Construction of a Mutant Bacillus Subtilis Strain for High Purity Poly-γ-Glutamic Acid Production

Purpose To construct a Bacillus subtilis strain for improved purity of poly-γ-glutamic acid. Results The construction of strain GH16 was achieved by knocking out five extracellular protein genes and an operon from Bacillus subtilis G423. Then we analyzed the protein content in the γ-PGA produced by the resultant strain GH16/pHPG which decreased by 6.08%. Subsequently the fla-che operon, PBSX and the yrpD, ywoF and yclQ genes were knocked out successively and the mutant strain GH17, GH18, and GH19 was obtained. Ultimately, the protein content was reduced by 43.9%. In addition, the polysaccharide content in the γ-PGA was decreased from 2.21–1.93% due to the epsA-O operon was knocked. Conclusion γ-PGA has potential applications as a drug carrier, sustained-releasing agent and medical composite in medicine. To our knowledge, this is the first report of engineered Bacillus subtilis strains which can produce γ-PGA with a purity higher than 97%. Our results confirmed that this upstream strategy significantly enhanced specific protein purity by the removal of extracellular protein genes in Bacillus subtilis, and it is promising in other protein purification.

Biodegradation Capacity and Activity Enzymatic of Bacillus subtilis against Low-Density Polyethylene

Aims: The objective of this work was to determine the degradation capacity of low-density polyethylene by the bacterium Bacillus subtilis and analyze the production of extracellular laccase activity. Methodology: The experiments was realized in 50 mL of culture medium, added with a fragment of known dry weight (1 cm2 colorless polyethylene bag squares), and were incubated at 28°C, pH 6.5, for 6 months under static conditions, determining the growth of the bacterium by dry weight (68, 75, and 91 mg), the production of extracellular protein (271, 234, and 326.1 mg/mL), and the degradation of the substrate by dry biodegraded (8.57%, 5.88%, and 11.76%). Results: The production of extracellular laccase enzyme was analyzed in presence of polyethylene, finding an enzymatic activity of laccase of 2.06, 1.49, and 2,03 U/mL, while in the control without substrate, no enzymatic activity was observed, which suggests that this enzyme may participate in the degradation of polyethylene. In addition, some characteristics of the extracellular enzymatic activities were analyzed, such as stability at 4oC and 28oC, optimal pH and temperature, the effect of protein and substrate concentration. Conclusion: The extracellular protein production and dry weight of the bacterium are higher in the presence of low-density polyethylene. The laccase activity is very stable at 4oC and 28oC, the most effective pH and temperature, were 4.5 and 28oC, and present an incubation time of 5 minutes, and this data suggest that this enzymatic activitiy may participate in the degradation of low density polyethylene.

Unbiased Identification of Extracellular Protein–Protein Interactions for Drug Target and Biologic Drug Discovery

Technological improvements in unbiased screening have accelerated drug target discovery. In particular, membrane-embedded and secreted proteins have gained attention because of their ability to orchestrate intercellular communication. Dysregulation of their extracellular protein–protein interactions (ePPIs) underlies the initiation and progression of many human diseases. Practically, ePPIs are also accessible for modulation by therapeutics since they operate outside of the plasma membrane. Therefore, it is unsurprising that while these proteins make up about 30% of human genes, they encompass the majority of drug targets approved by the FDA. Even so, most secreted and membrane proteins remain uncharacterized in terms of binding partners and cellular functions. To address this, a number of approaches have been developed to overcome challenges associated with membrane protein biology and ePPI discovery. This chapter will cover recent advances that use high-throughput methods to move towards the generation of a comprehensive network of ePPIs in humans for future targeted drug discovery.

MO063DESCRIPTION OF GENETIC VARIANTS IN A COHORT OF TOLVAPTAN ADPKD PATIENTS

Background and Aims Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited renal cystic disease. It is genetically heterogeneous: 72-75% of ADPKD cases are related to mutations in the PKD1, 15-18 % to PKD2 and the remaining 7–10% of affected are genetically unresolved (GUR). Recent years, new drugs have emerged as promising agents that may retard the progression of ADPKD, such as Tolvaptan. In Italy Tolvaptan is available since 2016 and commonly used since 2017 in ADPKD patients, which fulfill the criteria of “rapid disease progression”, according to the European recommendations. High intra-interfamilial variability in pedigrees was observed, despite the same germ-line mutation. This could be explained by other clinical or genetic factors (environmental, modifier genes, etc), that may affect disease severity. The aim of the study is to describe the genetic variants in a cohort of Tolvaptan ADPKD patients (pts) referral to Renal Genetic Disease Ambulatory of Nephrology Department. Method Patients with ADPKD and in Tolvaptan treatment were enrolled. Diagnosis of ADPKD was made upon the revised Ravine’s criteria and Eligibility Criteria for Tolvaptan was made upon Italian indication for Tolvaptan prescription according to Italian Medicine Agency (AIFA) and European Medicines Agency (EMA). We performed genetic analysis (PKD1, PKD2 and PKHD1 genes) to identify mutations by NGS capture-based target enrichment kit (Sophia Genetic™), sequencing on Illumina MySeq Platform® and Sanger Sequencing on 3500 Series Genetic Analyzer (Applied Biosystems™). Results Eighteen pts [median age 46 (IQR 39-48) yrs ], 12 male, were included in the analysis. We manage to perform genetic analysis in all pts. Genetic analysis was essential in 4 patients without family history for Tolvaptan eligibility. Sixteen pts (88,9%) have mutations in PKD1, confirming what is already known from the literature for rapid progressor subjects; 2 pts are characterized by PKD2 mutations, both truncating. In only one pt, concomitant with a PKD1 mutation, also a PKHD1 mutation was found. In order to better characterize the cohort it was decided to subdivide the pts into 3 groups, by gene involvement and mutation type: 1st group: 12 Subjects with truncated PKD1 mutation (66.7%). In 7 pts (58,3%) the mutations are within exons (5 and from exon 11 to 15 inclusive) that encode for Immunoglobulin-like repeats or PKD domain of Polycystin 1 (PC1). 2nd group: 4 Subjects with non-truncated PKD1 mutations (22.2%). 3 pts (75.0%) are characterized by missense variants, as previous studies highlighted (a higher percentage of missense mutations in subjects with non-truncating mutations). In 2 pts (50%) the mutations are within exons (2 and 6) that encode for C-type lectin domain (CTLs) di PC1 and typical domain of extracellular protein. 3rd group: 2 Subjects with a PKD2 mutation (11.1%), both truncating. These data confirmed the lower mutation rate of PKD2 compared to PKD1 and highlighted an effective prevalence of truncation mutations in rapid disease progressors as previous reported. Conclusion Although our cohort of patients is small, we manage to perform genetic analysis in all pts reaching a detection rate of 100%. In 9 of 16 pts (56,3%) with PKD1 mutation the presence of mutations in exons coding PKD domain in PC1 or Immunoglobulin-like repeats or typical domain of extracellular protein allows us to hypothesize that the resulting alteration of the polycystin-mediated cell recognition and communication processes play a crucial role in the pathogenesis of ADPKD.

Lignocellulolytic activity of Pleurotus ostreatus under solid state fermentation using silage, stover, and cobs of maize

Lignocellulolytic white-rot fungi allow the bioconversion of agricultural wastes into value-added products that are used in a myriad of applications. The aim of this work was to use corn residues (Zea mays L.) to produce valuable products under solid-state fermentation (SSF) with Pleurotus ostreatus. White-rot fungus P. ostreatus was isolated from maize silage (MS) and thereafter it was inoculated on MS as substrate and compared with maize stover (MSt) and maize cobs (MC) to determine the best lignocellulosic substrate for the production of lignocellulolytic enzymes and extracellular protein. The MS gave the highest productivity of CMCase (368.2 U/mL), FPase (170.5 U/mL), laccase (11.4 U/mL), and MnPase (6.6 U/mL). This is compared to productivity on MSt of 222 U/mL, 50.2 U/mL, 4.55 U/mL, and 2.57 U/mL, respectively; and productivity on MC at the same incubation period as 150.5 U/mL, 48.2 U/mL, 3.58 U/mL, and 2.5 U/mL, respectively. The levels of enzyme production declined with increasing incubation period after 15 and 20 days using MS and MC, respectively, as substrates. Maximum liberated extracellular protein content (754 to 878 µg/mL) was recorded using MS, while a low amount (343 to 408 µg/mL) was liberated with using MSt and MC.

The Constitutive Extracellular Protein Release by Acute Myeloid Leukemia Cells—A Proteomic Study of Patient Heterogeneity and Its Modulation by Mesenchymal Stromal Cells

Extracellular protein release is important both for the formation of extracellular matrix and for communication between cells. We investigated the extracellular protein release by in vitro cultured normal mesenchymal stem cells (MSCs) and by primary human acute myeloid leukemia (AML) cells derived from 40 consecutive patients. We observed quantifiable levels of 3082 proteins in our study; for the MSCs, we detected 1446 proteins, whereas the number of released proteins for the AML cells showed wide variation between patients (average number 1699, range 557–2380). The proteins were derived from various cellular compartments (e.g., cell membrane, nucleus, and cytoplasms), several organelles (e.g., cytoskeleton, endoplasmatic reticulum, Golgi apparatus, and mitochondria) and had various functions (e.g., extracellular matrix and exosomal proteins, cytokines, soluble adhesion molecules, protein synthesis, post-transcriptional modulation, RNA binding, and ribonuclear proteins). Thus, AML patients were very heterogeneous both regarding the number of proteins and the nature of their extracellularly released proteins. The protein release profiles of MSCs and primary AML cells show a considerable overlap, but a minority of the proteins are released only or mainly by the MSC, including several extracellular matrix molecules. Taken together, our observations suggest that the protein profile of the extracellular bone marrow microenvironment differs between AML patients, these differences are mainly caused by the protein release by the leukemic cells but this leukemia-associated heterogeneity of the overall extracellular protein profile is modulated by the constitutive protein release by normal MSCs.

Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials

ABSTRACT The bacterial extracellular matrix forms autonomously, giving rise to complex material properties and multicellular behaviors. Synthetic matrix analogues can replicate these functions but require exogenously added material or have limited programmability. Here, we design a two-strain bacterial system that self-synthesizes and structures a synthetic extracellular matrix of proteins. We engineered Caulobacter crescentus to secrete an extracellular matrix protein composed of an elastin-like polypeptide (ELP) hydrogel fused to supercharged SpyCatcher [SC(−)]. This biopolymer was secreted at levels of 60 mg/liter, an unprecedented level of biomaterial secretion by a native type I secretion apparatus. The ELP domain was swapped with either a cross-linkable variant of ELP or a resilin-like polypeptide, demonstrating this system is flexible. The SC(−)-ELP matrix protein bound specifically and covalently to the cell surface of a C. crescentus strain that displays a high-density array of SpyTag (ST) peptides via its engineered surface layer. Our work develops protein design guidelines for type I secretion in C. crescentus and demonstrates the autonomous secretion and assembly of programmable extracellular protein matrices, offering a path forward toward the formation of cohesive engineered living materials. IMPORTANCE Engineered living materials (ELM) aim to mimic characteristics of natural occurring systems, bringing the benefits of self-healing, synthesis, autonomous assembly, and responsiveness to traditional materials. Previous research has shown the potential of replicating the bacterial extracellular matrix (ECM) to mimic biofilms. However, these efforts require energy-intensive processing or have limited tunability. We propose a bacterially synthesized system that manipulates the protein content of the ECM, allowing for programmable interactions and autonomous material formation. To achieve this, we engineered a two-strain system to secrete a synthetic extracellular protein matrix (sEPM). This work is a step toward understanding the necessary parameters to engineering living cells to autonomously construct ELMs.