scholarly journals Efficient Bioconversion of Raspberry Ketone in Escherichia Coli Using Fatty Acids Feedstocks

2020 ◽  
Author(s):  
Chen Chang ◽  
Bo Liu ◽  
Yihong Bao ◽  
Yong Tao ◽  
Weifeng Liu
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
High Titer ◽  
Low Cost ◽  
Fine Tuning ◽  
Promising Alternative ◽  
Raspberry Ketone ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Coa Ligase

Abstract BackgroundPhenylpropanoid including raspberry ketone, is a kind of important natural plant product and widely used in pharmaceuticals, chemicals, cosmetics, and healthcare products. Bioproduction of phenylpropanoid in Escherichia coli and other microbial cell factories is an attractable approach considering the low phenylpropanoid contents in plants. However, it is usually difficult to produce high titer phenylpropanoid production when fermentation using glucose as carbon source. Developing novel bioprocess using alternative sources might provide a solution to this problem. In this study, typical phenylpropanoid raspberry ketone was used as the target product to develop a biosynthesis pathway for phenylpropanoid production from fatty acids, a promising alternative low-cost feedstock. ResultsA raspberry ketone biosynthesis module was developed and optimized by introducing 4-coumarate-CoA ligase (4CL), benzalacetone synthase (BAS), and raspberry ketone reductase (RZS) in Escherichia coli strains CR1-CR4. Then strain CR5 was developed by introducing raspberry ketone biosynthesis module into a fatty acids-utilization chassis FA09 to achieve production of raspberry ketone from fatty acids feedstock. However, the production of raspberry ketone was still limited by the low biomass and unable to substantiate whole-cell bioconversion process. Thus, a process by coordinately using fatty-acids and glycerol was developed. In addition, we systematically screened and optimized fatty acids-response promoters. The optimized promoter Pfrd3 was then successfully used for the efficient expression of key enzymes of raspberry ketone biosynthesis module during bioconversion from fatty acids. The final engineered strain CR8 could efficiently produce raspberry ketone repeatedly using bioconversion from fatty acids feedstock strategy, and about 291.3 mg/L raspberry ketone was produced.ConclusionMetabolically engineered Escherichia coli strains were successfully developed for raspberry ketone production from fatty acids using several strategies, including optimization of bioconversion process and fine-tuning key enzyme expression. This study provides an essential reference to establish the low-cost biological manufacture of phenylpropanoids compounds.

scholarly journals Efficient bioconversion of raspberry ketone in Escherichia coli using fatty acids feedstocks

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Chen Chang ◽  
Bo Liu ◽  
Yihong Bao ◽  
Yong Tao ◽  
Weifeng Liu
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
High Titer ◽  
Low Cost ◽  
Fine Tuning ◽  
Promising Alternative ◽  
Raspberry Ketone ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Coa Ligase

Abstract Background Phenylpropanoid including raspberry ketone, is a kind of important natural plant product and widely used in pharmaceuticals, chemicals, cosmetics, and healthcare products. Bioproduction of phenylpropanoid in Escherichia coli and other microbial cell factories is an attractive approach considering the low phenylpropanoid contents in plants. However, it is usually difficult to produce high titer phenylpropanoid production when fermentation using glucose as carbon source. Developing novel bioprocess using alternative sources might provide a solution to this problem. In this study, typical phenylpropanoid raspberry ketone was used as the target product to develop a biosynthesis pathway for phenylpropanoid production from fatty acids, a promising alternative low-cost feedstock. Results A raspberry ketone biosynthesis module was developed and optimized by introducing 4-coumarate-CoA ligase (4CL), benzalacetone synthase (BAS), and raspberry ketone reductase (RZS) in Escherichia coli strains CR1–CR4. Then strain CR5 was developed by introducing raspberry ketone biosynthesis module into a fatty acids-utilization chassis FA09 to achieve production of raspberry ketone from fatty acids feedstock. However, the production of raspberry ketone was still limited by the low biomass and unable to substantiate whole-cell bioconversion process. Thus, a process by coordinately using fatty-acids and glycerol was developed. In addition, we systematically screened and optimized fatty acids-response promoters. The optimized promoter Pfrd3 was then successfully used for the efficient expression of key enzymes of raspberry ketone biosynthesis module during bioconversion from fatty acids. The final engineered strain CR8 could efficiently produce raspberry ketone repeatedly using bioconversion from fatty acids feedstock strategy, and was able to produce raspberry ketone to a concentration of 180.94 mg/L from soybean oil in a 1-L fermentation process. Conclusion Metabolically engineered Escherichia coli strains were successfully developed for raspberry ketone production from fatty acids using several strategies, including optimization of bioconversion process and fine-tuning key enzyme expression. This study provides an essential reference to establish the low-cost biological manufacture of phenylpropanoids compounds.

scholarly journals Genome-scale target identification in Escherichia coli for high-titer production of free fatty acids

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lixia Fang ◽  
Jie Fan ◽  
Shulei Luo ◽  
Yaru Chen ◽  
Congya Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Free Fatty Acids ◽  
Stress Responses ◽  
Metabolic Networks ◽  
Target Identification ◽  
High Titer ◽  
Cell Factory ◽  
Microbial Cell Factory ◽  
Ffa Metabolism

AbstractTo construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cellular potential by identifying and engineering beneficial gene targets in sophisticated metabolic networks. Here, we take advantage of CRISPR interference (CRISPRi) and omics analyses to systematically identify beneficial genes that can be engineered to promote free fatty acids (FFAs) production in Escherichia coli. CRISPRi-mediated genetic perturbation enables the identification of 30 beneficial genes from 108 targets related to FFA metabolism. Then, omics analyses of the FFAs-overproducing strains and a control strain enable the identification of another 26 beneficial genes that are seemingly irrelevant to FFA metabolism. Combinatorial perturbation of four beneficial genes involving cellular stress responses results in a recombinant strain ihfAL−-aidB+-ryfAM−-gadAH−, producing 30.0 g L−1 FFAs in fed-batch fermentation, the maximum titer in E. coli reported to date. Our findings are of help in rewiring cellular metabolism and interwoven intracellular processes to facilitate high-titer production of biochemicals.

scholarly journals Redirection of lipid flux toward phospholipids in yeast increases fatty acid turnover and secretion

2018 ◽  
Vol 115 (6) ◽  
pp. 1262-1267 ◽  
Author(s):  
Raphael Ferreira ◽  
Paulo Gonçalves Teixeira ◽  
Verena Siewers ◽  
Jens Nielsen
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Lipid Metabolism ◽  
Metabolic Network ◽  
Feedback Regulation ◽  
Acid Activation ◽  
Phospholipid Hydrolysis ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
High Level

Bio-based production of fatty acids and fatty acid-derived products can enable sustainable substitution of petroleum-derived fuels and chemicals. However, developing new microbial cell factories for producing high levels of fatty acids requires extensive engineering of lipid metabolism, a complex and tightly regulated metabolic network. Here we generated a Saccharomyces cerevisiae platform strain with a simplified lipid metabolism network with high-level production of free fatty acids (FFAs) due to redirected fatty acid metabolism and reduced feedback regulation. Deletion of the main fatty acid activation genes (the first step in β-oxidation), main storage lipid formation genes, and phosphatidate phosphatase genes resulted in a constrained lipid metabolic network in which fatty acid flux was directed to a large extent toward phospholipids. This resulted in simultaneous increases of phospholipids by up to 2.8-fold and of FFAs by up to 40-fold compared with wild-type levels. Further deletion of phospholipase genes PLB1 and PLB2 resulted in a 46% decrease in FFA levels and 105% increase in phospholipid levels, suggesting that phospholipid hydrolysis plays an important role in FFA production when phospholipid levels are increased. The multiple deletion mutant generated allowed for a study of fatty acid dynamics in lipid metabolism and represents a platform strain with interesting properties that provide insight into the future development of lipid-related cell factories.

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 Reverse engineering of fatty acid-tolerant Escherichia coli identifies design strategies for robust microbial cell factories

2020 ◽  
Vol 61 ◽  
pp. 120-130 ◽  
Author(s):  
Yingxi Chen ◽  
Erin E. Boggess ◽  
Efrain Rodriguez Ocasio ◽  
Aric Warner ◽  
Lucas Kerns ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Reverse Engineering ◽  
Microbial Cell ◽  
Design Strategies ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Acid Tolerant

scholarly journals Genome-wide targets identification by CRISPRi-Omics for high-titer production of free fatty acids in Escherichia coli

2020 ◽  
Author(s):  
Lixia Fang ◽  
Jie Fan ◽  
Congya Wang ◽  
Yingxiu Cao ◽  
Hao Song
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Free Fatty Acids ◽  
Metabolic Networks ◽  
High Titer ◽  
Cell Structure ◽  
Cell Factory ◽  
Microbial Cell Factory ◽  
Cell Potential ◽  
E Coli

AbstractTo construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cell potential via identifying and engineering beneficial gene targets in the sophisticated metabolic networks. Here, we develop an approach that integrates CRISPR interference (CRISPRi) to readily modulate genes expression and omics analyses to identify potential targets in multiple cellular processes, enabling systematical discovery of beneficial chromosomal gene targets that can be engineered to optimize free fatty acids (FFAs) production in Escherichia coli. We identify 56 beneficial genes via synergistic CRISPRi-Omics strategy, including 46 novel targets functioning in cell structure and division, and signaling transduction that efficiently facilitate FFAs production. Upon repressing ihfA and overexpressing aidB and tesA’ in E. coli, the recombinant strain LihfA-OaidB results in a FFAs titer of 21.6 g L-1 in fed-batch fermentation, which, to our best knowledge, is the maximum FFAs titer by the recombinant E. coli reported to date.

scholarly journals The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters

1998 ◽  
Vol 64 (1) ◽  
pp. 82-87 ◽  
Author(s):  
Peter Ruhdal Jensen ◽  
Karin Hammer
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Dna Sequencing ◽  
Lactococcus Lactis ◽  
Entire Range ◽  
Fine Tuning ◽  
Activity Increase ◽  
Consensus Sequences ◽  
Synthetic Promoters ◽  
Cell Factories

ABSTRACT We constructed a library of synthetic promoters forLactococcus lactis in which the known consensus sequences were kept constant while the sequences of the separating spacers were randomized. The library consists of 38 promoters which differ in strength from 0.3 up to more than 2,000 relative units, the latter among the strongest promoters known for this organism. The ranking of the promoter activities was somewhat different when assayed inEscherichia coli, but the promoters are efficient for modulating gene expression in this bacterium as well. DNA sequencing revealed that the weaker promoters (which had activities below 5 relative units) all had changes either in the consensus sequences or in the length of the spacer between the −35 and −10 sequences. The promoters in which those features were conserved had activities from 5 to 2,050 U, which shows that by randomizing the spacers, at least a 400-fold change in activity can be obtained. Interestingly, the entire range of promoter activities is covered in small steps of activity increase, which makes these promoters very suitable for quantitative physiological studies and for fine-tuning of gene expression in industrial bioreactors and cell factories.

scholarly journals ESCHERICHIA COLI REDOX MUTANTS AS MICROBIAL CELL FACTORIES FOR THE SYNTHESIS OF REDUCED BIOCHEMICALS

2012 ◽  
Vol 3 (4) ◽  
pp. e201210019 ◽  
Author(s):  
Jimena A. Ruiz ◽  
Alejandra de Almeida ◽  
Manuel S. Godoy ◽  
Mariela P. Mezzina ◽  
Gonzalo N. Bidart ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Microbial Cell ◽  
Microbial Cell Factories ◽  
Cell Factories

scholarly journals Conversion of no/low value waste frying oils into biodiesel and polyhydroxyalkanoates

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marco Vastano ◽  
Iolanda Corrado ◽  
Giovanni Sannia ◽  
Daniel K. Y. Solaiman ◽  
Cinzia Pezzella
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Process Optimization ◽  
Biodiesel Production ◽  
Microbial Fermentation ◽  
Frying Oils ◽  
Medium Chain Length ◽  
Cell Factories ◽  
Waste Oils ◽  
Waste Frying Oils

Abstract A sustainable bioprocess was developed for the valorization of a no/low value substrate, i.e. waste frying oils (WFOs) with high content of free fatty acids (FFAs), otherwise unsuitable for biodiesel production. The bioprocess was verified using both recombinant (Escherichia coli) and native (Pseudomonas resinovorans) polyhydroxyalkanoates (PHAs) producing cell factories. Microbial fermentation of WFOs provided a 2-fold advantage: i) the reduction of FFAs content resulting into an upgrading of the “exhausted waste oils” and ii) the production of a bio-based microbial polymer. Proper strain designing and process optimization allowed to achieve up to 1.5 g L−1 of medium chain length, mcl-PHAs, together with an efficient conversion (80% yield) of the treated WFO into biodiesel.

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

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