Volumetric Product Yield Research Articles

Development of a streptokinase expression platform using the native signal sequence of the protein with internal repeats 1 (PIR1) in P. pastoris: Gene dosage optimization and cell retention strategies

Abstract Streptokinase is a therapeutically important thrombolytic agent which is toxic to its heterologous expression hosts and also prone to proteolytic degradation. In the current study, its secretory expression was targeted in P. pastoris using the native protein with internal repeats 1 (PIR1) secretion signal where the optimization of streptokinase gene dosage resulted in an increase in the volumetric product yield from 339.69 to 1206.48 mg/L. The cell retention strategy was applied during the induction phase to maintain sustained productivity where up to a 2.4 fold enhancement in the overall product yield was achieved at shake flask to a level of 3049.53 mg/L. Moreover, the cell recycling approach prevented the streptokinase proteolytic degradation and also helped in achieving a high specific product yield (YP/X) in a range of 167.65–176.44 mg/g DCW even after five cycles. Similarly, the cell recycling strategy was also employed using S. cerevisiae α-mating factor (α-MF) signal sequence which also resulted in improved product stability and volumetric product concentration confirming its wide applicability in obtaining enhanced product quality and yield. The recombinant streptokinase produced using PIR1 signal sequence was biologically active possessing a specific activity of 62,782.40 IU/mg and was predicted to contain 10.45% α-helix and 29.09% β-sheet.

β-Glucosidase from Thermotoga naphthophila RKU-10 for exclusive synthesis of galactotrisaccharides: Kinetics and thermodynamics insight into reaction mechanism

This work reports a novel thermophilic β-glucosidase (TN0602) from Thermotoga naphthophila RKU-10, demonstrating exceptionally high catalytic selectivity (100%) for the exclusive synthesis of prebiotic galactotrisaccharides (GOS3) in a high volumetric production yield of 23.28gL−1h−1 (higher than the highest value ever reported) at pH 6.5 and 75°C, with milk processing waste lactose as both the galactosyl donor and acceptor. A comparative study with commercial β-galactosidase from Aspergillus oryzae (AO) with respect to reaction kinetics, enzyme-substrate thermodynamic binding (substrate induced fluorescence quenching) and molecular docking simulation studies showed that β-glucosidase TN0602 has a deep catalytic “pocket” with a narrow entrance that prevents simultaneous access of lactose and GOS3 to the catalytic site, explaining its distinct catalytic specificity and reaction kinetics. The findings revealed in this work offer an improved understanding of how enzyme protein structure determines catalytic specificity, which serves as new knowledge to engineer β-glucosidase for the biosynthesis of designer GOS.

Performance loss of Corynebacterium glutamicum cultivations under scale‐down conditions using complex media

Substrate and oxygen gradients appear in industrial‐scale fed‐batch processes due to limitations in the achievable power input and concomitantly increased mixing times. In order to mimic these gradients at lab scale, scale‐down reactors are applied. Previous studies in such reactor systems suggest that Corynebacterium glutamicum is robust against oscillatory oxygen and substrate availability in relation to growth and side product accumulation. Usually, defined mineral salt media are applied contrary to the industrial case, in which complex media containing different carbon sources are used. Therefore, this study investigated the cultivation performance using complex medium based on sucrose, molasses, and corn steep liquor in a three‐compartment scale‐down reactor. The reactor consisted of a stirred tank and two plug flow reactor modules. This approach was applied based on assumptions of gradient distributions in bottom‐fed bioreactors. A drastic reduction of growth and volumetric product yield of a cadaverine producing strain was observed while several short chain fatty acids accumulated, among them l‐lactate and acetate. Growth was depleted after several hours of cultivation, while the substrate uptake rate was reduced by 20%. Hence, the main carbon source sucrose accumulated after 10 h of fed‐batch cultivation. Despite growth cessation, neither reduction of cell vitality nor increased cell lysis were observed.

Soluble expression, purification and functional characterization of a coil peptide composed of a positively charged and hydrophobic motif.

A de novo heterodimeric coiled-coil system formed by the association of two synthetic peptides, the Ecoil and Kcoil, has been previously designed and proven to be an excellent and versatile tool for various biotechnology applications. However, based on the challenges encountered during its chemical synthesis, the Kcoil peptide has been designated as a "difficult peptide". In this study, we explore the expression of the Kcoil peptide by a bacterial system as well as its subsequent purification. The maximum expression level was observed when the peptide was fused to thioredoxin and the optimized purification process consisted of three chromatographic steps: immobilized-metal affinity chromatography followed by cation-exchange chromatography and, finally, a reverse-phase high-performance liquid chromatography. This entire process led to a final volumetric production yield of 1.5mg of pure Kcoil peptide per liter of bacterial culture, which represents a significant step towards the cost-effective production and application of coiled-coil motifs. Our results thus demonstrate for the first time that bacterial production is a viable alternative to the chemical synthesis of de novo designed coil peptides.

High‐titer production and strong antimicrobial activity of sophorolipids from Rhodotorula bogoriensis

Rhodotorula bogoriensis produces sophorolipids (SLs) that contain 13-hydroxydocosanoic acid (OH-C22 ) as the lipid moiety. A systematic study was conducted to further understand the fermentative production of SLs containing OH-C22 (C22 -SL) by R. bogoriensis. Shake-flask studies showed that R. bogoriensis consumed glucose at a slow pace. HPLC analysis of the C22 -SL products from shake-flask fermentations at different glucose concentrations showed a correlation between glucose depletion and the extent of C22 -SL deacetylation. A large-scale bioreactor fermentation resulted in the isolation of C22 -SL at a volumetric product yield of 51 g/L. HPLC analysis of C22 -SL product from the bioreactor fermentation corroborated the finding that glucose depletion correlated with extensive deacetylation of C22 -SL. The antimicrobial activity of C22 -SL was established for the first time to be stronger than the C18 -SL from Candida bombicola against Propionibacterium acnes in a plate assay.

Effective enhancement of Pseudomonas stutzeri D-phenylglycine aminotransferase functional expression in Pichia pastoris by co-expressing Escherichia coli GroEL-GroES

BackgroundD-phenylglycine aminotransferase (D-PhgAT) of Pseudomonas stutzeri ST-201 catalyzes the reversible stereo-inverting transamination potentially useful in the application for synthesis of D-phenylglycine and D-4-hydroxyphenylglycine using L-glutamate as a low cost amino donor substrate in one single step. The enzyme is a relatively hydrophobic homodimeric intracellular protein difficult to express in the soluble functionally active form. Over-expression of the dpgA gene in E. coli resulted in the majority of the D-PhgAT aggregated into insoluble inclusion bodies that failed to be re-natured. Expression in Pichia pastoris was explored as an alternative route for high level production of the D-PhgAT.ResultsIntracellular expression of the codon-optimized synthetic dpgA gene under the PAOX1 promoter in P. pastoris resulted in inactive D-PhgAT associated with insoluble cellular fraction and very low level of D-PhgAT activity in the soluble fraction. Manipulation of culture conditions such as addition of sorbitol to induce intracellular accumulation of osmolytes, addition of benzyl alcohol to induce chaperone expression, or lowering incubation temperature to slow down protein expression and folding rates all failed to increase the active D-PhgAT yield. Co-expression of E. coli chaperonins GroEL-GroES with the D-PhgAT dramatically improved the soluble active enzyme production. Increasing gene dosage of both the dpgA and those of the chaperones further increased functional D-PhgAT yield up to 14400-fold higher than when the dpgA was expressed alone. Optimization of cultivation condition further increased D-PhgAT activity yield from the best co-expressing strain by 1.2-fold.ConclusionsThis is the first report on the use of bacterial chaperones co-expressions to enhance functional intracellular expression of bacterial enzyme in P. pastoris. Only two bacterial chaperone genes groEL and groES were sufficient for dramatic enhancement of functionally active D-PhgAT expression in this yeast. With the optimized gene dosage and chaperone combinations, P. pastoris can be attractive for intracellular expression of bacterial proteins since it can grow to a very high cell density which is translated into the higher volumetric product yield than the E. coli or other bacterial systems.

Oxygen transfer rate control in the production of human‐like collagen by recombinant Escherichia coli

The effects of different methods for elevating the OTR (oxygen transfer rate) during foreign gene expression and the cell growth of recombinant Escherichia coli BL21 were investigated. Two strategies were introduced to control DO (dissolved oxygen) levels in the fermentation broth: (i) increasing fermentor pressure and (ii) supplying oxygen-enriched air. These two methods were compared with the glucose feedback model, which acted as the control. By adopting a fed-batch method of cultivation, the cell yield coefficient (YX/S), accumulation of acetic acid and volumetric product yield (Yp) were measured or estimated. Adoption of these two methods led to an improvement in the OTR. The cell density and volumetric product yield in the cultivation controlled by increasing the fermentor pressure reached 77 g x l(-1) (dry cell weight) and 14 g x l(-1) respectively, which were much higher than those obtained with the strategy of supplying oxygen-enriched air (48 and 6 g x l(-1) respectively) and in the control (46 and 7 g x l(-1) respectively). The results indicate that increasing fermentor pressure is an effective way to enhance the OTR and recombinant protein (human-like collagen) productivity.

Enzyme controlled glucose auto-delivery for high cell density cultivations in microplates and shake flasks

BackgroundHere we describe a novel cultivation method, called EnBase™, or enzyme-based-substrate-delivery, for the growth of microorganisms in millilitre and sub-millilitre scale which yields 5 to 20 times higher cell densities compared to standard methods. The novel method can be directly applied in microwell plates and shake flasks without any requirements for additional sensors or liquid supply systems. EnBase is therefore readily applicable for many high throughput applications, such as DNA production for genome sequencing, optimisation of protein expression, production of proteins for structural genomics, bioprocess development, and screening of enzyme and metagenomic libraries.ResultsHigh cell densities with EnBase are obtained by applying the concept of glucose-limited fed-batch cultivation which is commonly used in industrial processes. The major difference of the novel method is that no external glucose feed is required, but glucose is released into the growth medium by enzymatic degradation of starch. To cope with the high levels of starch necessary for high cell density cultivation, starch is supplied to the growing culture suspension by continuous diffusion from a storage gel.Our results show that the controlled enzyme-based supply of glucose allows a glucose-limited growth to high cell densities of OD600 = 20 to 30 (corresponding to 6 to 9 g l-1 cell dry weight) without the external feed of additional compounds in shake flasks and 96-well plates. The final cell density can be further increased by addition of extra nitrogen during the cultivation. Production of a heterologous triosphosphate isomerase in E. coli BL21(DE3) resulted in 10 times higher volumetric product yield and a higher ratio of soluble to insoluble product when compared to the conventional production method.ConclusionThe novel EnBase method is robust and simple-to-apply for high cell density cultivation in shake flasks and microwell plates. The potential of the system is that the microbial growth rate and oxygen consumption can be simply controlled by the amount (and principally also by the activity) of the starch-degrading enzyme. This solves the problems of uncontrolled growth, oxygen limitation, and severe pH drop in shaken cultures. In parallel the method provides the basis for enhanced cell densities. The feasibility of the new method has been shown for 96-well plates and shake flasks and we believe that it can easily be adapted to different microwell and deepwell plate formats and shake flasks. Therefore EnBase will be a helpful tool especially in high throughput applications.

Overexpression and purification of recombinant human interferon alpha2b in Escherichia coli

Overexpression of rhIFN-α2b was obtained by synthesizing a codon optimized gene for IFN-α2b and expressing it in the form of inclusion bodies (IBs) in Escherichia coli. The recombinant plasmid pRSET-IFNα, which had the IFN-α2b gene under the T7 promoter, was coexpressed with plasmid pGP1-2, which carried the gene for T7 RNA polymerase under the heat inducible λP L promoter. This two plasmid expression system was optimized with respect to heat shock time, media, and time of induction in shake flask cultures. This was then scaled up into a bioreactor to get a maximum volumetric product yield of 5.2 g/L at a final OD 600 of 67. At this point, the IBs represented ∼40% of the total cellular protein. This high specific product yields eased the further downstream processing steps and improved product recoveries. The IBs were isolated and purified through ion exchange followed by step refolding to give a final product yield of ∼3 g/L, which is maximum reported in the literature. The bioassay of the refolded protein gave a specific activity of ∼3 × 10 9 IU/mg protein.