an irreversible reaction catalyzed bytrans-enoyl-coenzyme A (CoA) reductase (Ter) and created NADH and acetyl-CoA driving forces to direct the flux. We achieved high-titer (30 g/liter) and high-yield (70 to 88% of the theoretical) production of 1-butanol anaerobically, comparable to or exceeding the levels demonstrated by native producers. Without the NADH and acetyl-CoA driving forces, the Ter reaction alone only achieved about 1/10 the level of production. The engineered host platform also enables the selection of essential enzymes with better catalytic efficiency or expression by anaerobic growth rescue. These results demonstrate the importance of driving forces in the efficient production of nonnative products.

scholarly journals KONSTRUKSI VEKTOR BINER UNTUK EKSPRESI GEN dip22 (YANG DIISOLASI DARI TEBU VARIETAS M 442-51) PADA TANAMAN

2007 ◽  
Vol 13 (1) ◽  
pp. 15-26
Author(s):  
Wiwit Budi Widyasari ◽  
Sony Suhandono
Keyword(s):  
Escherichia Coli ◽  
Water Stress ◽  
Recombinant Plasmid ◽  
Binary Vector ◽  
Restriction Enzymes ◽  
Coli Strain ◽  
High Yield ◽  
Over Expression ◽  
Number Of Genes ◽  
Inducible Protein

Sugarcane is the principle plant for producing sugar in Indonesia. Water supply is one key element in the agronomy of sugarcane. Sugarcane is a high biomass crop which requires large amounts of water. Low yields of sugar observed in water stressed plants indicate that sugarcane is very sensititive to drought. A number of genes that respond to drought, salt, and cold stress at the trasnscriptional level have been reported. dip22 (drought inducible protein) protein isolated from drought resistance variety M 442-51 was predicted to be a protein regulator to water stress in sugarcane. Increasing of tolerance to water stress by over expression of dip22 genes in high yield sugarcane variety hopefully will maintain sugar production. The goal of this research was to construct a binary vector for dip22 gene expression in plant. dip22 gene from mutated PCR was cloned to pGEM®–T Easy and transformed to Escherichia coli strain DH5a. And then, these gene was isolated again from pGEM®–T Easy-dip22 (pGdip) plasmid using restriction enzymes NcoI and PmlI. pCAMBIA 1303 plasmid is an expression vector which has the constitutive promoter CaMV35S. Recombinant plasmid was transformed to Escherichia coli strain DH5a for plasmid propagation through DNA replication. Recombinant plasmid was isolated, and digested with NcoI and PmlI to examine the presence of dip22 gene in the pCAMBIA 1303 plasmid. The recombinant plasmid was transformed to A. tumefaciens strain LBA 4404. Plasmid isolated from A. tumefaciens was digested with Bst XI and Bst EII to examine the similarity between pCAMBIA 1303-dip22 (pCdip) from Escherichia coli and A. tumefaciens. The result by electrophoresis showed that both plasmids had the same size after digested. It was concluded that the transformed A. tumefaciens strain LBA 4404 bacteria has pCAMBIA 1303-dip22 (pCdip) plasmid indeed. Therefore, this construct of dip22 gene in binary vector can be used for improving drought tolerance in plant.

scholarly journals Media optimization for SHuffle T7 Escherichia coli expressing SUMO-Lispro proinsulin by response surface methodology

2022 ◽  
Vol 22 (1) ◽  
Author(s):  
Aida Bakhshi Khalilvand ◽  
Saeed Aminzadeh ◽  
Mohammad Hossein Sanati ◽  
Fereidoun Mahboudi
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Shake Flask ◽  
High Cell Density ◽  
Fold Increase ◽  
Soluble Expression ◽  
High Yield ◽  
High Cell ◽  
E Coli ◽  
High Cell Density Fermentation

Abstract Background SHuffle is a suitable Escherichia coli (E. coli) strain for high yield cytoplasmic soluble expression of disulfide-bonded proteins such as Insulin due to its oxidative cytoplasmic condition and the ability to correct the arrangement of disulfide bonds. Lispro is an Insulin analog that is conventionally produced in E. coli as inclusion bodies (IBs) with prolonged production time and low recovery. Here in this study, we aimed to optimize cultivation media composition for high cell density fermentation of SHuffle T7 E. coli expressing soluble Lispro proinsulin fused to SUMO tag (SU-INS construct) to obtain high cell density fermentation. Results Factors including carbon and nitrogen sources, salts, metal ions, and pH were screened via Plackett–Burman design for their effectiveness on cell dry weight (CDW) as a measure of cell growth. The most significant variables of the screening experiment were Yeast extract and MgCl2 concentration, as well as pH. Succeedingly, The Central Composite Design was utilized to further evaluate and optimize the level of significant variables. The Optimized media (OM-I) enhanced biomass by 2.3 fold in the shake flask (2.5 g/L CDW) that reached 6.45 g/L (2.6 fold increase) when applied in batch culture fermentation. The efficacy of OM-I media for soluble expression was confirmed in both shake flask and fermentor. Conclusion The proposed media was suitable for high cell density fermentation of E. coli SHuffle T7 and was applicable for high yield soluble expression of Lispro proinsulin.

scholarly journals Improved production of Humira antibody in the genetically engineered Escherichia coli SHuffle, by co-expression of human PDI-GPx7 fusions

2020 ◽  
Vol 104 (22) ◽  
pp. 9693-9706
Author(s):  
Marine Lénon ◽  
Na Ke ◽  
Cecily Szady ◽  
Hassan Sakhtah ◽  
Guoping Ren ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Lines ◽  
Shake Flask ◽  
Eukaryotic Cell ◽  
Coli Strain ◽  
Genetically Engineered ◽  
High Density ◽  
In Vivo Studies ◽  
Efficient Production

Abstract Microbial production of antibodies offers the promise of cheap, fast, and efficient production of antibodies at an industrial scale. Limiting this capacity in prokaryotes is the absence of the post-translational machinery, present in dedicated antibody producing eukaryotic cell lines, such as B cells. There has been few and limited success in producing full-length, correctly folded, and assembled IgG in the cytoplasm of prokaryotic cell lines. One such success was achieved by utilizing the genetically engineered Escherichia coli strain SHuffle with an oxidative cytoplasm. Due to the genetic disruption of reductive pathways, SHuffle cells are under constant oxidative stress, including increased levels of hydrogen peroxide (H2O2). The oxidizing capacity of H2O2 was linked to improved disulfide bond formation, by expressing a fusion of two endoplasmic reticulum-resident proteins, the thiol peroxidase GPx7 and the protein disulfide isomerase, PDI. In concert, these proteins mediate disulfide transfer from H2O2 to target proteins via PDI-Gpx7 fusions. The potential of this new strain was tested with Humira, a blockbuster antibody usually produced in eukaryotic cells. Expression results demonstrate that the new engineered SHuffle strain (SHuffle2) could produce Humira IgG four-fold better than the parental strain, both in shake-flask and in high-density fermentation. These preliminary studies guide the field in genetically engineering eukaryotic redox pathways in prokaryotes for the production of complex macromolecules. Key points • A eukaryotic redox pathway was engineered into the E. coli strain SHuffle in order to improve the yield of the blockbuster antibody Humira. • The best peroxidase-PDI fusion was selected using bioinformatics and in vivo studies. • Improved yields of Humira were demonstrated at shake-flask and high-density fermenters.

scholarly journals Biocatalytic Conversion of Short-Chain Fatty Acids to Corresponding Alcohols in Escherichia coli

Processes ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 973
Author(s):  
Li-Jen Lin ◽  
Mukesh Saini ◽  
Chung-Jen Chiang ◽  
Yun-Peng Chao
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Clostridium Acetobutylicum ◽  
Short Chain Fatty Acids ◽  
Coli Strain ◽  
Short Chain ◽  
Glycolytic Flux ◽  
Pentanoic Acid ◽  
Biocatalytic Production ◽  
Do So

Advanced biofuels possess superior characteristics to serve for gasoline substitutes. In this study, a whole cell biocatalysis system was employed for production of short-chain alcohols from corresponding fatty acids. To do so, Escherichia coli strain was equipped with a biocatalytic pathway consisting of endogenous atoDA and Clostridium acetobutylicum adhE2. The strain was further reprogrammed to improve its biocatalytic activity by direction the glycolytic flux to acetyl-CoA and recycling acetate. The production of 1-propanol and n-pentanol were exemplified with the engineered strain. By substrate (glucose and propionate) feeding, the strain enabled production of 5.4 g/L 1-propanol with productivity reaching 0.15 g/L/h. In addition, the strain with a heavy inoculum was implemented for the n-pentanol production from n-pentanoic acid. The production titer and productivity finally attained 4.3 g/L and 0.86 g/L/h, respectively. Overall, the result indicates that this developed system is useful and effective for biocatalytic production of short-chain alcohols.

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