Downscaling screening cultures in a multifunctional bioreactor array-on-a-chip for speeding up optimization of yeast-based lactic acid bioproduction

A key challenge for bioprocess engineering is the identification of the optimum process conditions for the production of biochemical and biopharmaceutical compounds using prokaryotic as well as eukaryotic cell factories. Shake flasks and bench-scale bioreactor systems are still the golden standard in the early stage of bioprocess development, though they are known to be expensive, time-consuming, and labor-intensive as well as lacking the throughput for efficient production optimizations. To bridge the technological gap between bioprocess optimization and upscaling, we have developed a microfluidic bioreactor array to reduce time and costs, and to increase throughput compared with traditional lab-scale culture strategies. We present a multifunctional microfluidic device containing 12 individual bioreactors (V_t = 15 µl) in a 26 mm × 76 mm area with in-line biosensing of dissolved oxygen and biomass concentration. Following initial device characterization, the bioreactor lab-on-a-chip was used in a proof-of-principle study to identify the most productive cell line for lactic acid production out of two engineered yeast strains, evaluating whether it could reduce the time needed for collecting meaningful data compared with shake flasks cultures. Results of the study showed significant difference in the strains' productivity within 3 hr of operation exhibiting a 4- to 6-fold higher lactic acid production, thus pointing at the potential of microfluidic technology as effective screening tool for fast and parallelizable industrial bioprocess development.     

Multiple factors influence the role of arbuscular mycorrhizal fungi in soil aggregation—a meta-analysis

Background and aims

Soil aggregation is a crucial aspect of ecosystem functioning in terrestrial ecosystems. Arbuscular mycorrhizal fungi (AMF) play a key role in soil aggregate formation and stabilization. Here we quantitatively analyzed the importance of experimental settings as well as biotic and abiotic factors for the effectiveness of AMF to stabilize soil macroaggregates.

Methods

We gathered 35 studies on AMF and soil aggregation and tested 13 predictor variables for their relevance with a boosted regression tree analysis and performed a meta-analysis, fitting individual random effects models for each variable.

Results and conclusions

The overall mean effect of inoculation with AMF on soil aggregation was positive and predictor variable means were all in the range of beneficial effects. Pot studies and studies with sterilized sandy soil, near neutral soil pH, a pot size smaller than 2.5 kg and a duration between 2.2 and 5 months were more likely to result in stronger effects of AMF on soil aggregation than experiments in the field, with non-sterilized or fine textured soil or an acidic pH. This is the first study to quantitatively show that the effect of AMF inoculation on soil aggregation is positive and context dependent. Our findings can help to improve the use of this important ecosystem process, e.g. for inoculum application in restoration sites.     

Honokiol: A non-adipogenic PPARγ agonist from nature

Background

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are clinically used to counteract hyperglycemia. However, so far experienced unwanted side effects, such as weight gain, promote the search for new PPARγ activators.

Methods

We used a combination of in silico, in vitro, cell-based and in vivo models to identify and validate natural products as promising leads for partial novel PPARγ agonists.

Results

The natural product honokiol from the traditional Chinese herbal drug Magnolia bark was in silico predicted to bind into the PPARγ ligand binding pocket as dimer. Honokiol indeed directly bound to purified PPARγ ligand-binding domain (LBD) and acted as partial agonist in a PPARγ-mediated luciferase reporter assay. Honokiol was then directly compared to the clinically used full agonist pioglitazone with regard to stimulation of glucose uptake in adipocytes as well as adipogenic differentiation in 3T3-L1 pre-adipocytes and mouse embryonic fibroblasts. While honokiol stimulated basal glucose uptake to a similar extent as pioglitazone, it did not induce adipogenesis in contrast to pioglitazone. In diabetic KKAy mice oral application of honokiol prevented hyperglycemia and suppressed weight gain.

Conclusion

We identified honokiol as a partial non-adipogenic PPARγ agonist in vitro which prevented hyperglycemia and weight gain in vivo.

General significance

This observed activity profile suggests honokiol as promising new pharmaceutical lead or dietary supplement to combat metabolic disease, and provides a molecular explanation for the use of Magnolia in traditional medicine.     

A structural perspective on the PP-loop ATP pyrophosphatase family

Derived from an ancient ATP-hydrolyzing Rossmann-like fold protein, members of the PP-loop ATP pyrophosphatase family feature an absolutely conserved P-loop-like “SxGxDS/T” motif used for binding and presenting ATP for substrate adenylylation (AMPylation). Since the first family member was reported more than 20 years ago, numerous representatives catalyzing very diverse reactions have been characterized both functionally and structurally. The availability of more than 100 high quality structures in the protein data bank provides an excellent opportunity to gain structural insights into the generally conserved catalytic mechanism and the uniqueness of the reactions catalyzed by family members. In this work, we conducted a thorough database search for the PP-loop ATP pyrophosphatase family members, resulting in the most comprehensive and up-to-date collection that includes 18 enzyme families. Structure comparison of representative family members allowed us to identify common structure features in the core catalytic domain, as well as four highly variable regions that define the unique chemistry for each enzyme family. The newly identified enzymes, particularly those from pathogens, warrant further research to enlarge the scope of this ever-expanding and highly diverse enzyme superfamily for use in potential bioengineering and biomedical applications.     

Studies on water transport through the sweet cherry fruit surface: II. Conductance of the cuticle in relation to fruit development

Water conductance of the cuticular membrane (CM) of sweet cherry (Prunus avium L. cv. Sam) fruit during stages II and III (31-78 days after full bloom, DAFB) was investigated by gravimetrically monitoring water loss through segments of the exocarp. Segments were mounted in stainless-steel diffusion cells, filled with 0.5 ml of deionized water and incubated for 8 h at 25 +/- 2 degrees C over dry silica. Conductance was calculated by dividing the amount of water transpired per unit surface area and time by the difference in water vapor concentration across the segment (23.07 g m(-3) at 25 degrees C). Fruit mass and fruit surface area increased 4.9- and 2.8-fold between 31 and 78 DAFB, respectively. However, CM mass per unit area decreased from 3.9 to 1.5 g m(-2) and percentage of total wax content remained constant at about 31%. Stomatal density decreased from 0.8 to 0.2 mm(-2) (31-78 DAFB). Total conductance of the CM on the fruit cheek (gtot.) remained constant during stage II of development (approx. 1.38 x 10(-4) m s(-1) from 31 to 37 DAFB), increased to 1.73 x 10(-4) m s(-1) during early stage III of fruit growth (43-64 DAFB) then decreased to 0.95 x 10(-4) m s(-1) at maturity (78 DAFB). Partitioning gtot. into cuticular (gcut.) and stomatal conductance (gsto.) revealed that the relative contribution of gcut. to gtot. increased linearly from 30% to 87% of gtot. between 31 and 78 DAFB. respectively. On a whole-fruit basis, g,tot. and gcut. consistently increased up to 64 DAFB, and decreased thereafter. A significant negative linear relationship was obtained between gcut. and CM thickness, but not between the permeability coefficient (p) and CM thickness. Further, p was positively related to strain rate, suggesting that strain associated with expansion of the fruit surface increased p.     

Evolutionary Diversification Indicated by Compensatory Base Changes in ITS2 Secondary Structures in a Complex Fungal Species, <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The rRNA cistron (18S–ITS1–5.8S–ITS2–28S) is used widely for phylogenetic analyses. Recent studies show that compensatory base changes (CBC) in the secondary structure of ITS2 correlate with genetic incompatibility between organisms. Rhizoctonia solani consists of genetically incompatible strain groups (anastomosis groups, AG) distinguished by lack of anastomosis between hyphae of strains. Phylogenetic analysis of internal transcribed spacer (ITS) sequences shows a strong correlation with AG determination. In this study, ITS sequences were reannotated according to the flanking 5.8S and 28S regions which interact during ribogenesis. One or two CBCs were detected between the ITS2 secondary structure of AG-3 potato strains as compared to AG-3 tobacco strains, and between these two strains and all other AGs. When a binucleate Rhizoctonia species related to Ceratobasidiaceae was compared to the AGs of R. solani, which were multinucleate (3–21 nuclei per cell), 1–3 CBCs were detected. The CBCs in potato strains of AG-3 distinguish them from AG-3 tobacco strains and other AGs yielding further evidence that the potato strains of AG-3 originally described as R. solani are a species distinct from other AGs. The ITS1–5.8S–ITS2 sequences were analyzed by direct sequencing of PCR products from 497 strains of AG-3 isolated from potato. The same 10 and 4 positions in ITS1 and ITS2, respectively, contained variability in 425 strains (86%). Nine different unambiguous ITS sequences (haplotypes) could be detected in a single strain by sequencing cloned PCR products indicating that concerted evolution had not homogenized the rRNA cistrons in many AG-3 strains. Importantly, the sequence variability did not affect the secondary structure of ITS2 and CBCs in AG-3. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Association of fucoxanthin chlorophyll a/c-binding polypeptides with photosystems and phosphorylation in the centric diatom Cyclotella cryptica

Solubilization of thylakoid membranes of Cyclotella cryptica with dodecyl-beta maltoside followed by sucrose density gradient centrifugation or deriphate polyacrylamide gel electrophoresis resulted in the isolation of pigment protein complexes. These complexes were characterized by absorption and fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western immunoblotting using antisera against fucoxanthin chlorophyll a/c-binding proteins and the reaction center protein D2 of photosystem II. Sucrose density gradient centrifugation yielded four bands. Band 1 consisted of free pigments with minor amounts of fucoxanthin chlorophyll a/c-binding proteins. Bands 2, 3, and 4 represented a major fucoxanthin chlorophyll a/c-binding protein fraction, photosystem II, and photosystem I, respectively. Deriphate polyacrylamide gel electrophoresis gave rise to five bands, representing photosystem I, photosystem II, two fucoxanthin chlorophyll a/c-binding protein complexes, and a band mostly consisting of free pigments. In the Western immunoblotting experiments, the specific association of two fucoxanthin chlorophyll a/c-binding proteins, Fcp2 and Fcp4, to the photosystems could be demonstrated. In vivo experiments using antibodies against phosphothreonine residues and in vitro studies using [gamma-32P]ATP showed that fucoxanthin chlorophyll a/c binding-proteins of 22 kDa became phosphorylated.     

Structural basis of Rnd1 binding to plexin Rho GTPase binding domains (RBDs)

Plexin receptors regulate cell adhesion, migration, and guidance. The Rho GTPase binding domain (RBD) of plexin-A1 and -B1 can bind GTPases, including Rnd1. By contrast, plexin-C1 and -D1 reportedly bind Rnd2 but associate with Rnd1 only weakly. The structural basis of this differential Rnd1 GTPase binding to plexin RBDs remains unclear. Here, we solved the structure of the plexin-A2 RBD in complex with Rnd1 and the structures of the plexin-C1 and plexin-D1 RBDs alone, also compared with the previously determined plexin-B1 RBD.Rnd1 complex structure. The plexin-A2 RBD·Rnd1 complex is a heterodimer, whereas plexin-B1 and -A2 RBDs homodimerize at high concentration in solution, consistent with a proposed model for plexin activation. Plexin-C1 and -D1 RBDs are monomeric, consistent with major residue changes in the homodimerization loop. In plexin-A2 and -B1, the RBD β3-β4 loop adjusts its conformation to allow Rnd1 binding, whereas minimal structural changes occur in Rnd1. The plexin-C1 and -D1 RBDs lack several key non-polar residues at the corresponding GTPase binding surface and do not significantly interact with Rnd1. Isothermal titration calorimetry measurements on plexin-C1 and -D1 mutants reveal that the introduction of non-polar residues in this loop generates affinity for Rnd1. Structure and sequence comparisons suggest a similar mode of Rnd1 binding to the RBDs, whereas mutagenesis suggests that the interface with the highly homologous Rnd2 GTPase is different in detail. Our results confirm, from a structural perspective, that Rnd1 does not play a role in the activation of plexin-C1 and -D1. Plexin functions appear to be regulated by subfamily-specific mechanisms, some of which involve different Rho family GTPases.     

The maize lipoxygenase,ZmLOX10, mediates green leaf volatile,jasmonate and herbivore‐induced plant volatile production for defense against insect attack

Fatty acid derivatives are of central importance for plant immunity against insect herbivores; however, major regulatory genes and the signals that modulate these defense metabolites are vastly understudied, especially in important agro‐economic monocot species. Here we show that products and signals derived from a single Zea mays (maize) lipoxygenase (LOX), ZmLOX10, are critical for both direct and indirect defenses to herbivory. We provide genetic evidence that two 13‐LOXs, ZmLOX10 and ZmLOX8, specialize in providing substrate for the green leaf volatile (GLV) and jasmonate (JA) biosynthesis pathways, respectively. Supporting the specialization of these LOX isoforms, LOX8 and LOX10 are localized to two distinct cellular compartments, indicating that the JA and GLV biosynthesis pathways are physically separated in maize. Reduced expression of JA biosynthesis genes and diminished levels of JA in lox10 mutants indicate that LOX10‐derived signaling is required for LOX8‐mediated JA. The possible role of GLVs in JA signaling is supported by their ability to partially restore wound‐induced JA levels in lox10 mutants. The impaired ability of lox10 mutants to produce GLVs and JA led to dramatic reductions in herbivore‐induced plant volatiles (HIPVs) and attractiveness to parasitoid wasps. Because LOX10 is under circadian rhythm regulation, this study provides a mechanistic link to the diurnal regulation of GLVs and HIPVs. GLV‐, JA‐ and HIPV‐deficient lox10 mutants display compromised resistance to insect feeding, both under laboratory and field conditions, which is strong evidence that LOX10‐dependent metabolites confer immunity against insect attack. Hence, this comprehensive gene to agro‐ecosystem study reveals the broad implications of a single LOX isoform in herbivore defense.