Engineered Deregulation of Expression in Yeast with Designed Hybrid-Promoter Architectures in Coordination with Discovered Master Regulator Transcription Factor.

Engineered promoters are key components in the cell-factory design, allowing precise and enhanced expression of genes. Promoters having exceptional strength are attractive candidates for designing metabolic engineering strategies for tailoring de novo production strategies that require directed evolution methods by engineering with de novo synthetic biology tools. Here, the custom-designed AOX1 hybrid-promoter architectures in coordination with targeted transcription factors are shown, transcriptionally rewired the expression over methanol-free substrate-utilization pathway(s) and converted methanol-dependent Pichia pastoris alcohol oxidase 1(AOX1) promoter (PAOX1 ) expression into a non-toxic carbon-source-regulated system. AOX1 promoter variants are engineered by replacing specified cis-regulatory DNA elements with synthetic Adr1, Cat8, and Aca2 cis-acting DNA elements for Mxr1, Cat8, and Aca1 binding, respectively. Applications of the engineered-promoters are validated for eGFP expression and extracellular human serum albumin production. The hybrid-promoter architecture designed with single Cat8 cis-acting DNA element deregulates the expression on ethanol. Compared with PAOX1 on methanol, the expression on ethanol is increased with i) PAOX1/Cat8-L3 (designed with single Cat8 cis-acting element) to 74%, ii) PAOX1/Adr1-L3/Cat8-L3 (designed with single- Cat8 and Adr1 cis-acting elements) to 85%, and for further consolidation of deregulated expression iii) PeAOX1 (designed with triplet- Cat8 and Adr1 cis-acting elements) 1.30-fold, at t = 20 h of batch cultivations.

Feeding strategy design for recombinant human growth hormone production by Bacillus subtilis.

Defined and semi-defined medium-based feeding strategies were developed to enhance recombinant human growth hormone (rhGH) production by Bacillus subtilis BGSC-1A178 (scoC (-)) strain carrying pMK4::pre(subC)::hGH. Defined medium-based feeding strategies were designed by exponential feeding of glucose and (NH4)2HPO4 at two pre-determined specific growth rates, µ 0 = 0.10 and 0.17 h(-1). Semi-defined medium-based feeding strategies were designed by exponential feeding of substrate solution consisting of glucose, (NH4)2HPO4, peptone, and trace salt solution (PTM1) at three pre-determined specific growth rates, µ 0 = 0.10, 0.17, and 0.25 h(-1). At all the strategies applied, transition cultivation time from batch to fed-batch operation was t T = 4 h. The highest rhGH concentration was obtained as C rhGH = 0.5 g L(-1) with semi-defined medium-based feeding strategy designed with µ 0 = 0.25 h(-1) using feed substrate stock solution containing 200 g L(-1) glucose, 117 g L(-1) (NH4)2HPO4, 100 g L(-1) peptone, and 5 mL L(-1) PTM1 at t = 22 h when the cell concentration reached to C X = 8.29 g L(-1). The overall product and cell yields on glucose were obtained as [Formula: see text] = 7.21 mg g(-1) and [Formula: see text] = 0.12 g g(-1), respectively. The results indicate the requirement of designing continuous feed stream in fed-batch production to enhance rhGH production by r-B. subtilis.

Human growth hormone-specific aptamer identification using improved oligonucleotide ligand evolution method.

LETEG is a method developed and used for the separation and purification of proteins employing a single-step ligand (aptamers) evolution in which aptamers are eluted with an increasing temperature gradient. Using recombinant human growth hormone (rhGH) as the test purification target, and after avoiding cross reactions of aptamers with Bacillus subtilis extracellular proteins by negative SELEX, the effects of time and pH on aptamer binding to rhGH were investigated. The highest binding efficiency of aptamers on rhGH-immobilized microparticles was obtained at pH 7.0. The aptamers that interacted with rhGH were eluted by a multi-stage step-up temperature gradient in DeltaT=10 degrees C increments within the range T=55-95 degrees C; and the strongest affinity binding was disrupted at T=85 degrees C where C(Apt)=0.16muM was eluted. The equilibrium binding data obtained was described by a Langmuir-type isotherm; where the affinity constant was K(D)=218nM rhGH. RhGH was separated from the fermentation broth with 99.8% purity, indicating that the method developed is properly applicable even for an anionic protein.

Microarray Studies in Bacillus subtilis.

This review focuses on the construction of a global, comprehensive understanding of Bacillus subtilis through microarray studies. The microarray studies in B. subtilis were analysed based on the theme of the work, by mentioning the growth media, bioreactor operation conditions, RNA isolation method, number of data points analysed in exponential or stationary phases, compared genotypes, induction and repression ratios, investigated gene(s) and their positive and/or negative influences. Based on the theme and scope of the studies, the articles were reviewed under seven thematic sections, i.e., effects of gene deletion(s) or overexpression, effects of overexression of heterologous genes, comparison of global gene expression between aerobic and anaerobic respiration, effects of temperature change, effects of transported molecules, effects of limitations and stress conditions, and other microarray studies in B. subtilis.

Biotechnology in Turkey: an overview.

The term biotechnology first appeared in the programs of the Scientific and Technological Research Council of Turkey (TUBITAK) in 1982. The State Planning Organization (SPO) in 1988 defined biotechnology and the scientific fields. Moreover, it put forward an institutional framework and suggested priority areas for research and development. Turkey has been researching and investing in biotechnology for almost four decades. This review covers the development of science and technology policy with its history, consensus and consequences, bio-industries in Turkey, and research activities in biotechnology at Turkish Universities. Details are provided by the research groups in response to a common request for information on their activities and major publications in the field. The information provided has been grouped under thematic topics within the broad theme of biotechnology, and summarized within these topics. Although many aspects of biotechnological research are being pursued in Turkey, it appears that the most common research activities of the field are in fermentation processes, environmental biotechnology, and biomedical engineering.

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 C(rhGH) = 70 mg L(-1) with a product yield on substrate Y(rhGH/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.

The influence of carbon sources on recombinant-human- growth-hormone production by Pichia pastoris is dependent on phenotype: a comparison of Muts and Mut+ strains.

The influence of carbon sources on rhGH (recombinant human growth hormone) production by two Pichia pastoris strains having different methanol utilization phenotypes (P. pastoris-hGH-Mut(+) and P. pastoris-hGH-Mut(s)) was investigated using batch bioreactors. The effect of methanol concentration (C(MeOH)) in defined and complex media, and further glycerol/methanol mixed defined media, was analysed systematically over a wide range. With methanol as the sole carbon source, strain Mut(s) grew only slightly, whereas with Mut(+), a cell concentration (C(X)) of 6.0 g of dry cells/dm(3) was obtained and an rhGH concentration (C(rhGH)) of 0.032 g/dm(3) was produced. In complex medium without glycerol at a C(MeOH) of 2% (v/v), a C(rhGH) of 0.16 g of rhGH/dm(3) was produced by Mut(s), a value 3-fold higher than that produced by Mut(+), despite the fact that the C(X) of Mut(+) (6.1 g/dm(3)) was 2-fold higher than that of Mut(s) (3.0 g/dm(3)). In a glycerol/methanol mixed defined medium, methanol consumption began when glycerol was totally depleted, indicating that glycerol is a repressor of the AOX1 (alcohol oxidase-1 gene) promoter. With strain Mut(s) at a glycerol concentration (C(Gly)) of 30 g/dm(3) and a C(MeOH) of 1% (v/v), the C(rhGH) produced was 0.11 g/dm(3), whereas, with the Mut(+) strain, a C(rhGH) of 0.06 g/dm(3) was obtained at a C(Gly) of 30 g/dm(3) and a C(MeOH) of 4%. As methanol is not consumed by Mut(s) strain effectively and the presence of methanol in the fermentation broth triggers induction of the AOX1 promoter, our results encourage the use of the Mut(s) strain for rhGH production. In addition to rhGH production, the specific cell growth rates, specific methanol and/or glycerol utilization rates and maintenance coefficients in methanol- and glycerol-based defined media were determined. With a methanol-based defined medium and using the Mut(+) strain, a higher specific growth rate (mu) of approx. 0.14 h(-1) was observed during the exponential cell growth phase at a C(MeOH) of

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 pPICZalphaA 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.

Effect of ionic environments on the adsorption and diffusion characteristics of serine alkaline protease enzyme in polyethersulfone ultrafiltration membranes.

Static adsorption of serine alkaline protease (SAP) enzyme on hydrophobic polyether sulfone (PES) ultrafiltration membranes in different ionic environments was investigated. The amount of SAP adsorbed on membranes was the lowest at its isoelectric point (IEP) where the maximum adsorption was obtained below the IEP of the enzyme. The extent of SAP adsorption in the phosphate buffer solutions including different salts followed the order: (NH4)2HPO4 > KH2PO4 > Na2HPO4-NaH2PO4 (buffer) > CaCl2 > ((NH4)2HPO4 + H2PO4 + CaCl2), which was consistent with the Hofmeister series. The zeta potentials of membranes contacted with the ionic species were calculated by streaming potential measurements and found that the increase in ionic strength decreased the electrical double layer thickness leading to a decrease in adsorption. A model based on mass balance was developed to calculate the diffusion coefficient of SAP in PES membranes. Employing experimental data evaluated in a diffusion cell along with the data of adsorption isotherms, diffusion coefficients of SAP in PES membranes in the presence of different ionic species were calculated. To detect the structural changes occurred, membrane surfaces were analysed by Fourier transform infrared-attenuated total reflectance (FTIR-ATR) measurements.

Regulatory effects of alanine-group amino acids on serine alkaline protease production by recombinant Bacillus licheniformis.

Influences of the concentration and addition time of alanine-group amino acids, i.e. alanine, leucine and valine, on serine alkaline protease (SAP) synthesis were investigated by Bacillus licheniformis (DSM 1969) carrying pHV1431:: sub C in a defined medium to identify the amino acids creating intracellular reaction-rate limitation in SAP production. While the precursors of alanine-group amino acids, pyruvate and alanine, did not affect SAP production considerably within the range 0-15 mM, the addition of leucine decreased both SAP production and cell formation, because of the inhibition of valine synthesis. Although valine inhibits reactions starting with pyruvate towards 2-oxo-isovalerate, due to conversion of valine into 2-oxo-isovalerate and from 2-oxo-isovalerate to leucine, valine did not inhibit leucine synthesis. Val (7.5 mM) supply at t =0 h increased SAP activity to an activity of 1070 units.cm(-3) which was 1.3-fold higher than that of the reference production medium. The highest cell growth yield on substrate (Y (X/S)) was obtained as 0.24 g.g(-1) with the supply of alanine; and the highest product formation yield on substrate was obtained as 0.134 units.g(-1) with the supply of valine. By using the results obtained, strategies for increasing SAP production and complex medium design were also discussed.