A novel dominant selection system for plant transgenics based on phosphite metabolism catalyzed by bacterial alkaline phosphatase

Selective markers are generally indispensable in plant genetic transformation, of which the frequently used are of antibiotic or herbicide resistance. However, the increasing concerns on transgenic biosafety have encouraged many new and safe selective markers emerging, with an eminent representative as phosphite (Phi) in combination to its dehydrogenase (PTDH, e.g. PtxD). As bacterial alkaline phosphatase (BAP) can resemble PtxD to oxidatively convert toxic Phi into metabolizable phosphate (Pi), herein we harnessed it as the substitute of PtxD to develop an alternative Phi-based selection system. We first validated the Escherichia coli BAP (EcBAP) did own an extra enzymatic activity of oxidizing Phi to Pi. We further revealed EcBAP could be used as a dominant selective marker for Agrobacterium-mediated tobacco transformation. Although the involved Phi selection for transformed tobacco cells surprisingly required the presence of Pi, it showed a considerable transformation efficiency and dramatically accelerated transformation procedure, as compared to the routine kanamycin selection and the well-known PtxD/Phi system. Moreover, the EcBAP transgenic tobaccos could metabolize toxic Phi as a phosphorus (P) fertilizer thus underlying Phi-resistance, and competitively possess a dominant growth over wild-type tobacco and weeds under Phi stress. Therefore, this novel BAP/Phi-coupled system, integrating multiple advantages covering biosafe dominant selective marker, plant P utilization and weed management, can provide a PTDH-bypass technological choice to engineer transgenic plant species, especially those of great importance for sustainable agriculture.

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Faculty Opinions recommendation of A new activity for an old enzyme: Escherichia coli bacterial alkaline phosphatase is a phosphite-dependent hydrogenase.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1019084.214581 ◽

2004 ◽

Author(s):

Edward Stiefel

Keyword(s):

Escherichia Coli ◽

Alkaline Phosphatase ◽

Bacterial Alkaline Phosphatase

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Dynamic consolidated bioprocessing for innovative lab-scale production of bacterial alkaline phosphatase from Bacillus paralicheniformis strain APSO

Scientific Reports ◽

10.1038/s41598-021-85207-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Soad A. Abdelgalil ◽

Nadia A. Soliman ◽

Gaber A. Abo-Zaid ◽

Yasser R. Abdel-Fattah

Keyword(s):

Alkaline Phosphatase ◽

Consolidated Bioprocessing ◽

Black Liquor ◽

Batch Cultivation ◽

Efficient Production ◽

Scale Production ◽

Market Demand ◽

Stirred Tank Bioreactor ◽

Bacterial Alkaline Phosphatase ◽

Bacillus Paralicheniformis

AbstractTo meet the present and forecasted market demand, bacterial alkaline phosphatase (ALP) production must be increased through innovative and efficient production strategies. Using sugarcane molasses and biogenic apatite as low-cost and easily available raw materials, this work demonstrates the scalability of ALP production from a newfound Bacillus paralicheniformis strain APSO isolated from a black liquor sample. Mathematical experimental designs including sequential Plackett–Burman followed by rotatable central composite designs were employed to select and optimize the concentrations of the statistically significant media components, which were determined to be molasses, (NH4)2NO3, and KCl. Batch cultivation in a 7-L stirred-tank bioreactor under uncontrolled pH conditions using the optimized medium resulted in a significant increase in both the volumetric and specific productivities of ALP; the alkaline phosphatase throughput 6650.9 U L−1, and µ = 0.0943 h−1; respectively, were obtained after 8 h that, ameliorated more than 20.96, 70.12 and 94 folds compared to basal media, PBD, and RCCD; respectively. However, neither the increased cell growth nor enhanced productivity of ALP was present under the pH-controlled batch cultivation. Overall, this work presents novel strategies for the statistical optimization and scaling up of bacterial ALP production using biogenic apatite.

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Folding and assembly of bacterial alkaline phosphatase in vitro and in vivo.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)53073-8 ◽

1993 ◽

Vol 268 (11) ◽

pp. 8146-8150

Author(s):

Y. Akiyama ◽

K. Ito

Keyword(s):

Alkaline Phosphatase ◽

Bacterial Alkaline Phosphatase

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Effect of elevated CO2 and temperature on phosphorus efficiency of wheat grown in an Inceptisol of subtropical India

Plant Soil and Environment ◽

10.17221/749/2011-pse ◽

2012 ◽

Vol 58 (No. 5) ◽

pp. 230-235 ◽

Cited By ~ 10

Author(s):

Manoj-Kumar ◽

A. Swarup ◽

A.K. Patra ◽

J.U. Chandrakala ◽

K.M. Manjaiah

Keyword(s):

Grain Yield ◽

Dry Weight ◽

P Uptake ◽

Utilization Efficiency ◽

Phosphorus Efficiency ◽

Total Biomass ◽

P Efficiency ◽

Plant Parts ◽

P Fertilizer ◽

P Utilization

In a phytotron experiment, wheat was grown under two levels of atmospheric CO2 [ambient (385 ppm) vs. elevated (650 ppm)], two levels of temperature (ambient vs. ambient +3°C) superimposed with three levels of phosphorus (P) fertilization: 0, 100, and 200% of recommended dose. Various measures of P acquisition and utilization efficiency were estimated at crop maturity. In general, dry matter yields of all plant parts increased under elevated CO2 (EC) and decreased under elevated temperature (ET); however, under concurrently elevated CO2 and temperature (ECT), root (+36%) and leaf (+14.7%) dry weight increased while stem (–12.3%) and grain yield (–17.3%) decreased, leading to a non-significant effect on total biomass yield. Similarly, total P uptake increased under EC and decreased under ET, with an overall increase of 17.4% under ECT, signifying higher P requirements by plants grown thereunder. Although recovery efficiency of applied P fertilizer increased by 27%, any possible benefit of this increase was negated by the reduced physiological P efficiency (PPE) and P utilization efficiency (PUtE) under ECT. Overall, there was ~17% decline in P use efficiency (PUE) (i.e. grain yield/applied P) of wheat under ECT.

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Bacterial alkaline phosphatase clonal variation in some Escherichia coli K-12 phoR mutant strains.

Journal of Bacteriology ◽

10.1128/jb.168.3.1366-1371.1986 ◽

1986 ◽

Vol 168 (3) ◽

pp. 1366-1371 ◽

Cited By ~ 7

Author(s):

B L Wanner

Keyword(s):

Escherichia Coli ◽

Alkaline Phosphatase ◽

Clonal Variation ◽

Bacterial Alkaline Phosphatase ◽

Mutant Strains ◽

K 12

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A Test for Bacterial Alkaline Phosphatase: Use in Rapid Identification of Serratia Organisms

Clinical Chemistry ◽

10.1093/clinchem/19.11.1248 ◽

1973 ◽

Vol 19 (11) ◽

pp. 1248-1249 ◽

Cited By ~ 10

Author(s):

Paul L Wolf ◽

Elisabeth Von der Muehll ◽

Karen Praisler

Keyword(s):

Alkaline Phosphatase ◽

Clinical Laboratory ◽

Rapid Identification ◽

Hospitalized Patients ◽

Coagulase Negative Staphylococcus ◽

Blue Color ◽

Bacterial Alkaline Phosphatase ◽

Life Threatening ◽

Life Threatening Complication ◽

Coagulase Positive Staphylococcus

Abstract This investigation concerns identification of alkaline phosphatase production by bacterial organisms, as detected by a blue color resulting from conversion of indolyl phosphate to indigo. Coagulase-positive Staphylococcus produced alkaline phosphatase; coagulase-negative Staphylococcus did not. Serratia did not produce alkaline phosphatase; those Enterobacteriaceae we tested did. Thus, this test rapidly differentiates these organisms, diminishing the time for identification of Serratia in the clinical laboratory by 48 h. Identification of Serratia should not be ignored, because it is a life-threatening complication for certain hospitalized patients.

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Phytoplankton and bacterial alkaline phosphatase activities in relation to phosphate and DOP availability within the Gironde plume waters (Bay of Biscay)

Journal of Experimental Marine Biology and Ecology ◽

10.1016/j.jembe.2004.12.017 ◽

2005 ◽

Vol 318 (2) ◽

pp. 213-225 ◽

Cited By ~ 104

Author(s):

C. Labry ◽

D. Delmas ◽

A. Herbland

Keyword(s):

Alkaline Phosphatase ◽

Bay Of Biscay ◽

Phosphatase Activities ◽

Bacterial Alkaline Phosphatase

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The Effect of Organic Matter (Centrosema pubescens) and Rock Phosphate Application on the Activity of Phosphatase and P Fraction of Latosol Soil in Darmaga, Bogor

Jurnal Ilmu Tanah dan Lingkungan ◽

10.29244/jitl.9.1.10-15 ◽

2007 ◽

Vol 9 (1) ◽

pp. 10-15

Author(s):

S Djuniwati ◽

H.B Pulunggono ◽

Suwarno .

Keyword(s):

Alkaline Phosphatase ◽

Organic Matter ◽

Acid Phosphatase ◽

Rock Phosphate ◽

Acid Soils ◽

Available P ◽

Inorganic P ◽

Two Factors ◽

P Fertilizer ◽

P Fraction

One of the problems of acid soils such as Latosol is very low in P-availabi/ity due to high P-j'vcation in those soils. Sincesoils is deficiency of P, adaptation of plants and microorganisms to overcome deficiency of P in soil is by producing phosphatase. Phosphatase. is an enzyme that able to catalize transformation of organic P to inorganic P, and produced by plant roots, residual of plants and microorganisms. Organic mailer as a source of P besides N and energy formicroorganisms, and then rock phosphate as an alternative P fertilizer were used in this research. The objective of theresearch was to study the effect of organic matter (Centrocema pubescent) and rock phosphate application on phosphataseactivity and P fraction of Latosol soil from Darmaga, Bogor. The experiment was designed by completely randomized designwith two factors and three replications. The first factor was application of organic matter with the rate of 0, 2.5, and 5 %, and the second factor was application of rock phosphate with the rate of 0, 20, and 40 ppm P. Soil equivalent to four hundred grams of oven dried soil, organic matter, and rock phosphate were mixed based on the treatments of the experiment, and were placed in plastic pots, and then were incubated for 8 weeks period After incubation period, the soil were air dried and analyzed for phosphatase activity, available P, and organic and inorganic P. The result showed that organic mailer increased activities of acid and alkaline phosphatase, organic and inorganic-P of soil. On the other hand, rocle phosphate increased inorganic P but decreased activities of acid phosphatase,. Application of organic matter or rock phosphate on activity of acid phosphatase was higher (2.3-2.6 times) than on alkaline phosphatase. There was no effect of combinationbetween organic matter and rock phosphate on the activity of phosphatase and organic/inorganic P. Combination betweenorganic matter and rock phosphate Significantly affected available P. In each rates of rock phosphate given, the increasing rate of organic matter increased available P in their interactions, on the contrary, in each rates of organic matter, the increasing rate of rock phosphate did not affect available P in their interactions. However it was tended to decrease in therate of 40 ppm P.

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Boosting toxic protein expression: transient in vivo inactivation of engineered bacterial alkaline phosphatase

10.21203/rs.3.rs-34692/v1 ◽

2020 ◽

Author(s):

Natalia Krawczun ◽

Marta Bielawa ◽

Kasjan Szemiako ◽

Beata Lubkowska ◽

Ireneusz Sobolewski ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Sulfhydryl Group ◽

Antibody Detection ◽

Bacterial Alkaline Phosphatase ◽

E Coli ◽

Metal Affinity ◽

Novel Approach ◽

Key Enzyme

Abstract Background:The biotechnology production of enzymes is often troubled by the toxicity of the recombinant products of cloned and expressed genes, which interferes with the recombinant hosts’ metabolism. Various approaches have been taken to overcome these limitations, exemplified by tight control of recombinant genes or secretion of recombinant proteins. An industrial approach to protein production demands maximum possible yields of biosynthesized proteins, balanced with the recombinant host’s viability. Bacterial alkaline phosphatase (BAP) from Escherichia coli ( E. coli ) is a key enzyme used in protein/antibody detection and molecular cloning. As it removes terminal phosphate from DNA, RNA and deoxyribonucleoside triphosphates, it is used to lower self-ligated vectors’ background. The precursor enzyme contains a signal peptide at the N-terminus and is secreted to the E. coli periplasm. Then, the leader is clipped off and dimers are formed upon oxidation.Results: We present a novel approach to phoA gene cloning, engineering, expression, purification and reactivation of the transiently inactivated enzyme. The recombinant bap gene was modified by replacing a secretion leader coding section with a N-terminal his6-tag, cloned and expressed in E. coli in a P BAD promoter expression vector. The expression was robust, resulting in accumulation of His6-BAP in the cytoplasm, exceeding 50% of total cellular proteins. The His6-BAP protein was harmless to the cells, as its natural toxicity was inhibited by the reducing environment within the E. coli cytoplasm, preventing formation of the active enzyme. A simple protocol based on precipitation and immobilized metal affinity chromatography (IMAC) purification yielded homogeneous protein, which was reactivated by dialysis into a redox buffer containing reduced and oxidized sulfhydryl group compounds, as well as the protein structure stabilizing cofactors Zn 2+ , Mg 2+ and phosphate. The reconstituted His6-BAP exhibited high activity and was used to develop an efficient protocol for all types of DNA termini, including problematic ones (blunt, 3’-protruding).Conclusions: The developed method appears well suited for the industrial production of ultrapure BAP. Further, the method of transient inactivation of secreted toxic enzymes by conducting their biosynthesis in an inactive state in the cytoplasm, followed by in vitro reactivation, can be generally applied to other problematic proteins.

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