Towards the characterization of squalene synthase activity in extracts of Zymomonas mobilis

Abstract Extracts of Zymomonas mobilis in the presence of NADPH converted tritium-labelled farnesyl diphosphate (FPP) into squalene, resulting from the activity of squalene synthase, as well as diploptene and diplopterol, derived from further squalene cyclisation. An unidentified isoprenoid representing up to 70% of the conversion products of FPP and different from presqualene alcohol was also formed, even in the absence of NADPH. Addition of squalestatin 1, an inhibitor of squalene synthase, blocked biosynthesis from FPP of the three former triterpenes, in accordance with the role of squalene synthase in their formation, as well as that of the unknown compound.     

Reduction of xylose to xylitol catalyzed by glucose–fructose oxidoreductase from Zymomonas mobilis

Genetic improvements of Zymomonas mobilis for pentose utilization have a huge potential in fuel ethanol production. The production of xylitol and the resulting growth inhibition by xylitol phosphate have been considered to be one of the important factors affecting the rates and yields from xylose metabolism by the recombinant Z. mobilis , but the mechanism of xylitol formation is largely unknown. Here, we reported that glucose–fructose oxidoreductase (GFOR), a periplasmic enzyme responsible for sorbitol production, catalyzed the reduction of xylose to xylitol in vitro , operating via a ping-pong mechanism similar to that in the formation of sorbitol. However, the specific activity of GFOR for sorbitol was higher than that for xylitol (68.39 vs. 1.102 μmol min⁻¹ mg⁻¹), and an apparent substrate-induced positive cooperativity occurred during the catalyzed formation of xylitol, with the Hill coefficient being about 2. While a change of the potential acid–base catalyst Tyr269 to Phe almost completely abolished the activity toward xylose as well as fructose, mutant S116D, which has been shown to lose tight cofactor binding, displayed an even slower catalytic process against xylose.     

Xanthan gum and ethanol production by Xanthomonas campestris and Zymomonas mobilis from peach pulp

The wild type of Xanthomonas campestris and a mutant strain of Zymomonas mobilis CP4, tolerant to sucrose up to 40% (w/v), were used to produce either xanthan gum or ethanol, respectively, from peach pulp supplemented with different salts. Both bacteria grew well (2.7 mg/ml for X. campestris and 1.45 mg/ml for Z. mobilis) in fine peach pulp and the production of xanthan gum or ethanol was 0.1–0.2 g/l or 110 g/l, respectively.     

Disruption of the Zymomonas mobilis extracellular sucrase gene (sacC) improves levan production

AIMS: Disruption of the extracellular Zymomonas mobilis sucrase gene (sacC) to improve levan production. METHODS AND RESULTS: A PCR-amplified tetracycline resistance cassette was inserted within the cloned sacC gene in pZS2811. The recombinant construct was transferred to Z. mobilis by electroporation. The Z. mobilis sacC gene, encoding an efficient extracellular sucrase, was inactivated. A sacC defective mutant of Z. mobilis, which resulted from homologous recombination, was selected and the sacC gene disruption was confirmed by PCR. Fermentation trials with this mutant were conducted, and levansucrase activity and levan production were measured. In sucrose medium, the sacC mutant strain produced threefold higher levansucrase (SacB) than the parent strain. This resulted in higher levels of levan production, whilst ethanol production was considerably decreased. CONCLUSIONS: Zymomonas mobilis sacC gene encoding an extracellular sucrase was inactivated by gene disruption. This sacC mutant strain produced higher level of levan in sucrose medium because of the improved levansucrase (SacB) than the parent strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The Z. mobilis CT2, sacC mutant produces high level of levansucrase (SacB) and can be used for the production of levan.     

Localisation of the glucose-fructose oxidoreductase in wild type and overproducing strains of Zymomonas mobilis

Abstract The enzyme glucose-fructose oxidoreductase (GFOR) from the Gram-negative ethanologenic bacterium Zymomonas mobilis was purified to homogeneity and was shown to be a tetrameric protein with a subunit size of M _r 42 500. Using immunogold-labelling in combination with electron microscopy, ultrathin sections of Z. mobilis wild type cells showed that the enzyme GFOR is located in the periplasm off the bacterial cells. Z. mobilis strains which carried the cloned gfo gene on plasmid pSUP104, had 5–6-fold increased GFOR enzyme activities. Moreover, these cells accumulated large amounts of a presumable unprocessed pre-GFOR protein ( M _r 48 000).     

Using the CRISPR/Cas9 system to eliminate native plasmids of Zymomonas mobilis ZM4

The CRISPR/Cas system can be used to simply and efficiently edit the genomes of various species, including animals, plants, and microbes. Zymomonas mobilis ZM4 is a highly efficient, ethanol-producing bacterium that contains five native plasmids. Here, we constructed the pSUZM2a-Cas9 plasmid and a single-guide RNA expression plasmid. The pSUZM2a-Cas9 plasmid was used to express the Cas9 gene cloned from Streptococcus pyogenes CICC 10464. The single-guide RNA expression plasmid pUC-T7sgRNA, with a T7 promoter, can be used for the in vitro synthesis of single-guide RNAs. This system was successfully employed to knockout the upp gene of Escherichia coli and the replicase genes of native Z. mobilis plasmids. This is the first study to apply the CRISPR/Cas9 system of S. pyogenes to eliminate native plasmids in Z. mobilis. It provides a new method for plasmid curing and paves the way for the genomic engineering of Z. mobilis.     

Biphasic aqueous/organic biotransformation of acetaldehyde and benzaldehyde by Zymomonas mobilis pyruvate decarboxylase

Zymomonas mobilis pyruvate decarboxylase (PDC) transformed acetaldehyde and benzaldehyde into (R)-phenylacetylcarbinol (PAC), the precursor for the synthesis of ephedrine and pseudoephedrine. Organic solvents were screened for a biphasic biotransformation with the enzyme in an aqueous phase and the toxic substrates delivered through the organic phase. In the absence of substrates a second phase of 1-pentanol, hexadecane or MTBE (methyl tertiary-butyl ether) stabilized the PDC activity in comparison to a control without added solvent. Organic phase solvents for optimal PAC production had partitioning coefficient (log P) values between 0.8 and 2.8 (production of more than 8 mg PAC/ U PDC), however there was no correlation between enzyme stability and log P. Best PAC formation was observed with the eight tested alcohols, which in contrast to the other solvents allowed lower initial concentrations of toxic acetaldehyde (54-81 mM) in the aqueous phase. 1-pentanol, 1-hexanol, and isobutanol resulted in the highest specific PAC production of 11 mg PAC /U PDC. Without the addition of an organic phase, only 1.2 mg/U was formed.     

Construction of a novel secretion expression system guided by native signal peptide of PhoD in Zymomonas mobilis

In the current study, three native signal peptides (SPs) from PhoC, PhoD, and ZMO0331were investigated and compared to construct novel secretion expression systems in Zymomonas mobilis. The secretion expression of target protein, α-amylase from Bacillus amyloliquefaciens (BAA), guided by PhoD’s SP resulted in more hydrolysis of starch than that by the other two SPs. Extracellular and intracellular α-amylase activities of the strain containing PhoD’s SP were also higher than the other two strains containing PhoC or ZMO0331’s SP. In addition, the evidence by alcohol dehydrogenase activity assay further confirmed that the starch hydrolysis was resulted from the secretion expression of BAA rather than the breakage of cells. Our results indicated that the SP of PhoD is able to serve as a promising candidate to assist secretion expression of heterogeneous genes in Z. mobilis. This will contribute to development of engineered Z. mobilis strains converting starch into ethanol.     

Inhibition analysis of inhibitors derived from lignocellulose pretreatment on the metabolic activity of Zymomonas mobilis biofilm and planktonic cells and the proteomic responses

Lignocellulose pretreatment produces various toxic inhibitors that affect microbial growth, metabolism, and fermentation. Zymomonas mobilis is an ethanologenic microbe that has been demonstrated to have potential to be used in lignocellulose biorefineries for bioethanol production. Z. mobilis biofilm has previously exhibited high potential to enhance ethanol production by presenting a higher viable cell number and higher metabolic activity than planktonic cells or free cells when exposed to lignocellulosic hydrolysate containing toxic inhibitors. However, there has not yet been a systematic study on the tolerance level of Z. mobilis biofilm compared to planktonic cells against model toxic inhibitors derived from lignocellulosic material. We took the first insight into the concentration of toxic compound (formic acid, acetic acid, furfural, and 5‐HMF) required to reduce the metabolic activity of Z. mobilis biofilm and planktonic cells by 25% (IC₂₅), 50% (IC₅₀), 75% (IC₇₅), and 100% (IC₁₀₀). Z. mobilis strains ZM4 and TISTR 551 biofilm were two‐ to three fold more resistant to model toxic inhibitors than planktonic cells. Synergetic effects were found in the presence of formic acid, acetic acid, furfural, and 5‐HMF. The IC₂₅ of Z. mobilis ZM4 biofilm and TISTR 551 biofilm were 57 mm formic acid, 155 mm acetic acid, 37.5 mm furfural and 6.4 mm 5‐HMF, and 225 mm formic acid, 291 mm acetic acid, 51 mm furfural and 41 mm 5‐HMF, respectively. There was no significant difference found between proteomic analysis of the stress response to toxic inhibitors of Z. mobilis biofilm and planktonic cells on ZM4. However, TISTR 551 biofilms exhibited two proteins (molecular chaperone DnaK and 50S ribosomal protein L2) that were up‐regulated in the presence of toxic inhibitors. TISTR 551 planktonic cells possessed two types of protein in the group of 30S ribosomal proteins and motility proteins that were up‐regulated.     

Mathematical modelling of ethanol production from glucose/xylose mixtures by recombinant Zymomonas mobilis

A model has been developed for the fermentation of mixtures of glucose and xylose by recombinant Zymomonas mobilis strain ZM4(pZB5), containing additional genes for xylose assimilation and metabolism. A two-substrate model based on substrate limitation, substrate inhibition, and product (ethanol) inhibition was evaluated, and experimental data was compared with model simulations using a Microsoft EXCEL based program and methods of statistical analysis for error minimization. From the results it was established that the model provides good predictions of experimental batch culture data for 25/25, 50/50, and 65/65 g l^–1 glucose/xylose media.     

Exceptional characteristics of heterotetrameric (α2β2) E1p of the pyruvate dehydrogenase complex from Zymomonas mobilis: expression from an own promoter and a lipoyl domain in E1β

In the pyruvate dehydrogenase complex (PDHC) of Zymomonas mobilis the beta subunit of the pyruvate dehydrogenase (E1p) as well as the acetyltransferase (E2p) contain an N-terminal lipoyl domain. Both lipoyl domains were acetylated in vitro using 2-14C-pyruvate as a substrate, demonstrating that both lipoyl domains can accept acetyl groups from the E1 component. As previously shown the structural genes (pdhA alpha beta, pdhB, lpd) encoding the pyruvate dehydrogenase complex of Z. mobilis are located in two distinct gene clusters, pdhA alpha beta and pdhB-orf2-lpd (U. Neveling et al. (1998) J. Bacteriol. 180, 1540-1548). Analysis of pdh gene expression using lacZ fusions revealed that the DNA fragments upstream of pdhA alpha, pdhB and lpd each have promoter activities. These pdh promoter activities were 7-30-fold higher in Z. mobilis than in Escherichia coli.     

Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging

Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K- and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH. Both single amino acid replacements, M304R- and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.     

Structural insights into quinolone antibiotic resistance mediated by pentapeptide repeat proteins: conserved surface loops direct the activity of a Qnr protein from a gram-negative bacterium

Quinolones inhibit bacterial type II DNA topoisomerases (e.g. DNA gyrase) and are among the most important antibiotics in current use. However, their efficacy is now being threatened by various plasmid-mediated resistance determinants. Of these, the pentapeptide repeat-containing (PRP) Qnr proteins are believed to act as DNA mimics and are particularly prevalent in gram-negative bacteria. Predicted Qnr-like proteins are also present in numerous environmental bacteria. Here, we demonstrate that one such, Aeromonas hydrophila AhQnr, is soluble, stable, and relieves quinolone inhibition of Escherichia coli DNA gyrase, thus providing an appropriate model system for gram-negative Qnr proteins. The AhQnr crystal structure, the first for any gram-negative Qnr, reveals two prominent loops (1 and 2) that project from the PRP structure. Deletion mutagenesis demonstrates that both contribute to protection of E. coli DNA gyrase from quinolones. Sequence comparisons indicate that these are likely to be present across the full range of gram-negative Qnr proteins. On this basis we present a model for the AhQnr:DNA gyrase interaction where loop1 interacts with the gyrase A 'tower' and loop2 with the gyrase B TOPRIM domains. We propose this to be a general mechanism directing the interactions of Qnr proteins with DNA gyrase in gram-negative bacteria.     

A new H2 / CO2-using acetogenic bacterium from the rumen: Description of Ruminococcus schinkii sp. nov.

Abstract Two strains of H2 / CO2-using acetogenic bacteria were isolated from the rumen of suckling lambs. Both strains displayed a coccobacillar morphology and possessed a Gram-positive type cell wall. Numerous organic substrates, including some O-methylated aromatic compounds, were used heterotrophically. 16S rRNA gene sequencing demonstrated that the two acetogenic isolates were phylogenetically identical and represent a new subline within Clostridium cluster XIVa. Based on phenotypic and phylogenetic considerations a new species, Ruminococcus schinkii sp. nov., is proposed.     

Mutagenesis by random cloning of an Escherichia coli kanamycin resistance gene into the genome of the cyanobacterium Synechocystis PCC 6803: Selection of mutants defective in photosynthesis

Summary Photosynthetic mutants of the cyanobacterium Synechocystis PCC 6803 were produced by a random cartridge mutagenesis method leading to gene inactivation. This procedure relies on random ligation of an Escherichia coli kanamycin resistance (Km^r) gene to restriction fragments of genomic DNA from the host. Then recombination occurring during transformation promotes integration of the marker gene into the genome of the recipient cells. Several mutants impaired in photosynthesis were obtained by this procedure. All are partially or totally defective in photosystem II activity and some of them also harbour a functionally modified photosystem I. Restriction and recombination data showed that one mutant (AK1) is best explained as an insertion of the Km^r gene into an AvaII restriction site of the gene psbD-1. All other harbour a deletion, ranging from at least 1.15 kb (AK3) to more than 50 kb (AK9), which partly or fully overlaps the genes psbB and/or psbD-1, depending on the mutant. A genetic-physical map of the more than 60 kb region of the cyanobacterial genome harbouring the genes psbB, psbC and psbD-1 was constructed by combining published sequence data on these genes with the results of recombination and restriction mapping.     

Biochemical assessment of the taxonomic diversity of the operculate land snail, Cyclophorus fulguratus (Gastropoda: Cyclophoridae), from Thailand

Allozyme variation was examined in 223 samples of the operculate land snail Cyclophorus fulguratus from 13 localities across three regions of Thailand. Using horizontal starch gel electrophoresis, 13 allozyme presumed loci (12 polymorphic) were screened. Heterozygosity was moderate in C. fulguratus (H_exp = 0.008–0.127) with a high genetic heterogeneity among samples (F_st = 0.734). Populations showed a greater genetic differentiation in central Thailand (F_st = 0.380) than in northeastern Thailand (F_st = 0.108), suggesting frequent gene flow among populations in northeastern Thailand. C. fulguratus exhibits a strong pattern of isolation by distance over the entire tested species range in Thailand and may potentially have been involved in an extensive local fragmentation. Results of the distance analysis revealed that large genetic divergence has occurred among the central, northeastern and eastern Thailand groups [D = 0.361–0.701], strongly suggesting populations from these three geographical regions may actually represent or else be evolving into separate species.     

Metabolism of cyclic ADP-ribose: Zinc is an endogenous modulator of the cyclase/NAD glycohydrolase ratio of a CD38-like enzyme from human seminal fluid

CD38, a bifunctional enzyme capable of both synthesis and hydrolysis of the second messenger cyclic ADP-ribose (cADPR). Using the natural substrate of the enzyme, NAD+, the ratio of ADP-ribosyl cyclase/NAD glycohydrolase of CD38 is about 1/100. Here we describe that human seminal fluid contain a soluble CD38 like enzyme with an apparent M.W. of 49 kDa. When purified this enzyme has a cyclase/NAD glycohydrolase ratio of about 1/120. However, the in situ cyclase/NAD glycohydrolase ratio measured in seminal plasma approaches 1/1. We also found that physiological concentrations of zinc present in the seminal fluid, in the range of 0.6 to 4 mM, are responsible for the modulation of the cyclase/NAD glycohydrolase ratio. This new information indicates that the cyclase/NAD glycohydrolase ratio can be modified in vivo.     

Relevance of metal ions for lipase stability: Structural rearrangements induced in the Burkholderia glumae lipase by calcium depletion

We have studied the accessibility of the structural calcium ion in the Burkholderia glumae lipase and the consequences of its removal on the protein conformation by different biophysical techniques (circular dichroism, fluorimetry, and mass spectrometry) and by molecular-dynamics simulations. We show that, in the native protein, calcium is not accessible unless specific flexible loops are displaced, for example, by a temperature increase. Such movements concern the whole calcium-binding pocket and particularly the environment of the coordinating aspartate residue 241. As a consequence of metal depletion the protein unfolds irreversibly and undergoes aggregation. The removal of the metal ion causes major structural transitions and leads to an increase in β-structure, in particular in protein regions that are largely unstructured in the native protein and encompass the calcium coordination residues.