d-2,3-Butanediol Production Due to Heterologous Expression of an Acetoin Reductase in Clostridium acetobutylicum

ABSTRACTAcetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions,Clostridium acetobutylicumATCC 824 (and strains derived from it) generates bothd- andl-stereoisomers of acetoin, but because of the absence of an ACR enzyme, it does not produce 2,3-butanediol. A gene encoding ACR fromClostridium beijerinckiiNCIMB 8052 was functionally expressed inC. acetobutylicumunder the control of two strong promoters, the constitutivethlpromoter and the late exponentialadcpromoter. Both ACR-overproducing strains were grown in batch cultures, during which 89 to 90% of the natively produced acetoin was converted to 20 to 22 mMd-2,3-butanediol. The addition of a racemic mixture of acetoin led to the production of bothd-2,3-butanediol andmeso-2,3-butanediol. A metabolic network that is in agreement with the experimental data is proposed. Native 2,3-butanediol production is a first step toward a potential homofermentative 2-butanol-producing strain ofC. acetobutylicum.

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Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

Applied and Environmental Microbiology ◽

10.1128/aem.00792-15 ◽

2015 ◽

Vol 81 (12) ◽

pp. 4173-4183 ◽

Cited By ~ 10

Author(s):

Akira Watanabe ◽

Kazumi Hiraga ◽

Masako Suda ◽

Hideaki Yukawa ◽

Masayuki Inui

Keyword(s):

Heterologous Expression ◽

Corynebacterium Glutamicum ◽

Abc Transporter ◽

Binding Protein ◽

Functional Characterization ◽

Product Inhibition ◽

Gene Encoding ◽

Content Type ◽

Industrial Strains ◽

Efficient Fermentation

ABSTRACTTheCorynebacterium alkanolyticumxylEFGDgene cluster comprises thexylDgene that encodes an intracellular β-xylosidase next to thexylEFGoperon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover forp-nitrophenyl-β-d-xylopyranoside of 111 ± 4 s−1), weak α-l-arabinofuranosidase activity (turnover forp-nitrophenyl-α-l-arabinofuranoside of 5 ± 1 s−1), and high tolerance to product inhibition (Kifor xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutivetacpromoter in theCorynebacterium glutamicumxylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended oncgR_2369, a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguousxylDgene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability ofC. glutamicumto take up xylooligosaccharides, an ability that is enhanced by in the presence of a functionalxylEFG-encoded xyloside ABC transporter. The finding thatxylEFGimparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates.

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Metabolic Engineering of Clostridium acetobutylicum ATCC 824 for Isopropanol-Butanol-Ethanol Fermentation

Applied and Environmental Microbiology ◽

10.1128/aem.06382-11 ◽

2011 ◽

Vol 78 (5) ◽

pp. 1416-1423 ◽

Cited By ~ 162

Author(s):

Joungmin Lee ◽

Yu-Sin Jang ◽

Sung Jun Choi ◽

Jung Ae Im ◽

Hyohak Song ◽

...

Keyword(s):

Metabolic Engineering ◽

Clostridium Acetobutylicum ◽

Secondary Alcohol ◽

Clostridium Beijerinckii ◽

Fuel Additive ◽

Gas Stripping ◽

Alcohol Fermentation ◽

Content Type ◽

Formation Pathway ◽

Total Alcohol

ABSTRACTClostridium acetobutylicumnaturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineeredC. acetobutylicumcan be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene fromClostridium beijerinckiiNRRL B-593 (i.e.,adhB-593) inC. acetobutylicumATCC 824. To increase the total alcohol titer, a synthetic acetone operon (actoperon;adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in thebukgene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged byin situremoval of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.

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Confirmation and Elimination of Xylose Metabolism Bottlenecks in Glucose Phosphoenolpyruvate-Dependent Phosphotransferase System-Deficient Clostridium acetobutylicum for Simultaneous Utilization of Glucose, Xylose, and Arabinose

Applied and Environmental Microbiology ◽

10.1128/aem.00644-11 ◽

2011 ◽

Vol 77 (22) ◽

pp. 7886-7895 ◽

Cited By ~ 99

Author(s):

Han Xiao ◽

Yang Gu ◽

Yuanyuan Ning ◽

Yunliu Yang ◽

Wilfrid J. Mitchell ◽

...

Keyword(s):

Clostridium Acetobutylicum ◽

Xylose Isomerase ◽

Cost Effective ◽

Pentose Phosphate ◽

Xylose Metabolism ◽

Phosphotransferase System ◽

Gene Encoding ◽

Content Type ◽

Sugar Mixtures ◽

Residual Glucose

ABSTRACTEfficient cofermentation ofd-glucose,d-xylose, andl-arabinose, three major sugars present in lignocellulose, is a fundamental requirement for cost-effective utilization of lignocellulosic biomass. The Gram-positive anaerobic bacteriumClostridium acetobutylicum, known for its excellent capability of producing ABE (acetone, butanol, and ethanol) solvent, is limited in using lignocellulose because of inefficient pentose consumption when fermenting sugar mixtures. To overcome this substrate utilization defect, a predictedglcGgene, encoding enzyme II of thed-glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS), was first disrupted in the ABE-producing model strainClostridium acetobutylicumATCC 824, resulting in greatly improvedd-xylose andl-arabinose consumption in the presence ofd-glucose. Interestingly, despite the loss of GlcG, the resulting mutant strain 824glcG fermentedd-glucose as efficiently as did the parent strain. This could be attributed to residual glucose PTS activity, although an increased activity of glucose kinase suggested that non-PTS glucose uptake might also be elevated as a result ofglcGdisruption. Furthermore, the inherent rate-limiting steps of thed-xylose metabolic pathway were observed prior to the pentose phosphate pathway (PPP) in strain ATCC 824 and then overcome by co-overexpression of thed-xylose proton-symporter (cac1345),d-xylose isomerase (cac2610), and xylulokinase (cac2612). As a result, an engineered strain (824glcG-TBA), obtained by integratingglcGdisruption and genetic overexpression of the xylose pathway, was able to efficiently coferment mixtures ofd-glucose,d-xylose, andl-arabinose, reaching a 24% higher ABE solvent titer (16.06 g/liter) and a 5% higher yield (0.28 g/g) compared to those of the wild-type strain. This strain will be a promising platform host toward commercial exploitation of lignocellulose to produce solvents and biofuels.

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Butanol Production from Crystalline Cellulose by Cocultured Clostridium thermocellum and Clostridium saccharoperbutylacetonicum N1-4

Applied and Environmental Microbiology ◽

10.1128/aem.00706-11 ◽

2011 ◽

Vol 77 (18) ◽

pp. 6470-6475 ◽

Cited By ~ 62

Author(s):

Shunichi Nakayama ◽

Keiji Kiyoshi ◽

Toshimori Kadokura ◽

Atsumi Nakazato

Keyword(s):

Clostridium Acetobutylicum ◽

Clostridium Thermocellum ◽

Clostridium Beijerinckii ◽

Crystalline Cellulose ◽

Butanol Production ◽

Content Type ◽

Coculture System

ABSTRACTWe investigated butanol production from crystalline cellulose by cocultured cellulolyticClostridium thermocellumand the butanol-producing strain,Clostridium saccharoperbutylacetonicum(strain N1-4). Butanol was produced from Avicel cellulose after it was incubated withC. thermocellumfor at least 24 h at 60°C before the addition of strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by coculturedClostridium acetobutylicumandClostridium beijerinckiithan by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this coculture system.

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PacBio Amplicon Sequencing Method To Measure Pilin Antigenic Variation Frequencies of Neisseria gonorrhoeae

mSphere ◽

10.1128/msphere.00562-19 ◽

2019 ◽

Vol 4 (5) ◽

Cited By ~ 1

Author(s):

Egon A. Ozer ◽

Lauren L. Prister ◽

Shaohui Yin ◽

Billy H. Ward ◽

Stanimir Ivanov ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Antigenic Variation ◽

Amplicon Sequencing ◽

Common Mechanism ◽

Macrophage Infection ◽

Gene Encoding ◽

Transmitted Infection ◽

Variable Regions ◽

Content Type ◽

Strain Fa1090

ABSTRACT Gene diversification is a common mechanism pathogens use to alter surface structures to aid in immune avoidance. Neisseria gonorrhoeae uses a gene conversion-based diversification system to alter the primary sequence of the gene encoding the major subunit of the pilus, pilE. Antigenic variation occurs when one of the nonexpressed 19 silent copies donates part of its DNA sequence to pilE. We have developed a method using Pacific Biosciences (PacBio) amplicon sequencing and custom software to determine pilin antigenic variation frequencies. The program analyzes 37 variable regions across the strain FA1090 1-81-S2 pilE gene and can be modified to determine sequence variation from other starting pilE sequences or other diversity generation systems. Using this method, we measured pilin antigenic variation frequencies for various derivatives of strain FA1090 and showed we can also analyze pilin antigenic variation frequencies during macrophage infection. IMPORTANCE Diversity generation systems are used by many unicellular organism to provide subpopulations of cell with different properties that are available when needed. We have developed a method using the PacBio DNA sequencing technology and a custom computer program to analyze the pilin antigenic variation system of the organism that is the sole cause of the sexually transmitted infection, gonorrhea.

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Defects in Mitochondrial and Peroxisomal β-Oxidation Influence Virulence in the Maize Pathogen Ustilago maydis

Eukaryotic Cell ◽

10.1128/ec.00129-12 ◽

2012 ◽

Vol 11 (8) ◽

pp. 1055-1066 ◽

Cited By ~ 27

Author(s):

Matthias Kretschmer ◽

Jana Klose ◽

James W. Kronstad

Keyword(s):

Ustilago Maydis ◽

Short Chain Fatty Acids ◽

Phytopathogenic Fungi ◽

Metabolic Adaptation ◽

In Planta ◽

Gene Encoding ◽

Disease Symptoms ◽

Content Type ◽

C Terminus ◽

Fungal Phytopathogens

ABSTRACTAn understanding of metabolic adaptation during the colonization of plants by phytopathogenic fungi is critical for developing strategies to protect crops. Lipids are abundant in plant tissues, and fungal phytopathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. Previously, we demonstrated a role for the peroxisomal β-oxidation enzyme Mfe2 in the filamentous growth, virulence, and sporulation of the maize pathogenUstilago maydis. However,mfe2mutants still caused disease symptoms, thus prompting a more detailed investigation of β-oxidation. We now demonstrate that a defect in thehad1gene encoding hydroxyacyl coenzyme A dehydrogenase for mitochondrial β-oxidation also influences virulence, although its paralog,had2, makes only a minor contribution. Additionally, we identified a gene encoding a polypeptide with similarity to the C terminus of Mfe2 and designated it Mfe2b; this gene makes a contribution to virulence only in the background of anmfe2Δ mutant. We also show that short-chain fatty acids induce cell death inU. maydisand that a block in β-oxidation leads to toxicity, likely because of the accumulation of toxic intermediates. Overall, this study reveals that β-oxidation has a complex influence on the formation of disease symptoms byU. maydisthat includes potential metabolic contributions to proliferationin plantaand an effect on virulence-related morphogenesis.

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Heterologous Expression of Lysergic Acid and Novel Ergot Alkaloids in Aspergillus fumigatus

Applied and Environmental Microbiology ◽

10.1128/aem.02137-14 ◽

2014 ◽

Vol 80 (20) ◽

pp. 6465-6472 ◽

Cited By ~ 21

Author(s):

Sarah L. Robinson ◽

Daniel G. Panaccione

Keyword(s):

Aspergillus Fumigatus ◽

Candidate Genes ◽

Mass Spectra ◽

Ergot Alkaloids ◽

Lysergic Acid ◽

Precursor Feeding ◽

Gene Encoding ◽

Content Type ◽

Multiple Reactions ◽

Important Pathway

ABSTRACTDifferent lineages of fungi produce distinct classes of ergot alkaloids. Lysergic acid-derived ergot alkaloids produced by fungi in the Clavicipitaceae are particularly important in agriculture and medicine. The pathway to lysergic acid is partly elucidated, but the gene encoding the enzyme that oxidizes the intermediate agroclavine is unknown. We investigated two candidate agroclavine oxidase genes from the fungusEpichloë festucaevar.lolii×Epichloë typhinaisolate Lp1 (henceforth referred to asEpichloësp. Lp1), which produces lysergic acid-derived ergot alkaloids. Candidate geneseasHandcloAwere expressed in a mutant strain of the moldAspergillus fumigatus, which typically produces a subclass of ergot alkaloids not derived from agroclavine or lysergic acid. Candidate genes were coexpressed with theEpichloësp. Lp1 allele ofeasA, which encodes an enzyme that catalyzed the synthesis of agroclavine from anA. fumigatusintermediate; the agroclavine then served as the substrate for the candidate agroclavine oxidases. Strains expressingeasAandcloAfromEpichloësp. Lp1 produced lysergic acid from agroclavine, a process requiring a cumulative six-electron oxidation and a double-bond isomerization. Strains that accumulated excess agroclavine (as a result ofEpichloësp. Lp1easAexpression in the absence ofcloA) metabolized it into two novel ergot alkaloids for which provisional structures were proposed on the basis of mass spectra and precursor feeding studies. Our data indicate that CloA catalyzes multiple reactions to produce lysergic acid from agroclavine and that combining genes from different ergot alkaloid pathways provides an effective strategy to engineer important pathway molecules and novel ergot alkaloids.

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Genomic Variation in IbA10G2 and Other Patient-Derived Cryptosporidium hominis Subtypes

Journal of Clinical Microbiology ◽

10.1128/jcm.01798-16 ◽

2016 ◽

Vol 55 (3) ◽

pp. 844-858 ◽

Cited By ~ 16

Author(s):

Per Sikora ◽

Sofia Andersson ◽

Jadwiga Winiecka-Krusnell ◽

Björn Hallström ◽

Cecilia Alsmark ◽

...

Keyword(s):

Parasite Burden ◽

Genomic Variation ◽

Loss Of Function ◽

Oocyst Wall ◽

Gene Encoding ◽

Cryptosporidium Hominis ◽

Content Type ◽

Successful Isolation ◽

Target Dna ◽

Stool Samples

ABSTRACTIn order to improve genotyping and epidemiological analysis ofCryptosporidiumspp., genomic data need to be generated directly from a broad range of clinical specimens. Utilizing a robust method that we developed for the purification and generation of amplified target DNA, we present its application for the successful isolation and whole-genome sequencing of 14 differentCryptosporidium hominispatient specimens. Six isolates of subtype IbA10G2 were analyzed together with a single representative each of 8 other subtypes: IaA20R3, IaA23R3, IbA9G3, IbA13G3, IdA14, IeA11G3T3, IfA12G1, and IkA18G1. Parasite burden was measured over a range of more than 2 orders of magnitude for all samples, while the genomes were sequenced to mean depths of between 17× and 490× coverage. Sequence homology-based functional annotation identified several genes of interest, including the gene encodingCryptosporidiumoocyst wall protein 9 (COWP9), which presented a predicted loss-of-function mutation in all the sequence subtypes, except for that seen with IbA10G2, which has a sequence identical to theCryptosporidium parvumreference Iowa II sequence. Furthermore, phylogenetic analysis showed that all the IbA10G2 genomes form a monophyletic clade in theC. hoministree as expected and yet display some heterogeneity within the IbA10G2 subtype. The current report validates the aforementioned method for isolating and sequencingCryptosporidiumdirectly from clinical stool samples. In addition, the analysis demonstrates the potential in mining data generated from sequencing multiple whole genomes ofCryptosporidiumfrom human fecal samples, while alluding to the potential for a higher degree of genotyping withinCryptosporidiumepidemiology.

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Osmoadaptation Strategy of the Most Halophilic Fungus, Wallemia ichthyophaga, Growing Optimally at Salinities above 15% NaCl

Applied and Environmental Microbiology ◽

10.1128/aem.02702-13 ◽

2013 ◽

Vol 80 (1) ◽

pp. 247-256 ◽

Cited By ~ 43

Author(s):

Janja Zajc ◽

Tina Kogej ◽

Erwin A. Galinski ◽

José Ramos ◽

Nina Gunde-Cimerman

Keyword(s):

Biomass Production ◽

Osmotic Shock ◽

Model Organism ◽

Salinity Range ◽

Growth Media ◽

Optimal Salinity ◽

Content Type ◽

Batch Cultures ◽

Specific Growth Rates ◽

Optimum Salinity

ABSTRACTWallemia ichthyophagais a fungus from the ancient basidiomycetous genusWallemia(Wallemiales, Wallemiomycetes) that grows only at salinities between 10% (wt/vol) NaCl and saturated NaCl solution. This obligate halophily is unique among fungi. The main goal of this study was to determine the optimal salinity range for growth of the halophilicW. ichthyophagaand to unravel its osmoadaptation strategy. Our results showed that growth on solid growth media was extremely slow and resulted in small colonies. On the other hand, in the liquid batch cultures, the specific growth rates ofW. ichthyophagawere higher, and the biomass production increased with increasing salinities. The optimum salinity range for growth ofW. ichthyophagawas between 15 and 20% (wt/vol) NaCl. At 10% NaCl, the biomass production and the growth rate were by far the lowest among all tested salinities. Furthermore, the cell wall content in the dry biomass was extremely high at salinities above 10%. Our results also showed that glycerol was the major osmotically regulated solute, since its accumulation increased with salinity and was diminished by hypo-osmotic shock. Besides glycerol, smaller amounts of arabitol and trace amounts of mannitol were also detected. In addition,W. ichthyophagamaintained relatively small intracellular amounts of potassium and sodium at constant salinities, but during hyperosmotic shock, the amounts of both cations increased significantly. Given our results and the recent availability of the genome sequence,W. ichthyophagashould become well established as a novel model organism for studies of halophily in eukaryotes.

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Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine, l-Valine, and 2-Ketoisovalerate

Applied and Environmental Microbiology ◽

10.1128/aem.01741-13 ◽

2013 ◽

Vol 79 (18) ◽

pp. 5566-5575 ◽

Cited By ~ 64

Author(s):

Jens Buchholz ◽

Andreas Schwentner ◽

Britta Brunnenkan ◽

Christina Gabris ◽

Simon Grimm ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Pyruvate Dehydrogenase ◽

Pyruvate Dehydrogenase Complex ◽

Fed Batch ◽

Complex Activity ◽

Gene Encoding ◽

Content Type ◽

Genes Encoding ◽

Cell Densities ◽

Dehydrogenase Complex

ABSTRACTExchange of the nativeCorynebacterium glutamicumpromoter of theaceEgene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutateddapApromoter variants led to a series ofC. glutamicumstrains with gradually reduced growth rates and PDHC activities. Upon overexpression of thel-valine biosynthetic genesilvBNCE, all strains producedl-valine. Among these strains,C. glutamicum aceEA16 (pJC4ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of thepqoandppcgenes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities,C. glutamicum aceEA16 Δpqo Δppc(pJC4ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter)l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression ofilvBNCDinstead ofilvBNCEtransformed thel-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with aYP/Sof 0.24 mol per mol of glucose and aQPof 6.9 mM per h [0.8 g/(liter × h)]. The replacement of theaceEpromoter by thedapA-A16 promoter in the twoC. glutamicuml-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate thatC. glutamicumstrains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

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