Construction of a High-Expression System in Bacillus through Transcriptomic Profiling and Promoter Engineering

Bacillus subtilis is an ideal host for secretion and expression of foreign proteins. The promoter is one of the most important elements to facilitate the high-level production of recombinant protein. To expand the repertoire of strong promoters for biotechnological applications in Bacillus species, 14 highly transcribed genes based on transcriptome profiling of B. pumilus BA06 were selected and evaluated for their promoter strength in B. subtilis. Consequently, a strong promoter P2069 was obtained, which could drive the genes encoding alkaline protease (aprE) and green fluorescent protein (GFP) to express more efficiency by an increase of 3.65-fold and 18.40-fold in comparison with the control promoter (PaprE), respectively. Further, promoter engineering was applied to P2069, leading to a mutation promoter (P2069M) that could increase GFP expression by 3.67-fold over the wild-type promoter (P2069). Moreover, the IPTG-inducible expression systems were constructed using the lac operon based on the strong promoters of P2069 and P2069M, which could work well both in B. subtilis and B. pumilus. In this study, highly efficient expression system for Bacillus was constructed based on transcriptome data and promoter engineering, which provide not only a new option for recombinant expression in B. subtilis, but also novel genetic tool for B. pumilus.

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Multicopy Integration and Expression of Heterologous Genes in Methylobacterium extorquens ATCC 55366

Applied and Environmental Microbiology ◽

10.1128/aem.72.1.753-759.2006 ◽

2006 ◽

Vol 72 (1) ◽

pp. 753-759 ◽

Cited By ~ 22

Author(s):

Young J. Choi ◽

Denis Bourque ◽

Lyne Morel ◽

Denis Groleau ◽

Carlos B. Míguez

Keyword(s):

Green Fluorescent Protein ◽

Target Genes ◽

Fluorescent Protein ◽

Expression System ◽

Attachment Site ◽

Methylobacterium Extorquens ◽

Gene Copies ◽

Multicopy Integration ◽

Green Fluorescent ◽

High Level

ABSTRACT High-level expression of chromosomally integrated genes in Methylobacterium extorquens ATCC 55366 was achieved under the control of the strong M. extorquens AM1 methanol dehydrogenase promoter (P mxaF ) using the mini-Tn7 transposon system. Stable maintenance and expression of the integrated genes were obtained in the absence of antibiotic selective pressure. Furthermore, using this technology, a multicopy integration protocol for M. extorquens was also developed. Chromosomal integration of one to five copies of the gene encoding the green fluorescent protein (gfp) was achieved. The multicopy-based expression system permitted expression of a preset number of gene copies. A unique specific Tn7 integration locus in the chromosome of M. extorquens, known as the Tn7 attachment site (attTn7 site), was identified. This single attTn7 site was identified in an intergenic region between glmS, which encodes the essential enzyme glucosamine-6-phosphate synthetase, and dhaT, which encodes 1,3-propanediol dehydrogenase. The fact that the integration event is site specific and the fact that the attTn7 site is a noncoding region of the chromosome make the mini-Tn7 transposon system very useful for insertion of target genes and subsequent expression. In all transformants tested, expression and segregation of the transforming gene were stable without generation of secondary mutations in the host. In this paper, we describe single and multicopy chromosome integration and stable expression of heterologous genes (bgl [β-galactosidase], est [esterase], and gfp [green fluorescent protein]) in M. extorquens.

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Metabolomic analysis of riboswitch containing E. coli recombinant expression system

Molecular BioSystems ◽

10.1039/c5mb00624d ◽

2016 ◽

Vol 12 (2) ◽

pp. 350-361 ◽

Cited By ~ 9

Author(s):

Howbeer Muhamadali ◽

Yun Xu ◽

Rosa Morra ◽

Drupad K. Trivedi ◽

Nicholas J. W. Rattray ◽

...

Keyword(s):

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Recombinant Expression ◽

Expression System ◽

Metabolic Effects ◽

Metabolomic Analysis ◽

E Coli ◽

System P ◽

Recombinant Expression System ◽

Green Fluorescent

In this study we have employed metabolomics approaches to understand the metabolic effects of producing enhanced green fluorescent protein (eGFP) as a recombinant protein inEscherichia colicells.

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Rotavirus Virus-Like Particles as Surrogates in Environmental Persistence and Inactivation Studies

Applied and Environmental Microbiology ◽

10.1128/aem.70.7.3904-3909.2004 ◽

2004 ◽

Vol 70 (7) ◽

pp. 3904-3909 ◽

Cited By ~ 27

Author(s):

Santiago Caballero ◽

F. Xavier Abad ◽

Fabienne Loisy ◽

Françoise S. Le Guyader ◽

Jean Cohen ◽

...

Keyword(s):

Fluorescent Protein ◽

Uv Light ◽

Expression System ◽

Baculovirus Expression System ◽

Virus Removal ◽

Virus Like Particles ◽

Reverse Transcription Pcr ◽

Actual Field ◽

Uv Dose ◽

Green Fluorescent

ABSTRACT Virus-like particles (VLPs) with the full-length VP2 and VP6 rotavirus capsid proteins, produced in the baculovirus expression system, have been evaluated as surrogates of human rotavirus in different environmental scenarios. Green fluorescent protein-labeled VLPs (GFP-VLPs) and particles enclosing a heterologous RNA (pseudoviruses), whose stability may be monitored by flow cytometry and antigen capture reverse transcription-PCR, respectively, were used. After 1 month in seawater at 20°C, no significant differences were observed between the behaviors of GFP-VLPs and of infectious rotavirus, whereas pseudovirus particles showed a higher decay rate. In the presence of 1 mg of free chlorine (FC)/liter both tracers persisted longer in freshwater at 20°C than infectious viruses, whereas in the presence of 0.2 mg of FC/liter no differences were observed between tracers and infectious rotavirus at short contact times. However, from 30 min of contact with FC onward, the decay of infectious rotavirus was higher than that of recombinant particles. The predicted Ct value for a 90% reduction of GFP-VLPs or pseudoviruses induces a 99.99% inactivation of infectious rotavirus. Both tracers were more resistant to UV light irradiation than infectious rotavirus in fresh and marine water. The effect of UV exposure was more pronounced on pseudovirus than in GFP-VLPs. In all types of water, the UV dose to induce a 90% reduction of pseudovirus ensures a 99.99% inactivation of infectious rotavirus. Recombinant virus surrogates open new possibilities for the systematic validation of virus removal practices in actual field situations where pathogenic agents cannot be introduced.

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Analysis of the Bacillus subtilis spoIIIJ Gene and Its Paralogue Gene, yqjG

Journal of Bacteriology ◽

10.1128/jb.184.7.1998-2004.2002 ◽

2002 ◽

Vol 184 (7) ◽

pp. 1998-2004 ◽

Cited By ~ 47

Author(s):

Takako Murakami ◽

Koki Haga ◽

Michio Takeuchi ◽

Tsutomu Sato

Keyword(s):

Bacillus Subtilis ◽

Membrane Proteins ◽

Vegetative Growth ◽

Fluorescent Protein ◽

Specific Expression ◽

Rich Media ◽

Green Fluorescent ◽

High Level ◽

Fluorescent Protein Reporter

ABSTRACT The Bacillus subtilis spoIIIJ gene, which has been proven to be vegetatively expressed, has also been implicated as a sporulation gene. Recent genome sequencing information in many organisms reveals that spoIIIJ and its paralogous gene, yqjG, are conserved from prokaryotes to humans. A homologue of SpoIIIJ/YqjG, the Escherichia coli YidC is involved in the insertion of membrane proteins into the lipid bilayer. On the basis of this similarity, it was proposed that the two homologues act as translocase for the membrane proteins. We studied the requirements for spoIIIJ and yqjG during vegetative growth and sporulation. In rich media, the growth of spoIIIJ and yqjG single mutants were the same as that of the wild type, whereas spoIIIJ yqjG double inactivation was lethal, indicating that together these B. subtilis translocase homologues play an important role in maintaining the viability of the cell. This result also suggests that SpoIIIJ and YqjG probably control significantly overlapping functions during vegetative growth. spoIIIJ mutations have already been established to block sporulation at stage III. In contrast, disruption of yqjG did not interfere with sporulation. We further show that high level expression of spoIIIJ during vegetative phase is dispensable for spore formation, but the sporulation-specific expression of spoIIIJ is necessary for efficient sporulation even at the basal level. Using green fluorescent protein reporter to monitor SpoIIIJ and YqjG localization, we found that the proteins localize at the cell membrane in vegetative cells and at the polar and engulfment septa in sporulating cells. This localization of SpoIIIJ at the sporulation-specific septa may be important for the role of spoIIIJ during sporulation.

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High level Purkinje cell specific expression of green fluorescent protein in transgenic mice

Histochemistry and Cell Biology ◽

10.1007/s004180100283 ◽

2001 ◽

Vol 115 (6) ◽

pp. 455-464 ◽

Cited By ~ 17

Author(s):

Xulun Zhang ◽

Stephan L. Baader ◽

Feng Bian ◽

Wolfgang Müller ◽

John Oberdick

Keyword(s):

Green Fluorescent Protein ◽

Transgenic Mice ◽

Purkinje Cell ◽

Fluorescent Protein ◽

Specific Expression ◽

Green Fluorescent ◽

High Level

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MS1, a direct target of MS188, regulates the expression of key sporophytic pollen coat protein genes in Arabidopsis

Journal of Experimental Botany ◽

10.1093/jxb/eraa219 ◽

2020 ◽

Vol 71 (16) ◽

pp. 4877-4889

Author(s):

Jie-Yang Lu ◽

Shuang-Xi Xiong ◽

Wenzhe Yin ◽

Xiao-Dong Teng ◽

Yue Lou ◽

...

Keyword(s):

Fluorescent Protein ◽

Pollen Wall ◽

Coat Proteins ◽

Related Family ◽

Regulatory Cascade ◽

Pollen Coat ◽

Green Fluorescent ◽

High Level ◽

And Function

Abstract Sporophytic pollen coat proteins (sPCPs) derived from the anther tapetum are deposited into pollen wall cavities and function in pollen–stigma interactions, pollen hydration, and environmental protection. In Arabidopsis, 13 highly abundant proteins have been identified in pollen coat, including seven major glycine-rich proteins GRP14, 16, 17, 18, 19, 20, and GRP–oleosin; two caleosin-related family proteins (AT1G23240 and AT1G23250); three lipase proteins EXL4, EXL5 and EXL6, and ATA27/BGLU20. Here, we show that GRP14, 17, 18, 19, and EXL4 and EXL6 fused with green fluorescent protein (GFP) are translated in the tapetum and then accumulate in the anther locule following tapetum degeneration. The expression of these sPCPs is dependent on two essential tapetum transcription factors, MALE STERILE188 (MS188) and MALE STERILITY 1 (MS1). The majority of sPCP genes are up-regulated within 30 h after MS1 induction and could be restored by MS1 expression driven by the MS188 promoter in ms188, indicating that MS1 is sufficient to activate their expression; however, additional MS1 downstream factors appear to be required for high-level sPCP expression. Our ChIP, in vivo transactivation assay, and EMSA data indicate that MS188 directly activates MS1. Together, these results reveal a regulatory cascade whereby outer pollen wall formation is regulated by MS188 followed by synthesis of sPCPs controlled by MS1.

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Multicopy Integration of Heterologous Genes, Using the Lactococcal Group II Intron Targeted to Bacterial Insertion Sequences

Applied and Environmental Microbiology ◽

10.1128/aem.02992-05 ◽

2006 ◽

Vol 72 (9) ◽

pp. 6088-6093 ◽

Cited By ~ 19

Author(s):

Helen Rawsthorne ◽

Kevin N. Turner ◽

David A. Mills

Keyword(s):

Fluorescent Protein ◽

Group Ii Intron ◽

Multicopy Plasmid ◽

Bacterial Genomes ◽

Gfp Expression ◽

High Frequencies ◽

Multicopy Integration ◽

Group Ii ◽

Green Fluorescent ◽

High Level

ABSTRACT Group II introns are mobile genetic elements that can be redirected to invade specific genes. Here we describe the use of the lactococcal group II intron, Ll.ltrB, to achieve multicopy delivery of heterologous genes into the genome of Lactococcus lactis IL1403-UCD without the need for selectable markers. Ll.ltrB was retargeted to invade three transposase genes, the tra gene found in IS904 (tra904), tra981, and tra983, of which 9, 10, and 14 copies, respectively, were present in IL1403-UCD. Intron invasion of tra904, tra981, and tra983 allele groups occurred at high frequencies, and individual segregants possessed anywhere from one to nine copies of intron in the respective tra alleles. To achieve multicopy delivery of a heterologous gene, a green fluorescent protein (GFP) marker was cloned into the tra904-targeted Ll.ltrB, and the resultant intron (Ll.ltrB::GFP) was induced to invade the L. lactis tra904 alleles. Segregants possessing Ll.ltrB::GFP in three, four, five, six, seven, and eight copies in different tra904 alleles were obtained. In general, increasing the chromosomal copy number of Ll.ltrB::GFP resulted in strains expressing successively higher levels of GFP. However, strains possessing the same number of Ll.ltrB::GFP copies within different sets of tra904 alleles exhibited differential GFP expression, and segregants possessing seven or eight copies of Ll.ltrB::GFP grew poorly upon induction, suggesting that GFP expression from certain combinations of alleles was detrimental. The highest level of GFP expression was observed from a specific six-copy variant that produced GFP at a level analogous to that obtained with a multicopy plasmid. In addition, the high level of GFP expression was stable for over 120 generations. This work demonstrates that stable multicopy integration of heterologous genes can be readily achieved in bacterial genomes with group II intron delivery by targeting repeated elements.

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Cell Death of Nicotiana benthamiana Is Induced by Secreted Protein NIS1 of Colletotrichum orbiculare and Is Suppressed by a Homologue of CgDN3

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-12-11-0316 ◽

2012 ◽

Vol 25 (5) ◽

pp. 625-636 ◽

Cited By ~ 33

Author(s):

Kae Yoshino ◽

Hiroki Irieda ◽

Fumie Sugimoto ◽

Hirofumi Yoshioka ◽

Tetsuro Okuno ◽

...

Keyword(s):

Cell Death ◽

Nicotiana Benthamiana ◽

Protein Gene ◽

Fluorescent Protein ◽

Expression System ◽

Functional Screening ◽

Secreted Protein ◽

Virus Induced Gene Silencing ◽

Colletotrichum Orbiculare ◽

Green Fluorescent

Colletotrichum orbiculare, the causal agent of cucumber anthracnose, infects Nicotiana benthamiana. Functional screening of C. orbiculare cDNAs in a virus vector-based plant expression system identified a novel secreted protein gene, NIS1, whose product induces cell death in N. benthamiana. Putative homologues of NIS1 are present in selected members of fungi belonging to class Sordariomycetes, Dothideomycetes, or Orbiliomycetes. Green fluorescent protein–based expression studies suggested that NIS1 is preferentially expressed in biotrophic invasive hyphae. NIS1 lacking signal peptide did not induce NIS1-triggered cell death (NCD), suggesting apoplastic recognition of NIS1. NCD was prevented by virus-induced gene silencing of SGT1 and HSP90, indicating the dependency of NCD on SGT1 and HSP90. Deletion of NIS1 had little effect on the virulence of C. orbiculare against N. benthamiana, suggesting possible suppression of NCD by C. orbiculare at the postinvasive stage. The CgDN3 gene of C. gloeosporioides was previously identified as a secreted protein gene involved in suppression of hypersensitive-like response in Stylosanthes guianensis. Notably, we found that NCD was suppressed by the expression of a CgDN3 homologue of C. orbiculare. Our findings indicate that C. orbiculare expresses NIS1 at the postinvasive stage and suggest that NCD could be repressed via other effectors, including the CgDN3 homologue.

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Iba1+/NG2+ Macrophage-Like Cells Expressing a Variety of Neuroprotective Factors Ameliorate Ischemic Damage of the Brain

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2009.233 ◽

2009 ◽

Vol 30 (3) ◽

pp. 603-615 ◽

Cited By ~ 51

Author(s):

Anna Smirkin ◽

Hiroaki Matsumoto ◽

Hisaaki Takahashi ◽

Akihiro Inoue ◽

Masahiko Tagawa ◽

...

Keyword(s):

Bone Marrow ◽

Fluorescent Protein ◽

Artery Occlusion ◽

Brain Lesions ◽

Ischemic Lesion ◽

Ischemic Brain ◽

Green Fluorescent ◽

High Level ◽

Cerebral Artery Occlusion ◽

The Brain

In a transient 90-min middle cerebral artery occlusion (MCAO) model of rats, a large ischemic lesion is formed where macrophage-like cells massively accumulate, many of which express a macrophage marker, Iba1, and an oligodendrocyte progenitor cell marker, NG2 chondroitin sulfate proteoglycan (NG2); therefore, the cells were termed BINCs (Brain Iba1+/NG2+Cells). A bone marrow transplantation experiment using green-fluorescent protein-transgenic rats showed that BINCs were derived from bone marrow. 5-Fluorouracil (5FU) injection at 2 days post reperfusion (2 dpr) markedly reduced the number of BINCs at 7 dpr, causing enlargement of necrotic volumes and frequent death of the rats. When isolated BINCs were transplanted into 5FU-aggravated ischemic lesion, the volume of the lesion was much reduced. Quantitative real-time RT-PCR showed that BINCs expressed mRNAs encoding bFGF, BMP2, BMP4, BMP7, GDNF, HGF, IGF-1, PDGF-A, and VEGF. In particular, BINCs expressed IGF-1 mRNA at a very high level. Immunohistochemical staining showed that IGF-1-expressing BINCs were found not only in rat but also human ischemic brain lesions. These results suggest that bone marrow-derived BINCs play a beneficial role in ischemic brain lesions, at least in part, through secretion of neuroprotective factors.

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