Diversity and function of microbial communities in the sand sheath of Agropyron cristatum by metagenomic analysis

The roots of most gramineous plants are surrounded by a variety of microorganisms; however, few studies have focused on the rhizosheath of psammophytes. Therefore, in this study, we used Illumina HiSeq high-throughput sequencing technology to analyse the composition and functional diversity of microbial communities in the rhizosheath of sand-grown Agropyron cristatum (L.) Gaertn. We found that the number of species and functions of microbial communities gradually decreased from the rhizosheath to the bulk soil. Thus, the microbial composition of the rhizosheath was richer and more diverse, and the abundance of bacteria, including Sphingosinicella, Rhizorhabdus, Friedmanniella, Geodermatophilus, Blastococcus, and Oscillatoria, was higher, and the abundance of fungi, such as Mycothermus, was higher. The abundance of CO2 fixation-related genes (acsA, Pcc, and cbbL) in the carbon cycle; NO3–, NO2–, NH2OH, and N2 transformation genes (nrtP, nirS, hao, and nifK) in the nitrogen cycle; soxB/A/C, Sat, and dsrB genes in the sulphur cycle; and 1-phosphate mannitol dehydrogenase (MtlD) gene and polyketide synthase gene (pks) were higher in the rhizosheath than in the bulk soil, as well as genes related to phosphorus uptake in the phosphorus cycle. Our findings showed that the rhizosheath may host the predominant microbial species related to the formation of a rhizosheath.

Model-Based Optimization of Mannitol Production by Using a Sequence of Batch Reactors for a Coupled Bi-Enzymatic Process—A Dynamic Approach

Multi-enzymatic reactions can successfully replace complex chemical syntheses, using milder reaction conditions, and generating less waste. The present model-based analysis compares the performances of several optimally operated Batch Reactors (BR) with those of an optimally operated serial Sequence of BRs (SeqBR). In multi-enzymatic systems, SeqBR could be more advantageous and flexible, allowing the optimization of costly enzymes amounts used in each BR in the series. Exemplification was made for the bi-enzymatic reduction of D-fructose to mannitol by using MDH (mannitol dehydrogenase) and the NADH cofactor, with the in situ continuous regeneration of NADH at the expense of formate degradation in the presence of FDH (formate dehydrogenase). For such coupled enzymatic systems, the model-based engineering evaluations are difficult tasks, because they must account for the common species’ initial levels, their interaction, and their dynamics. The determination of optimal operating modes of sole BR or of a SeqBR turns into a multi-objective optimization problem with multiple constraints to be solved for every particular system. The study presents multiple elements of novelty: (i) the proof of higher performances of an optimal SeqBR (including N-BRs) compared to a sole optimal BR operated for N-number of runs and (ii) the effect of using a multi-objective optimization criteria on SeqBR adjustable dynamics.

Promising Pathway of Thermostable Mannitol Dehydrogenase (MtDH) from Caldicellulosiruptor hydrothermalis 108 for D-Mannitol Synthesis

In this study, we conducted the characterization and purification of the thermostable mannitol dehydrogenase (MtDH) from Caldicellulosiruptor hydrothermalis 108. Furthermore, a coupling-enzyme system was designed using (MtDH) from Caldicellulosiruptor hydrothermalis 108 and formate dehydrogenase (FDH) from Ogataea parapolymorpha. The biotransformation system was constructed using Escherichia coli whole cells. The purified enzyme native and subunit molecular masses were 76.7 and 38 kDa, respectively, demonstrating that the enzyme was a dimer. The purified and couple enzyme system results were as follows; the optimum pH for the reduction and the oxidation was 7.0 and 8.0, the optimum temperature was 60 °C, the enzyme activity was inhibited by EDTA and restored by zinc. Additionally, no activity was detected with NADPH and NADP. The purified enzyme showed high catalytic efficiency Kcat 385 s−1, Km 31.8 mM, and kcat/Km 12.1 mM−1 s−1 for D-fructose reduction. Moreover, the purified enzyme retained 80%, 75%, 60%, and 10% of its initial activity after 4 h at 55, 60, 65, and 75 °C, respectively. D-mannitol yield was achieved via HPLC. Escherichia coli are the efficient biotransformation mediator to produce D-mannitol (byproducts free) at high temperature and staple pH, resulting in a significant D-mannitol conversation (41 mg/mL) from 5% D-fructose.

Genetic evidences for the core biosynthesis pathway, regulation, transport and secretion of liamocins in yeast-like fungal cells

So far, it has been still unknown how liamocins are biosynthesized, regulated, transported and secreted. In this study, a highly reducing polyketide synthase (HR-PKS), a mannitol- 1-phosphate dehydrogenase (MPDH), a mannitol dehydrogenase (MtDH), an arabitol dehydrogenase (ArDH) and an esterase (Est1) were found to be closely related to core biosynthesis of extracellular liamocins in Aureobasidium melanogenum 6-1-2. The HR-PKS was responsible for biosynthesis of 3,5-dihydroxydecanoic acid. The MPDH and MtDH were implicated in mannitol biosynthesis and the ArDH was involved in arabitol biosynthesis. The Est1 catalyzed ester bond formation of them. A phosphopantetheine transferase (PPTase) activated the HR-PKS and a transcriptional activator Ga11 activated expression of the PKS1 gene. Therefore, deletion of the PKS1 gene, all the three genes encoding MPDH, MtDH and ArDH, the EST1, the gene responsible for PPTase and the gene for Ga11 made all the disruptants (Dpks13, Dpta13, Dest1, Dp12 and Dg11) totally lose the ability to produce any liamocins. A GLTP gene encoding a glycolipid transporter and a MDR1 gene encoding an ABC transporter took part in transport and secretion of the produced liamocins into medium. Removal of the GLTP gene and the MDR1 gene resulted in a Dgltp1 mutant and a Dmdr16 mutant, respectively, that lost the partial ability to secrete liamocins, but which cells were swollen and intracellular lipid accumulation was greatly enhanced. Hydrolysis of liamocins released 3,5-dihydroxydecanoic acid, mannitol, arabitol and acetic acid. We proposed a core biosynthesis pathway, regulation, transport and secretion of liamocins in A. melanogenum.

Fermentation conditions for mannitol biosynthesis by lactobacillus fermentum hf08

Mannitol is a six-carbon sugar alcohol that is claimed to have several health promoting effects (low-caloric, low-glycemic, low-insulinemic, anticariogenic, and prebiotic). Due to its low hygroscopic character, it widely used in food and pharmaceutical industries. In food industry, mannitol is used as sugar replacers because of their taste and sweetness. It is nonmetabolizable sweeteners which do not affect insulin levels making it applicable in diabetic food products. Among mannitol production methods including: chemical, enzyme and fermentation, the conversion of fructose to mannitol by lactic acid bacteria fermentation is the best way because of no requirement for highly purified substrates, making pure product and easy to produce in industry scale. There are many groups of microorganisms capable of fermenting mannitol biosynthesis, including lactic acid bacteria group, because of their conversion of fructose to mannitol by mannitol dehydrogenase with high mannitol content and low byproducts. In the study, we researched on conditions of fermentation for mannitol biosynthesis by Lactobacillus fermentum HF08. Mannitol production of the strain was reached to the maximum 93.1-93.2 g/l after 48 hours of fermentation in an appropriate medium (g/l): pepton 7.0; glucose/fructose 50/100; yeast extract 2.0; K2HPO4 2.0; MgSO4.5H2O 0.2; MnSO4 0.01. The pH of the medium fermentation for the mannitol production was 5.0-5.5. Suitable temperature for mannitol production was 35-37oC.

Modular Simulation to Determine the Optimal Operating Policy of a Batch Reactor for the Enzymatic Fructose Reduction to Mannitol with the in situ Continuous Enzymatic Regeneration of the NAD Cofactor

Novel coupled enzymatic systems reported important applications in the industrial bio-catalysis. Multi-enzymatic reactions can successfully replace complex chemical syntheses, using milder reaction conditions, and generating less waste. For such systems acting simultaneously, the model-based engineering calculations (design, reactor operation optimization) are difficult tasks, because they must account for interacting reactions, differences in enzymes optimal activity domains and deactivation kinetics. The determination of the optimal operating mode (enzyme ratios, enzyme feeding policy, temperature, pH) often turns into a difficult multi-objective optimization problem with multiple constraints to be solved for every particular system. The paper focuses on applying a modular screening procedure that can identify the optimal operating policy of an enzymatic reactor, which minimizes the enzyme consumption, given the process kinetic model, and an imposed production capacity. Following an optimization procedure, the process effectiveness is evaluated in a systematic approach, by including simple batch reactor (BR), batch with intermittent addition of the key-enzyme following certain optimal policies (BRP). Exemplification is made for the case of the enzymatic reduction of D-fructose to mannitol by using suspended MDH (mannitol dehydrogenase) and NADH (Nicotinamide adenine dinucleotide) cofactor, with the in-situ continuous regeneration of the cofactor by the expense of formate degradation in the presence of suspended FDH (Formate dehydrogenase).

A Genomic View of Lactobacilli and Pediococci Demonstrates that Phylogeny Matches Ecology and Physiology

ABSTRACTLactobacilli are used widely in food, feed, and health applications. The taxonomy of the genusLactobacillus, however, is confounded by the apparent lack of physiological markers for phylogenetic groups of lactobacilli and the unclear relationships between the diverse phylogenetic groups. This study used the core and pan-genomes of 174 type strains ofLactobacillusandPediococcusto establish phylogenetic relationships and to identify metabolic properties differentiating phylogenetic groups. The core genome phylogenetic tree separated homofermentative lactobacilli and pediococci from heterofermentative lactobacilli. Aldolase and phosphofructokinase were generally present in homofermentative but not in heterofermentative lactobacilli; a two-domain alcohol dehydrogenase and mannitol dehydrogenase were present in most heterofermentative lactobacilli but absent in most homofermentative organisms. Other genes were predominantly present in homofermentative lactobacilli (pyruvate formate lyase) or heterofermentative lactobacilli (lactaldehyde dehydrogenase and glycerol dehydratase). Cluster analysis of the phylogenomic tree and the average nucleotide identity grouped the genusLactobacillus sensu latointo 24 phylogenetic groups, including pediococci, with stable intra- and intergroup relationships. Individual groups may be differentiated by characteristic metabolic properties. The link between phylogeny and physiology that is proposed in this study facilitates future studies on the ecology, physiology, and industrial applications of lactobacilli.

Acquired Resistance to Mefenoxam in Sensitive Isolates of Phytophthora infestans

The systemic fungicide mefenoxam has been important in the control of late blight disease caused by Phytophthora infestans. This phenylamide fungicide has a negative effect on the synthesis of ribosomal RNA; however, the genetic basis for inherited field resistance is still not completely clear. We recently observed that a sensitive isolate became tolerant after a single passage on mefenoxam-containing medium. Further analyses revealed that all sensitive isolates tested (in three diverse genotypes) acquired this resistance equally quickly. In contrast, isolates that were “resistant” to mefenoxam in the initial assessment (stably resistant) did not increase in resistance upon further exposure. However, there appeared to be a cost associated with acquired resistance in the initially sensitive isolates, in that isolates with acquired resistance grew more slowly on mefenoxam-free medium than did the same isolates that had never been exposed to mefenoxam. The acquired resistance of the sensitive isolates declined slightly with subsequent culturing on medium free of mefenoxam. To investigate the mechanism of acquired resistance, we employed strand-specific RNA sequencing. Many differentially expressed genes were genotype specific, but one set of genes was differentially expressed in all genotypes. Among these were several genes (a phospholipase “Pi-PLD-like-3,” two ATP-binding cassette superfamily [ABC] transporters, and a mannitol dehydrogenase) that were up-regulated and whose function might contribute to a resistance phenotype.