Engineered promoters are key components that allow engineered expression of genes in the cell-factory design. 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. Engineered promoter variants (EPVs) of naturally occurring promoters (NOPs) can be designed using metabolic engineering strategies and synthetic biology tools if the genes encoding the activating transcription factors (TFs) exist in the genome and are expressed and synthesized at non-limiting concentrations within the cell.
View Article and Find Full Text PDFHybrid-architectured promoter design to deregulate expression in yeast under modulating power of carbon sources involves replacing native cis-acting DNA sequence(s) with de novo synthetic tools in coordination with master regulator transcription factor (TF) to alter crosstalk between signaling pathways, and consequently, transcriptionally rewire the expression. Hybrid-promoter architectures can be designed to mimic native promoter architectures in yeast's preferred carbon source utilization pathway. The method aims to generate engineered promoter variants (EPVs) that combine the advantages of being an exceptionally stronger EPV(s) than the naturally occurring promoters and permit "green-and-clean" production on a non-toxic carbon source.
View Article and Find Full Text PDFDouble-promoter expression system (DPES) design as de novo metabolic engineering strategy enables fine-tuned and enhanced gene expression. We constructed a collection of monodirectional hybrid-architectured DPESs with engineered promoter variants P and P and with the naturally occurring promoter P to enhance and upregulate-deregulated gene expressions in Pichia pastoris in methanol-free media. Reporter red fluorescent protein (mApple) and enhanced green fluorescent protein (eGFP) were expressed under P and P or P, respectively, enabling the determination of the transcription period and strength of each constituent in the DPESs.
View Article and Find Full Text PDFAppl Microbiol Biotechnol
September 2020
System-wide interactions in living cells and discovery of the diverse roles of transcriptional regulatory proteins that are mediator proteins with catalytic domains and regulatory subunits and transcription factors in the cellular pathways have become crucial for understanding the cellular response to environmental conditions. This review provides information for future metabolic engineering strategies through analyses on the highly interconnected regulatory networks in Saccharomyces cerevisiae and Pichia pastoris and identifying their components. We discuss the current knowledge on the carbon catabolite repression (CCR) mechanism, interconnecting regulatory system of the central metabolic pathways that regulate cell metabolism based on nutrient availability in the industrial yeasts.
View Article and Find Full Text PDFEngineered 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 (P ) expression into a non-toxic carbon-source-regulated system.
View Article and Find Full Text PDFAnalysis of RNA structuromes provides new insights into cellular processes, enabling systems biology and biotechnology researchers to calculate promoter and terminator strengths and to directly observe how differing circuit states impact host gene expression and the burdens imposed by the circuits. Such analysis, however, is crucially dependent on the availability of highly pure, intact RNA isolated from fresh or frozen cell cultures. RNA extraction from the yeast Pichia pastoris requires specific pretreatment steps to ensure the reproducibility of downstream applications, but current methods and extraction kits are generally adapted for the conventional yeast Saccharomyces cerevisiae, which has a different cell wall composition.
View Article and Find Full Text PDFThe aim of this work is to increase recombinant protein expression in Pichia pastoris over the ethanol utilization pathway under novel-engineered promoter variants (NEPVs) of alcohol dehydrogenase 2 promoter (P ) through the generation of novel regulatory circuits. The NEPVs were designed by engineering of transcription factor binding sites (TFBSs) determined by in silico analyses and manual curation systematically, by (a) single-handedly replacement of specified TFBSs with synthetic motifs for Mxr1, Cat8, and Aca1 binding, and synthetic TATA-box integration; and, (b) nucleosome optimization. P and P designed by the integration of synthetic Cat8 binding sites were superior, and then P .
View Article and Find Full Text PDFYeast was the first microorganism used by mankind for biotransformation of feedstock that laid the foundations of industrial biotechnology. Long historical use, vast amount of data, and experience paved the way for Saccharomyces cerevisiae as a first yeast cell factory, and still it is an important expression platform as being the production host for several large volume products. Continuing special needs of each targeted product and different requirements of bioprocess operations have led to identification of different yeast expression systems.
View Article and Find Full Text PDFThe Crabtree phenotype defines whether a yeast can perform simultaneous respiration and fermentation under aerobic conditions at high growth rates. It provides Crabtree positive yeasts an evolutionary advantage of consuming glucose faster and producing ethanol to outcompete other microorganisms in sugar rich environments. While a number of genetic events are associated with the emergence of the Crabtree effect, its evolution remains unresolved.
View Article and Find Full Text PDFAppl Microbiol Biotechnol
October 2017
Using double promoter expression systems is a promising approach to increase heterologous protein production. In this review, current double promoter expression systems for the production of recombinant proteins (r-proteins) by industrially important bacteria, Bacillus subtilis and Escherichia coli; and yeasts, Saccharomyces cerevisiae and Pichia pastoris, are discussed by assessing their potentials and drawbacks. Double promoter expression systems need to be designed to maintain a higher specific product formation rate within the production domain.
View Article and Find Full Text PDFThe constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (P ), which is one of the benchmark promoters of Pichia pastoris, was analyzed in terms of putative transcription factor binding sites. We constructed a synthetic library with distinct regulatory properties through deletion and duplication of these putative transcription factor binding sites and selected transcription factor (TF) genes were overexpressed or deleted to understand their roles on heterologous protein production. Using enhanced green fluorescent protein, an expression strength in a range between 0.
View Article and Find Full Text PDFBiotechnol Appl Biochem
November 2017
The aim of this work was to develop an effective fed-batch feeding strategy to enhance recombinant glucose isomerase (r-GI) production by recombinant Escherichia coli BL21 (DE3) pLysS on an industrially relevant feedstock without the application of an exogenous inducer. Following the batch operation (0 < t < 7 H), the effects of pulse and/or continuous feeding of hydrolyzed beet molasses were investigated under five different feeding strategies. The two most promising strategies with respect to r-GI activity were (i) PM-0.
View Article and Find Full Text PDFFor extracellular recombinant protein production, the efficiency of five endogenous secretion signal peptides (SPs) of Pichia pastoris, SP13 (MLSTILNIFILLLFIQASLQ), SP23 (MKILSALLLLFTLAFA), SP24 (MKVSTTKFLAVFLLVRLVCA), SP26 (MWSLFISGLLIFYPLVLG), SP34 (MRPVLSLLLLLASSVLA), selected based on their D-score which quantifies the signal peptide-ness of a given sequence segment, was investigated using recombinant human growth hormone (rhGH) as the model protein. The expression was conducted under glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP). The highest secretion efficiency among endogenous SPs was obtained by SP23 followed by SP24, SP34, SP13 and SP26, respectively.
View Article and Find Full Text PDFEffects of oxygen transfer on recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter were investigated. Recombinant glucose isomerase was chosen as the model protein. Two groups of oxygen transfer strategies were applied, one of which was based on constant oxygen transfer rate where aeration rate was Q O/V = 3 and 10 vvm, and agitation rate was N = 900 min(-1); while the other one was based on constant dissolved oxygen concentrations, C DO = 5, 10, 15, 20 and 40 % in the fermentation broth, by using predetermined exponential glucose feeding with μ o = 0.
View Article and Find Full Text PDFBioprocess Biosyst Eng
January 2016
In this review article, extremophilic lignocellulosic enzymes with special interest on xylanases, β-mannanases, laccases and finally cellulases, namely, endoglucanases, exoglucanases and β-glucosidases produced by Pichia pastoris are reviewed for the first time. Recombinant lignocellulosic extremozymes are discussed from the perspectives of their potential application areas; characteristics of recombinant and native enzymes; the effects of P. pastoris expression system on recombinant extremozymes; and their expression levels and applied strategies to increase the enzyme expression yield.
View Article and Find Full Text PDFBioprocess Biosyst Eng
October 2015
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.
View Article and Find Full Text PDFThe objectives of this work are the optimization of the codons of xylA gene from Thermus thermophilus to enhance the production of recombinant glucose isomerase (rGI) in P. pastoris and to investigate the effects of feeding strategies on rGI production. Codons of xylA gene from T.
View Article and Find Full Text PDFIn-silico identified novel secretory signal peptides (SPs) are required in vivo to achieve efficient transfer or to prevent other cellular proteins from interfering with the process in extracellular recombinant protein (r-protein) production. 56 endogenous and exogenous secretory SPs, have been used or having the potential to be used in Pichia pastoris for r-protein secretion, were analyzed in-silico using the softwares namely SignalP4.1, Phobius, WolfPsort0.
View Article and Find Full Text PDFThe influence of methanol feeding rate on intracellular reaction network of recombinant human growth hormone (rhGH) producing Pichia pastoris was investigated at three different specific growth rates, namely, 0.02 (MS-0.02), 0.
View Article and Find Full Text PDFIn this study, a new method combining magnetic separation (MS) and surface-enhanced Raman scattering (SERS) was developed to detect genetically modified organisms (GMOs). An oligonucleotide probe which is specific for 35 S DNA target was immobilized onto gold coated magnetic nanospheres to form oligonucleotide-coated nanoparticles. A self assembled monolayer was formed on gold nanorods using 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) and the second probe of the 35 S DNA target was immobilized on the activated nanorod surfaces.
View Article and Find Full Text PDFYeasts 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.
View Article and Find Full Text PDFThe 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.
View Article and Find Full Text PDFThis 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.
View Article and Find Full Text PDFBatch-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 (C(X) = 70 g CDW/l); (b) achieving 1.8-fold higher recombinant human erythropoietin (rHuEPO) (at t = 18 h); (c) reducing specific protease production 1.
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