Co-expression Of Molecular Chaperones Research Articles

Highly efficient expression of Rasamsonia emersonii lipase in Pichia pastoris: characterization and gastrointestinal simulated digestion in vitro.

Acidic lipases with high catalytic activities under acidic conditions have important application values in the food, feed and pharmaceutical industries. However, the availability of acidic lipases is still the main obstacle to their industrial applications. Although a novel acidic lipase Rasamsonia emersonii (LIPR) was heterologously expressed in Escherichia coli, the expression level was unsatisfactory. To achieve the high-efficiency expression and secretion of LIPR in Pichia pastoris GS115, the combinatorial optimization strategy was adopted including gene codon preference, signal peptide, molecular chaperone co-expression and disruption of vacuolar sorting receptor VPS10. The activity of the combinatorial optimization engineered strain in a shake flask reached 1480 U mL-1 , which was 8.13 times greater than the P. pastoris GS115 parental strain. After high-density fermentation in a 5-L bioreactor, the highest enzyme activity reached as high as 11 820 U mL-1 . LIPR showed the highest activity at 40 °C and pH 4.0 in the presence of Ca2+ ion. LIPR exhibited strong tolerance to methanol, indicating its potential application in biodiesel biosynthesis. Moreover, the gastrointestinal digestion simulation results demonstrated that LIPR was tolerant to pepsin and trypsin, but its activity was inhibited by sodium taurodeoxycholate. This study provided an effective approach for the high expression of acidic lipase LIPR. LIPR was more appropriate for lipid digestion in the stomach than in intestine according to the gastrointestinal digestion simulation results. © 2024 Society of Chemical Industry.

High-Level Secretory Production of Recombinant E2-Spy Antigen Protein via Combined Strategy in Pichia pastoris

E2-Spy (abbreviated as ES) plays a vital role as a component in the Bacterial-Like Particles (BLPs) vaccine against classical swine fever virus (CSFV). This vaccine demonstrates remarkable immunoprotection, highlighting the importance of augmenting ES production in the development of CSFV subunit vaccines. In this study, a Pichia pastoris strain capable of high-yield secretory production of ES was developed through signal peptide engineering, gene dosage optimization and co-expression of molecular chaperones. Initially, a hybrid signal peptide cSP3 was engineered, leading to a 3.38-fold increase in ES production when compared to the control strain 1-α-ES. Subsequently, cSP3 was evaluated for its expression efficiency alongside different commonly used signal peptides under multicopy conditions. SDS-PAGE analysis revealed that 2-αd14-ES exhibited the highest ES production, displaying a 4.38-fold increase in comparison to 1-α-ES. Afterwards, SSA1, YDJ1, BIP, LHS1, and their combinations were integrated into 2-αd14-ES, resulting in a 1.92-fold rise in ES production compared to 2-αd14-ES (equivalent to a 6.18-fold increase compared to 1-α-ES). The final yield of ES was evaluated as 168.3 mg/L through comparison with serially diluted BSA protein bands.

Novel recombinant aminoacylase from Paraburkholderia monticola capable of N-acyl-amino acid synthesis

N-Acyl-amino acids can act as mild biobased surfactants, which are used, e.g., in baby shampoos. However, their chemical synthesis needs acyl chlorides and does not meet sustainability criteria. Thus, the identification of biocatalysts to develop greener synthesis routes is desirable. We describe a novel aminoacylase from Paraburkholderia monticola DSM 100849 (PmAcy) which was identified, cloned, and evaluated for its N-acyl-amino acid synthesis potential. Soluble protein was obtained by expression in lactose autoinduction medium and co-expression of molecular chaperones GroEL/S. Strep-tag affinity purification enriched the enzyme 16-fold and yielded 15 mg pure enzyme from 100 mL of culture. Biochemical characterization revealed that PmAcy possesses beneficial traits for industrial application like high temperature and pH-stability. A heat activation of PmAcy was observed upon incubation at temperatures up to 80 °C. Hydrolytic activity of PmAcy was detected with several N-acyl-amino acids as substrates and exhibited the highest conversion rate of 773 U/mg with N-lauroyl-L-alanine at 75 °C. The enzyme preferred long-chain acyl-amino-acids and displayed hardly any activity with acetyl-amino acids. PmAcy was also capable of N-acyl-amino acid synthesis with good conversion rates. The best synthesis results were obtained with the cationic L-amino acids L-arginine and L-lysine as well as with L-leucine and L-phenylalanine. Exemplarily, L-phenylalanine was acylated with fatty acids of chain lengths from C8 to C18 with conversion rates of up to 75%. N-lauroyl-L-phenylalanine was purified by precipitation, and the structure of the reaction product was verified by LC–MS and NMR.Key points• A novel aminoacylase from Paraburkholderia monticola was cloned, expressed in E. coli and purified.• The enzyme PmAcy exhibits exceptional temperature and pH stability and a broad substrate spectrum.• Synthesis of acyl amino acids was achieved in good yields.

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.

Expression and characterisation of human glycerol kinase: the role of solubilising agents and molecular chaperones.

Glycerol kinase (GK; EC 2.7.1.30) facilitates the entry of glycerol into pathways of glucose and triglyceride metabolism and may play a potential role in Type 2 diabetes mellitus (T2DM). However, the detailed regulatory mechanisms and structure of the human GK are unknown. The human GK gene was cloned into the pET-24a(+) vector and over-expressed in Escherichia coli BL21 (DE3). Since the protein was expressed as inclusion bodies (IBs), various culture parameters and solubilising agents were used but they did not produce bioactive His-GK; however, co-expression of His-GK with molecular chaperones, specifically pKJE7, achieved expression of bioactive His-GK. The overexpressed bioactive His-GK was purified using coloumn chromatography and characterised using enzyme kinetics. The overexpressed bioactive His-GK was purified apparently to homogeneity (∼295-fold) and characterised. The native His-GK was a dimer with a monomeric molecular weight of ∼55 kDa. Optimal enzyme activity was observed in TEA buffer (50 mM) at 7.5 pH. K+ (40 mM) and Mg2+ (2.0 mM) emerged as prefered metal ions for His-GK activity with specific activity 0.780 U/mg protein. The purified His-GK obeyed standard Michaelis-Menten kinetics with Km value of 5.022 µM (R2=0.927) for its substrate glycerol; whereas, that for ATP and PEP was 0.767 mM (R2=0.928) and 0.223 mM (R2=0.967), respectively. Other optimal parameters for the substrate and co-factors were also determined. The present study demonstrates that co-expression of molecular chaperones assists with the expression of bioactive human GK for its characterisation.

Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli

BackgroundEndolysins are peptidoglycan hydrolases with promising use as environment-friendly antibacterials mainly when used topically. However, in general, endolysin expression is hampered by its low solubility. Thus, a critical point in endolysin industrial production is optimizing their expression, including improvement of solubility and recovery from cell extracts.ResultsWe report the expression of two endolysins encoded in the genome of phages infecting Staphylococcus aureus. Expression was optimized through changes in the concentration of the inducer and growth temperature during the expression. Usually, only 30–40% of the total endolysin was recovered in the soluble fraction. Co-expression of molecular chaperones (DnaK, GroEL) or N-term fusion tags endowed with increased solubility (DsbC, Trx, Sumo) failed to improve that yield substantially. Inclusion of osmolytes (NaCl, CaCl2, mannitol, glycine betaine, glycerol and trehalose) or tensioactives (Triton X-100, Tween 20, Nonidet P-40, CHAPS, N-lauroylsarcosine) in the cell disruption system (in the absence of any molecular chaperone) gave meager improvements excepted by N-lauroylsarcosine which increased recovery to 54% of the total endolysin content.ConclusionThis is the first attempt to systematically analyze methods for increasing yields of recombinant endolysins. We herein show that neither solubility tags nor molecular chaperones co-expression are effective to that end, while induction temperature, (His)6-tag location and lysis buffer additives (e.g.N-lauroylsarcosine), are sensible strategies to obtain higher levels of soluble S. aureus endolysins.

Enhanced Production of (R)-3-Hydroxybutyrate Oligomers by Coexpression of Molecular Chaperones in Recombinant Escherichia coli Harboring a Polyhydroxyalkanoate Synthase Derived from Bacillus cereus YB-4.

The biodegradable polyester poly-(R)-3-hydroxybutyrate [P(3HB)] is synthesized by a polymerizing enzyme called polyhydroxyalkanoate (PHA) synthase and accumulates in a wide variety of bacterial cells. Recently, we demonstrated the secretory production of a (R)-3HB oligomer (3HBO), a low-molecular-weight P(3HB), by using recombinant Escherichia coli expressing PHA synthases. The 3HBO has potential value as an antibacterial substance and as a building block for various polymers. In this study, to construct an efficient 3HBO production system, the coexpression of molecular chaperones and a PHA synthase derived from Bacillus cereus YB-4 (PhaRCYB4) was examined. First, genes encoding enzymes related to 3HBO biosynthesis (phaRCYB4, phaA and phaB derived from Ralstonia eutropha H16) and two types of molecular chaperones (groEL, groES, and tig) were introduced into the E. coli strains BW25113 and BW25113ΔadhE. As a result, coexpression of the chaperones promoted the enzyme activity of PHA synthase (approximately 2–3-fold) and 3HBO production (approximately 2-fold). The expression assay of each chaperone and PHA synthase subunit (PhaRYB4 and PhaCYB4) indicated that the combination of the two chaperone systems (GroEL-GroES and TF) supported the folding of PhaRYB4 and PhaCYB4. These results suggest that the utilization of chaperone proteins is a valuable approach to enhance the formation of active PHA synthase and the productivity of 3HBO.

Co-Expression of Chaperones for Improvement of Soluble Expression and Purification of An Anti-HER2 scFv in Escherichia Coli.

Single-chain fragment variable (scFv) is one of the most commonly used antibody fragments. They offer some advantages over full-length antibodies, including better penetration to target tissues. However, their functional production has been a challenge for manufacturers due to the potential misfolding and formation of inclusion bodies. Here we evaluated the soluble expression and purification of molecular chaperone co-expression. E. coli BL21(DE3) cells were co-transformed with the mixture of plasmids pKJE7 and pET22b-scFv by the electroporation method. First, L-arabinose was added to induce the expression of molecular chaperones, and then IPTG was used as an inducer to start the expression of anti-HER2 scFv. The effect of cultivation temperature and IPTG concentration on soluble expression of the protein with or without chaperones was evaluated. The soluble expressed protein was subjected to native purification using the Ni-NTA affinity column. SDS-PAGE analysis confirmed the successful co-expression of anti-HER2-scFv and DnaK/DnaJ/GrpE chaperones. Co-expression with chaperones and low-temperature cultivation synergistically improved the soluble expression of anti-HER2 scFv. Co-expression with chaperone also exhibited an approximately four-fold increase in the final yield of purified soluble protein. The combination of co-expression with chaperones and low temperature presented in this work may be useful for the improvement of commercial production of other scFvs in E. coli as functionally bioactive and soluble form.

Expression, isolation, and identification of an ethanol-resistant ethyl carbamate-degrading amidase from Agrobacterium tumefaciens d3

Ethyl carbamate (EC), widely found in alcoholic beverages, has been revealed to be a probable carcinogen in humans. Urethanase (EC 3.5.1.75) is an effective enzyme for the degradation of EC; however, the previously identified urethanases exhibited insufficient acid and alcohol resistance. In this study, an enantioselective amidase (AmdA) screened from Agrobacterium tumefaciens d3 exhibited urethanase activity with excellent alcohol resistance. AmdA was first overexpressed in Escherichia coli; however, the recombinant protein was primarily located in inclusion bodies, and thus, co-expression of molecular chaperones was used. The activity of AmdA increased 3.1 fold to 307 U/L, and the specific activity of urethanase with C-terminal His-tags reached 0.62 U/mg after purification through a Ni-NTA column. Subsequently, the enzymatic properties and kinetic constants of AmdA were investigated. The optimum temperature for AmdA was 55°C, it showed the highest activity at pH 7.5, and the Km was 0.964mM. Moreover, after 1h of heat treatment at 37°C in a 5-20% (v/v) ethanol solution, the residual urethanase activity was higher than 91%, considerably more than that reported thus far.

A Combinational Strategy for Effective Heterologous Production of Functional Human Lysozyme in Pichia pastoris.

Human lysozyme (hLYZ), known for its bacteriolytic activity, is widely applied in the food and pharmaceutical industries as an antimicrobial agent. However, its extensive application was limited by its low large-scale production efficiency. In this study, a combinational method of integrating codon optimization, multiple gene copies, and ER molecular chaperone co-expression was developed to improve the heterologous production of hLYZ in Pichia pastoris GS115. Our results showed that increasing the copy number of the optimized hLYZ gene in P. pastoris could enhance its secretory production level up to 1.57-fold. The recombinant opt-hLYZ-6C strain that contains six copies of opt-hLYZ gene exhibited the highest mRNA transcription levels, giving the highest production of 0.22 ± 0.02 mg/mL of hLYZ in the medium supernatant with a bacteriolytic activity of 14,680 ± 300 U/mL against Micrococcus lysodeikticus in the shaking flask experiment. Moreover, co-overexpression of ER retention molecular chaperones, such as Pdi1 or Ero1, in the recombinant opt-hLYZ-6C strain both presented positive effects on the secretory production of hLYZ. Our further characterization indicated that tandem co-expression of Ero1 and Pdi1 together presented an added-up effect. The secretory production of hLYZ in the medium supernatant reached 0.34 ± 0.02 mg/mL of the recombinant opt-hLYZ-6C-EP strain in the shaking flask experiment, with a bacteriolytic activity of 21,200 ± 400 U/mL. Compared to the recombinant opt-hLYZ-1C strain, these final improvements were calculated as 2.43-fold and 2.30-fold on secretory protein levels and antibacterial activity, respectively. Finally, the recombinant opt-hLYZ-6C-EP strain was applied for high-density cultivation in 5 L of fermenter, in which the secretory yield of hLYZ reached 2.34 ± 0.02 mg/mL in the medium supernatant, with a bacteriolytic activity of 1.76 ± 0.02 × 105 U/mL against M. lysodeikticus. All these numbers presented the highest heterologous production levels of hLYZ in microbial systems.

Chaperones Promote Remarkable Solubilization of Salmonella enterica serovar Enteritidis Flagellin Expressed in Escherichia coli.

Flagellin of Salmonella enterica serovar Enteritidis (SEF) stimulates immune responses to both itself and coapplied antigens. It is therefore used in vaccine development and immunotherapy. Removal of pathogenic S. enterica ser. Enteritidis from SEF production process is advantageous due to the process safety improvement. The protein solubility analysis using SDS-PAGE indicated that 53.49% of SEF expressed in Escherichia coli formed inclusion bodies. However, the protein recovery from inclusion bodies requires a complex process with a low yield. We thus aim to study possibility of enhancing SEF expression in E. coli in soluble form using chemical and molecular chaperones. Chemical chaperones including arginine, sorbitol, trehalose, sodium chloride and benzyl alcohol were used as cultivation medium additives during SEF expression. SEF solubilization by coexpression of molecular chaperones DnaK, DnaJ, and GrpE was also investigated. All of the chemical chaperones were effective in improving SEF solubility. However, sorbitol showed the most profound effect. SEF solubilization by molecular chaperones was slightly better than that using sorbitol and this approach enhanced noticeably SEF soluble concentration and SEF solubility percentage to almost two folds and 96.37% respectively. Results of limited proteolysis assay and native PAGE indicated similar conformational states and proper folding for SEF obtained without using chaperones and for those obtained using sorbitol and the molecular chaperones. However, the molecular chaperones based system was less costly than the sorbitol based system. The coexpression of molecular chaperones was then considered as the most appropriate approach for soluble SEF production. Therefore, SEF production for medical purposes is expected to be facilitated.

Efficient production of aggregation prone 4-α-glucanotransferase by combined use of molecular chaperones and chemical chaperones in Escherichia coli

In this study, a combined optimization strategy, based on co-expression of molecular chaperones and supplementation of osmolytes, was used to reduce the formation of inclusion bodies and enhance the expression of the soluble form of 4-α-glucanotransferase. The 4-α-glucanotransferase yield was enhanced by co-expression with pGro7 and supplementation of trimetlylamine oxide. Subsequently, the effects of process conditions (temperature, inducer concentration, and arabinose concentration) on cell growth and 4-α-glucanotransferase production were also investigated in shake flasks. In addition, a modified high-cell-density fermentation approach was proposed and applied in 3-L fermentor supplied with l-arabinose and trimetlylamine oxide, which achieved a dry cell weight of 65.92 g·L−1. Through this cultivation approach at 28 °C, the activity of 4-α-glucanotransferase reached 332.5 U·g−1 dry cell weight, which was 24.6-fold greater than the initial activity in shake flask cultivation. This combined strategy is expected to provide an efficient and economical approach to overproduction of aggregation prone proteins on a large scale.

High-level production of ChSase ABC I by co-expressing molecular chaperones in Escherichia coli

Chondroitinase ABC I (ChSase ABC I), as a polysaccharide lyase, can catalyze high molecular weight chondroitin sulfate (CS) to low molecular weight glycosaminoglycan which are easier to be absorbed and utilized by organisms. In this study, to enhance the production of ChSase ABC I and avoid the negative influence of tags on its catalytic efficiency, we employed molecular chaperones to co-express with ChSase ABC I. Firstly, different molecular chaperones and their combinations were screened and GroES exhibited the best positive effect. Consecutively, fermentation conditions were optimized to further improve the production. As a result, the production of ChSase ABC I was increased to 4640.44 ± 896.26 IU/g wet weight, a 2.15-fold higher value when compared with that of control in the same fermentation conditions. After that, to testify the influence of GroES on characterization of ChSase ABC I, the optimal pH and temperature, and kinetic parameters were confirmed. The affinity to substrate of ChSase ABC I with GroES assist was increased 7 folds as compared to the native ChSase ABC I, and ChSase ABC I with GroES co-expression still has high catalytic activity. This work not only presents to date the first achievement of ChSase ABC I high-level production with molecular chaperone co-expression, but also serves as a potential basis for its industrial application.

Polyionic Tags as Enhancers of Protein Solubility in Recombinant Protein Expression.

Since the introduction of recombinant protein expression in the second half of the 1970s, the growth of the biopharmaceutical field has been rapid and protein therapeutics has come to the foreground. Biophysical and structural characterisation of recombinant proteins is the essential prerequisite for their successful development and commercialisation as therapeutics. Despite the challenges, including low protein solubility and inclusion body formation, prokaryotic host systems and particularly Escherichia coli, remain the system of choice for the initial attempt of production of previously unexpressed proteins. Several different approaches have been adopted, including optimisation of growth conditions, expression in the periplasmic space of the bacterial host or co-expression of molecular chaperones, to assist correct protein folding. A very commonly employed approach is also the use of protein fusion tags that enhance protein solubility. Here, a range of experimentally tested peptide tags, which present specific advantages compared to protein fusion tags and the concluding remarks of these experiments are reviewed. Finally, a concept to design solubility-enhancing peptide tags based on a protein’s pI is suggested.

Effect of DnaK/DnaJ/GrpE and DsbC Chaperons on Periplasmic Expression of Fab Antibody by E. coli SEC Pathway

Expression of recombinant protein that possess disulfide bonds especially Fab antibody fragment in periplasm of E. coli is the most favorable platform. But formation of inclusion bodies and inefficient translocation of desired protein to the periplasm is considerable obstacle threatening the advantages of E. coli expression system. Co-expression with molecular chaperones is remarkable consideration to solve this problem. In this study we evaluated the effect of co-expression of molecular chaperones and optimization condition on efficient periplasmic translocation of Fab antibody fragment. Plasmid pKJE7 was used for co-expression of DnaK/DnaJ/GrpE in BL21 cells and SHuffle strain (constantly expressing DsbC chaperon) were used for evaluation of chaperones effects. Periplasmic fraction was prepared by osmotic shock method and ELISA test were used for activity measurement. The Fab antibody purified by IMAC chromatography. The results indicated that co-expression of anti-TNF-α Fab with DnaK/DnaJ/GrpE in BL21 cells had a marked effect on the yield of periplasmic fraction but DsbC had no effect on periplasmic translocation. The optimal culture condition was found 0.1 mM IPTG concentration and 25 °C temperatures. SDS-PAGE analysis indicated that solubility increased to 42% of total Fab in Bl21 co-expressing DnaKJE. This study reports that co-expression with DnaK/DnaJ/GrpE markedly increases soluble expression of Fab antibody fragment in optimized condition.

One-step purification and immobilization of extracellularly expressed sortase A by magnetic particles to develop a robust and recyclable biocatalyst

Sortase A (SrtA) is a transpeptidase widely used to site-specifically modify peptides and proteins and shows promise for industrial applications. In this study, a novel strategy was developed for constructing immobilized-SrtA as a robust and recyclable enzyme via direct immobilization of extracellularly expressed SrtA in the fermentation supernatant using magnetic particles. Efficient extracellular SrtA expression was achieved in Escherichia coli through molecular engineering, including manipulation of the protein transport pathway, codon optimization, and co-expression of molecular chaperones to promote expressed SrtA secretion into the medium at high levels. Subsequently, a simple one-step protocol was established for the purification and immobilization of SrtA containing a His-tag from the fermentation supernatant onto a nickel-modified magnetic particle. The immobilized SrtA was proved to retain full enzymatic activity for peptide-to-peptide ligation and protein modification, and was successfully reused for five cycles without obvious activity loss.

Expression and characterization of the key enzymes involved in 2-benzoxazolinone degradation by Pigmentiphaga sp. DL-8

In this study, the key enzymes involved in 2-benzoxazolinone (BOA) degradation by Pigmentiphaga sp. DL-8 were further verified and characterized in Escherichia coli. By codon optimization and co-expression of molecular chaperones in a combined strategy, recombinant BOA amidohydrolase (rCbaA) and 2-aminophenol (2-AP) 1,2-dioxygenase (rCnbCαCβ) were expressed and purified with the highest activity of 1934.6U·mgprotein−1 and 32.80U·mgprotein−1, respectively. BOA could be hydrolyzed to 2AP by rCbaA, which was further transformed to picolinic acid by rCnbCαCβ based on identified catalytic product. The optimal pH and temperature for rCbaA are 9.0 and 55°C with excellent stability for catalytic environments, and the residual activity was >50% after incubation at temperatures <45°C or at pH between 6.0 and 10.0 for 24h. On the contrary, rCnbCαCβ composed of α-subunit (33kDa) and β-subunit (38kDa) showed poor stability against environmental factors, including temperature, pH, metal ions and chemicals.

Strain engineering and process optimization for enhancing the production of a thermostable steryl glucosidase in Escherichia coli.

Biodiesels produced from transesterification of vegetable oils have a major problem in quality due to the presence of precipitates, which are mostly composed of steryl glucosides (SGs). We have recently described an enzymatic method for the efficient removal of SGs from biodiesel, based on the activity of a thermostable β-glycosidase from Thermococcus litoralis. In the present work, we describe the development of an Escherichia coli-based expression system and a high cell density fermentation process. Strain and process engineering include the assessment of different promoters to drive the expression of a codon-optimized gene, the co-expression of molecular chaperones and the development of a high cell density fermentation process. A 200-fold increase in the production titers was achieved, which directly impacts on the costs of the industrial process for treating biodiesel.

Soluble Expression of the Fucosyltransferase Gene from Helicobacter pylori in Escherichia coli by Co-expression of Molecular Chaperones

Soluble Expression of the Fucosyltransferase Gene from Helicobacter pylori in Escherichia coli by Co-expression of Molecular Chaperones

Overexpression of Candida rugosa lipase Lip1 via combined strategies in Pichia pastoris

In this study, combined strategies were employed to heterologously overexpress Candida rugosa lipase Lip1 (CRL1) in a Pichia pastoris system. The LIP1 gene was systematically codon-optimized and synthesized in vitro. The Lip1 activity of a recombinant strain harboring three copies of the codon-optimized LIP1 gene reached 1200U/mL in a shake flask culture. Higher lipase activity, 1450U/mL, was obtained using a five copy number construct. Co-expressing one copy of the ERO1p and BiP chaperones with Lip1p, the CRL1 lipase yield further reached 1758U/mL, which was significantly higher than that achieved by expressing Lip1p alone or only co-expressing one molecular chaperone. When cultivated in a 3L fermenter under optimal conditions, the recombinant strain GS115/87-ZA-ERO1p-BiP #7, expressing the molecular chaperones Ero1p and BiP, produced 13,490U/mL of lipase activity at 130h, which was greater than the 11,400U/mL of activity for the recombinant strain GS115/pAO815-α-mCRL1 #87, which did not express a molecular chaperone. This study indicates that a strategy of combining codon optimization with co-expression of molecular chaperones has great potential for the industrial-scale production of pure CRL1.