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

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.

Download Full-text

Metabolic engineering Escherichia coli for efficient production of icariside D2

Biotechnology for Biofuels ◽

10.1186/s13068-019-1601-x ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 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.

Download Full-text

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

10.1101/2020.03.03.974378 ◽

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.

Download Full-text

Modular, synthetic chromosomes as new tools for large scale engineering of metabolism

10.1101/2021.10.04.462994 ◽

2021 ◽

Author(s):

Eline Postma ◽

Else-Jasmijn Hassing ◽

Venda Mangkusaputra ◽

Jordi Geelhoed ◽

Pilar de la Torre ◽

...

Keyword(s):

Copy Number ◽

Large Scale ◽

Metabolic Networks ◽

De Novo ◽

Microbial Cell ◽

Cell Factory ◽

Microbial Cell Factory ◽

Microbial Cell Factories ◽

Cell Factories

The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.

Download Full-text

Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

10.1101/394338 ◽

2018 ◽

Author(s):

Huan Fang ◽

Dong Li ◽

Jie Kang ◽

Pingtao Jiang ◽

Jibin Sun ◽

...

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

Biosynthetic Pathway ◽

Rhodobacter Capsulatus ◽

De Novo ◽

Vitamin B ◽

E Coli ◽

De Novo Biosynthesis ◽

Optimization Of Fermentation ◽

Aerobic And Anaerobic

ABSTRACTThe only known source of vitamin B12 (adenosylcobalamin) is from bacteria and archaea, and the only unknown step in its biosynthesis is the production of the intermediate adenosylcobinamide phosphate. Here, using genetic and metabolic engineering, we generated an Escherichia coli strain that produces vitamin B12 via an engineered de novo aerobic biosynthetic pathway. Excitingly, the BluE and CobC enzymes from Rhodobacter capsulatus transform L-threonine into (R)-1-Amino-2-propanol O-2-Phosphate, which is then condensed with adenosylcobyric acid to yield adenosylcobinamide phosphate by either CobD from the aeroic R. capsulatus or CbiB from the anerobic Salmonella typhimurium. These findings suggest that the biosynthetic steps from co(II)byrinic acid a,c-diamide to adocobalamin are the same in both the aerobic and anaerobic pathways. Finally, we increased the vitamin B12 yield of a recombinant E. coli strain by more than ∼250-fold to 307.00 µg/g DCW via metabolic engineering and optimization of fermentation conditions. Beyond our scientific insights about the aerobic and anaerobic pathways and our demonstration of E. coli as a microbial biosynthetic platform for vitamin B12 production, our study offers an encouraging example of how the several dozen proteins of a complex biosynthetic pathway can be transferred between organisms to facilitate industrial production.

Download Full-text

Utilization of mechanocatalytic oligosaccharides by ethanologenic Escherichia coli as a model microbial cell factory

AMB Express ◽

10.1186/s13568-020-0965-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Tao Jin ◽

Mats Käldström ◽

Adriana Benavides ◽

Marcelo D. Kaufman Rechulski ◽

Laura R. Jarboe

Keyword(s):

Escherichia Coli ◽

Microbial Cell ◽

Cell Factory ◽

Microbial Cell Factory ◽

Ethanologenic Escherichia Coli

Download Full-text

Improved production of 2′-fucosyllactose in engineered Saccharomyces cerevisiae expressing a putative α-1, 2-fucosyltransferase from Bacillus cereus

Microbial Cell Factories ◽

10.1186/s12934-021-01657-5 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Mingyuan Xu ◽

Xiangfeng Meng ◽

Weixin Zhang ◽

Yu Shen ◽

Weifeng Liu

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Bacillus Cereus ◽

Infant Nutrition ◽

De Novo ◽

Cost Effective ◽

Fold Increase ◽

Microbial Cell ◽

Cell Factory ◽

Microbial Cell Factory

Abstract Background 2′-fucosyllactose (2′-FL) is one of the most abundant oligosaccharides in human milk. It constitutes an authorized functional additive to improve infant nutrition and health in manufactured infant formulations. As a result, a cost-effective method for mass production of 2′-FL is highly desirable. Results A microbial cell factory for 2′-FL production was constructed in Saccharomyces cerevisiae by expressing a putative α-1, 2-fucosyltransferase from Bacillus cereus (FutBc) and enhancing the de novo GDP-l-fucose biosynthesis. When enabled lactose uptake, this system produced 2.54 g/L of 2′-FL with a batch flask cultivation using galactose as inducer and carbon source, representing a 1.8-fold increase compared with the commonly used α-1, 2-fucosyltransferase from Helicobacter pylori (FutC). The production of 2′-FL was further increased to 3.45 g/L by fortifying GDP-mannose synthesis. Further deleting gal80 enabled the engineered strain to produce 26.63 g/L of 2′-FL with a yield of 0.85 mol/mol from lactose with sucrose as a carbon source in a fed-batch fermentation. Conclusion FutBc combined with the other reported engineering strategies holds great potential for developing commercial scale processes for economic 2′-FL production using a food-grade microbial cell factory.

Download Full-text

Engineering Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase for Fully Active Amidated NAD Biosynthesis

Applied and Environmental Microbiology ◽

10.1128/aem.00692-17 ◽

2017 ◽

Vol 83 (13) ◽

Cited By ~ 6

Author(s):

Xueying Wang ◽

Yongjin J. Zhou ◽

Lei Wang ◽

Wujun Liu ◽

Yuxue Liu ◽

...

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

Nicotinic Acid ◽

De Novo ◽

Colorimetric Assay ◽

Reduced Form ◽

Content Type ◽

E Coli ◽

Nad Biosynthesis ◽

Nicotinamide Mononucleotide

ABSTRACT NAD and its reduced form NADH function as essential redox cofactors and have major roles in determining cellular metabolic features. NAD can be synthesized through the deamidated and amidated pathways, for which the key reaction involves adenylylation of nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), respectively. In Escherichia coli, NAD de novo biosynthesis depends on the protein NadD-catalyzed adenylylation of NaMN to nicotinic acid adenine dinucleotide (NaAD), followed by NAD synthase-catalyzed amidation. In this study, we engineered NadD to favor NMN for improved amidated pathway activity. We designed NadD mutant libraries, screened by a malic enzyme-coupled colorimetric assay, and identified two variants, 11B4 (Y84V/Y118D) and 16D8 (A86W/Y118N), with a high preference for NMN. Whereas in the presence of NMN both variants were capable of enabling the viability of cells of E. coli BW25113-derived NAD-auxotrophic strain YJE003, for which the last step of the deamidated pathway is blocked, the 16D8 expression strain could grow without exogenous NMN and accumulated a higher cellular NAD(H) level than BW25113 in the stationary phase. These mutants established fully active amidated NAD biosynthesis and offered a new opportunity to manipulate NAD metabolism for biocatalysis and metabolic engineering. IMPORTANCE Adenylylation of nicotinic acid mononucleotide (NaMN) and adenylylation of nicotinamide mononucleotide (NMN), respectively, are the key steps in the deamidated and amidated pathways for NAD biosynthesis. In most organisms, canonical NAD biosynthesis follows the deamidated pathway. Here we engineered Escherichia coli NaMN adenylyltransferase to favor NMN and expressed the mutant enzyme in an NAD-auxotrophic E. coli strain that has the last step of the deamidated pathway blocked. The engineered strain survived in M9 medium, which indicated the implementation of a functional amidated pathway for NAD biosynthesis. These results enrich our understanding of NAD biosynthesis and are valuable for manipulation of NAD homeostasis for metabolic engineering.

Download Full-text

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

Nature Communications ◽

10.1038/s41467-021-25243-w ◽

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.

Download Full-text

Correlation of Intracellular Trehalose Concentration with Desiccation Resistance of Soil Escherichia coli Populations

Applied and Environmental Microbiology ◽

10.1128/aem.01904-12 ◽

2012 ◽

Vol 78 (20) ◽

pp. 7407-7413 ◽

Cited By ~ 37

Author(s):

Qian Zhang ◽

Tao Yan

Keyword(s):

Escherichia Coli ◽

De Novo ◽

Potassium Acetate ◽

Desiccation Stress ◽

Desiccation Resistance ◽

Content Type ◽

Soil Desiccation ◽

E Coli ◽

Organic Solute ◽

Trehalose Synthesis

ABSTRACTNaturalized soilEscherichia colipopulations need to resist common soil desiccation stress in order to inhabit soil environments. In this study, four representative soilE. colistrains and one lab strain, MG1655, were tested for desiccation resistance via die-off experiments in sterile quartz sand under a potassium acetate-induced desiccation condition. The desiccation stress caused significantly lower die-off rates of the four soil strains (0.17 to 0.40 day−1) than that of MG1655 (0.85 day−1). Cellular responses, including extracellular polymeric substance (EPS) production, exogenous glycine betaine (GB) uptake, and intracellular compatible organic solute synthesis, were quantified and compared under the desiccation and hydrated control conditions. GB uptake appeared not to be a specific desiccation response, while EPS production showed considerable variability among theE. colistrains. AllE. colistrains produced more intracellular trehalose, proline, and glutamine under the desiccation condition than the hydrated control, and only the trehalose concentration exhibited a significant correlation with the desiccation-contributed die-off coefficients (Spearman's ρ = −1.0;P= 0.02).De novotrehalose synthesis was further determined for 15E. colistrains from both soil and nonsoil sources to determine its prevalence as a specific desiccation response. MostE. colistrains (14/15) synthesized significantly more trehalose under the desiccation condition, and the soilE. colistrains produced more trehalose (106.5 ± 44.9 μmol/mg of protein [mean ± standard deviation]) than the nonsoil reference strains (32.5 ± 10.5 μmol/mg of protein).

Download Full-text

Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.180.7.1814-1821.1998 ◽

1998 ◽

Vol 180 (7) ◽

pp. 1814-1821 ◽

Cited By ~ 52

Author(s):

Yong Yang ◽

Ho-Ching Tiffany Tsui ◽

Tsz-Kwong Man ◽

Malcolm E. Winkler

Keyword(s):

Escherichia Coli ◽

De Novo ◽

Specific Activity ◽

Salvage Pathway ◽

Pyridoxal Kinase ◽

Triple Mutant ◽

Reading Frame ◽

E Coli ◽

Specific Activities ◽

K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

Download Full-text