scholarly journals Constructing an ethanol utilization pathway in Escherichia coli to produce acetyl-CoA derived compounds

2020 ◽  
Author(s):  
Hong Liang ◽  
Xiaoqiang Ma ◽  
Wenbo Ning ◽  
Yurou Liu ◽  
Anthony J. Sinskey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
Structural Similarity ◽  
List Type ◽  
Acetyl Coa ◽  
E Coli ◽  
Utilization Pathway ◽  
Aspartate Family

AbstractEngineering microbes to utilize non-conventional substrates could create short and efficient pathways to convert substrate into product. In this study, we designed and constructed a two-step heterologous ethanol utilization pathway (EUP) in Escherichia coli by using acetaldehyde dehydrogenase (encoded by ada) from Dickeya zeae and alcohol dehydrogenase (encoded by adh2) from Saccharomyces cerevisiae. This EUP can convert ethanol into acetyl-CoA without ATP consumption, and generate two molecules of NADH per molecule of ethanol. We optimized the expression of these two genes and found that ethanol consumption could be improved by expressing them in a specific order (ada-adh2) with a constitutive promoter (PgyrA). The engineered E. coli strain with EUP consumed approximately 8 g/L of ethanol in 96 hours when it was used as sole carbon source. Subsequently, we combined EUP with the biosynthesis of polyhydroxybutyrate (PHB), a biodegradable polymer derived from acetyl-CoA. The engineered E. coli strain carrying EUP and PHB biosynthetic pathway produced 1.1 g/L of PHB from 10 g/L of ethanol and 1 g/L of aspartate family amino acids in 96 hours. We also engineered E. coli strain to produced 24 mg/L of prenol from 10 g/L of ethanol in 48 hours, supporting the feasibility of converting ethanol into different classes of acetyl-CoA derived compounds.HighlightsEngineered Escherichia coli strains to grow on ethanol as sole carbon sourceDemonstrated that ethanol was converted into acetyl-CoA (AcCoA) through two pathways (acetaldehyde-acetate-AcCoA and acetaldehyde-AcCoA)Converted ethanol into two acetyl-CoA derived products with low structural similarity (polyhydroxybutyrate and prenol)Discovered that supplementation of the aspartate family amino acids can substantially improve cell growth on ethanol

scholarly journals Heterologous Production of 6-Deoxyerythronolide B in Escherichia coli through the Wood Werkman Cycle

Metabolites ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 228
Author(s):  
R. Axayacatl Gonzalez-Garcia ◽  
Lars K. Nielsen ◽  
Esteban Marcellin
Keyword(s):  
Escherichia Coli ◽  
Natural Products ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
Structural Diversity ◽  
Heterologous Production ◽  
E Coli ◽  
Anticancer Properties ◽  
Defined Media ◽  
Extender Unit

Polyketides are a remarkable class of natural products with diverse functional and structural diversity. The class includes many medicinally important molecules with antiviral, antimicrobial, antifungal and anticancer properties. Native bacterial, fungal and plant hosts are often difficult to cultivate and coax into producing the desired product. As a result, Escherichia coli has been used for the heterologous production of polyketides, with the production of 6-deoxyerythronolide B (6-dEB) being the first example. Current strategies for production in E. coli require feeding of exogenous propionate as a source for the precursors propionyl-CoA and S-methylmalonyl-CoA. Here, we show that heterologous polyketide production is possible from glucose as the sole carbon source. The heterologous expression of eight genes from the Wood-Werkman cycle found in Propionibacteria, in combination with expression of the 6-dEB synthases DEBS1, DEBS2 and DEBS3 resulted in 6-dEB formation from glucose as the sole carbon source. Our results show that the Wood-Werkman cycle provides the required propionyl-CoA and the extender unit S-methylmalonyl-CoA to produce up to 0.81 mg/L of 6-dEB in a chemically defined media.

scholarly journals Enzymic synthesis of N-acetyl-l-phenylalanine in Escherichia coli K12

1971 ◽  
Vol 124 (5) ◽  
pp. 905-913 ◽  
Author(s):  
R. V. Krishna ◽  
P. R. Krishnaswamy ◽  
D. Rajagopal Rao
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
E Coli ◽  
Enzymic Synthesis ◽  
Escherichia Coli K12

1. Cell-free extracts of Escherichia coli K12 catalyse the synthesis of N-acetyl-l-phenylalanine from acetyl-CoA and l-phenylalanine. 2. The acetyl-CoA–l-phenylalanine α-N-acetyltransferase was purified 160-fold from cell-free extracts. 3. The enzyme has a pH optimum of 8 and catalyses the acetylation of l-phenylalanine. Other l-amino acids such as histidine and alanine are acetylated at slower rates. 4. A transacylase was also purified from E. coli extracts and its substrate specificity studied. 5. The properties of both these enzymes were compared with those of other known amino acid acetyltransferases and transacylases.

scholarly journals Effect of deregulation of repressor-specific carbon catabolite repression on carbon source consumption in Escherichia coli

2021 ◽  
Vol 64 (1) ◽  
Author(s):  
Hyeon Jeong Seong ◽  
Yu-Sin Jang
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Catabolite Repression ◽  
Sole Carbon Source ◽  
Carbon Catabolite Repression ◽  
Cell Factory ◽  
Essential Information ◽  
E Coli ◽  
Marine Biomass ◽  
Galactose Utilization

AbstractEscherichia coli has been used as a host to construct the cell factory for biobased production of chemicals from renewable feedstocks. Because galactose is found in marine biomass as a major component, the strategy for galactose utilization in E. coli has been gained more attention. Although galactose and glucose co-fermentation has been reported using the engineered E. coli strain, few reports have covered fermentation supplemented with galactose as a sole carbon source in the mutant lacking the repressor-specific carbon catabolite repression (CCR). Here, we report the effects of the deregulation of the repressor-specific CCR (galR− and galS−) in fermentation supplemented with galactose as a sole carbon source, using the engineered E. coli strains. In the fermentation using the galR− and galS− double mutant (GR2 strain), an increase of rates in sugar consumption and cell growth was observed compared to the parent strain. In the glucose fermentation, wild-type W3110 and its mutant GR2 and GR2PZ (galR−, galS−, pfkA−, and zwf−) consumed sugar at a higher rate than those values obtained from galactose fermentation. However, the GR2P strain (galR−, galS−, and pfkA−) showed no difference between fermentations using glucose and galactose as a sole carbon source. This study provides essential information for galactose fermentation using the CCR-deregulated E. coli strains.

The function ofackAandptagenes is necessary for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in recombinantpha+Escherichia coli

1995 ◽  
Vol 41 (13) ◽  
pp. 200-206 ◽  
Author(s):  
Ho Gun Rhie ◽  
Douglas Dennis
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Kinase Activity ◽  
Acetate Kinase ◽  
Multicopy Plasmid ◽  
Biosynthesis Pathway ◽  
Acetyl Coa ◽  
E Coli ◽  
Acetyl Coa Synthesis ◽  
Copolymer Poly

In Escherichia coli carrying the poly(3-hydroxyalkanoate) (PHA) biosynthesis pathway on a plasmid (pha+), the function of the ackA (acetate kinase) and pta (phosphotransacetylase) genes is necessary for efficient incorporation of 3-hydroxyvalerate (3-HV) into the copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)). Recombinant pha+E. coli fadR atoC(Con) strains possessing mutations in ackA, pta, or both ackA and pta exhibited substantially reduced levels of 3-HV formation. Conversely, the same strains carrying the ackA gene on a multicopy plasmid exhibited an increase in 3-HV formation concomitant with a large increase in acetate kinase activity. However, if the strain possessing the multicopy ackA+plasmid was mutant at the pta locus, it lost the ability to incorporate significant amounts of 3-HV into P(3HB-co-3HV). In addition to the ackA pta pathway, there is an inducible activity that can also mediate the incorporation of 3-HV into P(3HB-co-3HV). This pathway is repressed by glucose and is not normally operative in P(3HB-co-3HV) production in recombinant pha+E. coli strains that are grown using glucose as the major carbon source. It appears likely that this activity is due to an inducible acetyl-CoA synthetase that converts propionate to propionyl-CoA.Key words: polyhydroxyalkanoates, acetate kinase, phosphotransacetylase, acetyl-CoA synthesis, propionyl-CoA synthesis.

scholarly journals DsdX Is the Second d-Serine Transporter in Uropathogenic Escherichia coli Clinical Isolate CFT073

2006 ◽  
Vol 188 (18) ◽  
pp. 6622-6628 ◽  
Author(s):  
Andrew T. Anfora ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Carbon Source ◽  
Clinical Isolate ◽  
Sole Carbon Source ◽  
Minimal Medium ◽  
Amino Acid Sequence Similarity ◽  
Predicted Amino Acid Sequence ◽  
E Coli ◽  
K 12

ABSTRACTd-Serine is an amino acid present in mammalian urine that is inhibitory toEscherichia colistrains lacking a functionaldsdAgene. Counterintuitively, adsdAstrain ofE. coliclinical isolate CFT073 hypercolonizes the bladder and kidneys of mice relative to wild type during a coinfection in the murine model of urinary tract infection. We are interested in the mechanisms for uptake ofd-serine in CFT073.d-Serine entersE. coliK-12 via CycA, thed-alanine transporter andd-cycloserine sensitivity locus. CFT073cycAcan grow on minimal medium withd-serine as a sole carbon source. ThedsdXgene of thedsdCXAlocus is a likely candidate for an additionald-serine transporter based on its predicted amino acid sequence similarity to gluconate transporters. In minimal medium, CFT073dsdXcan grow ond-serine as a sole carbon source; however, CFT073dsdX cycAcannot. Additionally, CFT073dsdXA cycAis not sensitive to inhibitory concentrations ofd-serine during growth on glycerol andd-serine minimal medium.d-[14C]serine uptake experiments with CFT073dsdX cycAharboringdsdXorcycArecombinant plasmids confirm thatd-serine is able to enterE. colicells via CycA or DsdX. In whole-celld-[14C]serine uptake experiments, DsdX has an apparentKmof 58.75 μM and aVmaxof 75.96 nmol/min/mg, and CycA has an apparentKmof 82.40 μM and aVmaxof 58.90 nmol/min/mg. Onlyd-threonine marginally inhibits DsdX-mediatedd-serine transport, whereasd-alanine, glycine, andd-cycloserine inhibit CycA-mediatedd-serine transport. DsdX or CycA is sufficient to transport physiological quantities ofd-serine, but DsdX is ad-serine-specific permease.

Comparison of glucose, acetate and ethanol as carbon resource for production of acetyl-CoA derived chemicals in engineered Escherichia coli

2020 ◽  
Author(s):  
Shenmei Sun ◽  
Yamei Ding ◽  
Min Liu ◽  
Mo Xian ◽  
guang zhao
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Reducing Power ◽  
High Yield ◽  
Metabolome Analysis ◽  
Acetyl Coa ◽  
E Coli ◽  
Decarboxylation Reaction ◽  
Acetyl Coa Synthesis ◽  
Carbon Resource

Abstract Background: Acetyl-CoA is a fundamental metabolite in Escherichia coli, and also a precursor for biosynthesis of chemicals and materials suitable for multiple applications. The acetyl-CoA synthesis route from glucose presents low atomic economy due to the release of CO2 in pyruvate decarboxylation reaction. Because ethanol and acetate, both ordinary and inexpensive chemicals, can be converted into acetyl-CoA directly, they could be alternative substrates for production of acetyl-CoA derivatives. Results: In this study, the bifunctional reductase AdhE mutant (A267T/E568K), which converts ethanol into acetyl-CoA, was used to enable E. coli to grow on ethanol, and AMP-forming acetyl-CoA synthetase ACS was employed to enhance the ability of E. coli to utilize acetate. Several products derived from acetyl-CoA, including polyhydroxybutyrate, 3-hydroxypropionate, and phloroglucinol, were produced from glucose, ethanol, and acetate, respectively, by engineered E. coli strains. Compared with glucose and acetate, the strains grown on ethanol presented the highest production and yield of carbon source, and metabolome analysis revealed the reasons of high yield from ethanol. Conclusions: The conversion of ethanol into acetyl-CoA presents high atomic economy along with generation of reducing power, and the yield of target chemical from ethanol is much higher than those from glucose and acetate. All these results suggested that ethanol could be a suitable carbon source for production of acetyl-CoA derived bioproducts.

scholarly journals Identification, Source, and Metabolism of N-Ethylglutamate in Escherichia coli

2010 ◽  
Vol 192 (20) ◽  
pp. 5437-5440 ◽  
Author(s):  
Robert H. White
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Yeast Extract ◽  
Sole Carbon Source ◽  
Isotope Dilution Analysis ◽  
Sole Nitrogen Source ◽  
Glucose Medium ◽  
Detectable Amount ◽  
Deuterated Water ◽  
E Coli

ABSTRACT N-Ethylglutamate (NEG) was detected in Escherichia coli BL21 cells grown on LB broth, and it was found to occur at a concentration of ∼4 mM in these cells under these conditions. The same cells grown on M9 glucose medium contained no detectable amount of NEG. Analysis of the LB broth showed the presence of NEG, a compound never before reported as a natural product. Isotope dilution analysis showed that it occurred at a concentration of 160 μM in LB broth. Analyses of yeast extract and tryptone, the organic components of LB broth, both showed the presence NEG. It was demonstrated that NEG can be generated during the autolysis of the yeast used in the preparation of the yeast extract. Growth of these E. coli cells in LB broth prepared in deuterated water showed no incorporation of deuterium into NEG, demonstrating that E. coli cells did not generate the NEG. Cell growth rates were not affected by the addition of 5 mM NEG to either LB or M9 glucose medium. l-[ethyl-2H4]NEG was found to be readily incorporated into the cells and metabolized by the cells. From these results, it was concluded that all of the NEG present in the cells was taken up from the medium. NEG could serve as the sole nitrogen source for E. coli when grown on M9 glucose medium in the presence of glucose but could not serve as the sole carbon source on M9 medium in the absence of glucose.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

scholarly journals Acetate and Formate Stress: Opposite Responses in the Proteome of Escherichia coli

2001 ◽  
Vol 183 (21) ◽  
pp. 6466-6477 ◽  
Author(s):  
Christopher Kirkpatrick ◽  
Lisa M. Maurer ◽  
Nikki E. Oyelakin ◽  
Yuliya N. Yoncheva ◽  
Russell Maurer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Opposite Effect ◽  
Stress Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acid Resistance ◽  
Luria Broth ◽  
Acetyl Coa ◽  
Fermentation Products ◽  
E Coli

ABSTRACT Acetate and formate are major fermentation products ofEscherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-ptastrain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of theackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins.

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