Microbial Utilization of Electrically Reduced Neutral Red as the Sole Electron Donor for Growth and Metabolite Production

Electrically reduced neutral red (NR) served as the sole source of reducing power for growth and metabolism of pure and mixed cultures of H₂-consuming bacteria in a novel electrochemical bioreactor system. NR was continuously reduced by the cathodic potential (−1.5 V) generated from an electric current (0.3 to 1.0 mA), and it was subsequently oxidized by Actinobacillus succinogenes or by mixed methanogenic cultures. The A. succinogenes mutant strain FZ-6 did not grow on fumarate alone unless electrically reduced NR or hydrogen was present as the electron donor for succinate production. The mutant strain, unlike the wild type, lacked pyruvate formate lyase and formate dehydrogenase. Electrically reduced NR also replaced hydrogen as the sole electron donor source for growth and production of methane from CO₂. These results show that both pure and mixed cultures can function as electrochemical devices when electrically generated reducing power can be used to drive metabolism. The potential utility of utilizing electrical reducing power in enhancing industrial fermentations or biotransformation processes is discussed.     

Increased transformation frequency and tagging of developmental genes in Aspergillus nidulans by restriction enzyme-mediated integration (REMI)

We have used a plasmid containing the argB gene to transform an Aspergillus nidulansargB-deleted strain in the presence of restriction enzymes and show a 20- to 60-fold increase in transformation frequency via restriction enzyme-mediated integration (REMI). This procedure was used to try to tag new genes involved in the asexual development of this fungus. More than 2000 transformants isolated following electroporation of conidia and ～3700 transformants recovered following protoplast fusion were screened for sporulation defects. Unexpectedly, developmental mutants were obtained only when the protoplast fusion approach was used. Southern blot analysis of these mutants, and of randomly selected transformants obtained by electroporation, was consistent with the occurrence of single plasmid integration events in 33 and 65% of the cases, respectively. The argB marker was shown to be tightly linked to the mutant phenotype in only 62% of the mutants analyzed by sexual crosses. Partial DNA sequencing of a tagged gene, whose mutation delays asexual sporulation and results in a fluffy phenotype, showed no homology to previously reported sequences. Our results indicate that REMI can be used in A.?nidulans to increase the transformation frequency and illustrate the advantages and potential problems when using REMI to tag genes of interest in this and other fungi.     

Global metabolic response of Escherichia coli to gnd or zwf gene-knockout, based on 13C-labeling experiments and the measurement of enzyme activities

An integrated study on cell growth, enzyme activities and carbon flux redistribution was made to investigate how the central metabolism of Escherichia coli changes with the knockout of genes in the oxidative pentose phosphate pathway (PPP). Mutants deficient in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were constructed by disrupting the zwf and gnd genes and were grown in minimal media with two different carbon sources, such as glucose or pyruvate. It was shown that the knockout of either gnd or zwf gene did not affect the cell growth rate significantly, but the cellular metabolism was changed. While the specific substrate uptake rate and the specific carbon dioxide evolution rate for either mutant grown on glucose were higher than those obtained for the parent strain, these two rates were markedly decreased in mutants grown on pyruvate. The measurement of enzyme activities implied a significant change in metabolism, when alternative pathways such as the Entner–Doudoroff pathway (EDP) and the malic enzyme pathway were activated in the gnd mutant grown on glucose. As compared with the parent strain, the activities of phosphoglucose isomerase were increased in mutants grown on glucose but decreased in mutants grown on pyruvate. The metabolic flux redistribution obtained based on ¹³C-labeling experiments further indicated that the direction of the flux through the non-oxidative PPP was reversed in response to the gene knockout. Moreover, the knockout of genes caused an increased flux through the tricarboxlic acid cycle in mutants grown on glucose but caused a decrease in the case of using pyruvate. There was also a negative correlation between the fluxes through malic enzyme and isocitrate dehydrogenase in the mutants; and a positive correlation was found between the fluxes through malic enzyme and phosphoenolpyruvate carboxylase.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Low-potential cytochrome b as an essential electron-transport component of menaquinone reduction by formate in Vibrio succinogenes

Incorporation of the electron-transport enzymes of Vibrio succinogenes into liposomes was used to investigate the question of whether, in this organism, a cytochrome b is involved in electron transport from formate to fumarate on the formate side of menaquinone. (1) Formate dehydrogenase lacking cytochrome b was prepared by splitting the cytochrome from the formate dehydrogenase complex. The enzyme consisted of two different subunits (M_r 110 000 and 20 000), catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone by formate, and could be incorporated into liposomes. (2) The modified enzyme did not restore electron transport from formate to fumarate when incorporated into liposomes together with vitamin K-1 (instead of menaquinone) and fumarate reductase complex. In contrast, restoration was observed in liposomes that contained formate dehydrogenase with cytochrome b (E_m = ?224 mV), in addition to the subunits mentioned above (formate dehydrogenase complex). (3) In the liposomes containing formate dehydrogenase complex and fumarate reductase complex, the response of the cytochrome b of the formate dehydrogenase complex was consistent with its interaction on the formate side of menaquinone in a linear sequence of the components. The low-potential cytochrome b associated with fumarate reductase complex was not reducible by formate under any condition. It is concluded that the low-potential cytochrome b of the formate dehydrogenase complex is an essential component in the electron transport from formate to menaquinone. The low-potential cytochrome b of the fumarate reductase complex could not replace the former cytochrome in restoring electron-transport activity.     

Facile construction of mutations in Haemophilus ducreyi using lacZ as a counter-selectable marker

Haemophilus ducreyi is a Gram-negative bacterium which is the causative agent of chancroid, an ulcerative sexually transmitted disease. In order to understand the pathogenesis of H. ducreyi disease, studies designed to identify potential virulence determinants and construct mutants deficient in the elaboration of these determinants have been undertaken in several laboratories. At the present time, construction of isogenic mutants is accomplished by electroporation of linearized DNA containing insertionally inactivated H. ducreyi genes followed by selection for the resistance marker encoded on the inactivated gene. In our experience, certain mutants are difficult to construct using this procedure. In the construction of strains containing lacZ as a reporter gene, we observed that the growth of lacZ expressing H. ducreyi was inhibited in the presence of X-gal. We have exploited this observation to develop a new strategy for the construction of isogenic H. ducreyi mutants.     

Co-targeting strategy for precise,scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

Programmable site-specific nucleases, such as the clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas (Chlamydomonas reinhardtii). However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the dsDNA breaks induced by the RNPs is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. Here, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker)—in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precise edits in the homolog of bacterial ftsY or the WD and TetratriCopeptide repeats protein 1 genes in ∼1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways, or structures.

A transgene-free strategy allows precise editing of genes lacking a selectable phenotype by electroporation of CRISPR/Cas9 ribonucleoproteins and single-stranded oligodeoxynucleotide templates.     

Four products from Escherichia coli pseudogenes increase hydrogen production

Pseudogenes are considered to be nonfunctional genes that lack a physiological role. By screening 3985 Escherichia coli mutants using chemochromic membranes, we found four pseudogenes involved in hydrogen metabolism. Knockouts of pseudogenes ydfW and ypdJ had a defective hydrogen phenotype on glucose and formate, respectively. Also, the knockout of pseudogene yqiG formed hydrogen from formate but not from glucose. For the yqiG mutant, 100% hydrogen recovery was obtained by the complementation of YqiG via a plasmid. The knockout of pseudogene ylcE showed hydrogen deficiency in minimal media which suggested that the role of YlcE is associated with cell growth. Hence, the products of these four pseudogenes play an important physiological role in hydrogen production in E. coli.     

Metabolism of One-Carbon Compounds by the Ruminal Acetogen Syntrophococcus sucromutans

Syntrophococcus sucromutans is the predominant species capable of O demethylation of methoxylated lignin monoaromatic derivatives in the rumen. The enzymatic characterization of this acetogen indicated that it uses the acetyl coenzyme A (Wood) pathway. Cell extracts possess all the enzymes of the tetrahydrofolate pathway, as well as carbon monoxide dehydrogenase, at levels similar to those of other acetogens using this pathway. However, formate dehydrogenase could not be detected in cell extracts, whether formate or a methoxyaromatic was used as electron acceptor for growth of the cells on cellobiose. Labeled bicarbonate, formate, [1-¹⁴C] pyruvate, and chemically synthesized O-[methyl-¹⁴C]vanillate were used to further investigate the catabolism of one-carbon (C₁) compounds by using washed-cell preparations. The results were consistent with little or no contribution of formate dehydrogenase and pointed out some unique features. Conversion of formate to CO₂ was detected, but labeled formate predominantly labeled the methyl group of acetate. Labeled CO₂ readily exchanged with the carboxyl group of pyruvate but not with formate, and both labeled CO₂ and pyruvate predominantly labeled the carboxyl group of acetate. No CO₂ was formed from O demethylation of vanillate, and the acetate produced was position labeled in the methyl group. The fermentation pattern and specific activities of products indicated a complete synthesis of acetate from pyruvate and the methoxyl group of vanillate.     

A reusable method for construction of non-marker large fragment deletion yeast auxotroph strains: A practice in Torulopsis glabrata

A reusable method for construction of yeast auxotrophic mutants with large deletions that do not contain markers was developed and successfully applied to a pyruvate overproducing yeast strain, Torulopsis glabrata CCTCC M202019. A URA3 knockout fragment with a large deletion of the open reading frame and long homologous arms was constructed by fusion PCR and transformed into the parent strain by high-efficiency electroporation. A high concentration of transformed yeast was achieved by subsequently culturing in rich medium for 24 h and in nitrogen-free minimal medium for 4 h. Potential Deltaura3 auxotrophic mutants were enriched by treatment with nystatin and selection on uracil-limited medium. Mutants were confirmed by streaking cultures and colony PCR. Deltaarg8 and Deltaura3Deltaarg8 double auxotroph mutants were also successfully constructed using this method.     

Substrate and product inhibition kinetics in succinic acid production by Actinobacillus succinogenes

The inhibition of substrate and products on the growth of Actinobacillus succinogenes in fermentation using glucose as the major carbon source was studied. A. succinogenes tolerated up to 143 g/L glucose and cell growth was completely inhibited with glucose concentration over 158 g/L. Significant decrease in succinic acid yield and prolonged lag phase were observed with glucose concentration above 100 g/L. Among the end-products investigated, formate was found to have the most inhibitory effect on succinic acid fermentation. The critical concentrations of acetate, ethanol, formate, pyruvate and succinate were 46, 42, 16, 74, 104 g/L, respectively. A growth kinetic model considering both substrate and product inhibition is proposed, which adequately simulates batch fermentation kinetics using both semi-defined and wheat-derived media. The model accurately describes the inhibitory kinetics caused by both externally added chemicals and the same chemicals produced during fermentation. This paper provides key insights into the improvement of succinic acid production and the modelling of inhibition kinetics.     

sacB-5-Fluoroorotic Acid-pyrE-Based Bidirectional Selection for Integration of Unmarked Alleles into the Chromosome of Rhodobacter capsulatus

The gram-negative, purple nonsulfur, facultative photosynthetic bacterium Rhodobacter capsulatus is a widely used model organism and has well-developed molecular genetics. In particular, interposon mutagenesis using selectable gene cartridges is frequently employed for construction of a variety of chromosomal knockout mutants. However, as the gene cartridges are often derived from antibiotic resistance-conferring genes, their numbers are limited, which restricts the construction of multiple knockout mutants. In this report, sacB—5-fluoroorotic acid (5FOA)—pyrE-based bidirectional selection that facilitates construction of unmarked chromosomal knockout mutations is described. The R. capsulatus pyrE gene encoding orotate phosphoribosyl transferase, a key enzyme of the de novo pyrimidine nucleotide biosynthesis pathway, was used as an interposon in a genetic background that is auxotrophic for uracil (Ura⁻) and hence resistant to 5FOA (5FOA^r). Although Ura⁺ selection readily yielded chromosomal allele replacements via homologous recombination, selection for 5FOA^r to replace pyrE with unmarked alleles was inefficient. To improve the latter step, 5FOA^r selection was combined with sucrose tolerance selection using a suicide plasmid carrying the Bacillus subtilis sacB gene encoding levansucrase that induces lethality upon exposure to 5% (wt/vol) sucrose in the growth medium. Sucrose-tolerant, 5FOA^r colonies that were obtained carried chromosomal unmarked mutant alleles of the target gene via double crossovers between the resident pyrE-marked and incoming unmarked alleles. The effectiveness of this double selection was proven by seeking insertion and deletion alleles of helC involved in R. capsulatus cytochrome c biogenesis, which illustrated the usefulness of this system as a genetic means for facile construction of R. capsulatus unmarked chromosomal mutants.     

Construction and Characterization of Shuttle Vectors for Succinic Acid-Producing Rumen Bacteria

Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 × 10⁶ and 7.1 × 10⁶ transformants/μg DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.     

Altered redox status in Escherichia coli cells enhances pyruvate production in pH-adjusting culture with a fermenter

Improvements in pyruvate production process were examined using Escherichia coli BW25113?pta/pHfdh strain carrying the formate dehydrogenase gene of Mycobacterium vaccae to change the redox status of the cells. Glucose and formate concentrations, and oxygenation levels determined previously in a shake-flask culture were applied for pyruvate production in a 1 l fermenter. However, pyruvate was not produced under the examined conditions. Detailed pH measurements during the fermenter culture using CaCO₃ revealed that maintaining the pH value around 6.0 plays an important role in stabilizing the pyruvate accumulation. In the pH-adjusting culture around 6.0 with NaOH solution, the concentration and yield of pyruvate were 8.96 g l^?1 and 0.48 g pyruvate g glucose^?1, respectively, which were significantly higher than the values reported in the shake-flask culture (6.79 g l^?1 and 0.32 g pyruvate g glucose^?1).     

The maize CorA/MRS2/MGT-type Mg transporter,ZmMGT10, responses to magnesium deficiency and confers low magnesium tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Key message

A new selectable marker gene for stable transformation of the plastid genome was developed that is similarly efficient as the aadA, and produces no background of spontaneous resistance mutants.

Abstract

More than 25 years after its development for Chlamydomonas and tobacco, the transformation of the chloroplast genome still represents a challenging technology that is available only in a handful of species. The vast majority of chloroplast transformation experiments conducted thus far have relied on a single selectable marker gene, the spectinomycin resistance gene aadA. Although a few alternative markers have been reported, the aadA has remained unrivalled in efficiency and is, therefore, nearly exclusively used. The development of new marker genes for plastid transformation is of crucial importance to all efforts towards extending the species range of the technology as well as to those applications in basic research, biotechnology and synthetic biology that involve the multistep engineering of plastid genomes. Here, we have tested a bifunctional resistance gene for its suitability as a selectable marker for chloroplast transformation. The bacterial enzyme aminoglycoside acetyltransferase(6′)-Ie/aminoglycoside phosphotransferase(2″)-Ia possesses an N-terminal acetyltransferase domain and a C-terminal phosphotransferase domain that can act synergistically and detoxify aminoglycoside antibiotics highly efficiently. We report that, in combination with selection for resistance to the aminoglycoside tobramycin, the aac(6′)-Ie/aph(2″)-Ia gene represents an efficient marker for plastid transformation in that it produces similar numbers of transplastomic lines as the spectinomycin resistance gene aadA. Importantly, no spontaneous antibiotic resistance mutants appear under tobramycin selection.

     

Improved gene targeting in C. elegans using counter-selection and Flp-mediated marker excision

Gene targeting is widely used for the precise manipulation of genes. However, in the model organism Caenorhabditis elegans non-transposon mediated gene targeting remains laborious, and as a result has not been widely used. One obstacle to the wider use of this approach is the difficulty of identifying homologous recombination events amongst non-specific events. To improve gene targeting in C. elegans, we used a counter-selection approach to reduce the number of false positives; this involved using unc-119 as a positive-selection marker and GFP as a counter-selection marker which is lost during homologous recombination. This method of gene targeting allows straightforward screening for homologous events using a dissecting microscope equipped for fluorescence. In addition, to improve the final engineered product, we utilised Flp recombinase to remove the unc-119 selection marker, in somatic cells, producing clean knockouts in these cells. Using this strategy we have produced a knockout of the plc-4 gene, which encodes phospholipase C-δ in C. elegans, and demonstrated that conditional gene knockout is feasible in C. elegans.     

Metabolic engineering of Torulopsis glabrata for improved pyruvate production

During pyruvate production, ethanol is produced as a by-product, which both decreases the amount of pyruvate and makes the recovery of pyruvate more difficult. Pyruvate decarboxylase (PDC, EC 4.1.1.1), which degrades pyruvate to acetaldehyde and ultimately to ethanol, is a key enzyme in the pyruvate metabolism of yeast. Therefore, to order to increase the yield of pyruvate in Torulopsis glabrata, targeted PDC-disrupted strains were metabolically engineered. First, T. glabrata ura3 strains that were suitable for genetic transformation were isolated and identified through ethyl methansulfonate mutagenesis, 5-fluoroortic acid media selection, and Sacchramyces cerevisiae URA3 complement. Next, the PDC gene in T. glabrata was specifically disrupted through homologous recombinant with the S. cerevisiae URA3 gene as the selective marker. The PDC activity of the disruptants was about 33% that of the parent strain. Targeted PDC gene disruption in T. glabrata was also confirmed by PCR amplification and sequencing of the PDC gene and its mutants, PDC activity staining, and PDC Western blot. The disruptants displayed higher pyruvate accumulation and less ethanol production. Under basal fermentation conditions (see Section 2), the disruptants accumulated about 20 g/L of pyruvate with 4.6 g/L of ethanol, whereas the parental strain (T. glabrata IFO005) only accumulated 7–8 g/L of pyruvate with 7.4 g/L of ethanol. Under favorable conditions in jar fermentation, the disruptants accumulated 82.2 g/L of pyruvate in 52 h.     

Hydrogen Metabolism in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultative sediment microorganism which uses diverse compounds, such as oxygen and fumarate, as well as insoluble Fe(III) and Mn(IV) as electron acceptors. The electron donor spectrum is more limited and includes metabolic end products of primary fermenting bacteria, such as lactate, formate, and hydrogen. While the utilization of hydrogen as an electron donor has been described previously, we report here the formation of hydrogen from pyruvate under anaerobic, stationary-phase conditions in the absence of an external electron acceptor. Genes for the two S. oneidensis MR-1 hydrogenases, hydA, encoding a periplasmic [Fe-Fe] hydrogenase, and hyaB, encoding a periplasmic [Ni-Fe] hydrogenase, were found to be expressed only under anaerobic conditions during early exponential growth and into stationary-phase growth. Analyses of ΔhydA, ΔhyaB, and ΔhydA ΔhyaB in-frame-deletion mutants indicated that HydA functions primarily as a hydrogen-forming hydrogenase while HyaB has a bifunctional role and represents the dominant hydrogenase activity under the experimental conditions tested. Based on results from physiological and genetic experiments, we propose that hydrogen is formed from pyruvate by multiple parallel pathways, one pathway involving formate as an intermediate, pyruvate-formate lyase, and formate-hydrogen lyase, comprised of HydA hydrogenase and formate dehydrogenase, and a formate-independent pathway involving pyruvate dehydrogenase. A reverse electron transport chain is potentially involved in a formate-hydrogen lyase-independent pathway. While pyruvate does not support a fermentative mode of growth in this microorganism, pyruvate, in the absence of an electron acceptor, increased cell viability in anaerobic, stationary-phase cultures, suggesting a role in the survival of S. oneidensis MR-1 under stationary-phase conditions.     

Establishment of neomycin phosphotransferase II (<Emphasis Type="Italic">nptII</Emphasis>) selection for transformation of soybean somatic embryogenic cultures

Soybean transformation is limited by the lack of multiple efficient selectable marker systems. Biolistic transformation of somatic proliferative embryogenic cultures, one of the commonly used soybean transformation methods, relies largely on hygromycin phosphotransferase II (hptII) selection. The purpose of the present study was to establish another efficient selectable marker system to facilitate multiple gene transformations of soybean. We tested neomycin phosphotransferase II (nptII) that has been used successfully in cotyledonary node transformation, but with limited success in transformation of embryogenic cultures. Transgenic events were obtained using nptII with improved G418 selection without generating escapes. G418 selection required longer recovery and selection periods, and resulted in a lower efficiency of initial transformants compared to hygromycin selection. Six independent fertile transgenic plants were recovered using nptII and G418, a frequency similar to that obtained with hygromycin selection. Soybean embryogenic cultures co-transformed with the hptII and nptII markers showed resistance to both hygromycin B and G418, while regeneration and plant fertility were not adversely affected. The nptII will be useful as a second selectable marker for multiple gene transformations in basic and applied soybean research.