Metabolomic and Transcriptomic Analyses of Escherichia coli for Efficient Fermentation of L-Fucose

L-Fucose, one of the major monomeric sugars in brown algae, possesses high potential for use in the large-scale production of bio-based products. Although fucose catabolic pathways have been enzymatically evaluated, the effects of fucose as a carbon source on intracellular metabolism in industrial microorganisms such as Escherichia coli are still not identified. To elucidate the effects of fucose on cellular metabolism and to find clues for efficient conversion of fucose into bio-based products, comparative metabolomic and transcriptomic analyses were performed on E. coli on L-fucose and on D-glucose as a control. When fucose was the carbon source for E. coli, integration of the two omics analyses revealed that excess gluconeogenesis and quorum sensing led to severe depletion of ATP, resulting in accumulation and export of fucose extracellularly. Therefore, metabolic engineering and optimization are needed for E. coil to more efficiently ferment fucose. This is the first multi-omics study investigating the effects of fucose on cellular metabolism in E. coli. These omics data and their biological interpretation could be used to assist metabolic engineering of E. coli producing bio-based products using fucose-containing brown macroalgae.

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Media Optimization for Production of Recombinant Carrier Protein (CRM197) in Escherichia coli

Journal of Scientific Research ◽

10.3329/jsr.v13i1.48996 ◽

2021 ◽

Vol 13 (1) ◽

pp. 283-297

Author(s):

S. Shukla ◽

D. Mishra

Keyword(s):

Escherichia Coli ◽

Large Scale ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Luria Bertani ◽

Lag Phase ◽

Carrier Protein ◽

Scale Production ◽

E Coli ◽

Defined Media

Since the advent of vaccines, the mankind has benefited from the same and has been able to curb the mortality rate around the globe. Amongst different types of available vaccines, polysaccharide based vaccines are very widely used against various infectious diseases. The polysaccharide vaccines need to be conjugated with a carrier protein to make the vaccine more immunogenic. Recombinant Escherichia coli cells are the organism of choice for large scale production of a carrier protein because of its widely studied scientific aspects. In the present study, for proof of concept, the recombinant E. coli cells were cultured in Luria-Bertani media to check the expression of rCRM197. At 80L scale, it was observed that when recombinant E. coli cells were grown in a chemically defined media, it resulted in inconsistent growth and a long lag phase. When the defined media was supplemented with yeast extract, the lag phase of the culture was substantially reduced and the maximum growth of the culture was achieved. Protein expression was checked using SDS PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) and Western blot technique. The optimized media resulted in a robust fermentation process to achieve high cell density and maximum biomass for the production of recombinant protein.

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Implementation of a novel self-induced promoter for the expression of pharmaceutical peptides in Escherichia coli: YY(3-36) peptide

Hormone Molecular Biology and Clinical Investigation ◽

10.1515/hmbci-2018-0056 ◽

2019 ◽

Vol 0 (0) ◽

Author(s):

Amir Hossein Momen ◽

Naser Harzandi ◽

Azam Haddadi ◽

Bijan Bambai

Keyword(s):

Escherichia Coli ◽

Large Scale ◽

Peptide Yy ◽

Signal Sequence ◽

Expression System ◽

Promoter Sequence ◽

Scale Production ◽

Signal Sequences ◽

T7 Promoter ◽

E Coli

Abstract Background Increasing the expression rate of recombinant mammalian hormones in Escherichia coli by combining efficient promoters and signal sequences is a never ending process. A self-induced promoter will have some beneficial gains compared to the classical T7 promoter or its variants with isopropyl β-D-1-thiogalactopyranoside (IPTG) as the inducer. Obesity is the prime suspect in widespread frequency of diabetes type II and cardiovascular diseases worldwide. YY (tyrosine-tyrosine) peptide is a local acting hormone, controlling appetite. Excitingly, it was has been shown that a truncated version of the YY peptide, YY(3-36) peptide, has potential as a worthy biopharmaceutical agent in the fight against obesity. Materials and methods To develop an economical expression system for the large scale production of the peptide in Gram-negative bacteria, we introduced a promoter sequence upstream of a chimeric gene for the extracellular expression of this peptide with the assistance of a signal sequence of asparaginase II from E. coli. This system has the advantage of producing a complete sequence of a truncated YY peptide, YY(3-36), without any extra tags that would require further removal with the assistance of expensive specific proteases and reduced the downstream steps, significantly. Results Recombinant production of YY(3-36) peptide under a self-induced promoter proves the efficacy of the asparaginase II signal sequence as a communicator of foreign peptides and proteins into the extracellular space of E. coli. Conclusions The application of fusion protein expression of biopharmaceuticals, especially mammalian hormones, in prokaryotic systems with the help of native signal sequences makes some common tags with expensive proteases for the removal of the attached protein Tag redundant.

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Enhanced Silver Nanoparticle Synthesis by Escherichia Coli Transformed with Candida Albicans Metallothionein Gene

Materials ◽

10.3390/ma12244180 ◽

2019 ◽

Vol 12 (24) ◽

pp. 4180 ◽

Cited By ~ 2

Author(s):

Qunying Yuan ◽

Manjula Bomma ◽

Zhigang Xiao

Keyword(s):

Escherichia Coli ◽

Silver Nanoparticles ◽

Candida Albicans ◽

Silver Nanoparticle ◽

Large Scale ◽

Nanoparticle Synthesis ◽

Scale Production ◽

E Coli ◽

Metallothionein Gene ◽

Silver Nanoparticle Synthesis

In this study, the metallothionein gene of Candida albicans (C. albicans) was assembled by polymerase chain reaction (PCR), inserted into pUC19 vector, and further transformed into Escherichia coli (E. coli) DH5α cells. The capacity of these recombinant E. coli DH5α cells to synthesize silver nanoparticles was examined. Our results demonstrated that the expression of C. albicans metallothionein in E. coli promoted the bacterial tolerance to metal ions and increased yield of silver nanoparticle synthesis. The compositional and morphological analysis of the silver nanoparticles revealed that silver nanoparticles synthesized by the engineered E. coli cells are around 20 nm in size, and spherical in shape. Importantly, the silver nanoparticles produced by the engineered cells were more homogeneous in shape and size than those produced by bacteria lack of the C. albicans metallothionein. Our study provided preliminary information for further development of the engineered E. coli as a platform for large-scale production of uniform nanoparticles for various applications in nanotechnology.

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Iron Acquisition of Urinary Tract Infection Escherichia coli Involves Pathogenicity in Caenorhabditis elegans

Microorganisms ◽

10.3390/microorganisms9020310 ◽

2021 ◽

Vol 9 (2) ◽

pp. 310

Author(s):

Masayuki Hashimoto ◽

Yi-Fen Ma ◽

Sin-Tian Wang ◽

Chang-Shi Chen ◽

Ching-Hao Teng

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Caenorhabditis Elegans ◽

Large Scale ◽

Iron Acquisition ◽

Infection Model ◽

High Throughput Analysis ◽

E Coli ◽

C Elegans ◽

Tract Infections

Uropathogenic Escherichia coli (UPEC) is a major bacterial pathogen that causes urinary tract infections (UTIs). The mouse is an available UTI model for studying the pathogenicity; however, Caenorhabditis elegans represents as an alternative surrogate host with the capacity for high-throughput analysis. Then, we established a simple assay for a UPEC infection model with C. elegans for large-scale screening. A total of 133 clinically isolated E. coli strains, which included UTI-associated and fecal isolates, were applied to demonstrate the simple pathogenicity assay. From the screening, several virulence factors (VFs) involved with iron acquisition (chuA, fyuA, and irp2) were significantly associated with high pathogenicity. We then evaluated whether the VFs in UPEC were involved in the pathogenicity. Mutants of E. coli UTI89 with defective iron acquisition systems were applied to a solid killing assay with C. elegans. As a result, the survival rate of C. elegans fed with the mutants significantly increased compared to when fed with the parent strain. The results demonstrated, the simple assay with C. elegans was useful as a UPEC infectious model. To our knowledge, this is the first report of the involvement of iron acquisition in the pathogenicity of UPEC in a C. elegans model.

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Cloning, expression, and analysis of the group 2 allergen from Dermatophagoides farinae from China

Anais da Academia Brasileira de Ciências ◽

10.1590/s0001-37652010000400017 ◽

2010 ◽

Vol 82 (4) ◽

pp. 941-951 ◽

Cited By ~ 2

Author(s):

Cui Yu-bao ◽

Ying Zhou ◽

Shi Weihong ◽

Ma Guifang ◽

Li Yang ◽

...

Keyword(s):

Large Scale ◽

Alpha Helix ◽

Random Coil ◽

Reference Sequence ◽

Scale Production ◽

Dermatophagoides Farinae ◽

E Coli ◽

Large Scale Production ◽

Solid Foundation ◽

Group 2

To obtain the recombinant group 2 allergen product of Dermatophagoides farinae (Der f 2), the Der f 2 gene was synthesized by RT-PCR. The full-length cDNA comprised 441 nucleotides and was 99.3% identical to the reference sequence (GenBank AB195580). The cDNA was bound to vector pET28a to construct plasmid pET28a(+)-Der f 2, which was transformed into E. coli BL21 and induced by IPTG. SDS-PAGE showed a specific band of about 14kDa in the hole cell lysate. s estiated by chroatography, about 3.86 g of the recobinant product as obtained, which conjugated with serum IgE from asthmatic children. The protein had a signal peptide of 17 amino acids. Its secondary structure comprised an alpha helix (19.86%), an extended strand (30.82%), and a random coil (49.32%). The subcellular localization of this allergen was predicted to be at mitochondria. Furthermore, its function was shown to be associated with an MD-2-related lipid-recognition (ML) domain. The results of this study provide a solid foundation for large-scale production of the allergen for clinical diagnosis and treatent of allergic disorders.

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Large-scale production of citrate synthase from a cloned gene

Canadian Journal of Biochemistry ◽

10.1139/o82-146 ◽

1982 ◽

Vol 60 (12) ◽

pp. 1143-1147 ◽

Cited By ~ 12

Author(s):

Harry W. Duckworth ◽

Alexander W. Bell

Keyword(s):

Escherichia Coli ◽

Large Scale ◽

Citrate Synthase ◽

Specific Activity ◽

Tetracycline Resistance ◽

Scale Production ◽

Ampicillin Resistance ◽

Colicin E1 ◽

Large Scale Production ◽

Cloned Gene

Starting with a colicin E1 resistance recombinant plasmid which contains gltA, the gene for citrate synthase in Escherichia coli, we have constructed an ampicillin-resistance plasmid containing the gltA region as a 2.9-kilobase-pair insert in the tetracycline-resistance region of pBR322. Escherichia coli HB101 harbouring this plasmid, when grown on rich medium containing ampicillin, contains citrate synthase as about 8% of its soluble protein. The enzyme has been purified from this rich source and is identical to the chromosomal enzyme prepared previously in every property tested, except for specific activity, which is 64 U∙mg−1 as compared with 45–50 U∙mg−1 previously obtained. The N-terminal sequences of both enzymes are reported, and they are identical up to residue 16 at least. The overall yield of pure enzyme, starting with the cells grown in 15 L of medium, is 600–800 mg.

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Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach

Microbial Cell Factories ◽

10.1186/s12934-021-01615-1 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Zhenning Liu ◽

Xue Zhang ◽

Dengwei Lei ◽

Bin Qiao ◽

Guang-Rong Zhao

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

De Novo ◽

Microbial Cell ◽

Cell Factory ◽

Phosphopantetheinyl Transferase ◽

Chromosome Engineering ◽

Microbial Cell Factory ◽

E Coli ◽

Artificial Pathway

Abstract Background 3-Phenylpropanol with a pleasant odor is widely used in foods, beverages and cosmetics as a fragrance ingredient. It also acts as the precursor and reactant in pharmaceutical and chemical industries. Currently, petroleum-based manufacturing processes of 3-phenypropanol is environmentally unfriendly and unsustainable. In this study, we aim to engineer Escherichia coli as microbial cell factory for de novo production of 3-phenypropanol via retrobiosynthesis approach. Results Aided by in silico retrobiosynthesis analysis, we designed a novel 3-phenylpropanol biosynthetic pathway extending from l-phenylalanine and comprising the phenylalanine ammonia lyase (PAL), enoate reductase (ER), aryl carboxylic acid reductase (CAR) and phosphopantetheinyl transferase (PPTase). We screened the enzymes from plants and microorganisms and reconstructed the artificial pathway for conversion of 3-phenylpropanol from l-phenylalanine. Then we conducted chromosome engineering to increase the supply of precursor l-phenylalanine and combined the upstream l-phenylalanine pathway and downstream 3-phenylpropanol pathway. Finally, we regulated the metabolic pathway strength and optimized fermentation conditions. As a consequence, metabolically engineered E. coli strain produced 847.97 mg/L of 3-phenypropanol at 24 h using glucose-glycerol mixture as co-carbon source. Conclusions We successfully developed an artificial 3-phenylpropanol pathway based on retrobiosynthesis approach, and highest titer of 3-phenylpropanol was achieved in E. coli via systems metabolic engineering strategies including enzyme sources variety, chromosome engineering, metabolic strength balancing and fermentation optimization. This work provides an engineered strain with industrial potential for production of 3-phenylpropanol, and the strategies applied here could be practical for bioengineers to design and reconstruct the microbial cell factory for high valuable chemicals.

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Escherichia coli as a platform microbial host for systems metabolic engineering

Essays in Biochemistry ◽

10.1042/ebc20200172 ◽

2021 ◽

Author(s):

Dongsoo Yang ◽

Cindy Pricilia Surya Prabowo ◽

Hyunmin Eun ◽

Seon Young Park ◽

In Jin Cho ◽

...

Keyword(s):

Escherichia Coli ◽

Natural Products ◽

Metabolic Engineering ◽

Food Ingredients ◽

E Coli ◽

Renewable Biomass ◽

Microbial Cell Factories ◽

Cell Factories ◽

Systems Metabolic Engineering ◽

Microbial Host

Abstract Bio-based production of industrially important chemicals and materials from non-edible and renewable biomass has become increasingly important to resolve the urgent worldwide issues including climate change. Also, bio-based production, instead of chemical synthesis, of food ingredients and natural products has gained ever increasing interest for health benefits. Systems metabolic engineering allows more efficient development of microbial cell factories capable of sustainable, green, and human-friendly production of diverse chemicals and materials. Escherichia coli is unarguably the most widely employed host strain for the bio-based production of chemicals and materials. In the present paper, we review the tools and strategies employed for systems metabolic engineering of E. coli. Next, representative examples and strategies for the production of chemicals including biofuels, bulk and specialty chemicals, and natural products are discussed, followed by discussion on materials including polyhydroxyalkanoates (PHAs), proteins, and nanomaterials. Lastly, future perspectives and challenges remaining for systems metabolic engineering of E. coli are discussed.

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Increased Production of Recombinant O-Phospho-L-Serine Sulfhydrylase from the Hyperthermophilic Archaeon Aeropyrum pernix K1 Using Escherichia coli

Current Biotechnology ◽

10.2174/2211550108666190418125138 ◽

2019 ◽

Vol 8 (1) ◽

pp. 15-23

Author(s):

Takashi Nakamura ◽

Emi Takeda ◽

Tomoko Kiryu ◽

Kentaro Mori ◽

Miyu Ohori ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Industrial Production ◽

Codon Optimization ◽

Scale Production ◽

Enzymatic Production ◽

Hyperthermophilic Archaeon ◽

E Coli ◽

Aeropyrum Pernix ◽

Natural Amino Acids

Background: O-phospho-L-serine sulfhydrylase from the hyperthermophilic archaeon Aeropyrum pernix K1 (ApOPSS) is thermostable and tolerant to organic solvents. It can produce nonnatural amino acids in addition to L-cysteine. Objective: We aimed to obtain higher amounts of ApOPSS compared to those reported with previous methods for the convenience of research and for industrial production of L-cysteine and non-natural amino acids. Method: We performed codon optimization of cysO that encodes ApOPSS, for optimal expression in Escherichia coli. We then examined combinations of conditions such as the host strain, plasmid, culture medium, and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration to improve ApOPSS yield. Results and Discussion: E. coli strain Rosetta (DE3) harboring the expression plasmid pQE-80L with the codon-optimized cysO was cultured in Terrific broth with 0.01 mM IPTG at 37°C for 48 h to yield a 10-times higher amount of purified ApOPSS (650 mg·L-1) compared to that obtained by the conventional method (64 mg·L-1). We found that the optimal culture conditions along with codon optimization were essential for the increased ApOPSS production. The expressed ApOPSS had a 6-histidine tag at the N-terminal, which did not affect its activity. This method may facilitate the industrial production of cysteine and non-natural amino acids using ApOPSS. Conclusion: We conclude that these results could be used in applied research on enzymatic production of L-cysteine in E. coli, large scale production of non-natural amino acids, an enzymatic reaction in organic solvent, and environmental remediation by sulfur removal.

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Heterologous Production of 6-Deoxyerythronolide B in Escherichia coli through the Wood Werkman Cycle

Metabolites ◽

10.3390/metabo10060228 ◽

2020 ◽

Vol 10 (6) ◽

pp. 228

Author(s):

R. Axayacatl Gonzalez-Garcia ◽

Lars K. Nielsen ◽

Esteban Marcellin

Keyword(s):

Escherichia Coli ◽

Natural Products ◽

Carbon Source ◽

Sole Carbon Source ◽

Structural Diversity ◽

Heterologous Production ◽

E Coli ◽

Anticancer Properties ◽

Defined Media ◽

Extender Unit

Polyketides are a remarkable class of natural products with diverse functional and structural diversity. The class includes many medicinally important molecules with antiviral, antimicrobial, antifungal and anticancer properties. Native bacterial, fungal and plant hosts are often difficult to cultivate and coax into producing the desired product. As a result, Escherichia coli has been used for the heterologous production of polyketides, with the production of 6-deoxyerythronolide B (6-dEB) being the first example. Current strategies for production in E. coli require feeding of exogenous propionate as a source for the precursors propionyl-CoA and S-methylmalonyl-CoA. Here, we show that heterologous polyketide production is possible from glucose as the sole carbon source. The heterologous expression of eight genes from the Wood-Werkman cycle found in Propionibacteria, in combination with expression of the 6-dEB synthases DEBS1, DEBS2 and DEBS3 resulted in 6-dEB formation from glucose as the sole carbon source. Our results show that the Wood-Werkman cycle provides the required propionyl-CoA and the extender unit S-methylmalonyl-CoA to produce up to 0.81 mg/L of 6-dEB in a chemically defined media.

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