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

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.

scholarly journals Membrane Stresses Induced by Overproduction of Free Fatty Acids in Escherichia coli

2011 ◽  
Vol 77 (22) ◽  
pp. 8114-8128 ◽  
Author(s):  
Rebecca M. Lennen ◽  
Max A. Kruziki ◽  
Kritika Kumar ◽  
Robert A. Zinkel ◽  
Kristin E. Burnum ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Free Fatty Acids ◽  
Unsaturated Fatty Acid ◽  
High Energy ◽  
High Energy Density ◽  
Content Type ◽  
E Coli ◽  
Expression Of Genes

ABSTRACTMicrobially produced fatty acids are potential precursors to high-energy-density biofuels, including alkanes and alkyl ethyl esters, by either catalytic conversion of free fatty acids (FFAs) or enzymatic conversion of acyl-acyl carrier protein or acyl-coenzyme A intermediates. Metabolic engineering efforts aimed at overproducing FFAs inEscherichia colihave achieved less than 30% of the maximum theoretical yield on the supplied carbon source. In this work, the viability, morphology, transcript levels, and protein levels of a strain ofE. colithat overproduces medium-chain-length FFAs was compared to an engineered control strain. By early stationary phase, an 85% reduction in viable cell counts and exacerbated loss of inner membrane integrity were observed in the FFA-overproducing strain. These effects were enhanced in strains endogenously producing FFAs compared to strains exposed to exogenously fed FFAs. Under two sets of cultivation conditions, long-chain unsaturated fatty acid content greatly increased, and the expression of genes and proteins required for unsaturated fatty acid biosynthesis were significantly decreased. Membrane stresses were further implicated by increased expression of genes and proteins of the phage shock response, the MarA/Rob/SoxS regulon, and thenuoandcyooperons of aerobic respiration. Gene deletion studies confirmed the importance of the phage shock proteins and Rob for maintaining cell viability; however, little to no change in FFA titer was observed after 24 h of cultivation. The results of this study serve as a baseline for future targeted attempts to improve FFA yields and titers inE. coli.

scholarly journals Metabolic engineering Escherichia coli for efficient production of icariside D2

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Xue Liu ◽  
Lingling Li ◽  
Jincong Liu ◽  
Jianjun Qiao ◽  
Guang-Rong Zhao
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Carbon Sources ◽  
Gene Expression Pattern ◽  
Fine Tuning ◽  
Cell Factory ◽  
Efficient Production ◽  
Uridine Diphosphate ◽  
Microbial Cell Factory ◽  
E Coli

Abstract Background Icariside D2 is a plant-derived natural glycoside with pharmacological activities of inhibiting angiotensin-converting enzyme and killing leukemia cancer cells. Production of icariside D2 by plant extraction and chemical synthesis is inefficient and environmentally unfriendly. Microbial cell factory offers an attractive route for economical production of icariside D2 from renewable and sustainable bioresources. Results We metabolically constructed the biosynthetic pathway of icariside D2 in engineered Escherichia coli. We screened the uridine diphosphate glycosyltransferases (UGTs) and obtained an active RrUGT3 that regio-specifically glycosylated tyrosol at phenolic position to exclusively synthesize icariside D2. We put heterologous genes in E. coli cell for the de novo biosynthesis of icariside D2. By fine-tuning promoter and copy number as well as balancing gene expression pattern to decrease metabolic burden, the BMD10 monoculture was constructed. Parallelly, for balancing pathway strength, we established the BMT23–BMD12 coculture by distributing the icariside D2 biosynthetic genes to two E. coli strains BMT23 and BMD12, responsible for biosynthesis of tyrosol from preferential xylose and icariside D2 from glucose, respectively. Under the optimal conditions in fed-batch shake-flask fermentation, the BMD10 monoculture produced 3.80 g/L of icariside D2 using glucose as sole carbon source, and the BMT23–BMD12 coculture produced 2.92 g/L of icariside D2 using glucose–xylose mixture. Conclusions We for the first time reported the engineered E. coli for the de novo efficient production of icariside D2 with gram titer. It would be potent and sustainable approach for microbial production of icariside D2 from renewable carbon sources. E. coli–E. coli coculture approach is not limited to glycoside production, but could also be applied to other bioproducts.

Immunogenicity Evaluation of Chimeric Subunit Vaccine Comprising Adhesion Coli Surface Antigens from Enterotoxigenic Escherichia coli

2019 ◽  
Vol 29 (1-6) ◽  
pp. 91-100
Author(s):  
Dorna Khoobbakht ◽  
Shohreh Zare Karizi ◽  
Mohammad Javad  Motamedi ◽  
Rouhollah Kazemi ◽  
Pooneh Roghanian ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Subunit Vaccine ◽  
Fusion Gene ◽  
Intestinal Epithelial Cells ◽  
High Titer ◽  
Chimeric Protein ◽  
Amino Acid Sequences ◽  
Intestinal Epithelial ◽  
E Coli

Enterotoxigenic <i>Escherichia coli</i> (ETEC) is the most common agent of diarrhea morbidity in developing countries. ETEC adheres to host intestinal epithelial cells via various colonization factors. The CooD and CotD proteins play a significant role in bacteria binding to the intestinal epithelial cells as adhesin tip subunits of CS1 and CS2 pili. The purpose here was to design a new construction containing <i>cooD</i> and <i>cotD</i> genes and use several types of bioinformatics software to predict the structural and immunological properties of the designed antigen. The fusion gene was synthesized with codon bias of <i>E. coli</i> in order to increase the expression level of the protein. The amino acid sequences, protein structure, and immunogenicity properties of potential antigens were analyzed in silico. The chimeric protein was expressed in <i>E. coli</i>BL21 (DE3). The antigenicity of the recombinant proteins was verified by Western blotting and ELISA. In order to assess the induced immunity, the immunized mice were challenged with wild-type ETEC by an intraperitoneal route. Immunological analyses showed the production of a high titer of IgG serum with no sign of serum-mucosal IgA antibody response. The result of the challenge assay showed that 30% of immunized mice survived. The results of this study showed that CooD-CotD recombinant protein can stimulate immunity against ETEC. The designed chimera could be a prototype for the subunit vaccine, which is worthy of further consideration.

scholarly journals Multi-Path Optimization for Efficient Production of 2′-Fucosyllactose in an Engineered Escherichia coli C41 (DE3) Derivative

2020 ◽  
Vol 8 ◽  
Author(s):  
Zhijian Ni ◽  
Zhongkui Li ◽  
Jinyong Wu ◽  
Yuanfei Ge ◽  
Yingxue Liao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Batch Fermentation ◽  
Infant Nutrition ◽  
High Titer ◽  
Production Performance ◽  
Cell Factory ◽  
Efficient Production ◽  
Healthy Development ◽  
Fucosyltransferase Gene ◽  
Fed Batch Fermentation

2′-fucosyllactose (2′-FL), one of the simplest but most abundant oligosaccharides in human milk, has been demonstrated to have many positive benefits for the healthy development of newborns. However, the high-cost production and limited availability restrict its widespread use in infant nutrition and further research on its potential functions. In this study, on the basis of previous achievements, we developed a powerful cell factory by using a lacZ-mutant Escherichia coli C41 (DE3)ΔZ to ulteriorly increase 2′-FL production by feeding inexpensive glycerol. Initially, we co-expressed the genes for GDP-L-fucose biosynthesis and heterologous α-1,2-fucosyltransferase in C41(DE3)ΔZ through different plasmid-based expression combinations, functionally constructing a preferred route for 2′-FL biosynthesis. To further boost the carbon flux from GDP-L-fucose toward 2′-FL synthesis, deletion of chromosomal genes (wcaJ, nudD, and nudK) involved in the degradation of the precursors GDP-L-fucose and GDP-mannose were performed. Notably, the co-introduction of two heterologous positive regulators, RcsA and RcsB, was confirmed to be more conducive to GDP-L-fucose formation and thus 2′-FL production. Further a genomic integration of an individual copy of α-1,2-fucosyltransferase gene, as well as the preliminary optimization of fermentation conditions enabled the resulting engineered strain to achieve a high titer and yield. By collectively taking into account the intracellular lactose utilization, GDP-L-fucose availability, and fucosylation activity for 2′-FL production, ultimately a highest titer of 2′-FL in our optimized conditions reached 6.86 g/L with a yield of 0.92 mol/mol from lactose in the batch fermentation. Moreover, the feasibility of mass production was demonstrated in a 50-L fed-batch fermentation system in which a maximum titer of 66.80 g/L 2′-FL was achieved with a yield of 0.89 mol 2′-FL/mol lactose and a productivity of approximately 0.95 g/L/h 2′-FL. As a proof of concept, our preliminary 2′-FL production demonstrated a superior production performance, which will provide a promising candidate process for further industrial production.

scholarly journals Engineering Escherichia coli FAB system using synthetic plant genes for the production of long chain fatty acids

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Elias Kassab ◽  
Monika Fuchs ◽  
Martina Haack ◽  
Norbert Mehlmer ◽  
Thomas B. Brueck
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Production System ◽  
Biosynthetic Pathway ◽  
Long Chain Fatty Acids ◽  
E Coli ◽  
Fatty Acid Biosynthetic Pathway ◽  
Long Chain ◽  
Chain Fatty Acids

Abstract Background Sustainable production of microbial fatty acids derivatives has the potential to replace petroleum based equivalents in the chemical, cosmetic and pharmaceutical industry. Most fatty acid sources for production oleochemicals are currently plant derived. However, utilization of these crops are associated with land use change and food competition. Microbial oils could be an alternative source of fatty acids, which circumvents the issue with agricultural competition. Results In this study, we generated a chimeric microbial production system that features aspects of both prokaryotic and eukaryotic fatty acid biosynthetic pathways targeted towards the generation of long chain fatty acids. We redirected the type-II fatty acid biosynthetic pathway of Escherichia coli BL21 (DE3) strain by incorporating two homologues of the beta-ketoacyl-[acyl carrier protein] synthase I and II from the chloroplastic fatty acid biosynthetic pathway of Arabidopsis thaliana. The microbial clones harboring the heterologous pathway yielded 292 mg/g and 220 mg/g DCW for KAS I and KAS II harboring plasmids respectively. Surprisingly, beta-ketoacyl synthases KASI/II isolated from A. thaliana showed compatibility with the FAB pathway in E. coli. Conclusion The efficiency of the heterologous plant enzymes supersedes the overexpression of the native enzyme in the E. coli production system, which leads to cell death in fabF overexpression and fabB deletion mutants. The utilization of our plasmid based system would allow generation of plant like fatty acids in E. coli and their subsequent chemical or enzymatic conversion to high end oleochemical products.

Overproduction of free fatty acids in E. coli: Implications for biodiesel production

2008 ◽  
Vol 10 (6) ◽  
pp. 333-339 ◽  
Author(s):  
Xuefeng Lu ◽  
Harmit Vora ◽  
Chaitan Khosla
Keyword(s):  
Fatty Acids ◽  
Free Fatty Acids ◽  
Biodiesel Production ◽  
E Coli

scholarly journals An Unexpected Route to an Essential Cofactor:Escherichia coliRelies on Threonine for Thiamine Biosynthesis

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Jannell V. Bazurto ◽  
Kristen R. Farley ◽  
Diana M. Downs
Keyword(s):  
Escherichia Coli ◽  
Metabolic Network ◽  
Metabolic Networks ◽  
Thiamine Pyrophosphate ◽  
Metabolic Potential ◽  
E Coli ◽  
Regulatory Strategies ◽  
Phosphoribosylpyrophosphate Amidotransferase ◽  
Pyrimidine Moiety ◽  
Thiamine Biosynthesis

ABSTRACTMetabolism consists of biochemical reactions that are combined to generate a robust metabolic network that can respond to perturbations and also adapt to changing environmental conditions.Escherichia coliandSalmonella entericaare closely related enterobacteria that share metabolic components, pathway structures, and regulatory strategies. The synthesis of thiamine inS. entericahas been used to define a node of the metabolic network by analyzing alternative inputs to thiamine synthesis from diverse metabolic pathways. To assess the conservation of metabolic networks in organisms with highly conserved components, metabolic contributions to thiamine synthesis inE. coliwere investigated. Unexpectedly, we found that, unlikeS. enterica,E. colidoes not use the phosphoribosylpyrophosphate (PRPP) amidotransferase (PurF) as the primary enzyme for synthesis of phosphoribosylamine (PRA).In fact, our data showed that up to 50% of the PRA used byE. colito make thiamine requires the activities of threonine dehydratase (IlvA) and anthranilate synthase component II (TrpD). Significantly, the IlvA- and TrpD-dependent pathway to PRA functions inS. entericaonly in the absence of a functionalreactiveintermediatedeaminase (RidA) enzyme, bringing into focus how these closely related bacteria have distinct metabolic networks.IMPORTANCEIn most bacteria, includingSalmonellastrains andEscherichia coli, synthesis of the pyrimidine moiety of the essential coenzyme, thiamine pyrophosphate (TPP), shares enzymes with the purine biosynthetic pathway. Phosphoribosylpyrophosphate amidotransferase, encoded by thepurFgene, generates phosphoribosylamine (PRA) and is considered the first enzyme in the biosynthesis of purines and the pyrimidine moiety of TPP. We show here that, unlikeSalmonella,E. colisynthesizes significant thiamine from PRA derived from threonine using enzymes from the isoleucine and tryptophan biosynthetic pathways. These data show that two closely related organisms can have distinct metabolic network structures despite having similar enzyme components, thus emphasizing caveats associated with predicting metabolic potential from genome content.

scholarly journals Inhibition of Escherichia coli DNA topoisomerase I activity by phospholipids

1992 ◽  
Vol 285 (2) ◽  
pp. 503-506 ◽  
Author(s):  
T Mizushima ◽  
S Natori ◽  
K Sekimizu
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Topoisomerase I ◽  
Saturated Fatty Acids ◽  
Egg Yolk ◽  
Inhibitory Effect ◽  
Dna Topoisomerase I ◽  
Dna Topoisomerase ◽  
E Coli ◽  
Dna Relaxation

The DNA relaxation activity of Escherichia coli DNA topoisomerase I in vitro was greatly inhibited by cardiolipin. Inhibition also occurred to some extent with phosphatidylglycerol from egg yolk. Analysis with synthetic phospholipid revealed that phosphatidylglycerol containing unsaturated fatty acids exhibited a strong inhibitory effect, whereas inhibition by phosphatidylglycerol containing saturated fatty acids was weak. Phosphatidylethanolamine showed no inhibitory effect. Chlorpromazine, which interacts with phospholipids, suppressed the inhibitory effect of cardiolipin. Cardiolipin and phosphatidylglycerol with unsaturated fatty acid precipitated topoisomerase I even at low concentrations, whereas phosphatidylglycerol from egg yolk and a synthetic phosphatidylglycerol containing saturated fatty acids precipitated this enzyme only at high concentrations. One-third of the total topoisomerase I in E. coli was found in the membrane fraction. Treatment of E. coli cells with chlorpromazine resulted in relaxation of plasmid DNA. This DNA relaxation was not observed in a topA mutant, suggesting that this relaxation by chlorpromazine in vivo is catalysed by topoisomerase I.

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