Constructing an Antibiotic-Free Protein Expression System for Ovalbumin Biosynthesis in Probiotic Escherichia coli Nissle 1917.

Ovalbumin (OVA) is the principal protein constituent of eggs. As an alternative to eggs, cell-cultured OVA can reduce the environmental impact of global warming and land use. Escherichia coli Nissle 1917 (EcN), a probiotic with specific endogenous cryptic plasmids that stably exist in cells without the addition of antibiotics, was chosen as the host for the efficient heterologous expression of the OVA. OVA yield reached 20 mg·L-1 in shake flasks using the OVA expression cassette containing a tac promoter (Ptac) upstream of the OVA-coding sequences on the endogenous plasmid pMUT2. Subsequently, we improved the level of the expression of the OVA by employing a dual promoter (PP5 combined with Ptac via a sigma factor binding site 24) and ribosome binding site (RBS) substitution. These enhancements increased the level of production of OVA in shake flasks to 30 and 42 mg·L-1, respectively. OVA by EcNP-P28 harboring plasmid L28 equipped with both dual promoter and the strong RBS8 reached 3.70 g·L-1 in a 3 L bioreactor. Recombinant OVA and natural OVA showed similar biochemical characteristics, including secondary structure, isoelectric point, amino acid composition, and thermal stability. This is currently the highest OVA production reported among prokaryotes. We successfully constructed an antibiotic-free heterologous protein expression system for EcN.

A new look at the fluorescent protein-based approach for identifying optimal coding sequence for recombinant protein expression in E. coli.

Due to the degeneracy of the genetic code, most amino acids are encoded by several codons. The choice among synonymous codons at the N-terminus of genes has a profound effect on protein expression in Escherichia coli. This is often explained by the different contributions of synonymous codons to mRNA secondary structure formation. Strong secondary structures at the 5'-end of mRNA interfere with ribosome binding and affect the process of translation initiation. In silico optimization of the gene 5'-end can significantly increase the level of protein expression; however, this method is not always effective due to the uncertainty of the exact mechanism by which synonymous substitutions affect expression; thus, it may produce nonoptimal variants as well as miss some of the best producers. In this paper, an alternative approach is proposed based on screening a partially randomized library of expression constructs comprising hundreds of selected synonymous variants. The effect of such substitutions was evaluated using the gene of interest fused to the reporter gene of the fluorescent protein with subsequent screening for the most promising candidates according to the reporter's signal intensity. The power of the approach is demonstrated by a significant increase in the prokaryotic expression of three proteins: canine cystatin C, human BCL2-associated athanogene 3 and human cardiac troponin I. This simple approach was suggested which may provide an efficient, easy, and inexpensive optimization method for poorly expressed proteins in bacteria.

Genetic identification and expression optimization of a novel protease HapR from Bacillus velezensis.

Due to the broad application and substantial market demand for proteases, it was vital to explore the novel and efficient protease resources. The aim of this study was to identify the novel protease for tobacco protein degradation and optimize the expression levels. Firstly, the tobacco protein was used as the sole nitrogen resource for isolation of protease-producing strains, and a strain with high protease production ability was obtained, identified as Bacillus velezensis WH-7. Then, the whole genome sequencing was conducted on the strain B. velezensis WH-7, and 7 proteases genes were mined by gene annotation analysis. By further heterologous expression of the 7 protease genes, the key protease HapR was identified with the highest protease activity (144.19 U/mL). Moreover, the catalysis mechanism of HapR was explained by amino acid sequence analysis. The expression levels of protease HapR were further improved through optimization of promoter, signal peptide and host strain, and the maximum protease activity reaced 384.27 U/mL in WX-02/pHY-P43-SPyfkD-hapR, increased by 167% than that of initial recombinant strain HZ/pHY-P43-SPhapR-hapR. This study identified a novel protease HapR and the expression level was significantly improved, which provided an important enzyme resource for the development of enzyme preparations in tobacco protein degradation.

Setup and Applications of Modular Protein Expression Toolboxes (MoPET) for Mammalian Systems.

The design and generation of an optimal protein expression construct is the first and essential step in the characterization of any protein of interest. However, the exchange and modification of the coding and/or noncoding elements to analyze their effect on protein function or generating the optimal result can be a tedious and time-consuming process using standard molecular biology cloning methods. To streamline the process to generate defined expression constructs or libraries of otherwise difficult to express proteins, the Modular Protein Expression Toolbox (MoPET) has been developed (Weber E, PloS One 12(5):e0176314, 2017). The system applies Golden Gate cloning as an assembly method and follows the standardized modular cloning (MoClo) principle (Weber E, PloS One 6(2):e16765, 2011). This cloning platform allows highly efficient DNA assembly of pre-defined, standardized functional DNA modules effecting protein expression with a focus on minimizing the cloning burden in coding regions. The original MoPET system consists of 53 defined DNA modules divided into eight functional main classes and can be flexibly expanded dependent on the need of the experimenter and expression host. However, already with a limited set of only 53 modules, 792,000 different constructs can be rationally designed or used to generate combinatorial expression optimization libraries. We provide here a detailed protocol for the (1) design and generation of level 0 basic parts, (2) generation of defined expressions constructs, and (3) generation of combinatorial expression libraries.

[Optimization of retinin expression and the application with wax emulsion in nanocoatings].

Anti-reflective nanocoatings that mimic the eyes of fruit flies are biodegradable materials with great market potential for a variety of optical devices that require anti-reflective properties. Microbial expression of retinin provides a new idea for the preparation of nanocoatings under mild conditions compared to physicochemical methods. However, the current expression level of retinin, the key to anti-reflective coating, is low and difficult to meet mass production. In this study, we analyzed and screened the best expression hosts for Drosophila-derived retinin protein, and optimized its expression. Chinese hamster ovary (CHO) cells were identified as the efficient expression host of retinin, and purified retinin protein was obtained. At the same time, the preparation method of lanolin nanoemulsion was explored, and the best anti-reflective ability of the nano-coating was determined when the ratio of specific concentration of retinin protein and wax emulsion was 16:4, the pH of the nano-coating formation system was 7.0, and the temperature was 30 ℃. The enhanced antireflective ability and reduced production cost of artificial antireflective nanocoatings by determining the composition of nanocoatings and optimizing the concentration, pH and temperature of system components may facilitate future application of artificial green degradable antireflective coatings.

Multidimensional engineering of Escherichia coli for efficient biosynthesis of cis-3-hydroxypipecolic acid.

Cis-3-hydroxypipecolic acid (cis-3-HyPip) is the crucial part of many alkaloids and drugs. However, its bio-based industrial production remains challenging. Here, lysine cyclodeaminase from Streptomyces malaysiensis (SmLCD) and pipecolic acid hydroxylase from Streptomyces sp. L-49973 (StGetF) were screened to achieve the conversion of L-lysine to cis-3-HyPip. Considering the high-cost of cofactors, NAD(P)H oxidase from Lactobacillus sanfranciscensis (LsNox) was further overexpressed in chassis strain Escherichia coli W3110 ΔsucCD (α-ketoglutarate-producing strain) to construct the NAD+ regeneration system, thus realizing the bioconversion of cis-3-HyPip from low-cost substrate L-lysine without NAD+ and α-ketoglutarate addition. To further accelerate the transmission efficiency of cis-3-HyPip biosynthetic pathway, multiple-enzyme expression optimization and transporter dynamic regulation via promoter engineering were conducted. Through fermentation optimization, the final engineered strain HP-13 generated 78.4 g/L cis-3-HyPip with 78.9% conversion in a 5-L fermenter, representing the highest production level achieved so far. These strategies described herein show promising potentials for large-scale production of cis-3-HyPip.

Optimization, characterization, comparison of self-assembly VLP of capsid protein L1 in yeast and reverse vaccinology design against human papillomavirus type 52.

BACKGROUND: Vaccination is the one of the agendas of many countries to reduce cervical cancer caused by the Human papillomavirus. Currently, VLP-based vaccine is the most potent vaccine against HPV, which could be produced by a variety of expression systems. Our study focuses on a comparison of recombinant protein expression L1 HPV52 using two common yeasts, Pichia pastoris and Hansenula polymorpha that have been used for vaccine production on an industrial scale. We also applied bioinformatics approach using reverse vaccinology to design alternative multi-epitope vaccines in recombinant protein and mRNA types. RESULTS: Our study found that P. pastoris relatively provided higher level of L1 protein expression and production efficiency compared to H. polymorpha in a batch system. However, both hosts showed self-assembly VLP formation and stable integration during protein induction. The vaccine we have designed exhibited high immune activation and safe in computational prediction. It is also potentially suitable for production in a variety of expression systems. CONCLUSION: By monitoring the overall optimization parameter assessment, this study can be used as the basis reference for large-scale production of the HPV52 vaccine.

Optimization of Inclusion Body Formation and Purification in Multi-well Plates.

Heterologous expression has long been used for the efficient production of proteins and enzymes as it offers significant advantages over purification of proteins from their native organisms. When first established, great efforts have been made to heterologously express proteins with high yields in the soluble fraction, hence, avoiding protein aggregation. In recent decades, however, it has been shown that the formation of aggregates (inclusion bodies; IBs) can be beneficial. To recover active protein, however, proteins should have been refolded from IBs after purification. The discovery that IBs themselves can also be active has revolutionized the entire protein production field. Therefore, several approaches have been described to generate catalytically active IBs during heterologous expression. Since several extrinsic and intrinsic factors such as protein structure and toxicity, pH and temperature of expression, and the used media might influence the formation of IBs, it is time and material consuming to use shake flask to examine and optimize different expression conditions. However, by using multi-well plates, it is possible to rapidly develop an efficient protocol for the expression of catalytically active IBs in a rational approach. The presented protocol was used for the heterologous expression of a 5'-adenosine monophosphate phosphorylase which forms catalytically active aggregates during expression in E. coli.

Cost-effective, high-yield production of Pyrobaculum calidifontis DNA polymerase for PCR application.

Polymerase Chain Reaction (PCR) is widely used for cloning, genetic engineering, mutagenesis, detection and diagnosis. A thermostable DNA polymerase is required for PCR. Here we describe low-cost and high-recovery production of Pyrobaculum calidifontis DNA polymerase (Pca-Pol). The gene was cloned in pET-28a and expressed in Escherichia coli BL21CodonPlus. Gene expression conditions were optimized. Eventually, gene expression was induced with 0.1 mM IPTG for 3 hours at 37 °C. Recombinant Pca-Pol produced was purified to homogeneity by immobilized metal-ion affinity chromatography yielding around 9000 U of Pca-Pol per liter of the culture with a recovery of 92%. Stability and PCR amplification efficiency of Pca-Pol was tested under various storage conditions with highest efficiency in 25 mM Tris-Cl buffer (pH 8.5) containing 0.1% Tween 20, 0.2 mg/mL BSA and 20% glycerol. Under this condition, no loss in PCR activity of Pca-Pol was observed, even after one year of storage. Repeated freeze-thaw, however, deteriorated enzyme activity of Pca-Pol. 55% PCR amplification activity retained after 7 prolong freeze-thaw cycles (freezing overnight at -20 °C and thawing for 45 minutes at 28 °C). Purified Pca-Pol possessed 3'-5' exonuclease (proofreading) activity and is expected to have greater fidelity as compared to Taq polymerase which does not have proofreading activity.

Metabolic Engineering of Escherichia coli for Increased Bioproduction of N-Acetylneuraminic Acid.

N-Acetylneuraminic acid (NeuAc) is widely used in the food and pharmaceutical industries. Therefore, it is important to develop an efficient and eco-friendly method for NeuAc production. Here, we achieved de novo biosynthesis of NeuAc in an engineered plasmid-free Escherichia coli strain, which efficiently synthesizes NeuAc using glycerol as the sole carbon source, via clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9-based genome editing. NeuAc key precursor, N-acetylmannosamine (ManNAc; 0.40 g/L), was produced by expressing UDP-N-acetylglucosamine-2-epimerase and glucosamine-6-phosphate synthase (GlmS) mutants and blocking the NeuAc catabolic pathway in E. coli BL21 (DE3). The expression levels of GlmM and GlmU-GlmSA metabolic modules were optimized, significantly increasing the ManNAc titer to 8.95 g/L. Next, the expression levels of NeuAc synthase from different microorganisms were optimized, leading to the production of 6.27 g/L of NeuAc. Blocking the competing pathway of NeuAc biosynthesis increased the NeuAc titer to 9.65 g/L. In fed-batch culture in a 3 L fermenter, NeuAc titer reached 23.46 g/L with productivity of 0.69 g/L/h, which is the highest level achieved by microbial synthesis using glycerol as the sole carbon source in E. coli. The strategies used in our study can aid in the efficient bioproduction of NeuAc and its derivatives.

Modeling the effect of rRNA-mRNA interactions and mRNA folding on mRNA translation in chloroplasts.

The process of translation initiation in prokaryotes is mediated by the hybridization of the 16S rRNA of the small ribosomal subunit with the mRNA in a short region called the ribosomal binding site. However, translation initiation in chloroplasts, which have evolved from an ancestral bacterium, is not well understood. Some studies suggest that in many cases it differs from translation initiation in bacteria and involves various novel interactions of the mRNA structures with intracellular factors; however currently, there is no generic quantitative model related to these aspects in chloroplasts. We developed a novel computational pipeline and models that can be used for understanding and modeling translation regulation in chloroplasts. We demonstrate that local folding and co-folding energy of the rRNA and the mRNA correlates with codon usage estimators of expression levels (r = -0.63) and infer predictive models that connect these energies and codon usage to protein levels (with correlation up to 0.71). In addition, we demonstrate that the ends of the transcripts in chloroplasts are populated with various structural elements that may be functional. Furthermore, we report a database of 166 novel structures in the chloroplast transcripts that are predicted to be functional. We believe that the models reported here improve existing understandings of genomic evolution and the biophysics of translation in chloroplasts; as such, they can aid gene expression engineering in chloroplasts for various biotechnological objectives.

High-throughput strategy for identification of Mycobacterium tuberculosis membrane protein expression conditions using folding reporter GFP.

Mycobacterium tuberculosis membrane protein biochemistry and structural biology studies are often hampered by challenges in protein expression and selection for well-expressing protein candidates, suitable for further investigation. Here we present a folding reporter GFP (frGFP) assay, adapted for M. tuberculosis membrane protein screening in Escherichia coli Rosetta 2 (DE3) and Mycobacterium smegmatis mc24517. This method allows protein expression condition screening for multiple protein targets simultaneously by monitoring frGFP fluorescence in growing cells. We discuss the impact of common protein expression conditions on 42 essential M. tuberculosis H37Rv helical transmembrane proteins and establish the grounds for their further analysis. We have found that the basal expression of the lac operon in the T7-promoter expression system generally leads to high recombinant protein yield in M. smegmatis, and we suggest that a screening condition without the inducer is included in routine protein expression tests. In addition to the general observations, we describe conditions allowing high-level expression of more than 25 essential M. tuberculosis membrane proteins, containing 2 to 13 transmembrane helices. We hope that these findings will stimulate M. tuberculosis membrane protein research and aid the efforts in drug development against tuberculosis.

Overexpression and kinetic analysis of Ideonella sakaiensis PETase for polyethylene terephthalate (PET) degradation.

Ideonella sakaiensis PET hydrolase (IsPETase) is a well-characterized enzyme for effective PET biodegradation. However, the low soluble expression level of the enzyme hampers its practical implementation in the biodegradation of PET. Herein, the expression of IsPETaseMut, one of the most active mutants of IsPETase obtained so far, was systematically explored in E. coli by adopting a series of strategies. A notable improvement of soluble IsPETaseMut was observed by using chaperon co-expression and fusion expression systems. Under the optimized conditions, GroEL/ES co-expression system yielded 75 ± 3.4 mg·L-1 purified soluble IsPETaseMut (GroEL/ES), and NusA fusion expression system yielded 80 ± 3.7 mg·L-1 purified soluble NusA-IsPETaseMut, which are 12.5- and 4.6-fold, respectively, higher than its commonly expression in E. coli. The two purified enzymes were further characterized. The results showed that IsPETaseMut (GroEL/ES) displayed the same catalytic behavior as IsPETaseMut, while the fusion of NusA conferred new enzymatic properties to IsPETaseMut. Although NusA-IsPETaseMut displayed a lower initial hydrolysis capacity than IsPETaseMut, it showed a 1.4-fold higher adsorption constant toward PET. Moreover, the product inhibition effect of terephthalic acid (TPA) on IsPETase was reduced with NusA-IsPETaseMut. Taken together, the latter two catalytic properties of NusA-IsPETaseMut are more likely to contribute to the enhanced product release by NusA-IsPETaseMut PET degradation for two weeks.

Protein Expression Optimization Strategies in E. coli: A Tailored Approach in Strain Selection and Parallelizing Expression Conditions.

Escherichia coli remains a traditional and widely used host organism for recombinant protein production. Its well-studied genome, availability of vectors and strains, cheap and relatively straight-forward cultivation methods paired with reported high protein yields are reasons why E. coli is often the first-choice host expression system for recombinant protein production. The chapter enclosed here details protocols and design strategies in strain selection and methods on how to parallelize expression conditions to optimize for soluble target protein expression in E. coli. The methods described have been validated in a protein production research facility.

Rational design of a new variant of Reteplase with optimized physicochemical profile and large-scale production in Escherichia coli.

Structural engineering of the recombinant thrombolytic drug, Reteplase, and its cost-effective production are important goals in the pharmaceutical industry. In this study, a single-point mutant of the protein was rationally designed and evaluated in terms of physicochemical characteristics, enzymatic activity, as well as large-scale production settings. An accurate homology model of Reteplase was used as the input to appropriate tools to identify the aggregation-prone sites, while considering the structural stability. Selected variants underwent extensive molecular dynamic simulations (total 540 ns) to assess their solvation profile and their thermal stability. The Reteplase-fibrin interaction was investigated by docking. The best variant was expressed in E. coli, and Box-Behnken design was used through response surface methodology to optimize its expression conditions. M72R mutant demonstrated appropriate stability, enhanced enzymatic activity (p < 0.05), and strengthened binding to fibrin, compared to the wild type. The optimal conditions for the variant's production in a bioreactor was shown to be 37 ºC, induction with 0.5 mM IPTG, for 2 h of incubation. Under these conditions, the final amount of the produced enzyme was increased by about 23 mg/L compared to the wild type, with an increase in the enzymatic activity by about 2 IU/mL. This study thus offered a new Reteplase variant with nearly all favorable properties, except solubility. The impact of temperature and incubation time on its large-scale production were underlined as well.

Construction of a tunable promoter library to optimize gene expression in Methylomonas sp. DH-1, a methanotroph, and its application to cadaverine production.

BACKGROUND: As methane is 84 times more potent than carbon dioxide in exacerbating the greenhouse effect, there is an increasing interest in the utilization of methanotrophic bacteria that can convert harmful methane into various value-added compounds. A recently isolated methanotroph, Methylomonas sp. DH-1, is a promising biofactory platform because of its relatively fast growth. However, the lack of genetic engineering tools hampers its wide use in the bioindustry. RESULTS: Through three different approaches, we constructed a tunable promoter library comprising 33 promoters that can be used for the metabolic engineering of Methylomonas sp. DH-1. The library had an expression level of 0.24-410% when compared with the strength of the lac promoter. For practical application of the promoter library, we fine-tuned the expressions of cadA and cadB genes, required for cadaverine synthesis and export, respectively. The strain with PrpmB-cadA and PDnaA-cadB produced the highest cadaverine titre (18.12 ± 1.06 mg/L) in Methylomonas sp. DH-1, which was up to 2.8-fold higher than that obtained from a non-optimized strain. In addition, cell growth and lysine (a precursor of cadaverine) production assays suggested that gene expression optimization through transcription tuning can afford a balance between the growth and precursor supply. CONCLUSIONS: The tunable promoter library provides standard and tunable components for gene expression, thereby facilitating the use of methanotrophs, specifically Methylomonas sp. DH-1, as a sustainable cell factory.

Accelerating Whole-Cell Simulations of mRNA Translation Using a Dedicated Hardware.

In recent years, intracellular biophysical simulations have been used with increasing frequency not only for answering basic scientific questions but also in the field of synthetic biology. However, since these models include networks of interaction between millions of components, they are extremely time-consuming and cannot run easily on parallel computers. In this study, we demonstrate for the first time a novel approach addressing this challenge by using a dedicated hardware designed specifically to simulate such processes. As a proof of concept, we specifically focus on mRNA translation, which is the process consuming most of the energy in the cell. We design a hardware that simulates translation in Escherichia coli and Saccharomyces cerevisiae for thousands of mRNAs and ribosomes, which is in orders of magnitude faster than a similar software solution. With the sharp increase in the amount of genomic data available today and the complexity of the corresponding models inferred from them, we believe that the strategy suggested here will become common and can be used among others for simulating entire cells with all gene expression steps.

[Optimization of unnatural amino acid incorporation in collagen and the cross-linking through thioether bond].

The source of recombinant collagen is clean, and it has the advantages of flexible sequence design, high yield and high purity, so it has a wide application prospect as biomaterials in tissue engineering and other fields. However, how to promote the cross-linking of recombinant collagen molecules and make them form a more stable spatial structure is the difficulty to be overcome in the design of recombinant collagen nanomaterials. Unnatural amino acid O-(2-bromoethyl)-tyrosine was incorporated into collagen by two-plasmid expression system. The results showed that high-purity collagen incorporated with unnatural amino acid could be obtained by induction with final concentration of 0.5 mmol/L IPTG and 0.06% arabinose at 25 °C for 24 hours. The intermolecular cross-linking through thioether bond was formed between collagen molecule incorporated with unnatural amino acid and collagen molecule with cysteine mutation in pH 9.0 NH4HCO3 buffer, which formed aggregates with the largest molecular size up to 1 micrometre. The results pave the way for the design of recombinant collagen biomaterials.

Multi-level rebalancing of the naringenin pathway using riboswitch-guided high-throughput screening.

Recombinant microbes have emerged as promising alternatives to natural sources of naringenin-a key molecular scaffold for flavonoids. In recombinant strains, expression levels of the pathway genes should be optimized at both transcription and the translation stages to precisely allocate cellular resources and maximize metabolite production. However, the optimization of the expression levels of naringenin generally relies on evaluating a small number of variants from libraries constructed by varying transcription efficiency only. In this study, we introduce a systematic strategy for the multi-level optimization of biosynthetic pathways. We constructed a multi-level combinatorial library covering both transcription and translation stages using synthetic T7 promoter variants and computationally designed 5'-untranslated regions. Furthermore, we identified improved strains through high-throughput screening based on a synthetic naringenin riboswitch. The most-optimized strain obtained using this approach exhibited a 3-fold increase in naringenin production, compared with the parental strain in which only the transcription efficiency was modulated. Furthermore, in a fed-batch bioreactor, the optimized strain produced 260.2 mg/L naringenin, which is the highest concentration reported to date using glycerol and p-coumaric acid as substrates. Collectively, this work provides an efficient strategy for the expression optimization of the biosynthetic pathways.

A prokaryote system optimization for rMEPLox expression: A promising non-toxic antigen for Loxosceles antivenom production.

Loxoscelism is the most dangerous araneism form in Brazil and antivenom therapy is the recommended treatment. Antivenom is produced by horse immunization with Loxosceles spider venom, which is toxic for the producer animal. Moreover, due to the high amount of venom required for horse hyperimmunization, new strategies for antigens obtention have been proposed. In this sense, our research group has previously produced a non-toxic recombinant multiepitopic protein derived from Loxosceles toxins (rMEPLox). rMEPLox was a successful immunogen, being able to induce the production of neutralizing antibodies, which could be used in the Loxoscelism treatment. However, rMEPLox obtention procedure requires optimization, as its production needs to be scaled up to suit antivenom manufacture. Therefore, an effective protocol development for rMEPlox production would be advantageous. To achieve this objective, we evaluated the influence of different cultivation conditions for rMEPLox optimum expression. The optimum conditions to obtain large amounts of rMEPlox were defined as the use of C43(DE3)pLysS as a host strain, 2xTY medium, 0.6 mM IPTG, biomass pre induction of OD600nm = 0.4 and incubation at 30 °C for 16 h. Following the optimized protocol, 39.84 mg/L of soluble rMEPLox was obtained and tested as immunogen. The results show that the obtained rMEPLox preserved the previously described immunogenicity, and it was able to generate antibodies that recognize different epitopes of the main Loxosceles venom toxins, which makes it a promising candidate for the antivenom production for loxoscelism treatment.