Metabolic Engineering of Escherichia Coli BL21 Strain Using Simplified CRISPR-Cas9 and Asymmetric Homolog Arms Recombineering

2021 ◽  
Author(s):  
Sudha Shukal ◽  
Xiao Hui Lim ◽  
Congqiang Zhang ◽  
Xixian Chen
Keyword(s):  
Pcr Cloning ◽  
Bacterial Genomes ◽  
E Coli ◽  
Synthetic Dna ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Lycopene Production ◽  
One Step ◽  
The Cost ◽  
Template Dna

Abstract BackgroundThe recent CRISPR-Cas coupled with λ recombinase mediated genome recombineering has become a common laboratory practice to modify bacterial genomes. It requires supplying a template DNA or homolog arms for precise genome editing. However, it is often overlooked the process to generate the homolog arms which is a time-consuming, costly and inefficient step.ResultsIn this study, we first optimized CRISPR-Cas protocol in BL21 strain and successfully deleted 10 kb gene from the genome in one round of editing. To further simplify the protocol, asymmetric homolog arms as PCR fragments was used. It can be obtained by one-step PCR reaction with two primers and purified with desalting columns. Unlike conventional homolog arms that are prepared through overlapping PCR, cloning to plasmid or annealing synthetic DNA fragments, our method significantly shortened the time taken and reduced the cost to prepare the homolog arms. To test the robustness of the optimized workflow, we successfully deleted 26 / 27 genes across BL21 genome. Noteworthy, gRNA design is important for CRISPR-Cas system and a general heuristic gRNA design was proposed in the study. To apply our established protocol, we targeted 16 genes and iteratively deleted 7 genes from BL21 genome. The resulting strain increased lycopene production from ~15,000 ppm to > 40,000 ppm.ConclusionsOur work has optimized the homolog arms design for gene deletion in BL21 strains. The protocol efficiently edited BL21 strain to improve lycopene production. The same workflow is applicable to all E. coli strain which would be useful for genome rewiring to further increase metabolite production in microbial cell factories.

scholarly journals Complete biosynthesis of a sulfated chondroitin in Escherichia coli

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Abinaya Badri ◽  
Asher Williams ◽  
Adeola Awofiranye ◽  
Payel Datta ◽  
Ke Xia ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Scale Up ◽  
Production Methods ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Microbial Product ◽  
Dry Cell Weight ◽  
One Step ◽  
Dry Cell

AbstractSulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues. Although such methods are unsustainable and prone to contamination, animal-free production methods have not emerged as competitive alternatives due to complexities in scale-up, requirement for multiple stages and cost of co-factors and purification. Here, we demonstrate the development of single microbial cell factories capable of complete, one-step biosynthesis of chondroitin sulfate (CS), a type of GAG. We engineer E. coli to produce all three required components for CS production–chondroitin, sulfate donor and sulfotransferase. In this way, we achieve intracellular CS production of ~27 μg/g dry-cell-weight with about 96% of the disaccharides sulfated. We further explore four different factors that can affect the sulfation levels of this microbial product. Overall, this is a demonstration of simple, one-step microbial production of a sulfated GAG and marks an important step in the animal-free production of these molecules.

scholarly journals Metabolic Engineering Escherichia coli for the Production of Lycopene

Molecules ◽  
2020 ◽  
Vol 25 (14) ◽  
pp. 3136 ◽  
Author(s):  
Zhaobao Wang ◽  
JingXin Sun ◽  
Qun Yang ◽  
Jianming Yang
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Regulatory Networks ◽  
Carbon Sources ◽  
Operation Technique ◽  
Mva Pathway ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Lycopene Production

Lycopene, a potent antioxidant, has been widely used in the fields of pharmaceuticals, nutraceuticals, and cosmetics. However, the production of lycopene extracted from natural sources is far from meeting the demand. Consequently, synthetic biology and metabolic engineering have been employed to develop microbial cell factories for lycopene production. Due to the advantages of rapid growth, complete genetic background, and a reliable genetic operation technique, Escherichia coli has become the preferred host cell for microbial biochemicals production. In this review, the recent advances in biological lycopene production using engineered E. coli strains are summarized: First, modification of the endogenous MEP pathway and introduction of the heterogeneous MVA pathway for lycopene production are outlined. Second, the common challenges and strategies for lycopene biosynthesis are also presented, such as the optimization of other metabolic pathways, modulation of regulatory networks, and optimization of auxiliary carbon sources and the fermentation process. Finally, the future prospects for the improvement of lycopene biosynthesis are also discussed.

Escherichia coli as a platform microbial host for systems metabolic engineering

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.

scholarly journals Light-powered Escherichia coli cell division for chemical production

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Qiang Ding ◽  
Danlei Ma ◽  
Gao-Qiang Liu ◽  
Yang Li ◽  
Liang Guo ◽  
...  
Keyword(s):  
Cell Division ◽  
Blue Light ◽  
Ribonucleotide Reductase ◽  
Near Infrared ◽  
Infrared Light ◽  
Light Activation ◽  
Chemical Production ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories

Abstract Cell division can perturb the metabolic performance of industrial microbes. The C period of cell division starts from the initiation to the termination of DNA replication, whereas the D period is the bacterial division process. Here, we first shorten the C and D periods of E. coli by controlling the expression of the ribonucleotide reductase NrdAB and division proteins FtsZA through blue light and near-infrared light activation, respectively. It increases the specific surface area to 3.7 μm−1 and acetoin titer to 67.2 g·L−1. Next, we prolong the C and D periods of E. coli by regulating the expression of the ribonucleotide reductase NrdA and division protein inhibitor SulA through blue light activation-repression and near-infrared (NIR) light activation, respectively. It improves the cell volume to 52.6 μm3 and poly(lactate-co-3-hydroxybutyrate) titer to 14.31 g·L−1. Thus, the optogenetic-based cell division regulation strategy can improve the efficiency of microbial cell factories.

scholarly journals Engineering Escherichia coli biofilm to increase contact surface for shikimate and L-malate production

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Qiang Ding ◽  
Yadi Liu ◽  
Guipeng Hu ◽  
Liang Guo ◽  
Cong Gao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Contact Surface ◽  
Self Assembly ◽  
Cds Nanoparticles ◽  
Cellular Functions ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Nanoparticles Concentration ◽  
Definition Of

AbstractMicrobial organelles are a promising model to promote cellular functions for the production of high-value chemicals. However, the concentrations of enzymes and nanoparticles are limited by the contact surface in single Escherichia coli cells. Herein, the definition of contact surface is to improve the amylase and CdS nanoparticles concentration for enhancing the substrate starch and cofactor NADH utilization. In this study, two biofilm-based strategies were developed to improve the contact surface for the production of shikimate and L-malate. First, the contact surface of E. coli was improved by amylase self-assembly with a blue light-inducible biofilm-based SpyTag/SpyCatcher system. This system increased the glucose concentration by 20.7% and the starch-based shikimate titer to 50.96 g L−1, which showed the highest titer with starch as substrate. Then, the contact surface of E. coli was improved using a biofilm-based CdS-biohybrid system by light-driven system, which improved the NADH concentration by 83.3% and increased the NADH-dependent L-malate titer to 45.93 g L−1. Thus, the biofilm-based strategies can regulate cellular functions to increase the efficiency of microbial cell factories based on the optogenetics, light-driven, and metabolic engineering. Graphical Abstract

scholarly journals De novo Biosynthesis of Tyrosol Acetate and Hydroxytyrosol Acetate from Glucose in Engineered Escherichia Coli

2021 ◽  
Author(s):  
Daoyi Guo ◽  
Xiao Fu ◽  
Yue Sun ◽  
Xun Li ◽  
Hong Pan
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Virgin Olive Oil ◽  
Carbon Sources ◽  
Acetate Production ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Alcohol Acetyltransferase ◽  
First Time

Abstract Background: Tyrosol and hydroxytyrosol derived from virgin olive oil and olives extract, have wide applications both as functional food components and as nutraceuticals. However, they have low bioavailability due to their low absorption and high metabolism in human liver and small intestine. Acetylation of tyrosol and hydroxytyrosol can effectively improve their bioavailability and thus increase their potential use in the food and cosmeceutical industries. There is no report on the bioproductin of tyrosol acetate and hydroxytyrosol acetate so far. Thus, it is of great significance to develop microbial cell factories for achieving tyrosol acetate or hydroxytyrosol acetate biosynthesis.Results: In this study, two de novo biosynthetic pathways for the production of tyrosol acetate and hydroxytyrosol acetate were constructed in Escherichia coli. First, an engineered E. coli that allows production of tyrosol from simple carbon sources was established. Four aldehyde reductases were compared, and it was found that yeaE is the best aldehyde reductase for tyrosol accumulation. Subsequently, the pathway was extended for tyrosol acetate production by further overexpression of alcohol acetyltransferase ATF1 for the conversion of tyrosol to tyrosol acetate. Finally, the pathway was further extended for hydroxytyrosol acetate production by overexpression of 4-hydroxyphenylacetate 3-hydroxylase HpaBC.Conclusion: We have successfully established the artificial biosynthetic pathway of tyrosol acetate and hydroxytyrosol acetate from fermentable sugars and demonstrated for the first time the direct fermentative production of tyrosol acetate and hydroxytyrosol acetate from glucose in engineered E. coli

scholarly journals Reprogramming microbial populations using a programmed lysis system to improve chemical production

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wenwen Diao ◽  
Liang Guo ◽  
Qiang Ding ◽  
Cong Gao ◽  
Guipeng Hu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Division Of Labor ◽  
Microbial Populations ◽  
Chemical Production ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Phenotypic Structure ◽  
Colicin M ◽  
Butyrate Production

AbstractMicrobial populations are a promising model for achieving microbial cooperation to produce valuable chemicals. However, regulating the phenotypic structure of microbial populations remains challenging. In this study, a programmed lysis system (PLS) is developed to reprogram microbial cooperation to enhance chemical production. First, a colicin M -based lysis unit is constructed to lyse Escherichia coli. Then, a programmed switch, based on proteases, is designed to regulate the effective lysis unit time. Next, a PLS is constructed for chemical production by combining the lysis unit with a programmed switch. As a result, poly (lactate-co-3-hydroxybutyrate) production is switched from PLH synthesis to PLH release, and the content of free PLH is increased by 283%. Furthermore, butyrate production with E. coli consortia is switched from E. coli BUT003 to E. coli BUT004, thereby increasing butyrate production to 41.61 g/L. These results indicate the applicability of engineered microbial populations for improving the metabolic division of labor to increase the efficiency of microbial cell factories.

scholarly journals Insoluble proteins catch heterologous soluble proteins into inclusion bodies by intermolecular interaction of aggregating peptides.

2020 ◽  
Author(s):  
Jose Vicente Carratalá ◽  
Andrés Cisneros ◽  
Elijah Hellman ◽  
Antonio Villaverde ◽  
Neus Ferrer-Miralles
Keyword(s):  
Protein Aggregation ◽  
Inclusion Bodies ◽  
Protein Quality ◽  
Prion Diseases ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Protein Molecules ◽  
Biological Event ◽  
Protein Quality Control System

Abstract Background: Protein aggregation is a biological event observed in expression systems in which the recombinant protein is produced under stressful conditions surpassing the homeostasis of the protein quality control system. In addition, protein aggregation is related to conformational diseases in animals as transmissible prion diseases, and non-transmissible neurodegenerative diseases including Alzheimer, Parkinson's disease, amyloidosis and multiple system atrophy among others. At the molecular level, the presence of aggregating-prone domains in protein molecules act as seeding igniters to induce the accumulation of protein molecules in protease-resistant clusters by intermolecular interactions.Results: In this work the aggregating-prone performance of a small peptide (L6K2) with additional antimicrobial activity was studied and the relevance of the accompanying scaffold protein to enhance the aggregating profile of the fusion protein has been elucidated. Furthermore, it was demonstrated that the fusion of L6K2 to highly soluble recombinant proteins directs the protein to inclusion bodies (IBs) in E. coli through stereospecific interactions in the presence of an insoluble protein displaying the same aggregating-prone peptide (APP). Conclusions: These data suggest that the molecular bases of protein aggregation are related not only to the presence of aggregation-prone stretches, but to the net balance of protein aggregation potential. and not only to the presence of aggregation-prone stretches. This is ultimately presented as a generic platform to generate hybrid protein aggregates in microbial cell factories for biopharmaceutical and biotechnological applications.

scholarly journals Title: insoluble proteins catch heterologous soluble proteins into inclusion bodies by intermolecular interaction of aggregating peptides

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Jose Vicente Carratalá ◽  
Andrés Cisneros ◽  
Elijah Hellman ◽  
Antonio Villaverde ◽  
Neus Ferrer-Miralles
Keyword(s):  
Protein Aggregation ◽  
Inclusion Bodies ◽  
Protein Quality ◽  
Prion Diseases ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Protein Molecules ◽  
Biological Event ◽  
Protein Quality Control System

Abstract Background Protein aggregation is a biological event observed in expression systems in which the recombinant protein is produced under stressful conditions surpassing the homeostasis of the protein quality control system. In addition, protein aggregation is also related to conformational diseases in animals as transmissible prion diseases or non-transmissible neurodegenerative diseases including Alzheimer, Parkinson’s disease, amyloidosis and multiple system atrophy among others. At the molecular level, the presence of aggregation-prone domains in protein molecules act as seeding igniters to induce the accumulation of protein molecules in protease-resistant clusters by intermolecular interactions. Results In this work we have studied the aggregating-prone performance of a small peptide (L6K2) with additional antimicrobial activity and we have elucidated the relevance of the accompanying scaffold protein to enhance the aggregating profile of the fusion protein. Furthermore, we demonstrated that the fusion of L6K2 to highly soluble recombinant proteins directs the protein to inclusion bodies (IBs) in E. coli through stereospecific interactions in the presence of an insoluble protein displaying the same aggregating-prone peptide (APP). Conclusions These data suggest that the molecular bases of protein aggregation are related to the net balance of protein aggregation potential and not only to the presence of APPs. This is then presented as a generic platform to generate hybrid protein aggregates in microbial cell factories for biopharmaceutical and biotechnological applications.

scholarly journals Insoluble proteins catch heterologous soluble proteins into inclusion bodies by intermolecular interaction of aggregating peptides

2020 ◽  
Author(s):  
Jose Vicente Carratalá ◽  
Andrés Cisneros ◽  
Elijah Hellman ◽  
Antonio Villaverde ◽  
Neus Ferrer-Miralles
Keyword(s):  
Protein Aggregation ◽  
Inclusion Bodies ◽  
Protein Quality ◽  
Prion Diseases ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Protein Molecules ◽  
Biological Event ◽  
Protein Quality Control System

Abstract Background Protein aggregation is a biological event observed in expression systems in which the recombinant protein is produced under stressful conditions surpassing the homeostasis of the protein quality control system. In addition, protein aggregation is also related to conformational diseases in animals as transmissible prion diseases or non-transmissible neurodegenerative diseases including Alzheimer, Parkinson's disease, amyloidosis and multiple system atrophy among others. At the molecular level, the presence of aggregating-prone domains in protein molecules act as seeding igniters to induce the accumulation of protein molecules in protease-resistant clusters by intermolecular interactions. Results In this work we have studied the aggregating-prone performance of a small peptide (L6K2) with additional antimicrobial activity and we have elucidated the relevance of the accompanying scaffold protein to enhance the aggregating profile of the fusion protein. Furthermore, we demonstrated that the fusion of L6K2 to highly soluble recombinant proteins directs the protein to inclusion bodies (IB) in E. coli through stereospecific interactions in the presence of an insoluble protein displaying the same aggregating-prone peptide (APP). Conclusions These data suggest that the molecular bases of protein aggregation are related to the net balance of protein aggregation potential and not only to the presence of aggregation-prone stretches. This is then presented as a generic platform to generate hybrid protein aggregates in microbial cell factories for biopharmaceutical and biotechnological applications.

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