Biosynthesis of Gut-Microbiota-Derived Lantibiotics Reveals a Subgroup of S8 Family Proteases for Class III Leader Removal.

Lanthipeptides are a group of ribosomally synthesized and post-translationally modified peptides with diverse structural features and bioactivities. Gut-microbiota-derived lanthipeptides play important roles in gut homeostasis of the host. Herein, we report the discovery and biosynthesis of class III lantibiotics named amylopeptins, which are derived from the gut microbiota of Sprague-Dawley rats and display a narrow antimicrobial spectrum. In contrast to known class III lanthipeptides, the biosynthesis of amylopeptins employs AmyP, which belongs to a subgroup of S8 family serine proteases, to remove the leader of corresponding precursor peptides in a site-specific manner during the last step of their maturation. Overall, this study shows for the first time that S8 family proteases participate in the biosynthesis of class III lanthipeptides.

The preparation and antibacterial effect of egg yolk immunoglobulin (IgY) against the outer membrane proteins of Vibrio parahaemolyticus.

BACKGROUND: Vibrio parahaemolyticus causes not only various diseases in aquaculture animals but also seafood-borne illness in humans. Outer membrane proteins (OMPs) are species-specific proteins found in bilayer membranes of gram-negative bacteria. Egg yolk immunoglobulin (IgY) has been reported to serve as oral administration of antibodies against bacteria and virus. RESULTS: The present research extracted and identified OMPs from V. parahaemolyticus, and then the extracted OMPs were used to immunize hens to obtain specific IgY. The efficacy of IgY against V. parahaemolyticus were investigated in vitro and in vivo. The specific IgY effectively inhibited the growth of V. parahaemolyticus in liquid medium rather than Escherichia coli and Staphylococcus aureus. Specific IgY antibodies were incorporated into extruded food pellets and fed to bacteria-challenged white pacific shrimp to observe the anti-bacterial effect in vivo. The bacterial loads in muscles of V. parahaemolyticus infected shrimp fed with specific IgY-included diets were significantly fewer than those fed with non-specific IgY-included diets. The superoxide dismutase activities in muscles of infected shrimp fed with specific IgY-included diets were significantly higher than the control group. CONCLUSION: The results suggested that the specific IgY effectively inhibited the growth of V. parahaemolyticus and introduced passive immunity to shrimp. © 2018 Society of Chemical Industry.

Towards one sample per second for mass spectrometric screening of engineered microbial strains.

Microbial cell factories convert renewable feedstocks into desirable chemicals and materials. Due to the lack of predictive modeling, high-throughput screening remains essential for microbial strain engineering. Mass spectrometry (MS) is a label-free modality with superior sensitivity and chemical specificity. Critical advances in improving the throughput of MS assays on complex microbial samples include massively parallel cultivation, robotic sample preparation, and chromatography-free instrumentation. Here, we review the recent development and application of rapid MS assays in screening microbial libraries, achieving or approaching a rate of one sample per second. We conclude with unique challenges associated with MS screening of strain libraries and discuss future solutions.

Robotic Construction and Screening of Lanthipeptide Variant Libraries in Escherichia coli.

Lanthipeptides are a major class of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by thioether cross-links called lanthionine (Lan) and methyllanthionine (MeLan). Previously, we developed a method to produce mature lanthipeptides in recombinant Escherichia coli, but manual steps hinder large-scale analogue screening. Here we devised an automated workflow for creating and screening variant libraries of haloduracin, a two-component class II lanthipeptide. An integrated work cell of a synthetic biology foundry was programmed to robotically execute DNA library construction, host transformation, peptide production, mass spectrometry analysis, and activity screening by agar diffusion assay. For recombinantly produced Halα peptides, the sequence-activity relationship of 380 single-residue variants and >1300 triple-residue combinatorial variants were rapidly analyzed in microplates within weeks. The peptide expression levels in E. coli were also visualized via robotic creation and analysis of GFP-lanthipeptide fusions for select peptide mutants. Following shake-flask fermentation and purification, one Halα mutant was confirmed with enhanced specific antimicrobial activity relative to the wild-type peptide. Overall, this approach may be generally applicable for the high-throughput characterization and engineering of RiPP natural products.

Accelerating strain engineering in biofuel research via build and test automation of synthetic biology.

Biofuels are a type of sustainable and renewable energy. However, for the economical production of bulk-volume biofuels, biosystems design is particularly challenging to achieve sufficient yield, titer, and productivity. Because of the lack of predictive modeling, high-throughput screening remains essential. Recently established biofoundries provide an emerging infrastructure to accelerate biological design-build-test-learn (DBTL) cycles through the integration of robotics, synthetic biology, and informatics. In this review, we first introduce the technical advances of build and test automation in synthetic biology, focusing on the use of industry-standard microplates for DNA assembly, chassis engineering, and enzyme and strain screening. Proof-of-concept studies on prototypes of automated foundries are then discussed, for improving biomass deconstruction, metabolic conversion, and host robustness. We conclude with future challenges and opportunities in creating a flexible, versatile, and data-driven framework to support biofuel research and development in biofoundries.

Advances in RNAi-Assisted Strain Engineering in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a widely used eukaryotic model and microbial cell factory. RNA interference (RNAi) is a conserved regulatory mechanism among eukaryotes but absent from S. cerevisiae. Recent reconstitution of RNAi machinery in S. cerevisiae enables the use of this powerful tool for strain engineering. Here we first discuss the introduction of heterologous RNAi pathways in S. cerevisiae, and the design of various expression cassettes of RNAi precursor reagents for tunable, dynamic, and genome-wide regulation. We then summarize notable examples of RNAi-assisted functional genomics and metabolic engineering studies in S. cerevisiae. We conclude with the future challenges and opportunities of RNAi-based approaches, as well as the potential of other regulatory RNAs in advancing yeast engineering.