Heterologous Protein Production Research Articles

Application of SUMO fusion technology for the enhancement of stability and activity of lysophospholipase from Pyrococcus abyssi.

Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6H-S-PaLPL) works optimally at 35°C and pH 6.5 with remarkable thermostability at 35-95°C. Thermo-inactivation kinetics of 6H-S-PaLPL were also studied at 35-95°C with first order rate constant (kIN) of 5.58 × 10- 2 h-1 and half-life of 12 ± 0h at 95°C. Km and Vmax for the hydrolysis of 4-nitrophenyl butyrate were calculated to be 2 ± 0.015 mM and 3882 ± 22.368 U/mg, respectively. 2.4-fold increase in Vmax of Pa-LPL was observed after fusion of 6xHis-SUMO tag to its N-terminal. It is the first report on the utilization of SUMO fusion tag to enhance the overall stability and activity of Pa-LPL. Fusion of 6xHis-SUMO tag not only aided in the purification process but also played a crucial role in increasing the thermostability and activity of the enzyme. SUMO-fused enzyme, thus generated, can serve as an important candidate for degumming of vegetable oils at industrial scale.

Enhancing extracellular membrane vesicle productivity of Shewanella vesiculosa HM13, a prospective host for vesiculation-mediated protein secretion, by weakening outer membrane-peptidoglycan linkage

Shewanella vesiculosa HM13, a psychrotrophic gram-negative bacterium isolated from the intestinal contents of horse mackerel, produces abundant extracellular membrane vesicles (EMVs) by budding the outer membrane. The EMVs of this bacterium carry a single major cargo protein, P49, of unknown function, which may be useful as a carrier for the secretory production of heterologous proteins as cargoes of EMVs. In this study, to increase the utility of S.vesiculosa HM13 as a host for EMV-mediated protein production, we improved its EMV productivity by weakening the linkage between the outer membrane and underlying peptidoglycan layer. In gram-negative bacteria, the outer membrane is connected to peptidoglycans predominantly through Braun's lipoprotein (Lpp), and the formation of this linkage is catalyzed by an l,d-transpeptidase (Ldt). We constructed gene-disrupted mutants of Lpp and Ldt and assessed their EMV productivity. The EMVs of the lpp- and ldt-disrupted mutants grown at 18°C were evaluated using nanoparticle tracking analysis, and their morphologies were observed using transmission electron microscopy. As a result, an approximately 2.5-fold increase in EMV production was achieved, whereas the morphology of the EMVs of these mutants remained almost identical to that of the parent strain. In accordance with the increase in EMV production, the mutants secreted approximately 2-fold higher amounts of P49 than the parent strain into the culture broth as the EMV cargo. These findings will contribute to the development of an EMV-based secretory production system for heterologous proteins using S.vesiculosa HM13 as a host.

A novel chemically defined medium for the biotechnological and biomedical exploitation of the cell factory Leishmania tarentolae

The development of media for cell culture is a major issue in the biopharmaceutical industry, for the production of therapeutics, immune-modulating molecules and protein antigens. Chemically defined media offer several advantages, as they are free of animal-derived components and guarantee high purity and a consistency in their composition. Microorganisms of the genus Leishmania represent a promising cellular platform for production of recombinant proteins, but their maintenance requires supplements of animal origin, such as hemin and fetal bovine serum. In the present study, three chemically defined media were assayed for culturing Leishmania tarentolae, using both a wild-type strain and a strain engineered to produce a viral antigen. Among the three media, Schneider's Drosophila Medium supplemented with Horseradish Peroxidase proved to be effective for the maintenance of L. tarentolae promastigotes, also allowing the heterologous protein production by the engineered strain. Finally, the engineered strain was maintained in culture up to the 12th week without antibiotic, revealing its capability to produce the recombinant protein in the absence of selective pressure.

An endogenous promoter LpSUT2 discovered in duckweed: a promising transgenic tool for plants.

Promoters are one of the most critical elements in regulating gene expression. They are considered essential biotechnological tools for heterologous protein production. The one most widely used in plants is the 35S promoter from cauliflower mosaic virus. However, our study for the first time discovered the 35S promoter reduced the expression of exogenous proteins under increased antibiotic stress. We discovered an endogenous strong promoter from duckweed named LpSUT2 that keeps higher initiation activity under antibiotic stress. Stable transformation in duckweed showed that the gene expression of eGFP in the LpSUT2:eGFP was 1.76 times that of the 35S:eGFP at 100 mg.L-1 G418 and 6.18 times at 500 mg.L-1 G418. Notably, with the increase of G418 concentration, the gene expression and the fluorescence signal of eGFP in the 35S:eGFP were weakened, while the LpSUT2:eGFP only changed slightly. This is because, under high antibiotic stress, the 35S promoter was methylated, leading to the gene silencing of the eGFP gene. Meanwhile, the LpSUT2 promoter was not methylated and maintained high activity. This is a previously unknown mechanism that provides us with new insights into screening more stable promoters that are less affected by environmental stress. These outcomes suggest that the LpSUT2 promoter has a high capacity to initiate the expression of exogenous proteins. In conclusion, our study provides a promoter tool with potential application for plant genetic engineering and also provides new insights into screening promoters.

Exploiting Systematic Engineering of the Expression Cassette as a Powerful Tool to Enhance Heterologous Gene Expression in Trichoderma reesei.

Many endeavors in expressing a heterologous gene in microbial hosts rely on simply placing the gene of interest between a selected pair of promoters and terminator. However, although the expression efficiency could be improved by engineering the host cell, how modifying the expression cassette itself systematically would affect heterologous gene expression remains largely unknown. As the promoter and terminator bear plentiful cis-elements, herein using the Aspergillus niger mannanase with high application value in animal feeds and the eukaryotic filamentous fungus workhorse Trichoderma reesei as a model gene/host, systematic engineering of an expression cassette was investigated to decipher the effect of its mutagenesis on heterologous gene expression. Modifying the promoter, signal peptide, the eukaryotic-specific Kozak sequence, and the 3'-UTR could stepwise improve extracellular mannanase production from 17 U/mL to an ultimate 471 U/mL, representing a 27.7-fold increase in expression. The strategies can be generally applied in improving the production of heterologous proteins in eukaryotic microbial hosts.

Engineered Chimera Protein Constructs to Facilitate the Production of Heterologous Transmembrane Proteins in E. coli.

To delve into the structure-function relationship of transmembrane proteins (TMPs), robust protocols are needed to produce them in a pure, stable, and functional state. Among all hosts that express heterologous TMPs, E. coli has the lowest cost and fastest turnover. However, many of the TMPs expressed in E. coli are misfolded. Several strategies have been developed to either direct the foreign TMPs to E. coli's membrane or retain them in a cytosolic soluble form to overcome this deficiency. Here, we summarize protein engineering methods to produce chimera constructs of the desired TMPs fused to either a signal peptide or precursor maltose binding protein (pMBP) to direct the entire construct to the periplasm, therefore depositing the fused TMP in the plasma membrane. We further describe strategies to produce TMPs in soluble form by utilizing N-terminally fused MBP without a signal peptide. Depending on its N- or C-terminus location, a fusion to apolipoprotein AI can either direct the TMP to the membrane or shield the hydrophobic regions of the TMP, maintaining the soluble form. Strategies to produce G-protein-coupled receptors, TMPs of Mycobacterium tuberculosis, HIV-1 Vpu, and other TMPs are discussed. This knowledge could increase the scope of TMPs' expression in E. coli.

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.

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry.

In the 1980s, Escherichia coli was the preferred host for heterologous protein expression owing to its capacity for rapid growth in complex media; well-studied genetics; rapid and direct transformation with foreign DNA; and easily scalable fermentation. Despite the relative ease of use of E. coli for achieving the high expression of many recombinant proteins, for some proteins, e.g., membrane proteins or proteins of eukaryotic origin, this approach can be rather ineffective. Another microorganism long-used and popular as an expression system is baker's yeast, Saccharomyces cerevisiae. In spite of a number of obvious advantages of these yeasts as host cells, there are some limitations on their use as expression systems, for example, inefficient secretion, misfolding, hyperglycosylation, and aberrant proteolytic processing of proteins. Over the past decade, nontraditional yeast species have been adapted to the role of alternative hosts for the production of recombinant proteins, e.g., Komagataella phaffii, Yarrowia lipolytica, and Schizosaccharomyces pombe. These yeast species' several physiological characteristics (that are different from those of S. cerevisiae), such as faster growth on cheap carbon sources and higher secretion capacity, make them practical alternative hosts for biotechnological purposes. Currently, the K. phaffii-based expression system is one of the most popular for the production of heterologous proteins. Along with the low secretion of endogenous proteins, K. phaffii efficiently produces and secretes heterologous proteins in high yields, thereby reducing the cost of purifying the latter. This review will discuss practical approaches and technological solutions for the efficient expression of recombinant proteins in K. phaffii, mainly based on the example of enzymes used for the feed industry.

Antigen surface display in two novel whole genome sequenced food grade strains, Lactiplantibacillus pentosus KW1 and KW2

BackgroundUtilization of commensal bacteria for delivery of medicinal proteins, such as vaccine antigens, is an emerging strategy. Here, we describe two novel food-grade strains of lactic acid bacteria, Lactiplantibacillus pentosus KW1 and KW2, as well as newly developed tools for using this relatively unexplored but promising bacterial species for production and surface-display of heterologous proteins.ResultsWhole genome sequencing was performed to investigate genomic features of both strains and to identify native proteins enabling surface display of heterologous proteins. Basic characterization of the strains revealed the optimum growth temperatures for both strains to be 35–37 °C, with peak heterologous protein production at 33 °C (KW1) and 37 °C (KW2). Negative staining revealed that only KW1 produces closely bound exopolysaccharides. Production of heterologous proteins with the inducible pSIP-expression system enabled high expression in both strains. Exposure to KW1 and KW2 skewed macrophages toward the antigen presenting state, indicating potential adjuvant properties. To develop these strains as delivery vehicles, expression of the mycobacterial H56 antigen was fused to four different strain-specific surface-anchoring sequences.ConclusionAll experiments that enabled comparison of heterologous protein production revealed KW1 to be the better recombinant protein production host. Use of the pSIP expression system enabled successful construction of L. pentosus strains for production and surface display of an antigen, underpinning the potential of these strains as novel delivery vehicles.

Agrobacterium tumefaciens-mediated transformation for the genetic modification of the biotechnologically relevant fungus Aspergillus vadensis through synthetic biology

In the last years, many research efforts have been applied for the development of filamentous fungi as hosts for heterologous protein production. Aspergillus vadensis CBS 113365, a close relative of the industrial workhorse Aspergillus niger, has been suggested as a more suitable cell factory as it does not acidify the culture medium and produces very low levels of secreted proteases. Therefore, efficient methods and tools that allow the genetic manipulation and exploitation of this biotechnologically relevant fungus are needed. To date, only protoplast-mediated transformation and classical cloning strategies have been implemented for A. vadensis genetic modification, which decreases the exploitation capacity of this fungus at the industrial level. In this study, we have adapted and implemented an Agrobacterium tumefaciens-mediated transformation protocol for A. vadensis for the first time, and applied the FungalBraid system to genetically modify this species by means of synthetic biology. As proof of concept, we have successfully complemented and fluorescently labelled a uridine auxotrophic A. vadensis pyrA- strain and generated A. vadensis mutants carrying the Penicillium expansum-based expression cassette for the heterologous production of the antifungal protein PeAfpA from P. expansum. Even though we have yet to find the conditions that trigger PeAfpA production in this species, the implementation of the ATMT method reported here, along with the application of the FungalBraid system, will greatly aid in this task and will facilitate the exploitation of A. vadensis as a fungal workhorse for protein production for multiple biotechnological applications.

Recent developments in heterologous production of cellulases and xylanases using the Pichia pastoris expression system

AbstractThe yeast expression system, Pichia pastoris, is one of the most robust and versatile expression systems in biotechnology and molecular biology, generally considered as a safe host for heterologous protein expression, especially for producing cellulases and xylanases at the industrial level. Despite the high recombinant protein expression rate, the potential of the P. pastoris expression system has still not been fully explored. Cultivation of P. pastoris under optimized conditions greatly relies on the strain and is associated with certain problems such as promoter strength, sensitivity to methanol, and oxygen demand. To address these issues, different genetic engineering strategies have been employed. Advancements in promoter engineering, optimization of gene dosage and codon usage, recombinant plasmid engineering using CRISPR/Cas9 system, and directed evolution strategies have proven beneficial to the yield of cellulase expression levels. This study will systemically review recent progress in various genetic engineering strategies to enhance cellulase and xylanase expression in the P. pastoris expression system. The utilization of alcohol oxidase 1 promoter (pAOX1), methanol‐free system, and recombinant plasmid engineering for improved production of these enzymes are highlighted. Additionally, we discuss the recent advancements in the P. pastoris expression system toolbox for improved cellulase and xylanase production, thus providing a deep insight into how P. pastoris is becoming the indispensable platform for heterologous protein production. © 2023 Society of Chemical Industry (SCI).

Identification and investigation of the effects of N-acetylmuramoyl-L-alanine amidase in Bacillus amyloliquefaciens for the cell lysis and heterologous protein production

Bacillus amyloliquefaciens (BA) is considered as an important industrial strain for heterologous proteins production. However, its severe autolytic behavior leads to reduce the industrial production capacity of the chassis cells. In this study, we aimed to evaluate the autolysis of N-acetylmuranyl-L-alanine amidase in BA TCCC11018, and further slowed down the cell lysis for improved the heterologous protein production by a series of modifications. Firstly, we identified six N-acetylmuramic acid-L-alanines by bioinformatics, and analyzed the transcriptional levels at different culture time points by transcriptome and quantitative real-time PCR. Then, by establishing an efficient CRISPR-nCas9 gene editing method, N-acetylmuramic acid-L-alanine genes were knocked out or overexpressed to verify its effect on cell lysis. Then, by single or tandem knockout N-acetylmuramic acid-L-alanines, it was determined that the reasonable modification of LytH and CwlC1 can slow down cell lysis. After 48 h of culture, the autolysis rate of the mutant strain BA ΔlytH-cwlC1 decreased by 4.83 %, and the amylase activity reached 176 U/mL, which was 76.04 % higher than that of the control strain BA Δupp. The results provide a reference for mining the functional characteristics of autolysin in Bacillus spp., and provide from this study reveal valuable insights delaying the cell lysis and increasing heterologous proteins production.

Enhanced production of human interferon α2b in glycoengineered Pichia pastoris by robust control of methanol feeding and implications of various control strategies

Interferon α2b (IFNα2b), a prominent cytokine molecule, is bestowed with immunomodulatory, antiproliferative, and antiviral properties. Like any other recombinant protein produced in methylotrophic yeast Pichia pastoris, Human interferon α2b (huIFN α2b) production in P. pastoris depends on methanol utilization during the induction phase. This study investigates the impact of varying residual methanol concentrations on huIFNα2b productivity and assesses control strategies towards the tight control of residual methanol concentration in fed-batch experiments carried out in the fermentation calorimeter. Real-time monitoring of P. pastoris metabolism was facilitated by metabolic heat rate measurements and exhaust gas analyser. Optimal methanol concentration screening performed at different concentrations viz., 1.5, 3, 5, and 7 g/L, revealed the highest huIFNα2b productivity at 3 g/L (0.21 mg/g h). Methanol sensor provided input data for three different control strategies (on/off, proportional integral (PI), and model-based adaptive PI) to maintain optimal methanol levels (3 g/L). Model-based adaptive control resulted in the highest huIFNα2b yield, productivity (244.34 mg/L, 0.31 mg/g h), surpassing on/off (188.7 mg/L, 0.16 mg/g h) and PI (202.3 mg/L, 0.28 mg/g h) control strategies. The model-based adaptive PI control framework developed in this study could be employed for other heterologous protein production in P. pastoris.

Metabolic impact of heterologous protein production in Pseudomonas putida: Insights into carbon and energy flux control

For engineered microorganisms, the production of heterologous proteins that are often useless to host cells represents a burden on resources, which have to be shared with normal cellular processes. Within a certain metabolic leeway, this competitive process has no impact on growth. However, once this leeway, or free capacity, is fully utilized, the extra load becomes a metabolic burden that inhibits cellular processes and triggers a broad cellular response, reducing cell growth and often hindering the production of heterologous proteins. In this study, we sought to characterize the metabolic rearrangements occurring in the central metabolism of Pseudomonas putida at different levels of metabolic load. To this end, we constructed a P. putida KT2440 strain that expressed two genes encoding fluorescent proteins, one in the genome under constitutive expression to monitor the free capacity, and the other on an inducible plasmid to probe heterologous protein production. We found that metabolic fluxes are considerably reshuffled, especially at the level of periplasmic pathways, as soon as the metabolic load exceeds the free capacity. Heterologous protein production leads to the decoupling of anabolism and catabolism, resulting in large excess energy production relative to the requirements of protein biosynthesis. Finally, heterologous protein production was found to exert a stronger control on carbon fluxes than on energy fluxes, indicating that the flexible nature of P. putida's central metabolic network is solicited to sustain energy production.

Heterologous Single-Strand DNA-Annealing and Binding Protein Enhance CRISPR-Based Genome Editing Efficiency in Komagataella phaffii.

The industrial yeast Komagataella phaffii is a highly effective platform for heterologous protein production, owing to its high protein expression and secretion capacity. Heterologous genes and proteins are involved in multiple processes, including transcription, translation, protein folding, modification, transportation, and degradation; however, engineering these proteins and genes is challenging due to inefficient genome editing techniques. We employed Pseudomonas aeruginosa phage single-stranded DNA-annealing protein (SSAP) PapRecT and P. aeruginosa single-stranded DNA-binding protein (SSB) PaSSB to introduce SSAP-SSB-based homology recombination, which facilitated K. phaffii CRISPR-based genome engineering. Specifically, a host-independent method was developed by expressing sgRNA with PapRecT-PaSSB in a single plasmid, with which only a 50 bp short homologous arm (HA) reached a 100% positive rate for CRISPR-based gene insertion, reaching 18 colony-forming units (CFU) per μg of donor DNA. Single deletion using 1000 bp HA attained 100%, reaching 68 CFUs per μg of donor DNA. Using this efficient CRISPR-based genome editing tool, we integrated three genes (INO4, GAL4-like, and PAB1) at three different loci for overexpression to realize the collaborative regulation of human-lactalbumin (α-LA) production. Specifically, we strengthened phospholipid biosynthesis to facilitate endoplasmic reticulum membrane formation and enhanced recombinant protein transcription and translation by overexpressing transcription and translation factors. The final production of α-LA in the 3 L fermentation reached 113.4 mg L-1, two times higher than that of the strain without multiple site gene editing, which is the highest reported titer in K. phaffii. The CRISPR-based genome editing method developed in this study is suitable for the synergistic multiple-site engineering of protein and biochemical biosynthesis pathways to improve the biomanufacturing efficiency.

AOX1 promoter-driven expression of yeast-enhanced green fluorescent protein in Pichia pastoris

Green fluorescent protein (GFP) is a widely employed reporter for studying various cellular functions in genetic engineering. Methylotrophic yeast; Pichia pastoris is an excellent host for heterologous protein production. Codon optimized yeast-enhanced green fluorescent protein (yEGFP) has been reported to have higher expression as a yeast reporter which can be used to study many aspects of P. pastoris. The present investigation was focused on studying the yEGFP intracellular expression pattern in the P. pastoris GS115 strain. yEGFP is cloned into the pPICZ A vector under the control of an AOX1 (Alcohol Oxidase) promoter that was induced by adding methanol to the growth medium. However, methanol concentration and time of induction could affect the optimum expression of yEGFP. Hence, those conditions were tested for manifesting optimum yEGFP expression by monitoring the fluorescence intensities in minimal methanol histidine media supplemented with 0.5%, 1%, 2%, and 2.5% methanol. The pH of the media and the composition of the media (minimal or complex) also affect recombinant protein production. The effect of pH and the media composition was investigated in buffered media at pH 6 and unbuffered media of minimal methanol histidine media and methanol complex media. The results showed that the yEGFP expression increased up to the 5th day of induction and then decreased on the 6th day, 2% methanol showed the highest expression, and the pH and the media composition did not have any effect on the yEGFP expression. In conclusion, the optimum yEGFP expression can be obtained in 2% methanol on the 5th day of induction irrespective of minimal or complex media used.

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.

Heterologous protein production using Psychrobacter sp. PAMC 21119 analyzed with a green fluorescent protein-based reporter system

Insolubility and low expression are typical bottlenecks in the production of proteins for studying their function and structure using X-ray crystallography or nuclear magnetic resonance spectroscopy. Cold-active enzymes from polar microorganisms have unique structural features that render them unstable and thermolabile, and are responsible for decreased protein yield in heterologous expression systems. To address these challenges, we developed a heterologous protein expression system using a psychrophilic organism, Psychrobacter sp. PAMC 21119, as a protein expression host with its own promoter. We screened 11 promoters and evaluated their strength using quantitative real-time polymerase chain reaction and a reporter system harboring the SfGFP gene. The highest expression was achieved using promoters RH96_RS13655 (P21119_20930) and RH96_RS15090 (P21119_23410), regardless of the temperature used. The p20930 strain exhibited a maximum expression level 19.6-fold higher than that of its control at 20 °C and produced approximately 0.5 mg of protein per gram of dry cell weight. To our knowledge, this is the first report of a low-temperature recombinant protein expression system developed using Psychrobacter sp. that can be used to express various difficult-to-express and cold-active proteins.

Effects of Signal Peptide and Chaperone Co-Expression on Heterologous Protein Production in Escherichia coli.

Various host systems have been employed to increase the yield of recombinant proteins. However, some recombinant proteins were successfully produced at high yields but with no functional activities. To achieve both high protein yield and high activities, molecular biological strategies have been continuously developed. This work describes the effect of signal peptide (SP) and co-expression of molecular chaperones on the production of active recombinant protein in Escherichia coli. Extracellular enzymes from Bacillus subtilis, including β-1,4-xylanase, β-1,4-glucanase, and β-mannanase constructed with and without their signal peptides and intracellular enzymes from Pseudomonas stutzeri ST201, including benzoylformate decarboxylase (BFDC), benzaldehyde dehydrogenase (BADH), and d-phenylglycine aminotransferase (d-PhgAT) were cloned and overexpressed in E. coli BL21(DE3). Co-expression of molecular chaperones with all enzymes studied was also investigated. Yields of β-1,4-xylanase (Xyn), β-1,4-glucanase (Cel), and β-mannanase (Man), when constructed without their N-terminal signal peptides, increased 1112.61-, 1.75-, and 1.12-fold, respectively, compared to those of spXyn, spCel, and spMan, when constructed with their signal peptides. For the natural intracellular enzymes, the chaperones, GroEL-GroES complex, increased yields of active BFDC, BADH, and d-PhgAT, up to 1.31-, 4.94- and 37.93-fold, respectively, and also increased yields of Man and Xyn up to 1.53- and 3.46-fold, respectively, while other chaperones including DnaK-DnaJ-GrpE and Trigger factor (Tf) showed variable effects with these enzymes. This study successfully cloned and overexpressed extracellular and intracellular enzymes in E. coli BL21(DE3). When the signal peptide regions of the secretory enzymes were removed, yields of active enzymes were higher than those with intact signal peptides. In addition, a higher yield of active enzymes was obtained, in general, when these enzymes were co-expressed with appropriate chaperones. Therefore, E. coli can produce cytoplasmic and secretory enzymes effectively if only the enzyme coding sequence without its signal peptide is used and appropriate chaperones are co-expressed to assist in correct folding.

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.