Functional analysis of Lsm protein under multiple stress conditions in the extreme haloarchaeon Haloferax mediterranei.

The Sm, like-Sm, and Hfq proteins belonging to the Sm superfamily of proteins are represented in all domains of life. These proteins are involved in several RNA metabolism pathways. The functions of bacterial Hfq and eukaryotic Sm proteins have been described, but knowledge about the in vivo functions of archaeal Sm proteins remains limited. This study aims to improve the understanding of Lsm proteins and their role using the haloarchaeon Haloferax mediterranei as a model microorganism. The Haloferax mediterranei genome contains one lsm gene that overlaps with the rpl37e gene. To determine the expression of lsm and rpl37e genes and the co-transcription of both, reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed under different standard and stress conditions. The results suggest that the expression of lsm and rpl37e is constitutive. Co-transcription occurs at sub-optimal salt concentrations and temperatures, depending on the growth phase. The halophilic Lsm protein contains two Sm motifs, Sm1 and Sm2, and the sequence encoding the Sm2 motif also constitutes the promoter of the rpl37e gene. To investigate their biological functions, the lsm deletion mutant and the Sm1 motif deletion mutant, where the Sm2 motif remained intact, were generated and characterised. Comparison of the lsm deletion mutant, Sm1 deletion mutant, and the parental strain HM26 under standard and stress growth conditions revealed growth differences. Finally, swarming assays in complex and defined media showed greater swarming capacity in the deletion mutants.

Transcript Regulation of the Recoded Archaeal α-l-Fucosidase In Vivo.

Genetic decoding is flexible, due to programmed deviation of the ribosomes from standard translational rules, globally termed "recoding". In Archaea, recoding has been unequivocally determined only for termination codon readthrough events that regulate the incorporation of the unusual amino acids selenocysteine and pyrrolysine, and for -1 programmed frameshifting that allow the expression of a fully functional α-l-fucosidase in the crenarchaeon Saccharolobus solfataricus, in which several functional interrupted genes have been identified. Increasing evidence suggests that the flexibility of the genetic code decoding could provide an evolutionary advantage in extreme conditions, therefore, the identification and study of interrupted genes in extremophilic Archaea could be important from an astrobiological point of view, providing new information on the origin and evolution of the genetic code and on the limits of life on Earth. In order to shed some light on the mechanism of programmed -1 frameshifting in Archaea, here we report, for the first time, on the analysis of the transcription of this recoded archaeal α-l-fucosidase and of its full-length mutant in different growth conditions in vivo. We found that only the wild type mRNA significantly increased in S. solfataricus after cold shock and in cells grown in minimal medium containing hydrolyzed xyloglucan as carbon source. Our results indicated that the increased level of fucA mRNA cannot be explained by transcript up-regulation alone. A different mechanism related to translation efficiency is discussed.

Expression, purification, and characterization of a membrane-associated cyclic oligo-adenylate degrader from Sulfolobus islandicus.

Type III CRISPR-cas systems initiate cyclic oligo-adenylate (cOA) signaling to initiate immune response. Previously, we identified that a membrane-associated DHH-DHHA1 family protein from Sulfolobus islandicus efficiently degrades cOA. Here, we provide detailed protocols for expression and purification of the protein from its native host and a cOA degradation assay with the purified enzyme. The methodology should be of interest for researchers studying Sulfolobus, membrane-associated proteins, or type III CRISPR-cas systems. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020).

Optimal secretion of thermostable Beta-glucosidase in Bacillus subtilis by signal peptide optimization.

Commercial applications of ß-glucosidase (BGL) demands its purity and availability on a large scale. In the present study, we aim to optimize the expression and secretion of a thermostable BGL from Pyrococcus furiosus (PfuBGL) in B. subtilis strain RIK1285. Initial studies with base strain BV002 harboring aprE signal peptide (aprESP) showed PfuBGL yield of 0.743 ± 0.19 pNP U/ml only. A library of 173 different homologous SPs from B. subtilis 168 genome was fused with target PfuBGL gene (PF0073) in pBE-S vector and extracellularly expressed in RIK1285 strain to identify optimal SP for PfuBGL secretion. High-throughput screening of the resulting SP library for BGL activity with a synthetic substrate followed by systematic scaling of the clones yielded a gene construct with CitHSP reporting a sixteen fold enhancement of PfuBGL secretion in comparison to base strain. Batch fermentation (7.5 L scale) PfuBGL yield of the BV003 strain with CitHSP-PF0073 fusion was observed to be 12.08 ± 0.21 pNP U/ml with specific activity of 35.52 ± 0.53 U/mg. Thus, the study represents report on the secretory expression of thermostable PfuBGL using B. subtilis as a host organism and demonstrating its high potential for industrial production of any protein/enzyme.

Structural and Functional Insights into an Archaeal Lipid Synthase.

The UbiA superfamily of intramembrane prenyltransferases catalyzes an isoprenyl transfer reaction in the biosynthesis of lipophilic compounds involved in cellular physiological processes. Digeranylgeranylglyceryl phosphate (DGGGP) synthase (DGGGPase) generates unique membrane core lipids for the formation of the ether bond between the glycerol moiety and the alkyl chains in archaea and has been confirmed to be a member of the UbiA superfamily. Here, the crystal structure is reported to exhibit nine transmembrane helices along with a large lateral opening covered by a cytosolic cap domain and a unique substrate-binding central cavity. Notably, the lipid-bound states of this enzyme demonstrate that the putative substrate-binding pocket is occupied by the lipidic molecules used for crystallization, indicating the binding mode of hydrophobic substrates. Collectively, these structural and functional studies provide not only an understanding of lipid biosynthesis by substrate-specific lipid-modifying enzymes but also insights into the mechanisms of lipid membrane remodeling and adaptation.

Gene cloning, expression enhancement in Escherichia coli and biochemical characterization of a highly thermostable amylomaltase from Pyrobaculum calidifontis.

Pcal_0768 gene encoding an amylomaltase, a 4-α-glucanatransferase belonging to family 77 of glycosyl hydrolases, from Pyrobaculum calidifontis was cloned and expressed in Escherichia coli. The recombinant protein was produced in E. coli in soluble and active form. However, the expression level was not very high. Analysis of the mRNA of initial seven codons at the 5'-end of the gene revealed the presence of a hair pin like secondary structure. This secondary structure was removed by site directed mutagenesis, without altering the amino acids, which resulted in enhanced expression of the cloned gene. Recombinant Pcal_0768 exhibited optimal amylomaltase activity at 80 °C and pH 6.9. Under these conditions, the specific activity was 690 U/ mg. Recombinant Pcal_0768 was highly thermostable with a half-life of 6 h at 100 °C. It exhibited the highest kcat value among the characterized glucanotransferases. No metal ions were required for activity or stability of the enzyme. Recombinant Pcal_0768 was successfully employed in the synthesis of modified starch for producing thermoreversible gel. To the best of our knowledge, till now this is the most thermostable enzyme among the characterized amylomaltases. High thermostability and starch modification potential make it a novel and distinct amylomaltase.

Secretion of a low and high molecular weight ß-glycosidase by Yarrowia lipolytica.

BACKGROUND: The secretory production of recombinant proteins in yeast simplifies isolation and purification but also faces possible complications due to the complexity of the secretory pathway. Therefore, correct folding, maturation and intracellular transport of the recombinant proteins are important processing steps with a higher effort needed for complex and large proteins. The aim of this study was to elucidate the secretion potential of Yarrowia lipolytica for low and high molecular weight ß-glycosidases in a comparative cultivation approach. RESULTS: A low sized ß-glucosidase from Pyrococcus furiosus (CelB; 55 kDa) and a large sized ß-galactosidase isolated from the metagenome (M1; 120 kDa) were integrated into the acid extracellular protease locus using the CRISPR-Cas9 system to investigate the size dependent secretion of heterologous proteins in Y. lipolytica PO1f. The recombinant strains were cultivated in the bioreactor for 78 h and the extra- and intracellular enzyme activities were determined. The secretion of CelB resulted in an extracellular volumetric activity of 187.5 µkatoNPGal/Lmedium, while a volumetric activity of 2.98 µkatoNPGal/Lmedium was measured during the M1 production. However, when the amount of functional intra- and extracellular enzyme was investigated, the high molecular weight M1 (85%) was secreted more efficiently than CelB (27%). Real-time PCR experiments showed a linear correlation between the transcript level and extracellular activity for CelB, while a disproportional high mRNA level was observed regarding M1. Interestingly, mass spectrometry data revealed the unexpected secretion of two endogenous intracellular glycolytic enzymes, which is reported for the first time for Y. lipolytica. CONCLUSION: The results of this study provide deeper insights into the secretion potential of Y. lipolytica. A secretion limitation for the low-size CelB was observed, while the large size M1 enzyme was produced in lower amounts but was secreted efficiently. It was shown for the first time that Y. lipolytica is a promising host for the secretion of heterologous high molecular weight proteins (> 100 kDa), although the total secreted amount has to be increased further.

Antibiotics from Haloarchaea: What Can We Learn from Comparative Genomics?

The knowledge of antibiotics produced by Archaea (archaeocins) is still limited. So far, only two types of archaeocins are known: (i) sulfolobicins, produced by the extremely thermophilic Sulfolobus spp. and (ii) haloarcheocins, produced by halophilic archaea. Haloarcheocins were first discovered in the 1980s, but most of their characterisation was solely based on supernatant-based assays. Only a few were successfully purified and sequenced, and even fewer have a proposed biosynthetic model. Furthermore, their mode of action, ecological role and biotechnological potential are still to be explored. Haloarcheocin C8 (HalC8) is the best well-characterised haloarcheocin. We applied an approach of comparative genomics in order to go a step further in the knowledge of their biosynthetic clusters as well as the clusters encoding HalC8-like peptides. These peptides can be classified, at least, into 4 different clades, and there is low gene conservation between them. However, the putative function of some proteins is conserved. These include uncharacterized major facilitator superfamily proteins, transmembrane peptides, DNA-binding transcriptional regulators and proteins with extracellular domains. Our analysis reinforces the association of these proteins with HalC8/HalC8-like biosynthesis. Their functionality is unknown, and, in an era where it is known that haloarchaea are not confined to high salt habitats, the advance in the knowledge of their specialised metabolites will be imperative.

Affitins: Ribosome Display for Selection of Aho7c-Based Affinity Proteins.

Engineered protein scaffolds have made a tremendous contribution to the panel of affinity tools owing to their favorable biophysical properties that make them useful for many applications. In 2007, our group paved the way for using archaeal Sul7d proteins for the design of artificial affinity ligands, so-called Affitins. For many years, Sac7d and Sso7d have been used as molecular basis to obtain binders for various targets. Recently, we characterized their old gifted protein family and identified Aho7c, originating from Acidianus hospitalis, as the shortest member (60 amino-acids) with impressive stability (96.5 °C, pH 0-12). Here, we describe the construction of Aho7c combinatorial libraries and their use for selection of binders by ribosome display.

Transgenic Archaerhodopsin-3 Expression in Hypocretin/Orexin Neurons Engenders Cellular Dysfunction and Features of Type 2 Narcolepsy.

Narcolepsy, characterized by excessive daytime sleepiness, is associated with dysfunction of the hypothalamic hypocretin/orexin (Hcrt) system, either due to extensive loss of Hcrt cells (Type 1, NT1) or hypothesized Hcrt signaling impairment (Type 2, NT2). Accordingly, efforts to recapitulate narcolepsy-like symptoms in mice have involved ablating these cells or interrupting Hcrt signaling. Here, we describe orexin/Arch mice, in which a modified archaerhodopsin-3 gene was inserted downstream of the prepro-orexin promoter, resulting in expression of the yellow light-sensitive Arch-3 proton pump specifically within Hcrt neurons. Histological examination along with ex vivo and in vivo electrophysiological recordings of male and female orexin/Arch mice demonstrated silencing of Hcrt neurons when these cells were photoilluminated. However, high expression of the Arch transgene affected cellular and physiological parameters independent of photoillumination. The excitability of Hcrt neurons was reduced, and both circadian and metabolic parameters were perturbed in a subset of orexin/Arch mice that exhibited high levels of Arch expression. Orexin/Arch mice also had increased REM sleep under baseline conditions but did not exhibit cataplexy, a sudden loss of muscle tone during wakefulness characteristic of NT1. These aberrations resembled some aspects of mouse models with Hcrt neuron ablation, yet the number of Hcrt neurons in orexin/Arch mice was not reduced. Thus, orexin/Arch mice may be useful to investigate Hcrt system dysfunction when these neurons are intact, as is thought to occur in narcolepsy without cataplexy (NT2). These results also demonstrate the utility of extended phenotypic screening of transgenic models when specific neural circuits have been manipulated.SIGNIFICANCE STATEMENT Optogenetics has become an invaluable tool for functional dissection of neural circuitry. While opsin expression is often achieved by viral injection, stably integrated transgenes offer some practical advantages. Here, we demonstrate successful transgenic expression of an inhibitory opsin in hypocretin/orexin neurons, which are thought to promote or maintain wakefulness. Both brief and prolonged illumination resulted in inhibition of these neurons and induced sleep. However, even in the absence of illumination, these cells exhibited altered electrical characteristics, particularly when transgene expression was high. These aberrant properties affected metabolism and sleep, resulting in a phenotype reminiscent of the narcolepsy Type 2, a sleep disorder for which no good animal model currently exists.

Analysis of the neuronal network of the medullary respiratory center in transgenic rats expressing archaerhodopsin-3 in Phox2b-expressing cells.

Preinspiratory (Pre-I) neurons in the parafacial respiratory group (pFRG) comprise one of the respiratory rhythm generators in the medulla of the neonatal rat. A subgroup of pFRG/Pre-I neurons expresses the transcription factor Phox2b. To further analyze detailed neuronal mechanisms of respiratory rhythm generation in the neonatal rat, we developed a transgenic (Tg) rat line in which Phox2b-positive cells expressed archaerhodopsin-3 (Arch). Brainstem-spinal cord preparations were isolated from 0-2-day-old Tg newborn rats and were superfused with artificial cerebrospinal fluid equilibrated with 95% O2 and 5% CO2, pH 7.4, at 25-26 °C. Inspiratory fourth cervical ventral root (C4) activity was monitored, and membrane potentials of neurons in the pFRG including Pre-I and inspiratory neurons were recorded. Phox2b-positive cells in the Tg rats were essentially positive for enhanced green fluorescent protein (EGFP) signals (reporter for Arch) in the pFRG. Continuous photo-stimulation of the rostral ventral medulla for up to 90 s by covering the pFRG with green laser light (532 nm) induced a decrease of respiratory rate measured at C4 accompanied by membrane hyperpolarization of Phox2b-positive pFRG/Pre-I neurons. In contrast, Phox2b-negative inspiratory neurons were not hyperpolarized during the photo-stimulation. Our findings showed that Phox2b-expressing pFRG/Pre-I neurons are involved in the maintenance of the basic respiratory rhythm in neonatal rat.

Coexpression of 5-Aminolevulinic Acid Synthase Gene Facilitates Heterologous Production of Thermostable Cytochrome P450, CYP119, in Holo Form in Escherichia coli.

Cytochrome P450 enzymes are heme-containing monooxygenases that exhibit potential as biocatalysts for practical applications. The Escherichia coli expression system is frequently used for biocatalyst production; however, heterologous production of hemeproteins in their holo form is difficult due to insufficient heme synthesis by the host. In this study, 5-aminolevulinic acid synthase (ALAS) from Rhodobacter capsulatus is used to accelerate intracellular heme biosynthesis in E. coli; this demonstrates that coexpression of the ALAS gene (ALAS) improves the heterologous production of cytochrome P450, CYP119, from Sulfolobus acidocaldarius. Coexpression of ALAS increased the amount of heterologous CYP119 isolated and the ratio of its holo form. The ratio of holo-CYP119 resulting from the coexpression of ALAS in E. coli was 99 %, whereas that from cells expressing CYP119 exclusively was 66 %. Coexpression of ALAS is a promising alternative for the efficient heterologous production of hemeproteins by using E. coli.

Archaeal cell surface biogenesis.

Cell surfaces are critical for diverse functions across all domains of life, from cell-cell communication and nutrient uptake to cell stability and surface attachment. While certain aspects of the mechanisms supporting the biosynthesis of the archaeal cell surface are unique, likely due to important differences in cell surface compositions between domains, others are shared with bacteria or eukaryotes or both. Based on recent studies completed on a phylogenetically diverse array of archaea, from a wide variety of habitats, here we discuss advances in the characterization of mechanisms underpinning archaeal cell surface biogenesis. These include those facilitating co- and post-translational protein targeting to the cell surface, transport into and across the archaeal lipid membrane, and protein anchoring strategies. We also discuss, in some detail, the assembly of specific cell surface structures, such as the archaeal S-layer and the type IV pili. We will highlight the importance of post-translational protein modifications, such as lipid attachment and glycosylation, in the biosynthesis as well as the regulation of the functions of these cell surface structures and present the differences and similarities in the biogenesis of type IV pili across prokaryotic domains.

A transcriptional factor B paralog functions as an activator to DNA damage-responsive expression in archaea.

Previously it was shown that UV irradiation induces a strong upregulation of tfb3 coding for a paralog of the archaeal transcriptional factor B (TFB) in Sulfolobus solfataricus, a crenarchaea. To investigate the function of this gene in DNA damage response (DDR), tfb3 was inactivated by gene deletion in Sulfolobus islandicus and the resulting Δtfb3 was more sensitive to DNA damage agents than the original strain. Transcriptome analysis revealed that a large set of genes show TFB3-dependent activation, including genes of the ups operon and ced system. Furthermore, the TFB3 protein was found to be associated with DDR gene promoters and functional dissection of TFB3 showed that the conserved Zn-ribbon and coiled-coil motif are essential for the activation. Together, the results indicated that TFB3 activates the expression of DDR genes by interaction with other transcriptional factors at the promoter regions of DDR genes to facilitate the formation of transcription initiation complex. Strikingly, TFB3 and Ced systems are present in a wide range of crenarchaea, suggesting that the Ced system function as a primary DNA damage repair mechanism in Crenarchaeota. Our findings further suggest that TFB3 and the concurrent TFB1 form a TFB3-dependent DNA damage-responsive circuit with their target genes, which is evolutionarily conserved in the major lineage of Archaea.

Sequence analysis and biochemical properties of an acidophilic and hyperthermophilic amylopullulanase from Thermofilum pendens.

The acidophilic and thermophilic pullulanases have many potential applications in the processes of starch liquefaction and saccharification. In this study, a gene encoding an amylopullulanase from Thermofilum pendens (TPApu) was heterologously expressed in Escherichia coli. Although TPApu possessed the same continuous GH57N_Apu domain and the succeeding α-helical region as other two amylopullulanases from Staphylothermus marinus (SMApu) and Caldivirga maquilingensis (CMApu), it only showed maximal amino acid identities of 25.7-28.7% with CMApu and SMApu. The purified TPApu appeared as a single band of SDS-PAGE with a molecular mass of 65.5kDa and exhibited the maximal activity at pH3.5 and 95-100°C. TPApu had the highest catalytic efficiency towards pullulan (kcat/km, 8.79s-1mLmg-1) and α-cyclodextrin (kcat/km, 0.36s-1mM-1). In the initial stages, the ring-opening reactions of γ-cyclodextrin, 6-O-glucosyl-ß-cyclodextrin, 6-O-maltosyl-ß-cyclodextrin and the debranching reactions of 6-O-maltooctaosyl-ß-cyclodextrin were firstly catalyzed. In the subsequent reactions, a serial of maltooligosaccharides were produced. As the most acidophilic amylopullulanase among thermophilic pullulanases reported to date, TPApu preferred to debranch the DP6-12 side chains of amylopectin at pH4.5 and 100°C.

Laboratory Evolution of Bacillus circulans Xylanase Inserted into Pyrococcus furiosus Maltodextrin-Binding Protein for Increased Xylanase Activity and Thermal Stability Toward Alkaline pH.

High xylanase activity and stability toward alkaline pH is strongly desired for pulping and bleaching processes. We previously enhanced thermal stability of Bacillus circulans xylanase (BCX) by inserting into a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) (the resulting complex named as PfMBP-BCX165). In the present study, we aimed to evolve the inserted BCX domain within PfMBP-BCX165 for greater xylanase activity toward alkaline pH while maintaining enhanced thermal stability. No BCX sequence variation was required for the thermal stabilization, thus allowing us to explore the entire BCX sequence space for the evolution. Specifically, we randomized the BCX sequence within PfMBP-BCX165 and then screened the resulting libraries to identify a PfMBP-BCX165 variant, PfMBP-BCX165T50R. The T50R mutation enhanced xylanase activity of PfMBP-BCX165 toward alkaline pH without compromising thermal stability. When compared to PfMBP-BCX165T50R, the corresponding unfused BCX mutant, BCXT50R, exhibited similar pH dependence of xylanase activity, yet suffered from limited thermal stability. In summary, we showed that one can improve thermal stability and xylanase activity of BCX toward alkaline pH by inserting into PfMBP followed by sequence variation of the BCX domain. Our study also suggested that insertional fusion to PfMBP would be a useful stabilizing platform for evolving many proteins.

ATP-free biosynthesis of a high-energy phosphate metabolite fructose 1,6-diphosphate by in vitro metabolic engineering.

Fructose 1,6-diphosphate (FDP) is a widely used medicine and is also a precursor of two important three-carbon phosphates - glyceraldehyde 3-phosphate (GA3P) and dihydroxyacetone phosphate (DHAP) for the biosynthesis of numerous fine chemicals. An in vitro synthetic cofactor-free enzymatic pathway comprised of four hyperthermophilic enzymes was designed to produce FDP from starch and pyrophosphate. All of four hyperthermophilic enzymes (i.e., alpha-glucan phosphorylase from Thermotaga maritima, phosphoglucomutase from Thermococcus kodakarensis, glucose 6-phosphate isomerase from Thermus thermophilus, and pyrophosphate phosphofructokinase from T. maritima) were overexpressed in E. coli BL21(DE3) and purified by simple heat precipitation. The optimal pH and temperature of one-pot biosynthesis were 7.2 and 70°C, respectively. The optimal enzyme ratios of αGP, PGM, PGI and PFK were 2:2:1:2 in terms of units. Via step-wise addition of new substrates, up to 125 ± 4.6mM FDP was synthesized after 7-h reaction. This de novo ATP-free enzymatic pathway comprised of all hyperthermophilic enzymes could drastically decrease the manufacturing costs of FDP and its derivatives GA3P and DHAP, better than those catalyzed by ATP-regeneration cascade biocatalysis, the use of mesophilic enzymes, whole cell lysates, and microbial cell factories.

Efficient expression of recombinant human heavy chain ferritin (FTH1) with modified peptides.

Human heavy chain ferritin (FTH1) can self-assemble into a diameter of 12-nm spherical cage with an interior cavity of 8 nm in diameter. FTH1 has great potential as a nanocage in molecular imaging and drug delivery. Different peptides have been fused with FTH1 for targeting delivery; however, the expression of FTH1 modified with peptides in soluble form is not equivalent to natural FTH1. As shown in recent study, a novel scaffold protein --thioredoxin from the archaebacterium Pyrococcus furiosus (PfTrx)--exhibits a superior solubilization capacity and thermal stability [19]. Here we report a new construct (FTH1-PfTrx-His) that can be easily expressed and purified in Escherichia coli. Of note, different peptides inserted into FTH1-PfTrx-His did not influence the expression of proteins. Finally, the doxorubicin packaging ability of FTH1-PfTrx-His is comparable to natural FTH1. Our results showed that FTH1-PfTrx-His had a potential role as a novel peptide-modified ferritin carrier for drugs or imaging probes.

Optimization of thermophilic trans-isoprenyl diphosphate synthase expression in Escherichia coli by response surface methodology.

We optimized the heterologous expression of trans-isoprenyl diphosphate synthase (IDS), the key enzyme involved in the biosynthesis of trans-polyisoprene. trans-Polyisoprene is a particularly valuable compound due to its superior stiffness, excellent insulation, and low thermal expansion coefficient. Currently, trans-polyisoprene is mainly produced through chemical synthesis and no biotechnological processes have been established so far for its large-scale production. In this work, we employed D-optimal design and response surface methodology to optimize the expression of thermophilic enzymes IDS from Thermococcus kodakaraensis. The design of experiment took into account of six factors (preinduction cell density, inducer concentration, postinduction temperature, salt concentration, alternative carbon source, and protein inhibitor) and seven culture media (LB, NZCYM, TB, M9, Ec, Ac, and EDAVIS) at five different pH points. By screening only 109 experimental points, we were able to improve IDS production by 48% in close-batch fermentation.

Strategies for increasing heterologous expression of a thermostable esterase from Archaeoglobus fulgidus in Escherichia coli.

Heterologous proteins expressed in bacteria are used for numerous biotechnological applications. Escherichia coli is the most commonly used host for heterologous protein expression because of its many advantages. Researchers have been studying proteins from extremophiles heterologously expressed in E. coli because the proteins of extremophiles are strongly resistant to extreme conditions. In a previous study, a thermostable esterase Est-AF was isolated from Archaeoglobus fulgidus and expressed in E. coli. However, further studies of Est-AF were difficult owing to its low expression levels in E. coli. In this study, we used various strategies, such as changing the expression vector and host strain, codon optimization, and optimization of induction conditions, to increase the expression of Est-AF. Through codon optimization and by changing the vector and host strain, Est-AF expression was increased from 31.50 ± 0.35 mg/L to 61.75 ± 0.28 mg/L. The optimized expression system consisted of a codon-optimized Est-AF gene in a pET28a(+)-based expression plasmid in E. coli Rosetta cells. The expression level was further increased by optimizing the induction conditions. The optimized conditions were induction with 0.4 mM isopropyl-b-d-1-thiogalactoside (IPTG) at 37 °C for 5 h. Under these conditions, the expression level of Est-AF was increased from 31.5 ± 0.35 mg/L to 119.52 ± 0.34 mg/L.