scholarly journals Isobutanol Production by Autotrophic Acetogenic Bacteria

2021 ◽  
Vol 9 ◽  
Author(s):  
Sandra Weitz ◽  
Maria Hermann ◽  
Sonja Linder ◽  
Frank R. Bengelsdorf ◽  
Ralf Takors ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Acetolactate Synthase ◽  
Gene Clusters ◽  
Strain Engineering ◽  
Coenzyme Specificity ◽  
Ferredoxin Oxidoreductase ◽  
Clostridium Ljungdahlii ◽  
Acetogenic Bacteria ◽  
Genes Encoding ◽  
Atp Generation

Two different isobutanol synthesis pathways were cloned into and expressed in the two model acetogenic bacteria Acetobacterium woodii and Clostridium ljungdahlii. A. woodii is specialized on using CO2 + H2 gas mixtures for growth and depends on sodium ions for ATP generation by a respective ATPase and Rnf system. On the other hand, C. ljungdahlii grows well on syngas (CO + H2 + CO2 mixture) and depends on protons for energy conservation. The first pathway consisted of ketoisovalerate ferredoxin oxidoreductase (Kor) from Clostridium thermocellum and bifunctional aldehyde/alcohol dehydrogenase (AdhE2) from C. acetobutylicum. Three different kor gene clusters are annotated in C. thermocellum and were all tested. Only in recombinant A. woodii strains, traces of isobutanol could be detected. Additional feeding of ketoisovalerate increased isobutanol production to 2.9 mM under heterotrophic conditions using kor3 and to 1.8 mM under autotrophic conditions using kor2. In C. ljungdahlii, isobutanol could only be detected upon additional ketoisovalerate feeding under autotrophic conditions. kor3 proved to be the best suited gene cluster. The second pathway consisted of ketoisovalerate decarboxylase from Lactococcus lactis and alcohol dehydrogenase from Corynebacterium glutamicum. For increasing the carbon flux to ketoisovalerate, genes encoding ketol-acid reductoisomerase, dihydroxy-acid dehydratase, and acetolactate synthase from C. ljungdahlii were subcloned downstream of adhA. Under heterotrophic conditions, A. woodii produced 0.2 mM isobutanol and 0.4 mM upon additional ketoisovalerate feeding. Under autotrophic conditions, no isobutanol formation could be detected. Only upon additional ketoisovalerate feeding, recombinant A. woodii produced 1.5 mM isobutanol. With C. ljungdahlii, no isobutanol was formed under heterotrophic conditions and only 0.1 mM under autotrophic conditions. Additional feeding of ketoisovalerate increased these values to 1.5 mM and 0.6 mM, respectively. A further increase to 2.4 mM and 1 mM, respectively, could be achieved upon inactivation of the ilvE gene in the recombinant C. ljungdahlii strain. Engineering the coenzyme specificity of IlvC of C. ljungdahlii from NADPH to NADH did not result in improved isobutanol production.

scholarly journals Specialized Metabolites from Ribosome Engineered Strains of Streptomyces clavuligerus

Metabolites ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 239
Author(s):  
Arshad Ali Shaikh ◽  
Louis-Felix Nothias ◽  
Santosh K. Srivastava ◽  
Pieter C. Dorrestein ◽  
Kapil Tahlan
Keyword(s):  
In Silico Analysis ◽  
Cost Effective ◽  
Gene Clusters ◽  
Streptomyces Clavuligerus ◽  
Strain Engineering ◽  
Ribosome Recycling Factor ◽  
Metabolic Potential ◽  
Putative Gene ◽  
Specialized Metabolites ◽  
Streptomyces Spp

Bacterial specialized metabolites are of immense importance because of their medicinal, industrial, and agricultural applications. Streptomyces clavuligerus is a known producer of such compounds; however, much of its metabolic potential remains unknown, as many associated biosynthetic gene clusters are silent or expressed at low levels. The overexpression of ribosome recycling factor (frr) and ribosome engineering (induced rpsL mutations) in other Streptomyces spp. has been reported to increase the production of known specialized metabolites. Therefore, we used an overexpression strategy in combination with untargeted metabolomics, molecular networking, and in silico analysis to annotate 28 metabolites in the current study, which have not been reported previously in S. clavuligerus. Many of the newly described metabolites are commonly found in plants, further alluding to the ability of S. clavuligerus to produce such compounds under specific conditions. In addition, the manipulation of frr and rpsL led to different metabolite production profiles in most cases. Known and putative gene clusters associated with the production of the observed compounds are also discussed. This work suggests that the combination of traditional strain engineering and recently developed metabolomics technologies together can provide rapid and cost-effective strategies to further speed up the discovery of novel natural products.

scholarly journals Genome Reduction and Secondary Metabolism of the Marine Sponge-Associated Cyanobacterium Leptothoe

Marine Drugs ◽  
2021 ◽  
Vol 19 (6) ◽  
pp. 298
Author(s):  
Despoina Konstantinou ◽  
Rafael V. Popin ◽  
David P. Fewer ◽  
Kaarina Sivonen ◽  
Spyros Gkelis
Keyword(s):  
Natural Products ◽  
Microbial Communities ◽  
Genomic Analysis ◽  
Gene Clusters ◽  
Marine Sponges ◽  
Genome Reduction ◽  
Extracellular Polysaccharides ◽  
Comparative Genomic ◽  
Symbiotic Relationships ◽  
Genes Encoding

Sponges form symbiotic relationships with diverse and abundant microbial communities. Cyanobacteria are among the most important members of the microbial communities that are associated with sponges. Here, we performed a genus-wide comparative genomic analysis of the newly described marine benthic cyanobacterial genus Leptothoe (Synechococcales). We obtained draft genomes from Le. kymatousa TAU-MAC 1615 and Le. spongobia TAU-MAC 1115, isolated from marine sponges. We identified five additional Leptothoe genomes, host-associated or free-living, using a phylogenomic approach, and the comparison of all genomes showed that the sponge-associated strains display features of a symbiotic lifestyle. Le. kymatousa and Le. spongobia have undergone genome reduction; they harbored considerably fewer genes encoding for (i) cofactors, vitamins, prosthetic groups, pigments, proteins, and amino acid biosynthesis; (ii) DNA repair; (iii) antioxidant enzymes; and (iv) biosynthesis of capsular and extracellular polysaccharides. They have also lost several genes related to chemotaxis and motility. Eukaryotic-like proteins, such as ankyrin repeats, playing important roles in sponge-symbiont interactions, were identified in sponge-associated Leptothoe genomes. The sponge-associated Leptothoe stains harbored biosynthetic gene clusters encoding novel natural products despite genome reduction. Comparisons of the biosynthetic capacities of Leptothoe with chemically rich cyanobacteria revealed that Leptothoe is another promising marine cyanobacterium for the biosynthesis of novel natural products.

scholarly journals Side-by-Side Comparison of Clean and Biomass-Derived, Impurity-Containing Syngas as Substrate for Acetogenic Fermentation with Clostridium ljungdahlii

Fermentation ◽  
2020 ◽  
Vol 6 (3) ◽  
pp. 84
Author(s):  
Alba Infantes ◽  
Michaela Kugel ◽  
Klaus Raffelt ◽  
Anke Neumann
Keyword(s):  
Biomass Gasification ◽  
Product Formation ◽  
Inhibitory Effects ◽  
Fed Batch ◽  
Gaseous Mixtures ◽  
Clostridium Ljungdahlii ◽  
Acetogenic Bacteria ◽  
Fed Batch Fermentation ◽  
Total Productivity ◽  
The Impact

Syngas, the product of biomass gasification, can play an important role in moving towards the production of renewable chemical commodities, by using acetogenic bacteria to ferment those gaseous mixtures. Due to the complex and changing nature of biomass, the composition and the impurities present in the final biomass-derived syngas will vary. Because of this, it is important to assess the impact of these factors on the fermentation outcome, in terms of yields, productivity, and product formation and ratio. In this study, Clostridium ljungdahlii was used in a fed-batch fermentation system to analyze the effect of three different biomass-derived syngases, and to compare them to equivalent, clean syngas mixtures. Additionally, four other clean syngas mixtures were used, and the effects on product ratio, productivity, yield, and growth were documented. All biomass-derived syngases were suitable to be used as substrates, without experiencing any complete inhibitory effects. From the obtained results, it is clear that the type of syngas, biomass-derived or clean, had the greatest impact on product formation ratios, with all biomass-derived syngases producing more ethanol, albeit with lesser total productivity.

scholarly journals Two Gene Clusters Coordinate Galactose and Lactose Metabolism in Streptococcus gordonii

2012 ◽  
Vol 78 (16) ◽  
pp. 5597-5605 ◽  
Author(s):  
Lin Zeng ◽  
Nicole C. Martino ◽  
Robert A. Burne
Keyword(s):  
Gene Clusters ◽  
Selective Advantage ◽  
Streptococcus Gordonii ◽  
Phosphotransferase System ◽  
Content Type ◽  
High Affinity ◽  
Oral Biofilms ◽  
Genes Encoding ◽  
Complex Regulation ◽  
Galactose Utilization

ABSTRACTStreptococcus gordoniiis an early colonizer of the human oral cavity and an abundant constituent of oral biofilms. Two tandemly arranged gene clusters, designatedlacandgal, were identified in theS. gordoniiDL1 genome, which encode genes of the tagatose pathway (lacABCD) and sugar phosphotransferase system (PTS) enzyme II permeases. Genes encoding a predicted phospho-β-galactosidase (LacG), a DeoR family transcriptional regulator (LacR), and a transcriptional antiterminator (LacT) were also present in the clusters. Growth and PTS assays supported that the permease designated EIILactransports lactose and galactose, whereas EIIGaltransports galactose. The expression of the gene for EIIGalwas markedly upregulated in cells growing on galactose. Using promoter-catfusions, a role for LacR in the regulation of the expressions of both gene clusters was demonstrated, and thegalcluster was also shown to be sensitive to repression by CcpA. The deletion oflacTcaused an inability to grow on lactose, apparently because of its role in the regulation of the expression of the genes for EIILac, but had little effect on galactose utilization.S. gordoniimaintained a selective advantage overStreptococcus mutansin a mixed-species competition assay, associated with its possession of a high-affinity galactose PTS, althoughS. mutanscould persist better at low pHs. Collectively, these results support the concept that the galactose and lactose systems ofS. gordoniiare subject to complex regulation and that a high-affinity galactose PTS may be advantageous whenS. gordoniiis competing against the caries pathogenS. mutansin oral biofilms.

scholarly journals Reconstruction of an Acetogenic 2,3-Butanediol Pathway Involving a Novel NADPH-Dependent Primary-Secondary Alcohol Dehydrogenase

2014 ◽  
Vol 80 (11) ◽  
pp. 3394-3403 ◽  
Author(s):  
Michael Köpke ◽  
Monica L. Gerth ◽  
Danielle J. Maddock ◽  
Alexander P. Mueller ◽  
FungMin Liew ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Secondary Alcohol ◽  
Acetolactate Synthase ◽  
Chemical Production ◽  
Content Type ◽  
E Coli ◽  
Gas Fermentation ◽  
Secondary Alcohol Dehydrogenase ◽  
Clostridium Autoethanogenum ◽  
Butanediol Dehydrogenase

ABSTRACTAcetogenic bacteria use CO and/or CO2plus H2as their sole carbon and energy sources. Fermentation processes with these organisms hold promise for producing chemicals and biofuels from abundant waste gas feedstocks while simultaneously reducing industrial greenhouse gas emissions. The acetogenClostridium autoethanogenumis known to synthesize the pyruvate-derived metabolites lactate and 2,3-butanediol during gas fermentation. Industrially, 2,3-butanediol is valuable for chemical production. Here we identify and characterize theC. autoethanogenumenzymes for lactate and 2,3-butanediol biosynthesis. The putativeC. autoethanogenumlactate dehydrogenase was active when expressed inEscherichia coli. The 2,3-butanediol pathway was reconstituted inE. coliby cloning and expressing the candidate genes for acetolactate synthase, acetolactate decarboxylase, and 2,3-butanediol dehydrogenase. Under anaerobic conditions, the resultingE. colistrain produced 1.1 ± 0.2 mM 2R,3R-butanediol (23 μM h−1optical density unit−1), which is comparable to the level produced byC. autoethanogenumduring growth on CO-containing waste gases. In addition to the 2,3-butanediol dehydrogenase, we identified a strictly NADPH-dependent primary-secondary alcohol dehydrogenase (CaADH) that could reduce acetoin to 2,3-butanediol. Detailed kinetic analysis revealed that CaADH accepts a range of 2-, 3-, and 4-carbon substrates, including the nonphysiological ketones acetone and butanone. The high activity of CaADH toward acetone led us to predict, and confirm experimentally, thatC. autoethanogenumcan act as a whole-cell biocatalyst for converting exogenous acetone to isopropanol. Together, our results functionally validate the 2,3-butanediol pathway fromC. autoethanogenum, identify CaADH as a target for further engineering, and demonstrate the potential ofC. autoethanogenumas a platform for sustainable chemical production.

scholarly journals Genes Encoding Cher-TPR Fusion Proteins Are Predominantly Found in Gene Clusters Encoding Chemosensory Pathways with Alternative Cellular Functions

PLoS ONE ◽  
2012 ◽  
Vol 7 (9) ◽  
pp. e45810 ◽  
Author(s):  
Francisco Muñoz-Martínez ◽  
Cristina García-Fontana ◽  
Miriam Rico-Jiménez ◽  
Carlos Alfonso ◽  
Tino Krell
Keyword(s):  
Fusion Proteins ◽  
Gene Clusters ◽  
Cellular Functions ◽  
Genes Encoding ◽  
Chemosensory Pathways

scholarly journals The Ether-Cleaving Methyltransferase System of the Strict Anaerobe Acetobacterium dehalogenans: Analysis and Expression of the Encoding Genes

2008 ◽  
Vol 191 (2) ◽  
pp. 588-599 ◽  
Author(s):  
Anke Schilhabel ◽  
Sandra Studenik ◽  
Martin Vödisch ◽  
Sandra Kreher ◽  
Bernhard Schlott ◽  
...  
Keyword(s):  
Amino Acid Sequence Identity ◽  
Protein Complexes ◽  
Gene Clusters ◽  
Single Copy ◽  
Anaerobic Microorganisms ◽  
Sequence Identity ◽  
Copy Gene ◽  
Genes Encoding ◽  
Encoding Genes ◽  
Genetic Context

ABSTRACT Anaerobic O-demethylases are inducible multicomponent enzymes which mediate the cleavage of the ether bond of phenyl methyl ethers and the transfer of the methyl group to tetrahydrofolate. The genes of all components (methyltransferases I and II, CP, and activating enzyme [AE]) of the vanillate- and veratrol-O-demethylases of Acetobacterium dehalogenans were sequenced and analyzed. In A. dehalogenans, the genes for methyltransferase I, CP, and methyltransferase II of both O-demethylases are clustered. The single-copy gene for AE is not included in the O-demethylase gene clusters. It was found that AE grouped with COG3894 proteins, the function of which was unknown so far. Genes encoding COG3894 proteins with 20 to 41% amino acid sequence identity with AE are present in numerous genomes of anaerobic microorganisms. Inspection of the domain structure and genetic context of these orthologs predicts that these are also reductive activases for corrinoid enzymes (RACEs), such as carbon monoxide dehydrogenase/acetyl coenzyme A synthases or anaerobic methyltransferases. The genes encoding the O-demethylase components were heterologously expressed with a C-terminal Strep-tag in Escherichia coli, and the recombinant proteins methyltransferase I, CP, and AE were characterized. Gel shift experiments showed that the AE comigrated with the CP. The formation of other protein complexes with the O-demethylase components was not observed under the conditions used. The results point to a strong interaction of the AE with the CP. This is the first report on the functional heterologous expression of acetogenic phenyl methyl ether-cleaving O-demethylases.

scholarly journals Identification and Characterization of the Asperthecin Gene Cluster of Aspergillus nidulans

2008 ◽  
Vol 74 (24) ◽  
pp. 7607-7612 ◽  
Author(s):  
Edyta Szewczyk ◽  
Yi-Ming Chiang ◽  
C. Elizabeth Oakley ◽  
Ashley D. Davidson ◽  
Clay C. C. Wang ◽  
...  
Keyword(s):  
Secondary Metabolite ◽  
Polyketide Synthase ◽  
Gene Clusters ◽  
Laboratory Culture ◽  
Culture Conditions ◽  
Type I ◽  
Genes Encoding ◽  
Identification And Characterization ◽  
Normal Laboratory

ABSTRACT The sequencing of Aspergillus genomes has revealed that the products of a large number of secondary metabolism pathways have not yet been identified. This is probably because many secondary metabolite gene clusters are not expressed under normal laboratory culture conditions. It is, therefore, important to discover conditions or regulatory factors that can induce the expression of these genes. We report that the deletion of sumO, the gene that encodes the small ubiquitin-like protein SUMO in A. nidulans, caused a dramatic increase in the production of the secondary metabolite asperthecin and a decrease in the synthesis of austinol/dehydroaustinol and sterigmatocystin. The overproduction of asperthecin in the sumO deletion mutant has allowed us, through a series of targeted deletions, to identify the genes required for asperthecin synthesis. The asperthecin biosynthesis genes are clustered and include genes encoding an iterative type I polyketide synthase, a hydrolase, and a monooxygenase. The identification of these genes allows us to propose a biosynthetic pathway for asperthecin.

scholarly journals Characterization of Novel Carbazole Catabolism Genes from Gram-Positive Carbazole Degrader Nocardioides aromaticivorans IC177

2006 ◽  
Vol 72 (5) ◽  
pp. 3321-3329 ◽  
Author(s):  
Kengo Inoue ◽  
Hiroshi Habe ◽  
Hisakazu Yamane ◽  
Hideaki Nojiri
Keyword(s):  
Amino Acid ◽  
Gene Clusters ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Class Iii ◽  
Rt Pcr ◽  
Gram Positive ◽  
Mononuclear Iron ◽  
Reverse Transcription Pcr ◽  
Genes Encoding

ABSTRACT Nocardioides aromaticivorans IC177 is a gram-positive carbazole degrader. The genes encoding carbazole degradation (car genes) were cloned into a cosmid clone and sequenced partially to reveal 19 open reading frames. The car genes were clustered into the carAaCBaBbAcAd and carDFE gene clusters, encoding the enzymes responsible for the degradation of carbazole to anthranilate and 2-hydroxypenta-2,4-dienoate and of 2-hydroxypenta-2,4-dienoate to pyruvic acid and acetyl coenzyme A, respectively. The conserved amino acid motifs proposed to bind the Rieske-type [2Fe-2S] cluster and mononuclear iron, the Rieske-type [2Fe-2S] cluster, and flavin adenine dinucleotide were found in the deduced amino acid sequences of carAa, carAc, and carAd, respectively, which showed similarities with CarAa from Sphingomonas sp. strain KA1 (49% identity), CarAc from Pseudomonas resinovorans CA10 (31% identity), and AhdA4 from Sphingomonas sp. strain P2 (37% identity), respectively. Escherichia coli cells expressing CarAaAcAd exhibited major carbazole 1,9a-dioxygenase (CARDO) activity. These data showed that the IC177 CARDO is classified into class IIB, while gram-negative CARDOs are classified into class III or IIA, indicating that the respective CARDOs have diverse types of electron transfer components and high similarities of the terminal oxygenase. Reverse transcription-PCR (RT-PCR) experiments showed that the carAaCBaBbAcAd and carDFE gene clusters are operonic. The results of quantitative RT-PCR experiments indicated that transcription of both operons is induced by carbazole or its metabolite, whereas anthranilate is not an inducer. Biotransformation analysis showed that the IC177 CARDO exhibits significant activities for naphthalene, carbazole, and dibenzo-p-dioxin but less activity for dibenzofuran and biphenyl.

scholarly journals The Sodium-Driven Flagellar Motor Controls Exopolysaccharide Expression in Vibrio cholerae

2004 ◽  
Vol 186 (15) ◽  
pp. 4864-4874 ◽  
Author(s):  
Crystal M. Lauriano ◽  
Chandradipa Ghosh ◽  
Nidia E. Correa ◽  
Karl E. Klose
Keyword(s):  
Vibrio Cholerae ◽  
Gene Transcription ◽  
Biofilm Formation ◽  
Diarrheal Disease ◽  
Response Regulator ◽  
Gene Clusters ◽  
Signaling Cascade ◽  
Genes Encoding ◽  
Life Threatening

ABSTRACT Vibrio cholerae causes the life-threatening diarrheal disease cholera. This organism persists in aquatic environments in areas of endemicity, and it is believed that the ability of the bacteria to form biofilms in the environment contributes to their persistence. Expression of an exopolysaccharide (EPS), encoded by two vps gene clusters, is essential for biofilm formation and causes a rugose colonial phenotype. We previously reported that the lack of a flagellum induces V. cholerae EPS expression. To uncover the signaling pathway that links the lack of a flagellum to EPS expression, we introduced into a rugose flaA strain second-site mutations that would cause reversion back to the smooth phenotype. Interestingly, mutation of the genes encoding the sodium-driven motor (mot) in a nonflagellated strain reduces EPS expression, biofilm formation, and vps gene transcription, as does the addition of phenamil, which specifically inhibits the sodium-driven motor. Mutation of vpsR, which encodes a response regulator, also reduces EPS expression, biofilm formation, and vps gene transcription in nonflagellated cells. Complementation of a vpsR strain with a constitutive vpsR allele likely to mimic the phosphorylated state (D59E) restores EPS expression and biofilm formation, while complementation with an allele predicted to remain unphosphorylated (D59A) does not. Our results demonstrate the involvement of the sodium-driven motor and suggest the involvement of phospho-VpsR in the signaling cascade that induces EPS expression. A nonflagellated strain expressing EPS is defective for intestinal colonization in the suckling mouse model of cholera and expresses reduced amounts of cholera toxin and toxin-coregulated pili in vitro. Wild-type levels of virulence factor expression and colonization could be restored by a second mutation within the vps gene cluster that eliminated EPS biosynthesis. These results demonstrate a complex relationship between the flagellum-dependent EPS signaling cascade and virulence.

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