A unique microbiome that metabolizes lactate rather than ethanol for n-caproate production was obtained from a fermentation pit used for the production of Chinese strong-flavour liquor (CSFL). The microbiome was able to produce n-caproate at concentrations as high as 23.41 g/L at a maximum rate of 2.97 g/L/d in batch trials without in-line extraction. Compared with previous work using ethanol as the electron donor, the n-caproate concentration increased by 82.89%. High-throughput sequencing analysis showed that the microbiome was dominated by a Clostridium cluster IV, which accounted for 79.07% of total reads. A new process for n-caproate production was proposed, lactate oxidation coupled to chain elongation, which revealed new insight into the well-studied lactate conversion and carbon chain elongation. In addition, these findings indicated a new synthesis mechanism of n-caproate in CSFL. We believe that this efficient process will provide a promising opportunity for the innovation of waste recovery as well as for n-caproate biosynthesis.

The biological role played by non-BET bromodomains remains poorly understood, and it is therefore imperative to identify potent and highly selective inhibitors to effectively explore the biology of individual bromodomain proteins. A ligand-efficient nonselective bromodomain inhibitor was identified from a 6-methyl pyrrolopyridone fragment. Small hydrophobic substituents replacing the N-methyl group were designed directing toward the conserved bromodomain water pocket, and two distinct binding conformations were then observed. The substituents either directly displaced and rearranged the conserved solvent network, as in BRD4(1) and TAF1(2), or induced a narrow hydrophobic channel adjacent to the lipophilic shelf, as in BRD9 and CECR2. The preference of distinct substituents for individual bromodomains provided selectivity handles useful for future lead optimization efforts for selective BRD9, CECR2, and TAF1(2) inhibitors.

The ability to selectively degrade proteins with bifunctional small molecules has the potential to fundamentally alter therapy in a variety of diseases. However, the relatively large size of these chimeric molecules often results in challenging physico‐chemical properties (e. g., low aqueous solubility) and poor pharmacokinetics which may complicate their in vivo applications. We recently discovered an exquisitely potent chimeric BET degrader (GNE‐987) which exhibited picomolar cell potencies but also demonstrated low in vivo exposures. In an effort to improve the pharmacokinetic properties of this molecule, we discovered the first degrader‐antibody conjugate by attaching GNE‐987 to an anti‐CLL1 antibody via a novel linker. A single IV dose of the conjugate afforded sustained in vivo exposures that resulted in antigen‐specific tumor regressions. Enhancement of a chimeric protein degrader with poor in vivo properties through antibody conjugation thereby expands the utility of directed protein degradation as both a biological tool and a therapeutic possibility.

During two-phase sludge anaerobic digestion, sludge is usually hydrolyzed and acidified in the first phase, then methane is produced in the second stage. To get more methane from sludge, most studies in literature focused on the increase of sludge hydrolysis. In this paper a different sludge pretreatment method, i.e., pretreating sludge at pH 10 for 8 d is reported, by which both waste activated sludge hydrolysis and acidification were increased, and the methane production was significantly improved. First, the effect of different sludge pretreatment methods on methane yield was compared. The pH 10 pretreated sludge showed the highest accumulative methane yield (398 mL per g of volatile suspended solids), which was 4.4-, 3.5-, 3.1-, and 2.3-fold of the blank (unpretreated), ultrasonic, thermal, and thermal-alkaline pretreated sludge, respectively. Nevertheless, its total time involved in the first (hydrolysis and acidification) and second (methanogenesis) stages was 17 (8 + 9) d, which was almost the same as other pretreatments. Then, the mechanisms for pH 10 pretreatment significantly improving methane yield were investigated. It was found that pretreating sludge at pH 10 caused the greatest sludge hydrolysis, acidification, soluble C:N and C:P ratios, and Fe(3+) concentration with a suitable short-chain fatty acids composition in the first stage, which resulted in the highest microorganism activity (ATP) and methane production in the second phase. Further investigation on the second phase microorganisms with fluorescence in situ hybridization (FISH) and scanning electron microscopy (SEM) indicated that there were much greater active methanogenesis Archaea when methane was produced with the pH 10 pretreated sludge, and the predominant morphology of the microcolonies suggest a shift to Methanosarcina sp. like.

Background n-Caproic acid (CA), as a medium-chain carboxylic acid, is a valuable chemical feedstock for various industrial applications. The fermentative production of CA from renewable carbon sources has attracted a lot of attentions. Lactate is a significant intermediate waste in the anaerobic breakdown of carbohydrates that comprise 18–70% of the chemical oxygen demand (COD) in municipal and some industrial wastewaters. Recently, researchers (including our own group) reported the CA production using lactate as electron donor with newly identified microbiome systems. However, within such processes, it was hard to determine whether the CA production was completed by a single strain or by the co-metabolism of different microorganisms.ResultsHere, we report the CA production using lactate as electron donor using the strain CPB6, which we isolated from a microbiome for CA production as described previously. Strain CPB6 is affiliated with Clostridium cluster IV of the family of Ruminococcaceae based on 16S rRNA gene sequence analysis. The strain prefers acidic initial pH condition (pH 5.0–6.5), and the temperature ranging from 30 to 40 °C for CA production. In a fed-batch fermentation with non-sterilized lactate-containing organic wastewater as feedstock, strain CPB6 produced 16.6 g/L CA (from 45.1 g/L lactate) with a maximum productivity of 5.29 g/L/day. Enzyme assays with crude cell extract showed that CPB6 can metabolize acetate and butyryl-CoA to produce n-butyric acid, and acetate/n-butyrate and caproyl-CoA to produce CA, respectively.ConclusionThis study demonstrated that high concentration of CA production can be obtained by a newly isolated pure culture CPB6. This strain can be employed as a powerful workhorse for high-efficient CA recovery from lactate-containing waste streams. Our preliminary investigation suggested that the CA production from lactate in strain CPB6 might be via the chain elongation pathway of the reverse β-oxidation; the detailed mechanism, however, warrants further investigation using various molecular microbiology techniques.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0788-y) contains supplementary material, which is available to authorized users.

The biological and medicinal impacts of proteolysis-targeting chimeras (PROTACs) and related chimeric molecules that effect intracellular degradation of target proteins via ubiquitin ligase-mediated ubiquitination continue to grow. However, these chimeric entities are relatively large compounds that often possess molecular characteristics, which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. We therefore explored the conjugation of such molecules to monoclonal antibodies using technologies originally developed for cytotoxic payloads so as to provide alternate delivery options for these novel agents. In this report, we describe the first phase of our systematic development of antibody–drug conjugates (ADCs) derived from bromodomain-containing protein 4 (BRD4)-targeting chimeric degrader entities. We demonstrate the antigen-dependent delivery of the degrader payloads to PC3-S1 prostate cancer cells along with related impacts on MYC transcription and intracellular BRD4 levels. These experiments culminate with the identification of one degrader conjugate, which exhibits antigen-dependent antiproliferation effects in LNCaP prostate cancer cells.

Inhibition of the bromodomain of the transcriptional regulator CBP/P300 is an especially interesting new therapeutic approach in oncology. We recently disclosed in vivo chemical tool 1 (GNE-272) for the bromodomain of CBP that was moderately potent and selective over BRD4(1). In pursuit of a more potent and selective CBP inhibitor, we used structure-based design. Constraining the aniline of 1 into a tetrahydroquinoline motif maintained potency and increased selectivity 2-fold. Structure-activity relationship studies coupled with further structure-based design targeting the LPF shelf, BC loop, and KAc regions allowed us to significantly increase potency and selectivity, resulting in the identification of non-CNS penetrant 19 (GNE-781, TR-FRET IC = 0.94 nM, BRET IC = 6.2 nM; BRD4(1) IC = 5100 nΜ) that maintained good in vivo PK properties in multiple species. Compound 19 displays antitumor activity in an AML tumor model and was also shown to decrease Foxp3 transcript levels in a dose dependent manner.

SignificanceCellulose is synthesized at the plasma membrane by cellulose synthase complexes (CSCs). The ability to deliver CSCs to discrete sites at the cell surface is critical for cellulose synthesis. We discovered that the de novo secretion of CSCs is mediated by cooperation among a recently identified cellulose synthase interacting protein, exocyst complex, and a plant-specific protein PATROL1 in Arabidopsis thaliana. We present a timeline of events for exocytosis of CSCs. CSCs represent unique cargo proteins that are not conserved in the mammalian or yeast system. Plants offer unique opportunities to characterize the function and regulation of exocytosis that may provide insight into the evolution of exocytosis in eukaryotes.