Abstract:
BACKGROUND:The classic AOX1 replacement approach is still one of the most often used techniques for expression of recombinant proteins in the methylotrophic yeast Pichia pastoris. Although this approach is largely successful, it frequently delivers clones with unpredicted production characteristics and a work-intense screening process is required to find the strain with desired productivity. RESULTS:In this project 845 P. pastoris clones, transformed with a GFP expression cassette, were analyzed for their methanol-utilization (Mut)-phenotypes, GFP gene expression levels and gene copy numbers. Several groups of strains with irregular features were identified. Such features include GFP expression that is markedly higher or lower than expected based on gene copy number as well as strains that grew under selective conditions but where the GFP gene cassette and its expression could not be detected. From these classes of strains 31 characteristic clones were selected and their genomes sequenced. By correlating the assembled genome data with the experimental phenotypes novel insights were obtained. These comprise a clear connection between productivity and cassette-to-cassette orientation in the genome, the occurrence of false-positive clones due to a secondary recombination event, and lower total productivity due to the presence of untransformed cells within the isolates were discovered. To cope with some of these problems, the original vector was optimized by replacing the AOX1 terminator, preventing the occurrence of false-positive clones due to the secondary recombination event. CONCLUSIONS:Standard methods for transformation of P. pastoris led to a multitude of unintended and sometimes detrimental integration events, lowering total productivity. By documenting the connections between productivity and integration event we obtained a deeper understanding of the genetics of mutation in P. pastoris. These findings and the derived improved mutagenesis and transformation procedures and tools will help other scientists working on recombinant protein production in P. pastoris and similar non-conventional yeasts.
journal_name
Microb Cell Factjournal_title
Microbial cell factoriesauthors
Schwarzhans JP,Wibberg D,Winkler A,Luttermann T,Kalinowski J,Friehs Kdoi
10.1186/s12934-016-0486-7subject
Has Abstractpub_date
2016-05-20 00:00:00pages
84issn
1475-2859pii
10.1186/s12934-016-0486-7journal_volume
15pub_type
杂志文章abstract:BACKGROUND:Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by lignocellulose-degrading fungi including Pycnoporus cinnabarinus. We investigated the cellulolytic system of P. cinnabarinus, focusing on the involvement of CDH in the deconstruction of lignocellulosic biomass. RESULTS:First, P. ...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/1475-2859-10-113
更新日期:2011-12-28 00:00:00
abstract:BACKGROUND:Numerous studies have shown that stress induction and genetic engineering can effectively increase lipid accumulation, but lead to a decrease of growth in the majority of microalgae. We previously found that elevated CO2 concentration increased lipid productivity as well as growth in Phaeodactylum tricornutu...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-019-1214-x
更新日期:2019-09-23 00:00:00
abstract:BACKGROUND:Intracellular metabolism of glucocorticoid hormones plays an important role in the pathogenesis of metabolic syndrome and regulates, among many physiological processes, collagen metabolism in skin. At the peripheral level the concentration of active glucocorticoids is mainly regulated by the 11β-hydroxystero...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-016-0450-6
更新日期:2016-03-15 00:00:00
abstract:BACKGROUND:β-Ionone is a fragrant terpenoid that generates a pleasant floral scent and is used in diverse applications as a cosmetic and flavoring ingredient. A growing consumer desire for natural products has increased the market demand for natural β-ionone. To date, chemical extraction from plants remains the main ap...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-018-0984-x
更新日期:2018-09-01 00:00:00
abstract:BACKGROUND:Our laboratory has constructed a Bacillus stearothermophilus α-amylase (AmyS) derivative with excellent enzymatic properties. Bacillus subtilis is generally regarded as safe and has excellent protein secretory capability, but heterologous extracellular production level of B. stearothermophilus α-amylase in B...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-019-1119-8
更新日期:2019-04-11 00:00:00
abstract:BACKGROUND:The biocontrol strain Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere synthesizes three antifungal phenazine compounds, 2-Hydroxyphenazine (2-OH-PHZ), 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) and phenazine-1-carboxylic acid (PCA). PCA has been a commercialized antifungal pest...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-016-0529-0
更新日期:2016-07-28 00:00:00
abstract:BACKGROUND:Bioethanol produced by the yeast Saccharomyces cerevisiae is currently one of the most promising alternatives to conventional transport fuels. Lignocellulosic hemicelluloses obtained after hydrothermal pretreatment are important feedstock for bioethanol production. However, hemicellulosic materials cannot be...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-014-0145-9
更新日期:2014-10-12 00:00:00
abstract:BACKGROUND:The six-carbon circular non-proteinogenic compound L-pipecolic acid is an important chiral drug intermediate with many applications in the pharmaceutical industry. In the present study, we developed a metabolically engineered strain of Escherichia coli for the overproduction of L-pipecolic acid from glucose....
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-017-0666-0
更新日期:2017-03-27 00:00:00
abstract:BACKGROUND:To reduce the production cost of bioethanol obtained from fermentation of the sugars provided by degradation of lignocellulosic biomass (i.e., second generation bioethanol), it is necessary to screen for new enzymes endowed with more efficient biomass degrading properties. This demands the set-up of high-thr...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/1475-2859-9-58
更新日期:2010-07-16 00:00:00
abstract:BACKGROUND:Understanding the metabolic mechanism of sterols to produce valuable steroid intermediates in mycobacterium by a noncoding small RNA (sRNA) view is still limited. In the work, RNA-seq was implemented to investigate the noncoding transcriptome of Mycobacterium neoaurum (Mn) in the transformation process of st...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-016-0462-2
更新日期:2016-04-25 00:00:00
abstract:BACKGROUND:Ergot alkaloids are a group of highly bioactive molecules produced by a number of filamentous fungi. These compounds have been intensely studied for decades, mainly due to their deleterious effects in contaminated food and feeds, but also for their beneficial pharmaceutical and agricultural applications. Bio...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-014-0095-2
更新日期:2014-08-12 00:00:00
abstract:BACKGROUND:C4-dicarboxylic acids, including malic acid, fumaric acid and succinic acid, are valuable organic acids that can be produced and secreted by a number of microorganisms. Previous studies on organic acid production by Aspergillus carbonarius, which is capable of producing high amounts of citric acid from varie...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-017-0660-6
更新日期:2017-03-14 00:00:00
abstract:BACKGROUND:Terpenoids constitute a large family of natural products, attracting commercial interest for a variety of uses as flavours, fragrances, drugs and alternative fuels. Saccharomyces cerevisiae offers a versatile cell factory, as the precursors of terpenoid biosynthesis are naturally synthesized by the sterol bi...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/1475-2859-10-4
更新日期:2011-01-28 00:00:00
abstract:BACKGROUND:Histamine is a key mediator of the anti-inflammatory activity conferred by the probiotic organism Lactobacillus reuteri ATCC PTA 6475 in animal models of colitis and colorectal cancer. In L. reuteri, histamine synthesis and secretion requires L-histidine decarboxylase and a L-histidine/histamine exchanger. C...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-019-1264-0
更新日期:2019-12-12 00:00:00
abstract:BACKGROUND:A stereoisomer of inositol, scyllo-inositol (SI), has been regarded as a promising therapeutic agent for Alzheimer's disease. However, this compound is relatively rare, whereas another stereoisomer of inositol, myo-inositol (MI) is abundant in nature. Bacillus subtilis 168 has the ability to metabolize inosi...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-017-0682-0
更新日期:2017-04-21 00:00:00
abstract::Industrial development may result in the increase of environmental risks. The enzymatic transformation of polluting compounds to less toxic or even innocuous products is an alternative to their complete removal. In this regard, a number of different redox enzymes are able to transform a wide variety of toxic pollutant...
journal_title:Microbial cell factories
pub_type: 社论
doi:10.1186/1475-2859-3-10
更新日期:2004-08-02 00:00:00
abstract::Innovative new genome engineering technologies for manipulating chromosomes have appeared in the last decade. One of these technologies, recombination mediated genetic engineering (recombineering) allows for precision DNA engineering of chromosomes and plasmids in Escherichia coli. Single-stranded DNA recombineering (...
journal_title:Microbial cell factories
pub_type: 杂志文章,评审
doi:10.1186/1475-2859-13-S1-S10
更新日期:2014-08-29 00:00:00
abstract:BACKGROUND:A stereoisomer of inositol, scyllo-inositol, is known as a promising therapeutic agent for Alzheimer's disease, since it prevents the accumulation of beta-amyloid deposits, a hallmark of the disease. However, this compound is relatively rare in nature, whereas another stereoisomer of inositol, myo-inositol, ...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/1475-2859-10-69
更新日期:2011-09-07 00:00:00
abstract:BACKGROUND:Sesquiterpenes are a class of natural products with a diverse range of attractive industrial proprieties. Due to economic difficulties of sesquiterpene production via extraction from plants or chemical synthesis there is interest in developing alternative and cost efficient bioprocesses. The hydrocarbon α-sa...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/1475-2859-11-117
更新日期:2012-08-31 00:00:00
abstract:BACKGROUND:Cost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol®), remains limited despite growing global demands. Low yields of the critical taxadiene precursor remains a key bottleneck in microbial production. In this study, the key challenge of poor taxadiene synthase (TASY) solubili...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-020-01458-2
更新日期:2020-11-02 00:00:00
abstract:BACKGROUND:Ganoderic acids (GAs), derived from the medicinal mushroom Ganoderma lucidum, possess anticancer and other important pharmacological activities. To improve production of GAs, a homologous farnesyl diphosphate synthase (FPS) gene was overexpressed in G. lucidum. Moreover, the influence of FPS gene overexpress...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-019-1164-3
更新日期:2019-06-28 00:00:00
abstract:BACKGROUND:Mannosylglycerate (MG) is one of the most widespread compatible solutes among marine microorganisms adapted to hot environments. This ionic solute holds excellent ability to protect proteins against thermal denaturation, hence a large number of biotechnological and clinical applications have been put forward...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-018-1023-7
更新日期:2018-11-16 00:00:00
abstract:BACKGROUND:Glucose is the main carbon source of E. coli and a typical substrate in production processes. The main glucose uptake system is the glucose specific phosphotransferase system (Glc-PTS). The PTS couples glucose uptake with its phosphorylation. This is achieved by the concomitant conversion of phosphoenolpyruv...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-018-0980-1
更新日期:2018-08-28 00:00:00
abstract:BACKGROUND:The solubility of recombinant proteins expressed in bacteria is often disappointingly low. Several strategies have been developed to improve the yield and one of the most common strategies is the fusion of the target protein with a suitable partner. Despite several reports on the successful use of each of th...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/1475-2859-4-34
更新日期:2005-12-13 00:00:00
abstract:BACKGROUND:cis, cis-Muconic acid is an important chemical that can be biosynthesized from simple substrates in engineered microorganisms. Recently, it has been shown that engineering microbial cocultures is an emerging and promising approach for biochemical production. In this study, we aim to explore the potential of ...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-015-0319-0
更新日期:2015-09-15 00:00:00
abstract:BACKGROUND:Oxygen-evolving photoautotrophic organisms, like cyanobacteria, protect their photosynthetic machinery by a number of regulatory mechanisms, including alternative electron transfer pathways. Despite the importance in modulating the electron flux distribution between the photosystems, alternative electron tra...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-019-1238-2
更新日期:2019-11-05 00:00:00
abstract:BACKGROUND:The production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or cultur...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/1475-2859-8-10
更新日期:2009-01-29 00:00:00
abstract:BACKGROUND:The proteome reflects the available cellular machinery to deal with nutrients and environmental challenges. The most common E. coli strain BL21 growing in different, commonly employed media was evaluated using a detailed quantitative proteome analysis. RESULTS:The presence of preformed biomass precursor mol...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/1475-2859-13-45
更新日期:2014-03-23 00:00:00
abstract:BACKGROUND:The budding yeast Pichia pastoris is widely used for protein production. To determine the best suitable strategy for strain improvement, especially for high secretion, quantitative data of intracellular fluxes of recombinant protein are very important. Especially the balance between intracellular protein for...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/1475-2859-10-47
更新日期:2011-06-26 00:00:00
abstract:BACKGROUND:Numerous challenges remain to achieve industrially competitive space-time yields for bio-oxidations. The ability to rapidly screen bioconversion reactions for characterization and optimization is of major importance in bioprocess development and biocatalyst selection; studies at conventional lab scale are ti...
journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-017-0788-4
更新日期:2017-10-10 00:00:00