Secretion Of Heterologous Proteins Research Articles

Developing a platform for secretion of biomolecules in Mycoplasma feriruminatoris

BackgroundHaving a simple and fast dividing organism capable of producing and exposing at its surface or secreting functional complex biomolecules with disulphide bridges is of great interest. The mycoplasma bacterial genus offers a set of relevant properties that make it an interesting chassis for such purposes, the main one being the absence of a cell wall. However, due to their slow growth, they have rarely been considered as a potential platform in this respect. This notion may be challenged with the recent discovery of Mycoplasma feriruminatoris, a species with a dividing time close to that of common microbial workhorses. So far, no tools for heterologous protein expression nor secretion have been described for it.ResultsThe work presented here develops the fast-dividing M. feriruminatoris as a tool for secreting functional biomolecules of therapeutic interest that could be used for screening functional mutants as well as potentially for protein-protein interactions. Based on RNAseq, quantitative proteomics and promoter sequence comparison we have rationally designed optimal promoter sequences. Then, using in silico analysis, we have identified putative secretion signals that we validated using a luminescent reporter. The potential of the resulting secretion cassette has been shown with set of active clinically relevant proteins (interleukins and nanobodies).ConclusionsWe have engineered Mycoplasma feriruminatoris for producing and secreting functional proteins of medical interest.

Analysis of signal peptides and secreted proteins in the whole proteome of Pichia pastoris

The signal peptide is a key factor that affects the efficiency of protein secretion in Pichia pastoris. Currently, the most used signal peptide is the α-mating factor (MFα) pre-pro leader from Saccharomyces cerevisiae. This exogenous signal peptide has been successfully utilized to express and secret many heterologous proteins. However, MFα is not suitable for the secretory expression of all heterologous proteins. Many typical signal peptides are present in the secretory proteins of P. pastoris, which provides more options besides MFα. Therefore, it is necessary to analyze and identify more efficient endogenous signal peptides that can guide the secretion of heterologous proteins in P. pastoris. In this study, we employed bioinformatics tools such as SignalP, TMHMM, Phobius, WoLF PSORT, and NetGPI to predict endogenous signal peptides from the entire proteome of P. pastoris GS115 (ATCC 20864). Moreover, we analyzed the distribution, length, amino acid composition, and conservation of these signal peptides. Additionally, we screened 69 secreted proteins and their signal peptides, and through secretome validation, we identified 10 endogenous signal peptides that have potential to be used for exogenous protein expression. The endogenous signal peptides obtained in this study may serve as new valuable tools for the expression and secretion of heterologous proteins in P. pastoris.

Exploring the secretome of Corynebacterium glutamicum ATCC 13032.

The demand for alternative sources of food proteins is increasing due to the limitations and challenges associated with conventional food production. Advances in biotechnology have enabled the production of proteins using microorganisms, thus prompting the exploration of attractive microbial hosts capable of producing functional proteins in high titers. Corynebacterium glutamicum is widely used in industry for the production of amino acids and has many advantages as a host organism for recombinant protein production. However, its performance in this area is limited by low yields of target proteins and high levels of native protein secretion. Despite representing a challenge for heterologous protein production, the C. glutamicum secretome has not been fully characterized. In this study, state-of-the-art mass spectrometry-based proteomics was used to identify and analyze the proteins secreted by C. glutamicum. Both the wild-type strain and a strain that produced and secreted a recombinant β-lactoglobulin protein were analyzed. A total of 427 proteins were identified in the culture supernatants, with 148 predicted to possess a secretion signal peptide. MS-based proteomics on the secretome enabled a comprehensive characterization and quantification (based on abundance) of the secreted proteins through label-free quantification (LFQ). The top 12 most abundant proteins accounted for almost 80% of the secretome. These are uncharacterized proteins of unknown function, resuscitation promoting factors, protein PS1, Porin B, ABC-type transporter protein and hypothetical membrane protein. The data can be leveraged for protein production by, e.g., utilizing the signal peptides of the most abundant proteins to improve secretion of heterologous proteins. In addition, secretory stress can potentially be alleviated by inactivating non-essential secreted proteins. Here we provide targets by identifying the most abundant, secreted proteins of which majority are of unknown function. The data from this study can thus provide valuable insight for researchers looking to improve protein secretion and optimize C. glutamicum as a host for secretory protein production.

A simple and effective method to remove pigments from heterologous secretory proteins expressed in Pichia pastoris

Pichia pastoris is a popular yeast host for high-level heterologous expression of proteins on an industrial scale owing to its reliable expression, robust growth, high fermentation density, and easy genetic manipulation and cultivation at a relatively low cost. Of particular interest is its high secretion efficiency for small proteins including insulin, human serum albumin, vaccines, enzymes, and llama-derived heavy-chain only antibodies (nanobodies) for pharmaceutical and research applications. However, a recurring challenge in using P. pastoris heterologous secretory proteins is the co-purification of a sticky, yellow pigment which has been identified as a tetra-benzoyl disaccharide. Current methods for pigment removal involve crystallization of the heterologous secretory protein, active carbon absorption, and chromatography using cation exchange and hydrophobic interaction. Here, we present a simple and effective method to remove the yellow pigment, demonstrated with divalent nanobodies targeting SARS-CoV-2. The method entails capturing the nanobody on an affinity column and subsequent washing with the zwitterionic detergent lauryldimethylamine N-oxide (LDAO). We anticipate the method become generally useful to remove pigments from secretion proteins produced in P. pastoris, offering a practical solution to enhance the purity of heterologous proteins in various biotechnological applications.

Enhanced protein secretion in reduced genome strains of Streptomyces lividans

BackgroundS. lividans TK24 is a popular host for the production of small molecules and the secretion of heterologous protein. Within its large genome, twenty-nine non-essential clusters direct the biosynthesis of secondary metabolites. We had previously constructed ten chassis strains, carrying deletions in various combinations of specialized metabolites biosynthetic clusters, such as those of the blue actinorhodin (act), the calcium-dependent antibiotic (cda), the undecylprodigiosin (red), the coelimycin A (cpk) and the melanin (mel) clusters, as well as the genes hrdD, encoding a non-essential sigma factor, and matAB, a locus affecting mycelial aggregation. Genome reduction was aimed at reducing carbon flow toward specialized metabolite biosynthesis to optimize the production of secreted heterologous protein.ResultsTwo of these S. lividans TK24 derived chassis strains showed ~ 15% reduction in biomass yield, 2-fold increase of their total native secretome mass yield and enhanced abundance of several secreted proteins compared to the parental strain. RNAseq and proteomic analysis of the secretome suggested that genome reduction led to cell wall and oxidative stresses and was accompanied by the up-regulation of secretory chaperones and of secDF, a Sec-pathway component. Interestingly, the amount of the secreted heterologous proteins mRFP and mTNFα, by one of these strains, was 12 and 70% higher, respectively, than that secreted by the parental strain.ConclusionThe current study described a strategy to construct chassis strains with enhanced secretory abilities and proposed a model linking the deletion of specialized metabolite biosynthetic clusters to improved production of secreted heterologous proteins.

Enhancing bacterial fitness and recombinant enzyme yield by engineering the quality control protease HtrA of Bacillus subtilis.

Bacillus subtilis is a Gram-positive bacterium that is well-known for its high secretory capacity. Although secretion of homologous proteins is extremely efficient, secretion of heterologous proteins imposes what is known as "secretion stress" on the cells. This is not desirable in an industrial setting, because the bacterial secretion stress responses can adversely affect product yields. So far, the main approach to reduce such counterproductive secretion stress responses has been to remove components of the CssR-CssS two-component regulatory system, including the CssR-CssS-regulated quality control proteases HtrA and/or HtrB. However, there are several examples indicating that a complete loss of these proteases does not correlate with improved protein secretion or a reduced secretion stress response. In this study, an alternative approach to limit the effects of secretion stress by modulating the protease activity of HtrA rather than completely removing this protease was investigated. Indeed, expression of the proteolytically inactive HtrA led to a generally improved fitness of the bacteria when producing the heterologous α-amylase AmyQ. Moreover, compared to control strains, the strain expressing the mutant HtrA provided higher AmyQ yields, which correlated with superior α-amylase enzymatic activity in shake flasks and fermenter-scale cultivations. Proteome analysis of the engineered strains revealed critical differences in stress responses and metabolism. Altogether, our present findings show how proteolytically inactive HtrA can be employed to achieve improved bacterial fitness and higher enzyme yields. IMPORTANCE In the expanding market of recombinant proteins, microbial cell factories such as Bacillus subtilis are key players. Microbial cell factories experience secretion stress during high-level production of secreted proteins, which can negatively impact product yield and cell viability. The CssRS two-component system and CssRS-regulated quality control proteases HtrA and HtrB play critical roles in the secretion stress response. HtrA has a presumptive dual function in protein quality control by exerting both chaperone-like and protease activities. However, its potential role as a chaperone has not been explored in B. subtilis. Here, we describe for the first time the beneficial effects of proteolytically inactive HtrA on α-amylase yields and overall bacterial fitness.

An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system.

Saccharomyces cerevisiae is one of the most extensively used biosynthetic systems for the production of diverse bioproducts, especially biotherapeutics and recombinant proteins. Because the expression and insertion of foreign genes are always impaired by the endogenous factors of Saccharomyces cerevisiae and nonproductive procedures, various technologies have been developed to enhance the strength and efficiency of transcription and facilitate gene editing procedures. Thus, the limitations that block heterologous protein secretion have been overcome. Highly efficient promoters responsible for the initiation of transcription and the accurate regulation of expression have been developed that can be precisely regulated with synthetic promoters and double promoter expression systems. Appropriate codon optimization and harmonization for adaption to the genomic codon abundance of S. cerevisiae are expected to further improve the transcription and translation efficiency. Efficient and accurate translocation can be achieved by fusing a specifically designed signal peptide to an upstream foreign gene to facilitate the secretion of newly synthesized proteins. In addition to the widely applied promoter engineering technology and the clear mechanism of the endoplasmic reticulum secretory pathway, the innovative genome editing technique CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated system) and its derivative tools allow for more precise and efficient gene disruption, site-directed mutation, and foreign gene insertion. This review focuses on sophisticated engineering techniques and emerging genetic technologies developed for the accurate metabolic regulation of the S. cerevisiae expression system.

Engineering natural isolates of Saccharomyces cerevisiae for consolidated bioprocessing of cellulosic feedstocks

Saccharomyces cerevisiae has gained much attention as a potential host for cellulosic bioethanol production using consolidated bioprocessing (CBP) methodologies, due to its high-ethanol-producing titres, heterologous protein production capabilities, and tolerance to various industry-relevant stresses. Since the secretion levels of heterologous proteins are generally low in domesticated strains of S. cerevisiae, natural isolates may offer a more diverse genetic background for improved heterologous protein secretion, while also displaying greater robustness to process stresses. In this study, the potential of natural and industrial S. cerevisiae strains to secrete a core set of cellulases (CBH1, CBH2, EG2, and BGL1), encoded by genes integrated using CRISPR/Cas9 tools, was evaluated. High levels of heterologous protein production were associated with a reduced maximal growth rate and with slight changes in overall strain robustness, compared to the parental strains. The natural isolate derivatives YI13_BECC and YI59_BECC displayed superior secretion capacity for the heterologous cellulases at high incubation temperature and in the presence of acetic acid, respectively, compared to the reference industrial strain MH1000_BECC. These strains also exhibited multi-tolerance to several fermentation-associated and secretion stresses. Cultivation of the strains on crystalline cellulose in oxygen-limited conditions yielded ethanol concentrations in the range of 4–4.5 g/L, representing 35–40% of the theoretical maximum ethanol yield after 120 h, without the addition of exogenous enzymes. This study therefore highlights the potential of these natural isolates to be used as chassis organisms in CBP bioethanol production.Key points• Process-related fermentation stresses influence heterologous protein production.• Transformants produced up to 4.5 g/L ethanol, ~ 40% of the theoretical yield in CBP.• CRISPR/Cas9 was feasible for integrating genes in natural S. cerevisiae isolates.

Functional characterization of the Komagataella phaffii 1033 gene promoter and transcriptional terminator.

The methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris) is a widely used host for extracellularly producing heterologous proteins via an expression cassette integrated into the yeast genome. A strong promoter in the expression cassette is not always the most favorable choice for heterologous protein production, especially if the correct folding of the protein and/or post-translational processing is the limiting step. The transcriptional terminator is another regulatory element in the expression cassette that can modify the expression levels of the heterologous gene. In this work, we identified and functionally characterized the promoter (P1033) and transcriptional terminator (T1033) of a constitutive gene (i.e., the 1033 gene) with a weak non-methanol-dependent transcriptional activity. We constructed two K. phaffii strains with two combinations of the regulatory DNA elements from the 1033 and AOX1 genes (i.e., P1033-TAOX1 and P1033-T1033 pairs) and evaluated the impact of the regulatory element combinations on the transcript levels of the heterologous gene and endogenous 1033 and GAPDH genes in cells grown in glucose or glycerol, and on the extracellular product/biomass yield. The results indicate that the P1033 has a 2-3% transcriptional activity of the GAP promoter and it is tunable by cell growth and the carbon source. The combinations of the regulatory elements rendered different transcriptional activity of the heterologous and endogenous genes that were dependent on the carbon source. The promoter-terminator pair and the carbon source affected the heterologous gene translation and/or protein secretion pathway. Moreover, low heterologous gene-transcript levels along with glycerol cultures increased translation and/or protein secretion.

Efficient Autotransporter-Mediated Extracellular Secretion of a Heterologous Recombinant Protein by Escherichia coli.

The autotransporter protein secretion system has been used previously to target the secretion of heterologous proteins to the bacterial cell surface and the extracellular milieu at the laboratory scale. The platform is of particular interest for the production of "difficult" recombinant proteins that might cause toxic effects when produced intracellularly. One such protein is IrmA. IrmA is a vaccine candidate that is produced in inclusion bodies requiring refolding. Here, we describe the use and scale-up of the autotransporter system for the secretion of an industrially relevant protein (IrmA). A plasmid expressing IrmA was constructed such that the autotransporter platform could secrete IrmA into the culture supernatant fraction. The autotransporter platform was suitable for the production and purification of IrmA with comparable physical properties to the protein produced in the cytoplasm. The production of IrmA was translated to scale-up protein production conditions resulting in a yield of 29.3 mg/L of IrmA from the culture supernatant, which is consistent with yields of current industrial processes. IMPORTANCE Recombinant protein production is an essential component of the biotechnology sector. Here, we show that the autotransporter platform is a viable method for the recombinant production, secretion, and purification of a "difficult" to produce protein on an industrially relevant scale. Use of the autotransporter platform could reduce the number of downstream processing operations required, thus accelerating the development time and reducing costs for recombinant protein production.

Construction of a constitutively active type III secretion system for heterologous protein secretion.

Proteins comprise a multibillion-dollar industry in enzymes and therapeutics, but bacterial protein production can be costly and inefficient. Proteins of interest (POIs) must be extracted from lysed cells and inclusion bodies, purified, and resolubilized, which adds significant time and cost to the protein-manufacturing process. The Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS) has been engineered to address these problems by secreting soluble, active proteins directly into the culture media, reducing the number of purification steps. However, the current best practices method of T3SS pathway activation is not ideal for industrial scaleup. Previously, the T3SS was activated by plasmid-based overexpression of the T3SS transcriptional regulator, hilA, which requires the addition of a small molecule inducer (IPTG) to the culture media. IPTG adds significant cost to production and plasmid-based expression is subject to instability in large-scale fermentation. Here, we modulate the upstream transcriptional regulator, hilD, to activate the T3SS via three distinct methods. In doing so, we develop a toolbox of T3SS activation methods and construct constitutively active T3SS strains capable of secreting a range of heterologous proteins at titers comparable to plasmid-based hilA overexpression. We also explore how each activation method in our toolbox impacts the SPI-1 regulatory cascade and discover an epistatic relationship between T3SS regulators, hilE and the hilD 3' untranslated region (hilD 3'UTR). Together, these findings further our goal of making an industrially competitive protein production strain that reduces the challenges associated with plasmid induction and maintenance. KEY POINTS: • Characterized 3 new type III secretion system (T3SS) activation methods for heterologous protein secretion, including 2 constitutive activation methods. • Eliminated the need for a second plasmid and a small molecule inducer to activate the system, making it more suitable for industrial production. • Discovered new regulatory insights into the SPI-1 T3SS, including an epistatic relationship between regulators hilE and the hilD 3' untranslated region.

Pesticin-Like Effector VgrG3cp Targeting Peptidoglycan Delivered by the Type VI Secretion System Contributes to Vibrio cholerae Interbacterial Competition.

Vibrio cholerae can utilize a type VI secretion system (T6SS) to increase its intra- and interspecies competition. However, much still remains to be understood about the underlying mechanism of this intraspecies competition. In this study, we isolated an environmental V. cholerae strain E1 that lacked the typical virulence factors toxin-coregulated pilus and cholera toxin and that encoded a functional T6SS. We identified an evolved VgrG3 variant with a predicted C-terminal pesticin-like domain in V. cholerae E1, designated VgrG3cp. Using heterologous expression, protein secretion, and peptidoglycan-degrading assays, we demonstrated that VgrG3cp is a T6SS-dependent effector harboring cell wall muramidase activity and that its toxicity can be neutralized by cognate immunity protein TsiV3cp. Site-directed mutagenesis proved that the aspartic acid residue at position 867 is crucial for VgrG3cp-mediated antibacterial activity. Bioinformatic analysis showed that genes encoding VgrG3cp-like homologs are distributed in Vibrio species, are linked with T6SS structural genes and auxiliary genes, and the vgrG3cp-tsiV3cp gene pair of V. cholerae probably evolved from Vibrio anguillarum and Vibrio fluvialis via homologous recombination. Through a time-lapse microscopy assay, we directly determined that cells accumulating VgrG3cp disrupted bacterial division, while the cells continued to increase in size until the loss of membrane potential and cell wall breakage and finally burst. The results of the competitive killing assay showed that VgrG3cp contributes to V. cholerae interspecies competition. Collectively, our study revealed a novel T6SS E-I pair representing a new T6SS toxin family which allows V. cholerae to gain dominance within polymicrobial communities by T6SS. IMPORTANCE The type VI secretion system used by a broad range of Gram-negative bacteria delivers toxic proteins to target adjacent eukaryotic and prokaryotic cells. Diversification of effector proteins determines the complex bacterium-bacterium interactions and impacts the health of hosts and environmental ecosystems in which bacteria reside. This work uncovered an evolved valine-glycine repeat protein G3, carrying a C-terminal pesticin-like domain (VgrG3cp), which has been suggested to harbor cell wall hydrolase activity and is able to affect cell division and the integrity of cell wall structure. Pesticin-like homologs constitute a family of T6SS-associated effectors targeting bacterial peptidoglycan which are distributed in Vibrio species, and genetic loci of them are linked with T6SS structural genes and auxiliary genes. T6SS-delivered VgrG3cp mediated broad-spectrum antibacterial activity for several microorganisms tested, indicating that VgrG3cp-mediated antimicrobial activity is capable of conferring bacteria a competitive advantage over competitors in the same niches.

The α-mating factor secretion signals and endogenous signal peptides for recombinant protein secretion in Komagataella phaffii

BackgroundThe budding yeast Komagataella phaffii (Pichia pastoris) is widely employed to secrete proteins of academic and industrial interest. For secretory proteins, signal peptides are the sorting signal to direct proteins from cytosol to extracellular matrix, and their secretion efficiency directly impacts the yields of the targeted proteins in fermentation broth. Although the α-mating factor (MF) secretion signal from S. cerevisiae, the most common and widely used signal sequence for protein secretion, works in most cases, limitation exists as some proteins cannot be secreted efficiently. As the optimal choice of secretion signals is often protein specific, more secretion signals need to be developed to augment protein expression levels in K. phaffii.ResultsIn this study, the secretion efficiency of 40 α-MF secretion signals from various yeast species and 32 endogenous signal peptides from K. phaffii were investigated using enhanced green fluorescent protein (EGFP) as the model protein. All of the evaluated α-MF secretion signals successfully directed EGFP secretion except for the secretion signals of the yeast D. hansenii CBS767 and H. opuntiae. The secretion efficiency of α-MF secretion signal from Wickerhamomyces ciferrii was higher than that from S. cerevisiae. 24 out of 32 endogenous signal peptides successfully mediated EGFP secretion. The signal peptides of chr3_1145 and FragB_0048 had similar efficiency to S. cerevisiae α-MF secretion signal for EGFP secretion and expression.ConclusionsThe screened α-MF secretion signals and endogenous signal peptides in this study confer an abundance of signal peptide selection for efficient secretion and expression of heterologous proteins in K. phaffii.

Construction and development of a novel dual-gene coexpression system to promote heterologous protein secretion for Streptomyces.

Streptomyces lividans is a potent host for the extracellular overproduction of heterologous proteins. To further improve the usability and productivity of S. lividans, a dual gene expression vector of "pTSKr duet" containing two strong constitutive promoters, scmpPc and kasOp*, was constructed. The success in the overproduction of two secretory enzymes simultaneously without interference with each other indicated that the "pTSKr duet" vector can realize the coexpression of two genes simultaneously and independently. Further, using the two-gene coexpression vector, we screened the effects of the overexpression of five factors that possibly promote secretion on the extracellular overproduction of heterologous secretory proteins. Interestingly, the coexpression of a quality control regulator (CssR) promoted the overproduction level to 1.3-fold for a stable heterologous protein of SMTG (transglutaminase from S. mobaraensis), while other four factors limited the overproduction of SMTG at different degrees.

Identification of genetic variants of the industrial yeast Komagataella phaffii (Pichia pastoris) that contribute to increased yields of secreted heterologous proteins.

The yeast Komagataella phaffii (formerly called Pichia pastoris) is used widely as a host for secretion of heterologous proteins, but only a few isolates of this species exist and all the commonly used expression systems are derived from a single genetic background, CBS7435 (NRRL Y-11430). We hypothesized that other genetic backgrounds could harbor variants that affect yields of secreted proteins. We crossed CBS7435 with 2 other K. phaffii isolates and mapped quantitative trait loci (QTLs) for secretion of a heterologous protein, β-glucosidase, by sequencing individual segregant genomes. A major QTL mapped to a frameshift mutation in the mannosyltransferase gene HOC1, which gives CBS7435 a weaker cell wall and higher protein secretion than the other isolates. Inactivation of HOC1 in the other isolates doubled β-glucosidase secretion. A second QTL mapped to an amino acid substitution in IRA1 that tripled β-glucosidase secretion in 1-week batch cultures but reduced cell viability, and its effects are specific to this heterologous protein. Our results demonstrate that QTL analysis is a powerful method for dissecting the basis of biotechnological traits in nonconventional yeasts, and a route to improving their industrial performance.

The rational modification of the secretion pathway: The bidirectional grinding strategy on signal peptide and SecA in Bacillus subtilis

The secretory (Sec) pathway is essential for protein translocation in Bacillus subtilis. In contrast to signal peptides (SPs), the key components of the Sec pathway lack excessive attention. Herein, based on the structural and mechanistic insights of SecA with SPs, the “Embedded-Model” was proposed, and the matching behavior was “grinded” to boost heterologous protein secretion efficiency. The binding structure of SecA with a SP was established and analyzed using comprehensive computational biology methods. An interaction-based grinding approach was explored for SP and SecA rational modification. The corresponding AprE exhibited 117.2% and 22.0% higher activities than those obtained using wild-type SP and SecA, respectively. Furthermore, a bidirectional grinding strategy was developed for both SPs and SecA. The AprE activity was therefore remarkably increased up to 349.6%. This study provides a novel and promising strategy for optimizing the secretion system and enhancing the secretory production of valuable proteins in B. subtilis.

Comprehensive Analysis of Signal Peptides in Saccharomyces cerevisiae Reveals Features for Efficient Secretion.

Signal peptides (SPs) are N-terminus sequences on the nascent polypeptide for protein export or localization delivery, which are essential for maintaining cell function. SPs are also employed as a key element for industrial production of secreted recombinant proteins. Yet, detailed information and rules about SPs and their cellular interactions are still not well understood. Here, systematic bioinformatics analysis and secretion capacity measurement of genome-wide SPs from the model organism Saccharomyces cerevisiae is performed. Several key features of SPs, including region properties, consensus motifs, evolutionary relationships, codon bias, e.g., are successfully revealed. Diverse cell metabolism can be trigged by using different SPs for heterologous protein secretion. Influences on SPs with different properties by chaperones can cause different secretory efficiencies. Protein secretion by the SP NCW2 in SEC72 deletion strain is 10 times than the control. These findings provide insights into the properties and functions of SPs and contribute to both fundamental research and industrial application.

3D-Printable Cellular Composites for the Production of Recombinant Proteins.

The incorporation of living cells into materials promises both significant challenges and new possibilities. Although recent years have seen important advances in this field, there is still much to be learned about engineering interfaces between cells and materials. Here, we present a new class of 3D-printable materials, based on poly(N-hydroxymethylacrylamide) (PNHMAA), in which the spore-forming bacterium Bacillus subtilis is effectively cross-linked into the surrounding polymeric scaffold. After dehydration and subsequent re-swelling in nutrient-rich media, embedded cells and spores become metabolically active and are capable of heterologous protein production and secretion. Strikingly, the leak-free scaffold allows protein production while preventing escape of embedded cells. The successful construction of complex three-dimensional structures by stereolithographic printing of living PNHMAA composite materials suggests utility in a broad range of applications.

Cloning and Expression of Metagenomic DNA in Streptomyces lividans and Its Subsequent Fermentation for Optimized Production.

The choice of an expression system for the metagenomic DNA of interest is of vital importance for the detection of any particular gene or gene cluster. Most of the screens to date have used the Gram-negative bacterium Escherichia coli as a host for metagenomic gene libraries. However, the use of E. coli introduces a potential host bias since only 40% of the enzymatic activities may be readily recovered by random cloning in E. coli. To recover some of the remaining 60%, alternative cloning hosts such as Streptomyces spp. have been used. Streptomycetes are high-GC Gram-positive bacteria belonging to the Actinomycetales and they have been studied extensively for more than 25years as an alternative expression system. They are extremely well suited for the expression of DNA from other actinomycetes and genomes of high GC content. Furthermore, due to its high innate, extracellular secretion capacity, Streptomyces can be a better system than E. coli for the production of many extracellular proteins. In this article, an overview is given about the materials and methods for growth and successful expression and secretion of heterologous proteins from diverse origin using Streptomyces lividans as a host. More in detail, an overview is given about the protocols of transformation, type of plasmids used and of vectors useful for integration of DNA into the host chromosome, and accompanying cloning strategies. In addition, various control elements for gene expression including synthetic promoters are discussed, and methods to compare their strength are described. Stable and efficient marker-less integration of the gene of interest under the control of the promoter of choice into S. lividans chromosome via homologous recombination using pAMR23A-based system will be explained. Finally, a basic protocol for bench-top bioreactor experiments which can form the start in the production process optimization and up-scaling will be provided.

Eisosome disruption by noncoding RNA deletion increases protein secretion in yeast.

Noncoding RNAs (ncRNAs) regulate many aspects of gene expression. We investigated how ncRNAs affected protein secretion in yeast by large-scale screening for improved endogenous invertase secretion in ncRNA deletion strains with deletion of stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs), tRNAs, or snRNAs. We identified three candidate ncRNAs, SUT418, SUT390, and SUT125, that improved endogenous invertase secretion when deleted. As SUTs can affect expression of nearby genes, we quantified adjacent gene transcription and found that the PIL1 gene was down-regulated in the SUT125 deletion strain. Pil1 is a core component of eisosomes, nonmobile invaginations found throughout the plasma membrane. PIL1 knockout alone, or in combination with eisosome components LSP1 or SUR7, resulted in further increased secretion of invertase. Secretion of heterologous GFP was also increased upon PIL1 deletion, but this increase was signal sequence dependent. To reveal the potential for increased biopharmaceutical production, secretion of monoclonal antibody Pexelizumab scFv peptide was increased by PIL1 deletion. Global analysis of secreted proteins revealed that approximately 20% of secreted proteins, especially serine-enriched secreted proteins, including invertase, were increased upon eisosome disruption. Eisosomes are enriched with APC transporters and sphingolipids, which are essential components for secretory vesicle formation and protein sorting. Sphingolipid and serine biosynthesis pathways were up-regulated upon PIL1 deletion. We propose that increased secretion of endogenous and heterologous proteins upon PIL1 deletion resulted from sphingolipid redistribution in the plasma membrane and up-regulated sphingolipid biosynthesis. Overall, a new pathway to improve protein secretion in yeast via eisosome disruption has been identified.