Effect of Inducers on the Production of 5‐Aminolevulinic Acid by Recombinant Escherichia coli

Abstract

Aminolevulinic acid (ALA) was produced by recombinant Escherichia coli BL21(DE3) (pET28‐A.R‐hemA) harboring the ALA synthase gene (hemA) from Agrobacterium radiobacter zju‐0121. The effects of inducers on the ALA synthase activity and ALA productivity were evaluated. The results indicated that a low isopropyl‐β‐D‐thiogalactoside (IPTG) concentration (0.05 mmol/L) was favorable for high expression of ALA synthase, which resulted in higher ALA productivity. For metabolic engineering applications, lactose was a better substitute of IPTG for active enzyme expression. When lactose concentration was 5 mmol/L, the specific ALA synthase activity and ALA productivity reached 16.7 nmol/(min · mg of protein) and 1.15 g/L, respectively, which were about 15% and 43% higher than those induced by IPTG.     

Production of poly-β-hydroxybutyrate on molasses by recombinant Escherichia coli

Beet molasses successfully replaced glucose as sole carbon source to produce poly--hydroxybutyrate by a recombinant Escherichia coli strain (HMS174/pTZ18u-PHB). The fermentation with molasses was cheaper than with glucose. The final dry cell weight, PHB content and PHB productivity were 39.5 g/L, 80% (w/w) and 1 g/Lh, respectively, in a 5 L stirred tank fermenter after 31.5 h fed-batch fermentation with constant pH and dissolved O2 content. © Rapid Science Ltd. 1998     

Optimization of Recombinant Cytochrome P450 2C9 Protein Production in Escherichia coli DH5α by Statistically-Based Experimental Design

Cytochrome P450 (CYP) 2C9 is of major importance in drug metabolism. However, the low yield of recombinant CYP2C9 protein in E. coli strains prevents its extensive use in the study of in vitro drug metabolism. In the present study, Taguchi design and desirability function were first used to investigate the effect of medium components (glycerol, δ-ALA, IPTG, ampicillin, chloramphenicol, inoculum density, peptone, thiamine, trace elements, NH₄Cl, and MgSO₄) on recombinant human CYP2C9 production by E. coli DH5α. An L₁₂ (2¹¹) orthogonal array was used to design the experiments to screen out the most influential factors. The CYP concentration and the specific content of CYP were considered as two product quality variables. A desirability function was applied to combine these two qualities as a single objective function. Optimization via central composite design (CCD) was then undertaken to yield the best performance. The confirmation experiments indicated that the expression performance under the optimized conditions was better than those obtained under other conditions. A compromise between conflicting goals, such as achievement of good yield of recombinant CYP2C9 and facility of the following purification, was found by means of the desirability function D. This is the first report that combined Taguchi design and CCD, and performed experiments in a multiresponse framework to optimize the production of human CYP in a recombinant E. coli strain.     

Strategies for high titre plasmid DNA production in Escherichia coli DH5α

The production of plasmid pEGFP-N1 in Escherichia coli DH5α was optimised. A strategy evaluating different media components separately was not successful (OD < 2.5, low plasmid titres), a statistical approach via a Plackett Burman design (11 parameters) allowed some improvement (7 mg/L plasmid, OD₆₀₀ 8.5). Generally, high biomass did not correlate with high plasmid titres. When conditions were transferred to the bioreactor (batch operation) little improvement in plasmid titres (10 mg/L plasmid, OD₆₀₀ 20) was observed. By switching to a fed-batch procedure with linear feeding these values increased to 20 mg/L plasmid (OD₆₀₀ 50). By using an adaptive feeding strategy, plasmid titres could be increased to 50 mg/L. Finally, by combining a growth controlled (reduced temperature (35 °C), low dO₂) initial batch phase with an adaptive feeding strategy in the fed-batch phase (37 °C, glucose-/dO₂-limitation) we were reproducibly able to produce up to 250 mg/L of plasmid DNA in cultures that reached a final OD₆₀₀ of 80.     

Production of γ-aminobutyric acid in Escherichia coli by engineering MSG pathway

Abstract

The compound γ-aminobutyric acid (GABA) has many important physiological functions. The effect of glutamate decarboxylases and the glutamate/GABA antiporter on GABA production was investigated in Escherichia coli. Three genes, gadA, gadB, and gadC were cloned and ligated alone or in combination into the plasmid pET32a. The constructed plasmids were transformed into Escherichia coli BL21(DE3). Three strains, E. coli BL21(DE3)/pET32a-gadA, E. coli BL21(DE3)/pET32a-gadAB and E. coli BL21(DE3)/pET32a-gadABC were selected and identified. The respective titers of GABA from the three strains grown in shake flasks were 1.25, 2.31, and 3.98?g/L. The optimal titer of the substrate and the optimal pH for GABA production were 40?g/L and 4.2, respectively. The highest titer of GABA was 23.6?g/L at 36?h in batch fermentation and was 31.3?g/L at 57?h in fed-batch fermentation. This study lays a foundation for the development and use of GABA.     

Optimal Production of Poly-γ-glutamic Acid by Metabolically Engineered Escherichia coli

Metabolically-engineered Escherichia coli strains were developed by cloning poly-γ-glutamic acid (γ-PGA) biosynthesis genes, consisting of pgsB, pgsC and pgsA, from Bacillus subtilis The metabolic and regulatory pathways of γ-PGA biosynthesis in E. coli were analyzed by DNA microarray. The inducible trc promoter and a constitutive promoter (P_HCE) derived from the d-amino acid aminotransferase (D-AAT) gene of Geobacillus toebii were employed. The constitutive HCE promoter was more efficient than inducible trc promoter for the expression of γ-PGA biosynthesis genes. DNA microarray analysis showed that the expression levels of several NtrC family genes, glnA, glnK, glnG, yhdX, yhdY, yhdZ, amtB, nac, argT and cbl were up-regulated and sucA, B, C, D genes were down-regulated. When (NH₄)₂SO₄ was added at 40 g/l into the feeding solution, the final γ-PGA concentration reached 3.7 g/l in the fed-batch culture of recombinant E. coli/pCOpgs.     

Production of thermophilic α-amylase using immobilized transformed Escherichia coli by addition of glycine

Efficient production of thermophilic α-amylase from Bacillus stearothermophilus was investigated using recombinant Escherichia coli HB101/pH1301 immobilized with κ-carrageenan by the addition of glycine. The effects of glycine, the concentrations of κ-carrageenan and KCI on the production of the enzyme as well as the stability of plasmid pHI301 were studied. In the absence of glycine, the enzyme was localized in the periplasmic space of the recombinant E. coli cells and a small amount of the enzyme was liberated in the culture broth. Although the addition of glycine was very effective for release of α-amylase from the periplasm of E. coli entrapped in gel beads, a majority of the enzyme accumulated in the gel matrix. (In this paper, production of the enzyme from recombinant cells to an ambient is expressed by the term “release”, while diffusion-out from gel beads is referred to by the term “liberate”.) Concentrations of KCI and immobilizing support significantly affected on the liberation of α-amylase to the culture broth. Mutants which produced smaller amounts of the enzyme emerged during a successive culture of recombinant E. coli, even under selective pressure, and they predominated in the later period of the passages. The population of plasmid-lost segregants increased with cultivation time. The stability of pHI301 for the free cells was increased by the addition of 2% KCI, which is a hardening agent for carrageenan. Although the viability of cells and α-amylase activity in the beads decreased with cultivation time during the successive culture of the immobilized recombinant E. coli, the plasmid stability was increased successfully by immobilization. Efficient long-term production of α-amylase was attained by an iterative re-activation-liberation procedure using the immobilized recombinant cells. Although the viable cell number, plasmid stability and enzyme activity liberated in the glycine solution decreased at an early period in the cultivation cycles, the process attained steady state regardless of the addition of an antibiotic.     

Effects of plasmid presence on growth and enzyme activity of Escherichia coli DH5α

Summary The effects of recombinant DNA propagation and gene expression on the physiology of the host cell was investigated using a series of copy number mutant plasmids. The plasmids at copy numbers of 30, 57, 115 and 501 per chromosome equivalent encoded constitutive production of the enzyme -lactamase. Ribose phosphate isomerase activity was relatively unaffected by plasmid presence, and glucose-6-phosphate dehydrogenase, fructose 1,6-diphosphate aldolase and fructose 1,6-diphosphatase activities were lower in plasmid-containing cells than in the plasmid-free host strain. Increasing copy number resulted in increased depression of enzyme activity levels. The results indicate that plasmid presence mediates subtle changes in the net expression of host enzymes involved in carbon metabolism. Responses of Escherichia coli DH5 in Evans medium to these plasmids differed substantially from responses of E. coli HB101 in rich medium.Offprint requests to: J. E. Bailey     

Production of galactokinase by an 0°-mutant of the galactose operon in Escherichia coli

0°-mutans are operationally defined as revertible, pleiotropic mutations, which abolish the function of all genes of an operon in cis position. Initially it was thought that 0°-mutations are located in the operator, the region of an operon, which serves as the recognition site for the gene product of the corresponding regulator gene (Jacob and Monod, 1961). Recent evidence, however, mainly obtained in the lactose operon of E. coli, places the 0°-mutations in the first structural gene of the operon, while the operator region seems to be located in a segment of the operon distinct from any structural gene (Jacob et al., 1964). The 0°-mutations is thought to manifest itself as a defect at the translation step (Beckwith, 1964). In the present communication we want to report some experiments with an 0°-mutant of the galactose operon. These experiments make it likely that the 0°-mutation is also able to change the response of the operon towards the inducer.     

Production of Bioactive Human β-defensin-3 in Escherichia coli by Soluble Fusion Expression

A codon optimized mature human β-defensin-3 gene (smHBD3) was synthesized and fused with TrxA to construct pET32-smHBD3 vector, which was transformed into E. coli BL21(DE3) and cultured in MBL medium. The volumetric productivity of fusion protein reached 0.99 g fusion protein l⁻¹, i.e. 0.21 g mature HBD3 l⁻¹. Ninety-six percentage of the fusion protein was in a soluble form and constituted about 45% of the total soluble protein. After cell disruption, the soluble fusion protein was separated by affinity chromatography and cleaved by enterokinase, and then the mature HBD3 was purified by cationic ion exchange chromatography. The overall recovery ratio of HBD3 was 43%. The purified mature HBD3 demonstrated antimicrobial activity against E. coli. Revisions requested 13 December 2005; Revisions received 24 January 2006     

Extracellular Production of Human Tumor Necrosis Factor-α by Escherichia coli Using a Chemically-synthesized Gene

A DNA fragment of approximately 490 base pairs encoding human TNF was chemically synthesized and expressed within Escherichia coli cells. Furthermore, extracellular production of human TNF and several N-terminal deletion mutants of TNF was attempted using the excretion vector pEAP8. The TNF mutant with two N-terminal amino acids deleted (NΔ2-TNF) was efficiently excreted into the culture medium by E. coli carrying the plasmid pEXTNF3. In this clone, the signal peptide was correctly processed during the excretion. The E. coli-excreted NΔ2-TNF had higher antitumor activity than wild-type TNF or NΔ2-TNF produced intracellularly by E. coli.     

Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies

Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six double-stranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. We demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work.     

Transfection of restrictionless Escherichia coli by bacteriophage T7 DNA: Effect of in vitro erosion of DNA by λ exonuclease

T7 bacteriophage infects with equal efficiency restriction-proficient Escherichia coli K12 cells and the restriction-deficient mutants. To the contrary, the purified phage DNA transfects wild-type cells at a very low efficiency (10^?9 plaques/genome equivalent). Mutations in the recB recC (exonuclease V) and sbcB (exonuclease I) loci increase the transfecting efficiency tenfold. A 1000-fold increase is obtained with cells deficient in restriction. No further increase is observed in hosts carrying both sets of mutations. The transfecting activity of the DNA on restriction-deficient hosts increases another 20-fold (up to 4 × 10^?5 plaques/genome equivalent) by complete erosion of the redundant regions of DNA with λ exonuclease, both in rec⁺ and recB recC sbcB genotypes. Circles and linear oligomers arising from the annealing of eroded DNA show the same transfecting activity as the unannealed monomers. The terminal redundancy of the genome, as measured by the onset of annealability of eroded molecules, was found to comprise 50 to 100 base-pairs.     

Reactivation of Photoinactivated Single-Stranded DNA Bacteriophage φX174 by UV-Irradiated Escherichia coli Cells

Reactivation of single-stranded DNA phage, photodynamically inactivated in the presence of proflavine sulfate, by three isogenic Escherichia coli strains having different DNA repair capabilities has been studied. It was found that reactivation of photoinactivated phiX174 was possible only if the host cells were recombination proficient (recA(+)) and had been lightly irradiated with UV light prior to infection; the presence of the uvrA(+) gene was not essential. Only a small part of the proflavine-mediated photodynamic damage in phiX174 could be repaired in this fashion. Burst sizes of reactivated phages were, however, comparable to those of normal unirradiated phages.     

Over-production of human interferon-γ by HCDC of recombinant Escherichia coli

A simple fed-batch process was developed using a modified variable specific growth rate feeding strategy for high cell density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-gamma (hIFN-γ). The feeding rate was adjusted to achieve the maximum attainable specific growth rate during fed-batch cultivation. In this method, specific growth rate was changed from a maximum value of 0.55 h⁻¹ at the beginning of feeding and then it was reduced to 0.4 h⁻¹ at induction time.The final concentration of biomass and IFN-γ was reached to ∼115 g l⁻¹ (DCW) and 42.5 g(hIFN-γ) l⁻¹ after 16.5 h, also the final specific yield and overall productivity of recombinant hIFN-γ (rhIFN-γ) were obtained 0.37 g(hIFN-γ) g⁻¹ DCW and 2.57 g(hIFN-γ) l⁻¹ h⁻¹, respectively. According to available data this is the highest specific yield and productivity that has been reported for recombinant proteins production yet.