Engineering Extracellular Secretion Pathways In Escherichia Coli

Extracellular secretion is highly desirable in preparative protein production. The bacterium Escherichia coli is a commonly used for both laboratory- and industrial- scale biosynthesis of proteins, but it lacks many of the pathways for exporting proteins out of cells. This lack of a dedicated extracellular secretion system represents a major bottleneck across many biotechnology disciplines, in particular the bioprocessing of plant biomass where extracellular secretion of cellulase is required. Furthermore, the study and engineering of extracellular secretion systems is limited due to a lack of high-throughput screen to identify rare genetic conditions that affect secretion activity. Recently, it was discovered in E. coli that the YebF protein is secreted efficiently into the supernatant when over expressed, and YebF has been employed to carry heterologous proteins into the supernatant via C-terminal genetic fusions. Here, we harness the YebF pathway to simultaneously co-secrete active cellulases into the culture medium, which enabled non-cellulolytic E. coli cells to utilize and convert cellulose to bioenergy products. We also developed a universal approach to study and engineer YebF and other extracellular secretion pathways. This high-throughput screening platform was used to screen a genome-wide transposon insertion library for the isolation of gene deletions that upregulate the secretion of YebF and YebF fusions. We also developed an alternative strategy for engineering extracellular secretion systems by way of a genetic selection where the nonsecretory phenotype is lethal. Finally, we describe some of the physiological consequences to the bacterial host caused by heterologous protein secretion, in particular an envelope stress response that triggers CRISPR RNA-mediated DNA silencing. ii

Early-branching gut fungi possess a large, comprehensive array of biomass-degrading enzymes

available in PMC 2016 November 07The fungal kingdom is the source of almost all industrial enzymes in use for lignocellulose bioprocessing. We developed a systems-level approach that integrates transcriptomic sequencing, proteomics, phenotype, and biochemical studies of relatively unexplored basal fungi. Anaerobic gut fungi isolated from herbivores produce a large array of biomass-degrading enzymes that synergistically degrade crude, untreated plant biomass and are competitive with optimized commercial preparations from Aspergillus and Trichoderma. Compared to these model platforms, gut fungal enzymes are unbiased in substrate preference due to a wealth of xylan-degrading enzymes. These enzymes are universally catabolite-repressed and are further regulated by a rich landscape of noncoding regulatory RNAs. Additionally, we identified several promising sequence-divergent enzyme candidates for lignocellulosic bioprocessing.United States. Dept. of Energy. Office of Science (Biological and Environmental Research (BER) program)United States. Department of Energy (DOE Grant DE-SC0010352)United States. Department of Agriculture (Award 2011-67017-20459)Institute for Collaborative Biotechnologies (grant W911NF-09-0001

Genome-wide meta-analysis of 241,258 adults accounting for smoking behaviour identifies novel loci for obesity traits

Few genome-wide association studies (GWAS) account for environmental exposures, like smoking, potentially impacting the overall trait variance when investigating the genetic contribution to obesity-related traits. Here, we use GWAS data from 51,080 current smokers and 190,178 nonsmokers (87% European descent) to identify loci influencing BMI and central adiposity, measured as waist circumference and waist-to-hip ratio both adjusted for BMI. We identify 23 novel genetic loci, and 9 loci with convincing evidence of gene-smoking interaction (GxSMK) on obesity-related traits. We show consistent direction of effect for all identified loci and significance for 18 novel and for 5 interaction loci in an independent study sample. These loci highlight novel biological functions, including response to oxidative stress, addictive behaviour, and regulatory functions emphasizing the importance of accounting for environment in genetic analyses. Our results suggest that tobacco smoking may alter the genetic susceptibility to overall adiposity and body fat distribution.Peer reviewe

Designing chimeric enzymes inspired by fungal cellulosomes

Cellulosomes are synthesized by anaerobic bacteria and fungi to degrade lignocellulose via synergistic action of multiple enzymes fused to a protein scaffold. Through templating key protein domains (cohesin and dockerin), designer cellulosomes have been engineered from bacterial motifs to alter the activity, stability, and degradation efficiency of enzyme complexes. Recently a parts list for fungal cellulosomes from the anaerobic fungi (Neocallimastigomycota) was determined, which revealed sequence divergent fungal cohesin, dockerin, and scaffoldin domains that could be used to expand the available toolbox to synthesize designer cellulosomes. In this work, multi-domain carbohydrate active enzymes (CAZymes) from 3 cellulosome-producing fungi were analyzed to inform the design of chimeric proteins for synthetic cellulosomes inspired by anaerobic fungi. In particular, Piromyces finnis was used as a structural template for chimeric carbohydrate active enzymes. Recombinant enzymes with retained properties were engineered by combining thermophilic glycosyl hydrolase domains from Thermotoga maritima with dockerin domains from Piromyces finnis. By preserving the protein domain order from P. finnis, chimeric enzymes retained catalytic activity at temperatures over 80 °C and were able to associate with cellulosomes purified from anaerobic fungi. Fungal cellulosomes harbor a wide diversity of glycoside hydrolases, each representing templates for the design of chimeric enzymes. By conserving dockerin domain position within the primary structure of each protein, the activity of both the catalytic domain and dockerin domain was retained in enzyme chimeras. Taken further, the domain positioning inferred from native fungal cellulosome proteins can be used to engineer multi-domain proteins with non-native favorable properties, such as thermostability. Keywords: Cellulosome, Dockerin, Scaffoldin, Anaerobic fungi, Thermophile, Enzym

An Engineered Survival-Selection Assay for Extracellular Protein Expression Uncovers Hypersecretory Phenotypes in <i>Escherichia coli</i>

The extracellular expression of recombinant proteins using laboratory strains of <i>Escherichia coli</i> is now routinely achieved using naturally secreted substrates, such as YebF or the osmotically inducible protein Y (OsmY), as carrier molecules. However, secretion efficiency through these pathways needs to be improved for most synthetic biology and metabolic engineering applications. To address this challenge, we developed a generalizable survival-based selection strategy that effectively couples extracellular protein secretion to antibiotic resistance and enables facile isolation of rare mutants from very large populations (<i>i.e.</i>, 10^10–12 clones) based simply on cell growth. Using this strategy in the context of the YebF pathway, a comprehensive library of <i>E. coli</i> single-gene knockout mutants was screened and several gain-of-function mutations were isolated that increased the efficiency of extracellular expression without compromising the integrity of the outer membrane. We anticipate that this user-friendly strategy could be leveraged to better understand the YebF pathway and other secretory mechanismsenabling the exploration of protein secretion in pathogenesis as well as the creation of designer <i>E. coli</i> strains with greatly expanded secretomesall without the need for expensive exogenous reagents, assay instruments, or robotic automation

Production of Secretory and Extracellular N-Linked Glycoproteins in Escherichia coli▿ †

The Campylobacter jejuni pgl gene cluster encodes a complete N-linked protein glycosylation pathway that can be functionally transferred into Escherichia coli. In this system, we analyzed the interplay between N-linked glycosylation, membrane translocation and folding of acceptor proteins in bacteria. We developed a recombinant N-glycan acceptor peptide tag that permits N-linked glycosylation of diverse recombinant proteins expressed in the periplasm of glycosylation-competent E. coli cells. With this “glycosylation tag,” a clear difference was observed in the glycosylation patterns found on periplasmic proteins depending on their mode of inner membrane translocation (i.e., Sec, signal recognition particle [SRP], or twin-arginine translocation [Tat] export), indicating that the mode of protein export can influence N-glycosylation efficiency. We also established that engineered substrate proteins targeted to environments beyond the periplasm, such as the outer membrane, the membrane vesicles, and the extracellular medium, could serve as substrates for N-linked glycosylation. Taken together, our results demonstrate that the C. jejuni N-glycosylation machinery is compatible with distinct secretory mechanisms in E. coli, effectively expanding the N-linked glycome of recombinant E. coli. Moreover, this simple glycosylation tag strategy expands the glycoengineering toolbox and opens the door to bacterial synthesis of a wide array of recombinant glycoprotein conjugates

Universal Genetic Assay for Engineering Extracellular Protein Expression

A variety of strategies now exist for the extracellular expression of recombinant proteins using laboratory strains of Escherichia coli. However, secreted proteins often accumulate in the culture medium at levels that are too low to be practically useful for most synthetic biology and metabolic engineering applications. The situation is compounded by the lack of generalized screening tools for optimizing the secretion process. To address this challenge, we developed a genetic approach for studying and engineering protein-secretion pathways in E. coli<i>.</i> Using the YebF pathway as a model, we demonstrate that direct fluorescent labeling of tetracysteine-motif-tagged secretory proteins with the biarsenical compound FlAsH is possible <i>in situ</i> without the need to recover the cell-free supernatant. High-throughput screening of a bacterial strain library yielded superior YebF expression hosts capable of secreting higher titers of YebF and YebF-fusion proteins into the culture medium. We also show that the method can be easily extended to other secretory pathways, including type II and type III secretion, directly in E. coli. Thus, our FlAsH-tetracysteine-based genetic assay provides a convenient, high-throughput tool that can be applied generally to diverse secretory pathways. This platform should help to shed light on poorly understood aspects of these processes as well as to further assist in the construction of engineered E. coli strains for efficient secretory-protein production