Eda Çelik

Production of recombinant proteins by yeast cells

Yeasts are widely used in production of recombinant proteins of medical or industrial interest. For each individual product, the most suitable expression system has to be identified and optimized, both on the genetic and fermentative level, by taking into account the properties of the product, the organism and the expression cassette. There is a wide range of important yeast expression hosts including the species Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Schizosaccharomyces pombe, Yarrowia lipolytica and Arxula adeninivorans, with various characteristics such as being thermo-tolerant or halo-tolerant, rapidly reaching high cell densities or utilizing unusual carbon sources. Several strains were also engineered to have further advantages, such as humanized glycosylation pathways or lack of proteases. Additionally, with a large variety of vectors, promoters and selection markers to choose from, combined with the accumulated knowledge on industrial-scale fermentation techniques and the current advances in the post-genomic technology, it is possible to design more cost-effective expression systems in order to meet the increasing demand for recombinant proteins and glycoproteins. In this review, the present status of the main and most promising yeast expression systems is discussed.

Fed‐batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co‐substrate sorbitol

Batch‐wise sorbitol addition as a co‐substrate at the induction phase of methanol fed‐batch fermentation by Pichia pastoris (Mut+) was proposed as a beneficial recombinant protein production strategy and the metabolic responses to methanol feeding rate in the presence of sorbitol was systematically investigated. Adding sorbitol batch‐wise to the medium provided the following advantages over growth on methanol alone: (a) eliminating the long lag‐phase for the cells and reaching ‘high cell density production’ at t = 24 h of the process (CX = 70 g CDW/l); (b) achieving 1.8‐fold higher recombinant human erythropoietin (rHuEPO) (at t = 18 h); (c) reducing specific protease production 1.2‐fold; (d) eliminating the lactic acid build‐up period; (e) lowering the oxygen uptake rate two‐fold; and (f) obtaining 1.4‐fold higher overall yield coefficients. The maximum specific alcohol oxidase activity was not affected in the presence of sorbitol, and it was observed that sorbitol and methanol were utilized simultaneously. Thus, in the presence of sorbitol, 130 mg/l rHuEPO was produced at t = 24 h, compared to 80 mg/l rHuEPO (t = 24 h) on methanol alone. This work demonstrates not only the ease and efficiency of incorporating sorbitol to fermentations by Mut+ strains of P. pastoris for the production of any bio‐product, but also provides new insights into the metabolism of the methylotrophic yeast P. pastoris. Copyright

Production of Secretory and Extracellular N-Linked Glycoproteins in Escherichia coli†

ABSTRACT The Campylobacter jejuni pgl gene cluster encodes a complete N-linked protein glycosylation pathway that can be functionally transferred into Escherichia coli. In this system, we analyzed the interplay between N-linked glycosylation, membrane translocation and folding of acceptor proteins in bacteria. We developed a recombinant N-glycan acceptor peptide tag that permits N-linked glycosylation of diverse recombinant proteins expressed in the periplasm of glycosylation-competent E. coli cells. With this “glycosylation tag,” a clear difference was observed in the glycosylation patterns found on periplasmic proteins depending on their mode of inner membrane translocation (i.e., Sec, signal recognition particle [SRP], or twin-arginine translocation [Tat] export), indicating that the mode of protein export can influence N-glycosylation efficiency. We also established that engineered substrate proteins targeted to environments beyond the periplasm, such as the outer membrane, the membrane vesicles, and the extracellular medium, could serve as substrates for N-linked glycosylation. Taken together, our results demonstrate that the C. jejuni N-glycosylation machinery is compatible with distinct secretory mechanisms in E. coli, effectively expanding the N-linked glycome of recombinant E. coli. Moreover, this simple glycosylation tag strategy expands the glycoengineering toolbox and opens the door to bacterial synthesis of a wide array of recombinant glycoprotein conjugates.

Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: Effects of methanol feeding rate

The intracellular metabolic fluxes through the central carbon pathways in the bioprocess for recombinant human erythropoietin (rHuEPO) production by Pichia pastoris (Mut+) were calculated to investigate the metabolic effects of dual carbon sources (methanol/sorbitol) and the methanol feed rate, and to obtain a deeper understanding of the regulatory circuitry of P. pastoris, using the established stoichiometry‐based model containing 102 metabolites and 141 reaction fluxes. Four fed‐batch operations with (MS‐) and without (M‐) sorbitol were performed at three different constant specific growth rates (h−1), and denoted as M‐0.03, MS‐0.02, MS‐0.03, and MS‐0.04. Considering the methanol consumption pathway, the M‐0.03 and MS‐0.02 conditions produced similar effects and had >85% of formaldehyde flux towards the assimilatory pathway. In contrast, the use of the dual carbon source condition generated a shift in metabolism towards the dissimilatory pathway that corresponded to the shift in dilution rate from MS‐0.03 to MS‐0.04, indicating that the methanol feed exceeded the metabolic requirements at the higher µ0. Comparing M‐0.03 and MS‐0.03 conditions, which had the same methanol feeding rates, sorbitol addition increased the rHuEPO synthetic flux 4.4‐fold. The glycolysis, gluconeogenesis, and PPP pathways worked uninterruptedly only at MS‐0.02 condition. PPP and TCA cycles worked with the highest disturbances at MS‐0.04 condition, which shows the stress of increased feeding rates of methanol on cell metabolism. Biotechnol. Bioeng. 2010; 105: 317–329.

Expanding the glycoengineering toolbox: the rise of bacterial N-linked protein glycosylation

Glycosylation is the most prevalent post-translational modification found on proteins, occurring in all domains of life. Ever since the discovery of asparagine-linked (N-linked) protein glycosylation pathways in bacteria, major efforts have been made to harness these systems for the creation of novel therapeutics, vaccines, and diagnostics. Recent advances such as the ability to produce designer glycans in bacteria, some containing unnatural sugars, and techniques for evolving glycosylation enzymes have spawned an entirely new discipline known as bacterial glycoengineering. In addition to their biotechnological and therapeutic potential, bacteria equipped with recombinant N-linked glycosylation pathways are improving our understanding of the N-glycosylation process. This review discusses the key role played by microorganisms in glycosciences, particularly in the context of N-linked glycosylation.

Bioprocess parameters and oxygen transfer characteristics in β-lactamase production by Bacillus species

After screening potential β‐lactamase producers in a medium containing penicillin G, an inducible ( Bacillus subtilis NRS 1125) and a constitutive ( Bacillus licheniformis 749/C ATCC 25972) β‐lactamase producer were selected. As the highest enzyme activity was obtained with B. licheniformis 749/C, the effects of the concentration of carbon sources, i.e., glucose, fructose, sucrose, citric acid, and glycerol, and nitrogen sources, i.e., (NH4)2HPO4, NH4Cl, yeast extract, casamino acids and peptone, pH, and temperature on β‐lactamase production were investigated with B. licheniformis 749/C in laboratory scale bioreactors. Among the investigated media, the highest volumetric activity was obtained as 270 U cm−3 in the medium containing 10.0 kg m−3 glucose, 1.18 kg m−3 (NH4)2HPO4, 8.0 kg m−3 yeast extract, and the salt solution at 32 °C and pH0 = 6.0. By using the designed medium, fermentation and oxygen transfer characteristics of the bioprocess were investigated at V = 3.0 dm3 bioreactor systems with a VR = 1.65 dm3 working volume at QO/ VR = 0.5 vvm and N = 500 min‐1. At the beginning of the process the Damköhler number was <1, indicating that the process was at biochemical reaction limited condition; at t = 2–5 h both mass‐transfer and biochemical reaction resistances were effective; and at t = 6–10 h ( Da ≫1) the bioprocess was at mass transfer limited condition. Overall oxygen transfer coefficients ( KLa) varied between 0.01 and 0.03 s−1, enhancement factor ( KLa/ KLaO) varied between 1.2 and 2.3, and volumetric oxygen uptake rate varied between 0.001 and 0.003 mol m−3 s−1 throughout the bioprocess. The specific oxygen uptake and the specific substrate consumption rates were the highest at t = 2 h and then decreased with the cultivation. The maximum yield of cells on substrate and the maximum yield of cells on oxygen values were obtained, respectively, as YX/S = 0.34 and YX/O = 1.40, at t = 5 h, whereas the highest yield of substrate on oxygen was obtained as YS/O = 6.94 at t = 3.5 h. The rate of oxygen consumption for maintenance and the rate of substrate consumption for maintenance values were found, respectively, as mO = 0.13 kg kg−1 h−1 and mS = 3.02 kg kg−1 h−1.

Expression System for Synthesis and Purification of Recombinant Human Growth Hormone in Pichia pastoris and Structural Analysis by MALDI-ToF Mass Spectrometry

An expression system in Pichia pastoris for the production and purification of recombinant human growth hormone (rHGH) was designed and implemented. hGH cDNA sequence was cloned into pPICZαA vector under the control of AOX1 promoter, which included a polyhistidine‐tag on the amino terminal end to enable affinity purification and a target site for Factor Xa protease such that protease cleavage in vitro would produce rhGH without any non‐native N‐and C‐termini. Analyses of the affinity‐purified rhGH product by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) showed a spectral peak at m/ z 23699. Purified product digested with Factor Xa protease had a molecular mass of 22132 kDa. The molecular mass difference before and after Factor Xa protease digestion expectedly corresponds to the 12 amino acids in the rhGH amino terminus, which includes the EcoRI digestion site (Glu‐Phe), the 6xHis tag for affinity purification, and the Factor Xa protease recognition sequence (Ile‐Glu‐Gly‐Arg), a result that also indicates that the signal peptide was properly processed by P. pastoris. N‐Terminal sequence analysis of the Factor Xa protease trimmed recombinant product confirmed the mature hGH sequence. Thus, the system designed functioned with its intended purpose effectively in expression, cleavage, and purification of the recombinant product.

Expression system for recombinant human growth hormone production from Bacillus subtilis

We demonstrate for the first time, an expression system mimicking serine alkaline protease synthesis and secretion, producing native form of human growth hormone (hGH) from Bacillus subtilis. A hybrid‐gene of two DNA fragments, i.e., signal (pre‐) DNA sequence of B. licheniformis serine alkaline protease gene (subC) and cDNA encoding hGH, were cloned into pMK4 and expressed under deg‐promoter in B. subtilis. Recombinant‐hGH (rhGH) produced by B. subtilis carrying pMK4::pre(subC)::hGH was secreted. N‐terminal sequence and mass spectrometry analyses of rhGH confirm the mature hGH sequence, and indicate that the signal peptide was properly processed by B. subtilis signal‐peptidase. The highest rhGH concentration was obtained at t = 32 h as CrhGH = 70 mg L−1 with a product yield on substrate YrhGH/S = 9 g kg−1, in a glucose based defined medium. Fermentation characteristics and influence of hGH gene on the rhGH production were investigated by comparing B. subtilis carrying pMK4::pre(subC)::hGH with that of carrying merely pMK4. Excreted organic‐acid concentrations were higher by B. subtilis carrying pMK4::pre(subC)::hGH, whereas excreted amino‐acid concentrations were higher by B. subtilis carrying pMK4. The approach developed is expected to be applicable to the design of expression systems for heterologous protein production from Bacillus species.

A filamentous phage display system for N-linked glycoproteins

We have developed a filamentous phage display system for the detection of asparagine‐linked glycoproteins in Escherichia coli that carry a plasmid encoding the protein glycosylation locus (pgl) from Campylobacter jejuni. In our assay, fusion of target glycoproteins to the minor phage coat protein g3p results in the display of glycans on phage. The glyco‐epitope displayed on phage is the product of biosynthetic enzymes encoded by the C. jejuni pgl pathway and minimally requires three essential factors: a pathway for oligosaccharide biosynthesis, a functional oligosaccharyltransferase, and an acceptor protein with a D/E‐X1‐N‐X2‐S/T motif. Glycosylated phages could be recovered by lectin chromatography with enrichment factors as high as 2 × 105 per round of panning and these enriched phages retained their infectivity after panning. Using this assay, we show that desired glyco‐phenotypes can be reliably selected by panning phage‐displayed glycoprotein libraries on lectins that are specific for the glycan. For instance, we used our phage selection to identify permissible residues in the −2 position of the bacterial consensus acceptor site sequence. Taken together, our results demonstrate that a genotype–phenotype link can be established between the phage‐associated glyco‐epitope and the phagemid‐encoded genes for any of the three essential components of the glycosylation process. Thus, we anticipate that our phage display system can be used to isolate interesting variants in any step of the glycosylation process, thereby making it an invaluable tool for genetic analysis of protein glycosylation and for glycoengineering in E. coli cells.

Glycoarrays with engineered phages displaying structurally diverse oligosaccharides enable high-throughput detection of glycan-protein interactions

Glycan microarrays have become a powerful platform to investigate the interactions of carbohydrates with a variety of biomolecules. However, the number and diversity of glycans available for use in such arrays represent a key bottleneck in glycan array fabrication. To address this challenge, we describe a novel glycan array platform based on surface patterning of engineered glycophages that display unique carbohydrate epitopes. Specifically, we show that glycophages are compatible with surface immobilization procedures and that phage‐displayed oligosaccharides retain the ability to be recognized by different glycan‐binding proteins (e.g. antibodies and lectins) after immobilization. A key advantage of glycophage arrays is that large quantities of glycophages can be produced biosynthetically from recombinant bacteria and isolated directly from bacterial supernatants without laborious purification steps. Taken together, the glycophage array technology described here should help to expand the diversity of glycan libraries and provide a complement to the existing toolkit for high‐throughput analysis of glycan–protein interactions.