Cloning of heterologous genes specifying detrimental proteins on pUC-derived plasmids inEscherichia coli

A system is described that enables the cloning of genes specifying detrimental proteins inEscherichia coli. The system is based on pUC plasmids and was developed for the expression of theBacillus subtilis csaA gene, which is lethal when expressed at high levels. Suppressor strains that tolerate the presence of plasmids for high-level expression ofcsaA were isolated, which contained small cryptic deletion variants of the parental plasmid in high copy numbers. The cryptic plasmids consisted mainly of the pUC replication functions and lacked thecsaA region and selectable markers. The co-resident, incompatible, cryptic plasmids enabled the maintenance of thecsaA plasmids by reducing their copy number 20-fold, which resulted in a concomitant 3- to 7-fold reduction in the expression of plasmid-encoded genes. Strains carrying these cryptic endogenous plasmids proved to be useful for the construction of pUC-based recombinant plasmids carrying other genes, such as theskc gene ofStreptococcus equisimilis, which cannot be cloned in high copy numbers inE. coli. Several strategies to reduce production levels of heterologous proteins specified by plasmids are compared.     

Modulating secretory pathway pH by proton channel co‐expression can increase recombinant protein stability in plants

Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host‐cell engineering approaches are being developed to ameliorate host‐cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid‐susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co‐expression. The co‐expression of a heterologous ion channel to stabilize acid‐labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts.     

Efficient markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugation

The probiotic strain Escherichia coli Nissle 1917 (EcN) is a common bacterial chassis in synthetic biology developments for therapeutic applications given its long track record of safe administration in humans. Chromosomal integration of the genes of interest (GOIs) in the engineered bacterium offers significant advantages in genetic stability and to control gene dose, but common methodologies relying on the transformation of EcN are inefficient. In this work, we implement in EcN the use of bacterial conjugation in combination with markerless genome engineering to efficiently insert multiple GOIs at different loci of EcN chromosome, leaving no antibiotic resistance genes, vector sequences or scars in the modified bacterium. The resolution of cointegrants that leads to markerless insertion of the GOIs requires expression of I-SceI endonuclease and its efficiency is enhanced by λ Red proteins. We show the potential of this strategy by integrating different genes encoding fluorescent and bioluminescent reporters (i.e. GFP, mKate2, luxCDABE) both individually and sequentially. We also demonstrate its application for gene deletions in EcN (ΔflhDC) and to replace the endogenous regulation of chromosomal locus (i.e. flhDC) by heterologous regulatory elements (e.g. tetR-Ptet) in order to have an ectopic control of gene expression in EcN with an external inducer to alter bacterial behaviour (e.g. flagellar motility). Whole-genome sequencing confirmed the introduction of the designed modifications without off-target alterations in the genome. This straightforward approach accelerates the generation of multiple modifications in EcN chromosome for the generation of living bacterial therapeutics.     

Cloning of heterologous genes specifying detrimental proteins on pUC-derived plasmids inEscherichia coli

A system is described that enables the cloning of genes specifying detrimental proteins inEscherichia coli. The system is based on pUC plasmids and was developed for the expression of theBacillus subtilis csaA gene, which is lethal when expressed at high levels. Suppressor strains that tolerate the presence of plasmids for high-level expression ofcsaA were isolated, which contained small cryptic deletion variants of the parental plasmid in high copy numbers. The cryptic plasmids consisted mainly of the pUC replication functions and lacked thecsaA region and selectable markers. The co-resident, incompatible, cryptic plasmids enabled the maintenance of thecsaA plasmids by reducing their copy number 20-fold, which resulted in a concomitant 3- to 7-fold reduction in the expression of plasmid-encoded genes. Strains carrying these cryptic endogenous plasmids proved to be useful for the construction of pUC-based recombinant plasmids carrying other genes, such as theskc gene ofStreptococcus equisimilis, which cannot be cloned in high copy numbers inE. coli. Several strategies to reduce production levels of heterologous proteins specified by plasmids are compared.     

Oxidative stress-resistance assay for screening yeast strains overproducing heterologous proteins

Many natural proteins have been developed into drugs and produced for direct application. Identifying improved hosts to achieve high-level heterologous protein production is a challenge in the study of heterologous protein expression in recombinant yeast. In this study, a novel high-throughput assay to screen such overproducing Saccharomyces cerevisiae strains was systematically developed. The protocol designed was based on screening host strain derivatives with increased superoxide dismutase dependent resistance to oxidative stress. Yeast cells transformed with recombinant plasmid carrying SOD1 gene as a reporter responded exquisitely to oxidative stress induced by elevated concentrations of paraquat. Improved yeast strains resulting from screening clones subjected to genome shuffling through selective pressure argue for a more effective screening system compared with traditonal selection. Moreover, this approach can be employed in general biochemical analysis without utilization of flow cytometry or well plate reader. Therefore, it is expected that the high-throughput assay would make superior strains producing heterologous proteins.     

Versatile Expression and Secretion Vectors for Bacillus subtilis

Most expression systems are based on Escherichia coli as the host strain because of the large availability of all kinds of vector plasmids. However, aside from the obvious advantages of E. coli systems, serious problems can occur during the process of heterologous gene expression and purification. Therefore, low expression rates, formation of inclusion bodies, improper protein-folding, and/or toxicity problems might enforce changing the expression host. Here we describe the construction of two new vectors, pBSMuL1 and pBSMuL2, for overexpression and secretion of heterologous proteins in Bacillus subtilis as an alternative expression host. The new plasmids combine several advantages in comparison to available Bacillus expression systems: an appropriate multiple cloning site consisting of 13 unique restriction sites, one (pBSMuL1) or two (pBSMuL2) strong constitutive promoters, a high efficient signal sequence for protein secretion, and the possibility to express proteins as His-tagged fusions for easy detection and purification. We have demonstrated the applicability of the novel vector plasmids for the production and purification of the heterologous cutinase from Fusarium solani pisi.     

A rapid protein folding assay for the bacterial periplasm

An array of genetic screens and selections has been developed for reporting protein folding and solubility in the cytoplasm of living cells. However, there are currently no analogous folding assays for the bacterial periplasm, despite the significance of this compartment for the expression of recombinant proteins, especially those requiring important posttranslational modifications (e.g., disulfide bond formation). Here, we describe an engineered genetic selection for monitoring protein folding in the periplasmic compartment of Escherichia coli cells. In this approach, target proteins are sandwiched between an N‐terminal signal recognition particle (SRP)‐dependent signal peptide and a C‐terminal selectable marker, TEM‐1 β‐lactamase. The resulting chimeras are localized to the periplasmic space via the cotranslational SRP pathway. Using a panel of native and heterologous proteins, we demonstrate that the folding efficiency of various target proteins correlates directly with in vivo β‐lactamase activity and thus resistance to ampicillin. We also show that this reporter is useful for the discovery of extrinsic periplasmic factors (e.g., chaperones) that affect protein folding and for obtaining folding‐enhanced proteins via directed evolution. Collectively, these data demonstrate that our periplasmic folding reporter is a powerful tool for screening and engineering protein folding in a manner that does not require any structural or functional information about the target protein.     

Heterologous expression of nonribosomal peptide synthetases in B. subtilis: construction of a bi-functional B subtilis/E coli shuttle vector system

A major obstacle in investigating the biosynthesis of pharmacologically important peptide antibiotics is the heterologous expression of the giant biosynthetic genes. Recently, the genetically engineered strain Bacillus subtilis KE30 has been reported as an excellent surrogate host for the heterologous expression of an entire nonribosomal peptide synthetase (NRPS) gene cluster. In this study, we expand the applicability of this strain, by the development of four Escherichia coli/B. subtilis shuttle expression vectors. Comparative overproduction of hybrid NRPS proteins derived from both organisms revealed a significant beneficial effect of overproducing proteins in B. subtilis KE30 as underlined by the production of stable nondegradative proteins, as well as the formation of active phosphopantetheinylated holo-proteins.     

High‐level expression of Aspergillus niger β‐galactosidase in Ashbya gossypii

Ashbya gossypii has been recently considered as a host for the expression of recombinant proteins. The production levels achieved thus far were similar to those obtained with Saccharomyces cerevisiae for the same proteins. Here, the β‐galactosidase from Aspergillus niger was successfully expressed and secreted by A. gossypii from 2‐µm plasmids carrying the native signal sequence at higher levels than those secreted by S. cerevisiae laboratorial strains. Four different constitutive promoters were used to regulate the expression of β‐galactosidase: A. gossypii AgTEF and AgGPD promoters, and S. cerevisiae ScADH1 and ScPGK1 promoters. The native AgTEF promoter drove the highest expression levels of recombinant β‐galactosidase in A. gossypii, leading to 2‐ and 8‐fold higher extracellular activity than the AgGPD promoter and the heterologous promoters, respectively. In similar production conditions, the levels of active β‐galactosidase secreted by A. gossypii were up to 37 times higher than those secreted by recombinant S. cerevisiae and ～2.5 times higher than those previously reported for the β‐galactosidase‐high producing S. cerevisiae NCYC869‐A3/pVK1.1. The substitution of glucose by glycerol in the production medium led to a 1.5‐fold increase in the secretion of active β‐galactosidase by A. gossypii. Recombinant β‐galactosidase secreted by A. gossypii was extensively glycosylated, as are the native A. niger β‐galactosidase and recombinant β‐galactosidase produced by yeast. These results highlight the potential of A. gossypii as a recombinant protein producer and open new perspectives to further optimize recombinant protein secretion in this fungus. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:261–268, 2014     

Curing of four different plasmids in Yersinia pestis using plasmid incompatibility

Aims: Plasmids are critical for the pathogenicity of Yersinia pestis. In order to carry out a systematic investigation of their role in pathogenesis, we cured plasmids from Y. pestis. Methods and Results: Each plasmid’s replicon of Y. pestis was cloned into plasmid pEX18Gm containing a counter‐selectable sacB gene, and was then introduced into Y. pestis strain 201 by electroporation. Strains containing recombinant plasmids were cultivated under antibiotic selection. The resultant plasmid‐curing colonies, identified by specific polymerase chain reactions, were then cured off pEX18Gm under sucrose pressure. This method was used to successfully cure all four plasmids of Y. pestis, singly or in different combinations. Conclusions: Naturally evolving plasmids in Y. pestis are difficult to remove by conventional curing methods. We employed a method based on plasmid incompatibility to cure the plasmids from Y. pestis, which confirmed the efficacy of this method for curing plasmids with different types of replicons from one bacterium. Significance and Impact of the Study: There have been no reports on the curing of multiple plasmids by using replication mechanisms from one bacterium with this technique. In the present study, we were able to successfully apply this methodology to cure four plasmids from Y. pestis, confirming its feasibility.     

Genetic selection system for improving recombinant membrane protein expression in E. coli

A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C‐terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I‐CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75‐fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90‐fold.     

Proteomic analysis of outer membrane vesicles from the probiotic strain Escherichia coli Nissle 1917

Escherichia coli Nissle 1917 (EcN) is a probiotic used for the treatment of intestinal disorders. EcN improves gastrointestinal homeostasis and microbiota balance; however, little is known about how this probiotic delivers effector molecules to the host. Outer membrane vesicles (OMVs) are constitutively produced by Gram‐negative bacteria and have a relevant role in bacteria–host interactions. Using 1D SDS–PAGE and highly sensitive LC–MS/MS analysis we identified in this study 192 EcN vesicular proteins with high confidence in three independent biological replicates. Of these proteins, 18 were encoded by strain‐linked genes and 57 were common to pathogen‐derived OMVs. These proteins may contribute to the ability of this probiotic to colonize the human gut as they fulfil functions related to adhesion, immune modulation or bacterial survival in host niches. This study describes the first global OMV proteome of a probiotic strain and provides evidence that probiotic‐derived OMVs contain proteins that can target these vesicles to the host and mediate their beneficial effects on intestinal function. All MS data have been deposited in the ProteomeXchange with identifier PXD000367 ( http://proteomecentral.proteomexchange.org/dataset/PXD000367 ).     

Practical protocols for production of very high yields of recombinant proteins using Escherichia coli

The gram‐negative bacterium Escherichia coli offers a mean for rapid, high yield, and economical production of recombinant proteins. However, high‐level production of functional eukaryotic proteins in E. coli may not be a routine matter, sometimes it is quite challenging. Techniques to optimize heterologous protein overproduction in E. coli have been explored for host strain selection, plasmid copy numbers, promoter selection, mRNA stability, and codon usage, significantly enhancing the yields of the foreign eukaryotic proteins. We have been working on optimizations of bacterial expression conditions and media with a focus on achieving very high cell density for high‐level production of eukaryotic proteins. Two high‐cell‐density bacterial expression methods have been explored, including an autoinduction introduced by Studier (Protein Expr Purif 2005;41:207–234) recently and a high‐cell‐density IPTG‐induction method described in this study, to achieve a cell‐density OD₆₀₀ of 10–20 in the normal laboratory setting using a regular incubator shaker. Several practical protocols have been implemented with these high‐cell‐density expression methods to ensure a very high yield of recombinant protein production. With our methods and protocols, we routinely obtain 14–25 mg of NMR triple‐labeled proteins and 17–34 mg of unlabeled proteins from a 50‐mL cell culture for all seven proteins we tested. Such a high protein yield used the same DNA constructs, bacterial strains, and a regular incubator shaker and no fermentor is necessary. More importantly, these methods allow us to consistently obtain such a high yield of recombinant proteins using E. coli expression.     

Biosynthesis of paromamine derivatives in engineered Escherichia coli by heterologous expression

Aims: Paromamine is a vital and common intermediate in the biosynthesis of 4,5 and 4,6‐disubstituted 2‐deoxystreptamine (DOS)‐containing aminoglycosides. Our aim is to develop an engineered Escherichia coli system for heterologous production of paromamine. Methods and Results: We have constructed a mutant of E. coli BL21 (DE3) by disrupting glucose‐6‐phosphate isomerase (pgi) of primary metabolic pathway to increase glucose‐6‐phosphate pool inside the host. Disruption was carried out by λ Red/ET recombination following the protocol mentioned in the kit. Recombinants bearing 2‐deoxy‐scyllo‐inosose (DOI), DOS and paromamine producing genes were constructed from butirosin gene cluster and heterologously expressed in engineered host designed as E. coli BL21 (DE3) Δpgi. Secondary metabolites produced by the recombinants fermentated in 2YTG medium were extracted, and analysis of the extracts showed there is formation of DOI, DOS and paromamine. Conclusions: Escherichia coli system is engineered for heterologous expression of paromamine derivatives of aminoglycoside biosynthesis. Significance and Impact of the Study: This is the first report of heterologous expression of paromamine gene set in E. coli. Hence a new platform is established in E. coli system for the production of paromamine which is useful for the exploration of novel aminoglycosides by combinatorial biosynthesis of 4,5‐ and 4,6‐disubtituted route of DOS‐containing aminoglycosides.     

Scalable,two-stage,autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion

We report the scalable production of recombinant proteins in Escherichia coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in the stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales. Expression levels using this approach have reached as high as 55% of the total cellular protein. The initial use of the method in instrumented fed-batch fermentations enables cell densities of ∼30 gCDW/L and protein titers up to 8.1 ± 0.7 g/L (∼270 mg/gCDW). The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 μl (384-well plates), 100 μl (96-well plates), 20 ml, and 100 ml. In batch cultures, cell densities routinely reach ∼5–7 gCDW/L, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation.     

Recombinant production of bacterial toxins and their derivatives in the methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The methylotrophic yeast Pichia pastoris is a popular heterologous expression host for the recombinant production of a variety of prokaryotic and eukaryotic proteins. The rapid emergence of P. pastoris as a robust heterologous expression host was facilitated by the ease with which it can be manipulated and propagated, which is comparable to that of Escherichia coli and Saccharomyces cerevisiae. P. pastoris offers further advantages such as the tightly-regulated alcohol oxidase promoter that is particularly suitable for heterologous expression of foreign genes. While recombinant production of bacterial toxins and their derivatives is highly desirable, attempts at their heterologous expression using the traditional E. coli expression system can be problematic due to the formation of inclusion bodies that often severely limit the final yields of biologically active products. However, recent literature now suggests that P. pastoris may be an attractive alternative host for the heterologous production of bacterial toxins, such as those from the genera Bacillus, Clostridium, and Corynebacterium, as well as their more complex derivatives. Here, we review the recombinant production of bacterial toxins and their derivatives in P. pastoris with special emphasis on their potential clinical applications. Considering that de novo design and construction of synthetic toxin genes have often been necessary to achieve optimal heterologous expression in P. pastoris, we also present general guidelines to this end based on our experience with the P. pastoris expression of the Bacillus thuringiensis Cyt2Aa1 toxin.     

Production of complex viral glycoproteins in plants as vaccine immunogens

Plant molecular farming offers a cost‐effective and scalable approach to the expression of recombinant proteins which has been proposed as an alternative to conventional production platforms for developing countries. In recent years, numerous proofs of concept have established that plants can produce biologically active recombinant proteins and immunologically relevant vaccine antigens that are comparable to those made in conventional expression systems. Driving many of these advances is the remarkable plasticity of the plant proteome which enables extensive engineering of the host cell, as well as the development of improved expression vectors facilitating higher levels of protein production. To date, the only plant‐derived viral glycoprotein to be tested in humans is the influenza haemagglutinin which expresses at ~50 mg/kg. However, many other viral glycoproteins that have potential as vaccine immunogens only accumulate at low levels in planta. A critical consideration for the production of many of these proteins in heterologous expression systems is the complexity of post‐translational modifications, such as control of folding, glycosylation and disulphide bridging, which is required to reproduce the native glycoprotein structure. In this review, we will address potential shortcomings of plant expression systems and discuss strategies to optimally exploit the technology for the production of immunologically relevant and structurally authentic glycoproteins for use as vaccine immunogens.     

Engineered protein nano-compartments for targeted enzyme localization

Compartmentalized co-localization of enzymes and their substrates represents an attractive approach for multi-enzymatic synthesis in engineered cells and biocatalysis. Sequestration of enzymes and substrates would greatly increase reaction efficiency while also protecting engineered host cells from potentially toxic reaction intermediates. Several bacteria form protein-based polyhedral microcompartments which sequester functionally related enzymes and regulate their access to substrates and other small metabolites. Such bacterial microcompartments may be engineered into protein-based nano-bioreactors, provided that they can be assembled in a non-native host cell, and that heterologous enzymes and substrates can be targeted into the engineered compartments. Here, we report that recombinant expression of Salmonella enterica ethanolamine utilization (eut) bacterial microcompartment shell proteins in E. coli results in the formation of polyhedral protein shells. Purified recombinant shells are morphologically similar to the native Eut microcompartments purified from S. enterica. Surprisingly, recombinant expression of only one of the shell proteins (EutS) is sufficient and necessary for creating properly delimited compartments. Co-expression with EutS also facilitates the encapsulation of EGFP fused with a putative Eut shell-targeting signal sequence. We also demonstrate the functional localization of a heterologous enzyme (β-galactosidase) targeted to the recombinant shells. Together our results provide proof-of-concept for the engineering of protein nano-compartments for biosynthesis and biocatalysis.     

Metabolic profiling of recombinant Escherichia coli cultivations based on high‐throughput FT‐MIR spectroscopic analysis

Escherichia coli is one of the most used host microorganism for the production of recombinant products, such as heterologous proteins and plasmids. However, genetic, physiological and environmental factors influence the plasmid replication and cloned gene expression in a highly complex way. To control and optimize the recombinant expression system performance, it is very important to understand this complexity. Therefore, the development of rapid, highly sensitive and economic analytical methodologies, which enable the simultaneous characterization of the heterologous product synthesis and physiologic cell behavior under a variety of culture conditions, is highly desirable. For that, the metabolic profile of recombinant E. coli cultures producing the pVAX‐lacZ plasmid model was analyzed by rapid, economic and high‐throughput Fourier Transform Mid‐Infrared (FT‐MIR) spectroscopy. The main goal of the present work is to show as the simultaneous multivariate data analysis by principal component analysis (PCA) and direct spectral analysis could represent a very interesting tool to monitor E. coli culture processes and acquire relevant information according to current quality regulatory guidelines. While PCA allowed capturing the energetic metabolic state of the cell, e.g. by identifying different C‐sources consumption phases, direct FT‐MIR spectral analysis allowed obtaining valuable biochemical and metabolic information along the cell culture, e.g. lipids, RNA, protein synthesis and turnover metabolism. The information achieved by spectral multivariate data and direct spectral analyses complement each other and may contribute to understand the complex interrelationships between the recombinant cell metabolism and the bioprocess environment towards more economic and robust processes design according to Quality by Design framework. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:285–298, 2017