Genome-scale metabolic reconstruction and in silico analysis of methylotrophic yeast Pichia pastoris for strain improvement

<p>Abstract</p> <p>Background</p> <p><it>Pichia pastoris </it>has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive <it>in silico </it>model of <it>P. pastoris </it>can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.</p> <p>Results</p> <p>A fully compartmentalized metabolic model of <it>P. pastoris </it>(<it>iPP</it>668), composed of 1,361 reactions and 1,177 metabolites, was reconstructed based on its genome annotation and biochemical information. The constraints-based flux analysis was then used to predict achievable growth rate which is consistent with the cellular phenotype of <it>P. pastoris </it>observed during chemostat experiments. Subsequent <it>in silico </it>analysis further explored the effect of various carbon sources on cell growth, revealing sorbitol as a promising candidate for culturing recombinant <it>P. pastoris </it>strains producing heterologous proteins. Interestingly, methanol consumption yields a high regeneration rate of reducing equivalents which is substantial for the synthesis of valuable pharmaceutical precursors. Hence, as a case study, we examined the applicability of <it>P. pastoris </it>system to whole-cell biotransformation and also identified relevant metabolic engineering targets that have been experimentally verified.</p> <p>Conclusion</p> <p>The genome-scale metabolic model characterizes the cellular physiology of <it>P. pastoris</it>, thus allowing us to gain valuable insights into the metabolism of methylotrophic yeast and devise possible strategies for strain improvement through <it>in silico </it>simulations. This computational approach, combined with synthetic biology techniques, potentially forms a basis for rational analysis and design of <it>P. pastoris </it>metabolic network to enhance humanized glycoprotein production.</p

Expression, Immobilization and Enzymatic Properties of Glutamate Decarboxylase Fused to a Cellulose-Binding Domain

Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamic acid to gamma-aminobutyric acid (GABA), was fused to the cellulose-binding domain (CBD) and a linker of Trichoderma harzianum endoglucanase II. To prevent proteolysis of the fusion protein, the native linker was replaced with a S3N10 peptide known to be completely resistant to E. coli endopeptidase. The CBD-GAD expressed in E. coli was successfully immobilized on Avicel, a crystalline cellulose, with binding capacity of 33 ± 2 nmolCBD-GAD/gAvicel and the immobilized enzymes retained 60% of their initial activities after 10 uses. The results of this report provide a feasible alternative to produce GABA using immobilized GAD through fusion to CBD

Expression and purification of soluble and active human enterokinase light chain in Escherichia coli

Human enterokinase light chain (hEKL) specifically cleaves the sequence (Asp)4-Lys↓X (D4K), making this a frequently used enzyme for site-specific cleavage of recombinant fusion proteins. However, hEKL production from Escherichia coli is limited due to intramolecular disulphide bonds. Here, we present strategies to obtain soluble and active hEKL from E. coli by expressing the hEKL variant C112S fused with maltose-binding protein (MBP) through D4K and molecular chaperons including GroEL/ES. The fusion protein self-cleaved in vivo, thereby removing the MBP in the E. coli cells. Thus, the self-cleaved hEKL variant was released into the culture medium. One-step purification using HisTrap™ chromatography purified the hEKL variant exhibiting an enzymatic activity of 3.1 × 103 U/mL (9.934 × 105 U/mg). The approaches presented here greatly simplify the purification of hEKL from E. coli without requiring refolding processes

Complete genome sequence of the sulfur-oxidizing chemolithoautotrophic Sulfurovum lithotrophicum 42BKTT

Abstract A sulfur-oxidizing chemolithoautotrophic bacterium, Sulfurovum lithotrophicum 42BKTT, isolated from hydrothermal sediments in Okinawa, Japan, has been used industrially for CO2 bio-mitigation owing to its ability to convert CO2 into C5H8NO4 − at a high rate of specific mitigation (0.42 g CO2/cell/h). The genome of S. lithotrophicum 42BKTT comprised of a single chromosome of 2217,891 bp with 2217 genes, including 2146 protein-coding genes and 54 RNA genes. Here, we present its complete genome-sequence information, including information about the genes encoding enzymes involved in CO2 fixation and sulfur oxidation

Development of a promising microbial platform for the production of dicarboxylic acids from biorenewable resources

Abstract Background As a sustainable industrial process, the production of dicarboxylic acids (DCAs), used as precursors of polyamides, polyesters, perfumes, plasticizers, lubricants, and adhesives, from vegetable oil has continuously garnered interest. Although the yeast Candida tropicalis has been used as a host for DCA production, additional strains are continually investigated to meet productivity thresholds and industrial needs. In this regard, the yeast Wickerhamiella sorbophila, a potential candidate strain, has been screened. However, the lack of genetic and physiological information for this uncommon strain is an obstacle that merits further research. To overcome this limitation, we attempted to develop a method to facilitate genetic recombination in this strain and produce high amounts of DCAs from methyl laurate using engineered W. sorbophila. Results In the current study, we first developed efficient genetic engineering tools for the industrial application of W. sorbophila. To increase homologous recombination (HR) efficiency during transformation, the cell cycle of the yeast was synchronized to the S/G2 phase using hydroxyurea. The HR efficiency at POX1 and POX2 loci increased from 56.3% and 41.7%, respectively, to 97.9% in both cases. The original HR efficiency at URA3 and ADE2 loci was nearly 0% during the early stationary and logarithmic phases of growth, and increased to 4.8% and 25.6%, respectively. We used the developed tools to construct W. sorbophila UHP4, in which β-oxidation was completely blocked. The strain produced 92.5 g/l of dodecanedioic acid (DDDA) from methyl laurate over 126 h in 5-l fed-batch fermentation, with a productivity of 0.83 g/l/h. Conclusions Wickerhamiella sorbophila UHP4 produced more DDDA methyl laurate than C. tropicalis. Hence, we demonstrated that W. sorbophila is a powerful microbial platform for vegetable oil-based DCA production. In addition, by using the developed genetic engineering tools, this emerging yeast could be used for the production of a variety of fatty acid derivatives, such as fatty alcohols, fatty aldehydes, and ω-hydroxy fatty acids

Gamma-aminobutyric acid production using immobilized glutamate decarboxylase followed by downstream processing with cation exchange chromatography

Abstract: We have developed a gamma-aminobutyric acid (GABA) production technique using his-tag mediated immobilization of Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamate to GABA. The GAD was obtained at 1.43 g/L from GAD-overexpressed E. coli fermentation and consisted of 59.7 % monomer, 29.2 % dimer and 2.3 % tetramer with a 97.6 % soluble form of the total GAD. The harvested GAD was immobilized to metal affinity gel with an immobilization yield of 92%. Based on an investigation of specific enzyme activity and reaction characteristics, glutamic acid (GA) was chosen over monosodium glutamate (MSG) as a substrate for immobilized GAD, resulting in conversion of 2.17 M GABA in a 1 L reactor within 100 min. The immobilized enzymes retained 58.1 % of their initial activities after ten consecutive uses. By using cation exchange chromatography followed by enzymatic conversion, GABA was separated from the residual substrate and leached GAD. As a consequence, the glutamic acid was mostly removed with no detectable GAD, while 91.2 % of GABA was yielded in the purification step. Int. J. Mol. Sci. 2013, 14 172

Microcrystalline Cellulose for Delivery of Recombinant Protein-Based Antigen against Erysipelas in Mice

The study describes the development of a vaccine using microcrystalline cellulose (Avicel PH-101) as a delivery carrier of recombinant protein-based antigen against erysipelas. Recombinant SpaA, surface protective protein, from a gram-positive pathogen Erysipelothrix rhusiopathiae was fused to a cellulose-binding domain (CBD) from Trichoderma harzianum endoglucanase II through a S3N10 peptide. The fusion protein (CBD-SpaA) was expressed in Escherichia coli and was subsequently bound to Avicel PH-101. The antigenicity of CBD-SpaA bound to the Avicel was evaluated by enzyme-linked immunosorbent (ELISA) and confocal laser scanning microscope (CLSM) assays. For the examination of its immunogenicity, groups of mice were immunized with different constructs (soluble CBD-SpaA, Avicel coated with CBD-SpaA, whole bacterin of E. rhusiopathiae (positive control), and PBS (negative control)). In two weeks after immunization, mice were challenged with 1x107 CFU of E. rhusiopathiae and Avicel coated with CBD-SpaA induced protective immunity in mice. In conclusion, this study demonstrates the feasibility of microcrystalline cellulose as the delivery system of recombinant protein subunit vaccine against E. rhusiopathiae infection in mice

Effect of decanoic acid and 10-hydroxydecanoic acid on the biotransformation of methyl decanoate to sebacic acid

Abstract Biotransformation of fatty acid methyl esters to dicarboxylic acids has attracted much attention in recent years; however, reports of sebacic acid production using such biotransformation remain few. The toxicity of decanoic acid is the main challenge for this process. Decane induction has been reported to be essential to activate the enzymes involved in the α,ω-oxidation pathway before initiating the biotransformation of methyl decanoate to sebacic acid. However, we observed the accumulation of intermediates (decanoic acid and 10-hydroxydecanoic acid) during the induction period. In this study, we examined the effects of these intermediates on the biotransformation process. The presence of decanoic acid, even at a low concentration (0.2 g/L), inhibited the transformation of 10-hydroxydecanoic acid to sebacic acid. Moreover, about 24–32% reduction in the decanoic acid oxidation was observed in the presence of 0.5–1.5 g/L 10-hydroxydecanoic acid. To eliminate these inhibitory effects, we applied substrate-limiting conditions during the decane induction process, which eliminated the accumulation of decanoic acid. Although the productivity of sebacic acid (34.5 ± 1.10 g/L) was improved, by 28% over that achieved using the previously methods, after 54 h, the accumulation of 10-hydroxydecanoic acid was still detected. The accumulation of 10-hydroxydecanoic acid even under the decane limiting conditions could be an evidence that oxidation of 10-hydroxydecanoic acid could be the rate-limiting step in this process. The improvement of this reaction should be an important objective for further development of the production of sebacic acid using biotransformation