Metabolic Engineering of E. coli for Xylose Production from Glucose as the Sole Carbon Source

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.

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Characterization of L-serine deaminases, SdaA (PA2448) and SdaB (PA5379), and their potential role inPseudomonas aeruginosapathogenesis

10.1101/394957 ◽

2018 ◽

Cited By ~ 1

Author(s):

Sixto M. Leal ◽

Elaine Newman ◽

Kalai Mathee

Keyword(s):

Immune System ◽

Amino Acid ◽

Carbon Source ◽

Bacterial Infections ◽

Sole Carbon Source ◽

Opportunistic Pathogen ◽

Host Immune System ◽

E Coli ◽

Serine Deaminase

ABSTRACTRegardless of the site of infectivity, all pathogens require high energetic influxes. This energy is required to counterattack the host immune system and in the absence the bacterial infections are easily cleared by the immune system. This study is an investigation into one highly bioenergetic pathway inPseudomonas aeruginosainvolving the amino acid L-serine and the enzyme L-serine deaminase (L-SD).P. aeruginosais an opportunistic pathogen causing infections in patients with compromised immune systems as well as patients with cystic fibrosis. L-SD has been linked directly to the pathogenicity of several organisms including but not limited toCampylobacter jejuni, Mycobacterium bovis,Streptococcus pyogenes, andYersinia pestis. We hypothesized thatP. aeruginosaL-SD is likely to be critical for its virulence. The genome sequence analysis revealed the presence of two L-SD homologs encoded bysdaAandsdaB.We analyzed the ability ofP. aeruginosato utilize serine and the role of SdaA and SdaB in serine deamination by comparing mutant strains ofsdaA(PAOsdaA) andsdaB(PAOsdaB) with their isogenic parentP. aeruginosaPAO1. We demonstrate thatP. aeruginosais unable to use serine as a sole carbon source. However, serine utilization is enhanced in the presence of glycine. Both SdaA and SdaB contribute to L-serine deamination, 34 % and 66 %, respectively. Glycine was also shown to increase the L-SD activity especially from SdaB. Glycine-dependent induction requires the inducer serine. The L-SD activity from both SdaA and SdaB is inhibited by the amino acid L-leucine. These results suggest thatP. aeruginosaL-SD is quite different from the characterizedE. coliL-SD that is glycine-independent but leucine-dependent for activation. Growth mutants able to use serine as sole carbon source were isolated. In addition, suicide vectors were constructed which allow for selective mutation of thesdaAandsdaBgenes on anyP. aeruginosastrain of interest. Future studies with a double mutant will reveal the importance of these genes for pathogenicity.

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

Applied Biological Chemistry ◽

10.1186/s13765-021-00627-0 ◽

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.

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Host engineering for improved valerolactam production in Pseudomonas putida

10.1101/660795 ◽

2019 ◽

Cited By ~ 2

Author(s):

Mitchell G. Thompson ◽

Luis E. Valencia ◽

Jacquelyn M. Blake-Hedges ◽

Pablo Cruz-Morales ◽

Alexandria E. Velasquez ◽

...

Keyword(s):

Metabolic Engineering ◽

Carbon Source ◽

Pseudomonas Putida ◽

Aromatic Compounds ◽

Sole Carbon Source ◽

Carbon Sources ◽

Shotgun Proteomics ◽

Rich Media ◽

Amide Hydrolase

ABSTRACTPseudomonas putida is a promising bacterial chassis for metabolic engineering given its ability to metabolize a wide array of carbon sources, especially aromatic compounds derived from lignin. However, this omnivorous metabolism can also be a hindrance when it can naturally metabolize products produced from engineered pathways. Herein we show that P. putida is able to use valerolactam as a sole carbon source, as well as degrade caprolactam. Lactams represent important nylon precursors, and are produced in quantities exceeding one million tons per year[1]. To better understand this metabolism we use a combination of Random Barcode Transposon Sequencing (RB-TnSeq) and shotgun proteomics to identify the oplBA locus as the likely responsible amide hydrolase that initiates valerolactam catabolism. Deletion of the oplBA genes prevented P. putida from growing on valerolactam, prevented the degradation of valerolactam in rich media, and dramatically reduced caprolactam degradation under the same conditions. Deletion of oplBA, as well as pathways that compete for precursors L-lysine or 5-aminovalerate, increased the titer of valerolactam from undetectable after 48 hours of production to ~90 mg/L. This work may serve as a template to rapidly eliminate undesirable metabolism in non-model hosts in future metabolic engineering efforts.

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Biochemical and Molecular Characterization of theBacillus subtilis Acetoin Catabolic Pathway

Journal of Bacteriology ◽

10.1128/jb.181.12.3837-3841.1999 ◽

1999 ◽

Vol 181 (12) ◽

pp. 3837-3841 ◽

Cited By ~ 62

Author(s):

Min Huang ◽

Fred Bernd Oppermann-Sanio ◽

Alexander Steinbüchel

Keyword(s):

Bacillus Subtilis ◽

Carbon Source ◽

Sole Carbon Source ◽

Enzyme System ◽

Gene Clusters ◽

Catabolic Pathway ◽

E Coli ◽

Homologous Genes ◽

Dehydrogenase Enzyme

ABSTRACT A recent study indicated that Bacillus subtiliscatabolizes acetoin by enzymes encoded by the acu gene cluster (F. J. Grundy, D. A. Waters, T. Y. Takova, and T. M. Henkin, Mol. Microbiol. 10:259–271, 1993) that are completely different from those in the multicomponent acetoin dehydrogenase enzyme system (AoDH ES) encoded by aco gene clusters found before in all other bacteria capable of utilizing acetoin as the sole carbon source for growth. By hybridization with a DNA probe covering acoA and acoB of the AoDH ES from Clostridium magnum, genomic fragments from B. subtilis harboring acoA, acoB,acoC, acoL, and acoR homologous genes were identified, and some of them were functionally expressed inE. coli. Furthermore, acoA was inactivated inB. subtilis by disruptive mutagenesis; these mutants were impaired to express PPi-dependent AoDH E1 activity to remove acetoin from the medium and to grow with acetoin as the carbon source. Therefore, acetoin is catabolized in B. subtilis by the same mechanism as all other bacteria investigated so far, leaving the function of the previously described acu genes obscure.

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A synthetic Calvin cycle enables autotrophic growth in yeast

10.1101/862599 ◽

2019 ◽

Author(s):

Thomas Gassler ◽

Michael Sauer ◽

Brigitte Gasser ◽

Diethard Mattanovich ◽

Matthias G. Steiger

Keyword(s):

Metabolic Engineering ◽

Carbon Source ◽

Sole Carbon Source ◽

Heterologous Protein ◽

Calvin Cycle ◽

Methylotrophic Yeast ◽

Genes Encoding ◽

Endogenous Genes ◽

Multiple Cell ◽

New Yeast

AbstractThe methylotrophic yeast Pichia pastoris is frequently used for heterologous protein production and it assimilates methanol efficiently via the xylulose-5-phosphate pathway. This pathway is entirely localized in the peroxisomes and has striking similarities to the Calvin-Benson-Bassham (CBB) cycle, which is used by a plethora of organisms like plants to assimilate CO2 and is likewise compartmentalized in chloroplasts. By metabolic engineering the methanol assimilation pathway of P. pastoris was re-wired to a CO2 fixation pathway resembling the CBB cycle. This new yeast strain efficiently assimilates CO2 into biomass and utilizes it as its sole carbon source, which changes the lifestyle from heterotrophic to autotrophic.In total eight genes, including genes encoding for RuBisCO and phosphoribulokinase, were integrated into the genome of P. pastoris, while three endogenous genes were deleted to block methanol assimilation. The enzymes necessary for the synthetic CBB cycle were targeted to the peroxisome. Methanol oxidation, which yields NADH, is employed for energy generation defining the lifestyle as chemoorganoautotrophic. This work demonstrates that the lifestyle of an organism can be changed from chemoorganoheterotrophic to chemoorganoautotrophic by metabolic engineering. The resulting strain can grow exponentially and perform multiple cell doublings on CO2 as sole carbon source with a µmax of 0.008 h−1.Graphical Abstract

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

10.1101/2020.04.14.041889 ◽

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

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INTERRELATIONSHIPS BETWEEN THE TRICARBOXYLIC ACID AND GLYOXYLATE CYCLES STUDIED WITH BACTERIAL AUXOTROPHS

Canadian Journal of Microbiology ◽

10.1139/m62-030 ◽

1962 ◽

Vol 8 (2) ◽

pp. 241-247 ◽

Cited By ~ 1

Author(s):

Henry C. Reeves ◽

Samuel J. Ajl

Keyword(s):

Carbon Source ◽

Sole Carbon Source ◽

Tricarboxylic Acid Cycle ◽

Tricarboxylic Acid ◽

Mineral Salts ◽

Glyoxylate Bypass ◽

Acid Cycle ◽

E Coli ◽

Acetate Medium ◽

Metabolic Significance

An autotroph of Escherichia coli, E26-6, which is unable to grow aerobically in a simple mineral-salts medium with either acetate, glutamate, isocitrate, or any one of the C4 dicarboxylic acid intermediates of the tricarboxylic acid cycle as sole carbon source, has been investigated. The mutant is able to grow, however, in a mineral-salts acetate medium supplemented with any one of the above acids. The specific activities of the tricarboxylic acid cycle and glyoxylate bypass enzymes, with the exception of alpha-ketoglutaric dehydrogenase, which is greatly impaired in the auxotroph, were found to be essentially the same in both the parent and the mutant. Thus, the glyoxylate bypass alone is not capable of supplying sufficient C4 intermediates to allow the growth of E. coli on acetate. Further, there appear to be no other metabolic pathways leading to C4 production, which are of major metabolic significance during growth on acetate, other than the tricarboxylic and glyoxylate cycles. Finally, in conjunction with the tricarboxylic acid cycle, the malate synthetase and isocitritase reactions provide a mechanism which enables E. coli to grow on a medium containing acetate as the sole carbon source.

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Metabolomic and Transcriptomic Analyses of Escherichia coli for Efficient Fermentation of L-Fucose

Marine Drugs ◽

10.3390/md17020082 ◽

2019 ◽

Vol 17 (2) ◽

pp. 82 ◽

Cited By ~ 1

Author(s):

Jungyeon Kim ◽

Yu Cheong ◽

Inho Jung ◽

Kyoung Kim

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

Carbon Source ◽

Large Scale ◽

Cellular Metabolism ◽

Scale Production ◽

Brown Macroalgae ◽

E Coli ◽

Biological Interpretation ◽

Efficient Fermentation

L-Fucose, one of the major monomeric sugars in brown algae, possesses high potential for use in the large-scale production of bio-based products. Although fucose catabolic pathways have been enzymatically evaluated, the effects of fucose as a carbon source on intracellular metabolism in industrial microorganisms such as Escherichia coli are still not identified. To elucidate the effects of fucose on cellular metabolism and to find clues for efficient conversion of fucose into bio-based products, comparative metabolomic and transcriptomic analyses were performed on E. coli on L-fucose and on D-glucose as a control. When fucose was the carbon source for E. coli, integration of the two omics analyses revealed that excess gluconeogenesis and quorum sensing led to severe depletion of ATP, resulting in accumulation and export of fucose extracellularly. Therefore, metabolic engineering and optimization are needed for E. coil to more efficiently ferment fucose. This is the first multi-omics study investigating the effects of fucose on cellular metabolism in E. coli. These omics data and their biological interpretation could be used to assist metabolic engineering of E. coli producing bio-based products using fucose-containing brown macroalgae.

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