Sequence-based bioprospecting of myo-inositol oxygenase (Miox) reveals new homologues that increase glucaric acid production in Saccharomyces cerevisiae.

myo-Inositol oxygenase (Miox) is a rate-limiting enzyme for glucaric acid production via microbial fermentation. The enzyme converts myo-inositol to glucuronate, which is further converted to glucaric acid, a natural compound with industrial uses that range from detergents to pharmaceutical synthesis to polymeric materials. More than 2,000 Miox sequences are available in the Uniprot database but only thirteen are classified as reviewed in Swiss-Prot (August 2019). In this study, sequence similarity networks were used to identify new homologues to be expressed in Saccharomyces cerevisiae for glucaric acid production. The expression of four homologues did not lead to product formation. Some of these enzymes may have a defective "dynamic lid" - a structural feature important to close the reaction site - which might explain the lack of activity. Thirty-one selected Miox sequences did allow for product formation, of which twenty-five were characterized for the first time. Expression of Talaromyces marneffei Miox led to the accumulation of 1.76 ±â€¯0.33 g glucaric acid/L from 20 g glucose/L and 10 g/L myo-inositol. Specific glucaric acid titer with TmMiox increased 44 % compared to the often-used Arabidopsis thaliana variant AtMiox4 (0.258 vs. 0.179 g glucaric acid/g biomass). AtMiox4 activity decreased from 12.47 to 0.40 nmol/min/mg protein when cells exited exponential phase during growth on glucose, highlighting the importance of future research on Miox stability in order to further improve microbial production of glucaric acid.

Plasma metabolomic profiles differ at the time of artificial insemination based on pregnancy outcome, in Bos taurus beef heifers.

Infertility remains the most prevalent reason for cattle being removed from production environments. We utilized metabolomic profiling to identify metabolites in the blood plasma that may be useful in identifying infertile heifers at the time of artificial insemination (AI). Prior to AI, phenotypic parameters including body condition, weight, and reproductive organ measurements were collected. These were determined not effective at differentiating between fertile and infertile heifers. Analysis of the resulting metabolomic profiles revealed 15 metabolites at significantly different levels (T-test P ≤ 0.05), with seven metabolites having a greater than 2-fold difference (T-test P ≤ 0.05, fold change ≥2, ROC-AUC ≥ 0.80) between infertile and fertile heifers. We further characterized the utility of using the levels of these metabolites in the blood plasma to discriminate between fertile and infertile heifers. Finally, we investigated the potential role inflammation may play by comparing the expression of inflammatory cytokines in the white blood cells of infertile heifers to that of fertile heifers. We found significantly higher expression in infertile heifers of the proinflammatory markers tumor necrosis factor alpha (TNFα), interleukin 6 (IL6), and the C-X-C motif chemokine 5 (CXCL5). Our work offers potentially valuable information regarding the diagnosis of fertility problems in heifers undergoing AI.

Synthetic biology strategies for improving microbial synthesis of "green" biopolymers.

Polysaccharide-based biopolymers have many material properties relevant to industrial and medical uses, including as drug delivery agents, wound-healing adhesives, and food additives and stabilizers. Traditionally, polysaccharides are obtained from natural sources. Microbial synthesis offers an attractive alternative for sustainable production of tailored biopolymers. Here, we review synthetic biology strategies for select "green" biopolymers: cellulose, alginate, chitin, chitosan, and hyaluronan. Microbial production pathways, opportunities for pathway yield improvements, and advances in microbial engineering of biopolymers in various hosts are discussed. Taken together, microbial engineering has expanded the repertoire of green biological chemistry by increasing the diversity of biobased materials.

Phenotypic screening with oleaginous microalgae reveals modulators of lipid productivity.

Here we describe the first phenotypic screening with microalgae to study lipid metabolism and to discover organic small molecules as chemical triggers that increase growth and lipid production. A microplate assay has been developed for analysis of intracellular lipids using Nile Red fluorescence in order to screen a collection of diverse bioactive organic molecules (e.g., kinase inhibitors) with four strains of oleaginous microalgae (Nannochloropsis salina, Nannochloropsis oculata, Nannochloris sp., and Phaeodactylum tricornutum). Several small molecules identified in microplate screening increased lipid productivity >200% without decreasing growth and biomass production. Selected compounds were further investigated in the context of larger batch culture experiments (e.g., 500 mL) and demonstrated to increase lipid levels (up to 84%) while maintaining or increasing the specific growth rate. Bioactive molecules such as forskolin and quinacrine were identified as promising probes of microalgae lipid pathways. We have also determined that common antioxidants such as epigallocatechin gallate and butylated hydroxyanisole (BHA) increase lipid productivity and may represent new probes of oxidative signaling pathways for photooxidative protection.

Triacylglycerol profiling of marine microalgae by mass spectrometry.

We present a method for the determination of triacylglycerol (TAG) profiles of oleaginous saltwater microalgae relevant for the production of biofuels, bioactive lipids, and high-value lipid-based chemical precursors. We describe a technique to remove chlorophyll using quick, simple solid phase extraction (SPE) and directly compare the intact TAG composition of four microalgae species (Phaeodactylum tricornutum, Nannochloropsis salina, Nannochloropsis oculata, and Tetraselmis suecica) using MALDI time-of-flight (TOF) mass spectrometry (MS), ESI linear ion trap-orbitrap (LTQ Orbitrap) MS, and ¹H NMR spectroscopy. Direct MS analysis is particularly effective to compare the polyunsaturated fatty acid (PUFA) composition for triacylglycerols because oxidation can often degrade samples upon derivatization. Using these methods, we observed that T. suecica contains significant PUFA levels with respect to other microalgae. This method is applicable for high-throughput MS screening of microalgae TAG profiles and may aid in the commercial development of biofuels.

Three-component synthesis and anticancer evaluation of polycyclic indenopyridines lead to the discovery of a novel indenoheterocycle with potent apoptosis inducing properties.

A multicomponent reaction of indane-1,3-dione, an aldehyde and an amine-containing aromatic compound leading to the formation of indenopyridine-based heterocyclic medicinal scaffolds has been investigated. It was found that the yields significantly improve when oxygen gas is bubbled through the reaction mixture, facilitating the oxidation of the intermediate dihydropyridine-containing compounds to their aromatic counterparts. Investigation of the reaction scope revealed that formaldehyde, as well as various aliphatic, aromatic and heteroaromatic aldehydes, works well as the aldehyde component. In addition, substituted anilines and diverse aminoheterocycles can be utilized in this process as the amine-containing component. Preliminary biological evaluation of the synthesized library identified a pyrimidine-based polycycle, which rivals the anticancer drug etoposide in its toxicity and apoptosis inducing properties toward a human T-cell leukemia cell line.

P300 transcriptional repression is mediated by SUMO modification.

p300 and CREB binding protein can both activate and repress transcription. Here, we locate the CRD1 transcriptional repression domain between residues 1017 and 1029 of p300. This region contains two copies of the sequence psiKxE that are modified by the ubiquitin-like protein SUMO-1. Mutations that reduce SUMO modification increase p300-mediated transcriptional activity and expression of a SUMO-specific protease or catalytically inactive Ubc9 relieved repression, demonstrating that p300 repression was mediated by SUMO conjugation. SUMO-modified CRD1 domain bound HDAC6 in vitro, and p300 repression was relieved by histone deacetylase inhibition and siRNA-mediated ablation of HDAC6 expression. These results reveal a mechanism controlling p300 function and suggest that SUMO-dependent repression is mediated by recruitment of HDAC6.

Regulation of RelA (p65) function by the large subunit of replication factor C.

The RelA (p65) subunit of NF-kappaB is an important regulator of inflammation, proliferation, and apoptosis. We have discovered that the large subunit, p140, of replication factor C (RFC) can function as a regulator of RelA. RFC is a clamp loader, facilitating the addition and removal of proliferating-cell nuclear antigen from DNA during replication and repair but can also interact directly with the retinoblastoma tumor suppressor protein and the transcription factor C/EBPalpha. We find that RFC (p140) interacts with RelA both in vitro and in vivo and stimulates RelA transactivation. In contrast, coexpression of fragments of RFC (p140) that mediate the interaction with RelA results in transcriptional inhibition. The significance of this regulation was confirmed by using short interfering RNA oligonucleotides targeted to RFC (p140). Down regulation of endogenous RFC (p140) inhibits expression from a chromosomally integrated reporter plasmid induced by endogenous, TNF-alpha-activated NF-kappaB. Dominant negative fragments of RFC (p140) also cooperate with overexpressed RelA to induce cell death. Interestingly, RFC (p140) also interacts with the tumor suppressor p53. Taken together, these observations suggest that, in addition to its previously described function in DNA replication and repair, RFC (p140) has an important role as a regulator of transcription and NF-kappaB activity.

The large subunit of replication factor C interacts with the histone deacetylase, HDAC1.

Replication factor C (RFC) is a pentameric complex of five distinct subunits that functions as a clamp loader, facilitating the loading of proliferating cell nuclear antigen (PCNA) onto DNA during replication and repair. More recently the large subunit of RFC, RFC (p140), has been found to interact with the retinoblastoma (Rb) tumor suppressor and the CCAAT/enhancer-binding protein alpha (C/EBP alpha) transcription factor. We now report that RFC (p140) associates with histone deacetylase activity and interacts with histone deacetylase 1 (HDAC1). This complex is functional and when targeted to promoters as a Gal4 fusion, RFC (p140) is a strong, deacetylase-dependent repressor of transcription. Further analysis revealed that RFC (p140) contains two distinct transcriptional repression domains. Moreover, both of these domains interact separately with HDAC1.