Metabolic Engineering Escherichia coli for the Production of Lycopene

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.

Download Full-text

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.

Download Full-text

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

10.21203/rs.3.rs-287007/v1 ◽

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

Download Full-text

In Silico Design for Systems-Based Metabolic Engineering In Escherichia Coli for The Bioconversion of Aerobic Glycerol to Succinic Acid

10.21203/rs.3.rs-113854/v1 ◽

2020 ◽

Author(s):

Albert Enrique Tafur Rangel ◽

Wendy Lorena Rios Guzman ◽

Carmen Elvira Ojeda Cuella ◽

Daissy Esther Mejia Perez ◽

Ross Carlson ◽

...

Keyword(s):

Machine Learning ◽

Escherichia Coli ◽

Metabolic Engineering ◽

Succinic Acid ◽

In Silico ◽

Rational Design ◽

Industrial Biotechnology ◽

E Coli ◽

Cell Factories ◽

Transcriptomics Data

Abstract BackgroundGlycerol has become an interesting carbon source for industrial processes as consequence of the biodiesel business growth since it has shown promising results in terms of biomass/substrate yields. Selecting the appropriate metabolic targets to build efficient cell factories and maximize the desired chemical production in as little time as possible is a major challenge in industrial biotechnology. The engineering of microbial metabolism following rational design has been widely studied. However, it is a cost-, time-, and laborious-intensive process because of the cell network complexity; thus, to be proficient is needed known in advance the effects of gene deletions.ResultsAn in silico experiment was performed to model and understand the effects of metabolic engineering over the metabolism by transcriptomics data integration. In this study, systems-based metabolic engineering to predict the metabolic engineering targets was used in order to increase the bioconversion of glycerol to succinic acid by Escherichia coli. Transcriptomics analysis suggest insights of how increase the glycerol utilization of the cell for further design efficient cell factories. Three models were used; an E. coli core model, a model obtained after the integration of transcriptomics data obtained from E. coli growing in an optimized culture media, and a third one obtained after integration of transcriptomics data obtained from E. coli after adaptive laboratory evolution experiments. A total of 2402 strains were obtained from these three models. Fumarase and pyruvate dehydrogenase were frequently predicted in all the models, suggesting that these reactions are essential to increasing succinic acid production from glycerol. Finally, using flux balance analysis results for all the mutants predicted, a machine learning method was developed to predict new mutants as well as to propose optimal metabolic engineering targets and mutants based on the measurement of importance of each knockout’s (feature’s) contribution.ConclusionsThe combination of transcriptome, systems metabolic modeling, and machine learning analyses revealed versatile molecular mechanisms involved in the utilization of glycerol. These data provide a platform to improve the prediction of metabolic engineering targets to design efficient cell factories. Our results may also work a guide platform for the selection/engineering of microorganisms for production of interesting chemical compounds.

Download Full-text

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

Bioresources and Bioprocessing ◽

10.1186/s40643-021-00470-7 ◽

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

Download Full-text

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

Nature Communications ◽

10.1038/s41467-021-27226-3 ◽

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.

Download Full-text

Enhanced lycopene Production in Escherichia coli by Expression of Two MEP Pathway Enzymes from Vibrio sp. dhg

10.20944/preprints201910.0333.v1 ◽

2019 ◽

Author(s):

Min Jae Kim ◽

Myung Hyun Noh ◽

Sunghwa Woo ◽

Hyun Gyu Lim ◽

Gyoo Yeol Jung

Keyword(s):

Escherichia Coli ◽

Electron Transport Chain ◽

Farnesyl Diphosphate ◽

Mep Pathway ◽

Farnesyl Diphosphate Synthase ◽

Mva Pathway ◽

E Coli ◽

Transport Chain ◽

Lycopene Production ◽

Specific Activities

Microbial production is a promising method that can overcome major limitations in conventional methods of lycopene production, such as low yields and variations in product quality. Significant efforts have been made to improve lycopene production by engineering either the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway or mevalonate (MVA) pathway in microorganisms. To further improve lycopene production, it is critical to utilize metabolic enzymes with high specific activities. Two enzymes, 1-deoxy-D-xylulose-5-phosphate synthase (Dxs) and farnesyl diphosphate synthase (IspA), are required in lycopene production using MEP pathway. Here, we evaluated the activities of Dxs and IspA of Vibrio sp. dhg, a newly isolated and fast-growing microorganism. Considering that the MEP pathway is closely related to the cell membrane and electron transport chain, the activities of the two enzymes of Vibrio sp. dhg were expected to be higher than the enzymes of E. coli. We found that Dxs and IspA in Vibrio sp. dhg exhibited 1.08-fold and 1.38-fold higher catalytic efficiencies, respectively. Consequently, the heterologous overexpression improved the specific lycopene production by 1.88-fold. Our findings could be widely utilized to enhance production of lycopene and other carotenoids.

Download Full-text

Enhancement of β-Alanine Biosynthesis in Escherichia coli Based on Multivariate Modular Metabolic Engineering

Biology ◽

10.3390/biology10101017 ◽

2021 ◽

Vol 10 (10) ◽

pp. 1017

Author(s):

Jian Xu ◽

Li Zhou ◽

Zhemin Zhou

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

Industrial Production ◽

Metabolic Flux ◽

Coli Strain ◽

Biosynthesis Pathway ◽

Fed Batch ◽

Microbial Cell Factories ◽

Cell Factories ◽

Fed Batch Fermentation

β-alanine is widely used as an intermediate in industrial production. However, the low production of microbial cell factories limits its further application. Here, to improve the biosynthesis production of β-alanine in Escherichia coli, multivariate modular metabolic engineering was recruited to manipulate the β-alanine biosynthesis pathway through keeping the balance of metabolic flux among the whole metabolic network. The β-alanine biosynthesis pathway was separated into three modules: the β-alanine biosynthesis module, TCA module, and glycolysis module. Global regulation was performed throughout the entire β-alanine biosynthesis pathway rationally and systematically by optimizing metabolic flux, overcoming metabolic bottlenecks and weakening branch pathways. As a result, metabolic flux was channeled in the direction of β-alanine biosynthesis without huge metabolic burden, and 37.9 g/L β-alanine was generated by engineered Escherichia coli strain B0016-07 in fed-batch fermentation. This study was meaningful to the synthetic biology of β-alanine industrial production.

Download Full-text

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

10.21203/rs.3.rs-889623/v1 ◽

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.

Download Full-text

Metabolic Engineering of a Novel Muconic Acid Biosynthesis Pathway via 4-Hydroxybenzoic Acid in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01386-15 ◽

2015 ◽

Vol 81 (23) ◽

pp. 8037-8043 ◽

Cited By ~ 37

Author(s):

Sudeshna Sengupta ◽

Sudhakar Jonnalagadda ◽

Lakshani Goonewardena ◽

Veeresh Juturu

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

Carbon Sources ◽

Raw Material ◽

Hydroxybenzoic Acid ◽

Muconic Acid ◽

Content Type ◽

E Coli ◽

Catechol 1,2 Dioxygenase ◽

K 12

ABSTRACTcis,cis-Muconic acid (MA) is a commercially important raw material used in pharmaceuticals, functional resins, and agrochemicals. MA is also a potential platform chemical for the production of adipic acid (AA), terephthalic acid, caprolactam, and 1,6-hexanediol. A strain ofEscherichia coliK-12, BW25113, was genetically modified, and a novel nonnative metabolic pathway was introduced for the synthesis of MA from glucose. The proposed pathway converted chorismate from the aromatic amino acid pathway to MA via 4-hydroxybenzoic acid (PHB). Three nonnative genes,pobA,aroY, andcatA, coding for 4-hydroxybenzoate hydrolyase, protocatechuate decarboxylase, and catechol 1,2-dioxygenase, respectively, were functionally expressed inE. colito establish the MA biosynthetic pathway.E. colinative genesubiC,aroFFBR,aroE, andaroLwere overexpressed and the genesptsH,ptsI,crr, andpykFwere deleted from theE. coligenome in order to increase the precursors of the proposed MA pathway. The final engineeredE. colistrain produced nearly 170 mg/liter of MA from simple carbon sources in shake flask experiments. The proposed pathway was proved to be functionally active, and the strategy can be used for future metabolic engineering efforts for production of MA from renewable sugars.

Download Full-text

Complete biosynthesis of a sulfated chondroitin in Escherichia coli

Nature Communications ◽

10.1038/s41467-021-21692-5 ◽

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.

Download Full-text