scholarly journals Engineering of molybdenum-cofactor-dependent nitrate assimilation in Yarrowia lipolytica

2021 ◽  
Vol 21 (6) ◽  
Author(s):  
Thomas Perli ◽  
Irina Borodina ◽  
Jean-Marc Daran
Keyword(s):  
Nitrate Reductase ◽  
Yarrowia Lipolytica ◽  
Reductase Activity ◽  
Nitrate Assimilation ◽  
Fold Increase ◽  
Molybdenum Cofactor ◽  
Adaptive Laboratory Evolution ◽  
Synonymous Mutations ◽  
Enzyme Families ◽  
Microbial Host

ABSTRACT Engineering a new metabolic function in a microbial host can be limited by the availability of the relevant cofactor. For instance, in Yarrowia lipolytica, the expression of a functional nitrate reductase is precluded by the absence of molybdenum cofactor (Moco) biosynthesis. In this study, we demonstrated that the Ogataea parapolymorpha Moco biosynthesis pathway combined with the expression of a high affinity molybdate transporter could lead to the synthesis of Moco in Y. lipolytica. The functionality of Moco was demonstrated by expression of an active Moco-dependent nitrate assimilation pathway from the same yeast donor, O. parapolymorpha. In addition to 11 heterologous genes, fast growth on nitrate required adaptive laboratory evolution which, resulted in up to 100-fold increase in nitrate reductase activity and in up to 4-fold increase in growth rate, reaching 0.13h-1. Genome sequencing of evolved isolates revealed the presence of a limited number of non-synonymous mutations or small insertions/deletions in annotated coding sequences. This study that builds up on a previous work establishing Moco synthesis in S. cerevisiae demonstrated that the Moco pathway could be successfully transferred in very distant yeasts and, potentially, to any other genera, which would enable the expression of new enzyme families and expand the nutrient range used by industrial yeasts.

Nitrate Reductase from a Mutant Strain of Chlamydomonas reinhardii Incapable of Nitrate Assimilation

1983 ◽  
Vol 38 (5-6) ◽  
pp. 439-445 ◽  
Author(s):  
Emilio Fernández ◽  
Jacobo Cárdenas
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Wild Strain ◽  
Nitrate Assimilation ◽  
Pyridine Nucleotide ◽  
Spectral Studies ◽  
Binding Region ◽  
Chlamydomonas Reinhardii ◽  
Blue Sepharose

Nitrate reductase from mutant 305 of Chlamydomonas reinhardii has been purified about 90-fold and biochemically characterized. The enzyme can use reduced flavins and viologens as electron donors to reduce nitrate but, unlike the nitrate reductase complex from its parental wild strain, lacks NAD(P)H-nitrate reductase and NAD(P)H-cytochrome c reductase activities, does not bind to Blue-Agarose or Blue-Sepharose and exhibits a significantly lower molecular weight (177.000 vs. 241.000), whereas its kinetic characteristics and its sensitivity against several inhibitors and treatments are very similar to those of the terminal nitrate reductase activity of the wild strain complex. Spectral studies and antagonistic experiments with tungstate show the presence of cytochrome b557 and molybdenum. These facts lead us to propose that nitrate reductase from mutant 305 has a protein deletion which affects the pyridine nucleotide binding region of the diaphorase protein but without any effect on the terminal nitrate reductase activity.

Seasonal variation in nitrate reductase activity in needles of high-elevation red spruce trees

1992 ◽  
Vol 22 (3) ◽  
pp. 375-380 ◽  
Author(s):  
M.G. Tjoelker ◽  
S.B. McLaughlin ◽  
R.J. DiCosty ◽  
S.E. Lindberg ◽  
R.J. Norby
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Nitrate Assimilation ◽  
High Elevation ◽  
Preliminary Evidence ◽  
Air Pollutant ◽  
Red Spruce ◽  
Acid Vapor ◽  
Assimilation Capacity

To assess seasonal and site variation in foliar nitrate reductase activity and its utility as a biochemical marker for the uptake of nitrogen oxide pollutants in high-elevation forests, we measured nitrate reductase activity in current-year needles of red spruce (Picearubens Sarg.) saplings at two high-elevation stands (1935 and 1720 m) in the Great Smoky Mountains, North Carolina. Measurements spanned two growing seasons between September 1987 and September 1988. Nitrate reductase activity peaked near 60 nmol•g−1•h−1 at both sites in September and October 1987 and August 1988 and declined 80% in November 1987 and 65% in September 1988. Although nitrate reductase activity was 30% greater in saplings at the higher site relative to the lower site in September and October 1987, activity dropped to approximately 10 nmol•g−1•h−1 at both sites in November 1987. No differences among sites were evident the following year. Comparing deposition of nitric acid vapor at a nearby site to nitrate reductase activity suggests that needle nitrate reductase activity is not an unequivocal marker for foliar uptake of nitrogen oxides during air pollutant episodes. The changes in soil nitrate levels in this system provide preliminary evidence that foliar nitrate assimilation may, in part, include nitrate taken up from the soil, as the highest activity occurred during periods of higher A-horizon nitrate concentrations in 1988. These measurements of nitrate reductase activity suggest that red spruce are capable of assimilating nitrate in foliage in the field and that the nitrate assimilation capacity varies throughout the year.

scholarly journals Molecular Components of Nitrate and Nitrite Efflux in Yeast

2013 ◽  
Vol 13 (2) ◽  
pp. 267-278 ◽  
Author(s):  
Elisa Cabrera ◽  
Rafaela González-Montelongo ◽  
Teresa Giraldez ◽  
Diego Alvarez de la Rosa ◽  
José M. Siverio
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Uptake ◽  
Reductase Activity ◽  
Hansenula Polymorpha ◽  
Nitrate Assimilation ◽  
Nitrate Reductase Gene ◽  
Content Type ◽  
Nitrite Toxicity ◽  
Essential Components ◽  
Nitrate And Nitrite

ABSTRACTSome eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeastHansenula polymorphawas used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking eitherSSU2orNAR1along with the nitrate reductase geneYNR1showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-knownSaccharomyces cerevisiaesulfite efflux permease Ssu1 is also able to excrete nitrite and nitrate. These results characterize for the first time essential components of the nitrate/nitrite efflux system and their impact on net nitrate uptake and its regulation.

Contribution of the Root System to Nitrate Assimilation in Whole Cotton Plants.

1977 ◽  
Vol 4 (5) ◽  
pp. 811 ◽  
Author(s):  
JW Radin
Keyword(s):  
Nitrate Reductase ◽  
Root Growth ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Physiological Role ◽  
Nitrate Assimilation ◽  
Relative Magnitude ◽  
Balance Sheets ◽  
Cotton Plants ◽  
Reduced Nitrogen

Cotton (Gossypium hirsutum L.) is a species in which most nitrate is assimilated in the green shoot. A physiological role for the small amount of nitrate reductase activity in the roots can be questioned on the basis of relative magnitude. In this investigation, cotton plants were grown on nutrient solutions containing either 1 or 5 mM nitrate, and balance sheets were developed for the transport and metabolism of nitrate and reduced nitrogen in the root and shoot during exponential growth. At either nitrate level, assimilation in the roots was adequate to supply all the nitrogen for root growth. However, some of the reduced nitrogen was exported in the xylem, leaving a net deficit of about 10% at 1 mM nitrate and 36% at 5 mM nitrate. This deficit was presumably satisfied by reduced nitrogen from the shoot. Thus, at these two nitrate concentrations, root growth apparently depended more upon nitrogen assimilated in the roots themselves than upon nitrogen from the shoot. The different fates of nitrogen assimilated in the root and in the shoot may be related to the demonstrated differential regulation of nitrate reductase activity in these two sites.

scholarly journals Characteristics of Nicotiana tabacum nitrate reductase protein produced in Saccharomyces cerevisiae

1991 ◽  
Vol 278 (2) ◽  
pp. 393-397 ◽  
Author(s):  
H N Truong ◽  
C Meyer ◽  
F Daniel-Vedele
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nicotiana Tabacum ◽  
Cytochrome C ◽  
Nitrate Reductase ◽  
Higher Plants ◽  
Reductase Activity ◽  
Molybdenum Cofactor ◽  
Biochemical Data ◽  
Native Form ◽  
Nr Activity

Tobacco nitrate reductase (NR) produced in yeast retains cytochrome c reductase activity, but not NR activity. Biochemical data suggest that the haem and FAD domains are functional, and that the molybdenum cofactor (MoCo) domain is inactive owing to the absence of MoCo in yeast. The native form of the produced NR is dimeric. Thus MoCo is not involved in NR dimerization in higher plants, contrary to current assumptions.

scholarly journals Transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier Paracoccus denitrificans

2017 ◽  
Vol 474 (11) ◽  
pp. 1769-1787 ◽  
Author(s):  
Victor M. Luque-Almagro ◽  
Isabel Manso ◽  
Matthew J. Sullivan ◽  
Gary Rowley ◽  
Stuart J. Ferguson ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Mutational Analysis ◽  
Paracoccus Denitrificans ◽  
Reductase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Nitrate Assimilation ◽  
Transcriptional Unit ◽  
Regulation Mechanism ◽  
N Source

Transcriptional adaptation to nitrate-dependent anabolism by Paracoccus denitrificans PD1222 was studied. A total of 74 genes were induced in cells grown with nitrate as N-source compared with ammonium, including nasTSABGHC and ntrBC genes. The nasT and nasS genes were cotranscribed, although nasT was more strongly induced by nitrate than nasS. The nasABGHC genes constituted a transcriptional unit, which is preceded by a non-coding region containing hairpin structures involved in transcription termination. The nasTS and nasABGHC transcripts were detected at similar levels with nitrate or glutamate as N-source, but nasABGHC transcript was undetectable in ammonium-grown cells. The nitrite reductase NasG subunit was detected by two-dimensional polyacrylamide gel electrophoresis in cytoplasmic fractions from nitrate-grown cells, but it was not observed when either ammonium or glutamate was used as the N-source. The nasT mutant lacked both nasABGHC transcript and nicotinamide adenine dinucleotide (NADH)-dependent nitrate reductase activity. On the contrary, the nasS mutant showed similar levels of the nasABGHC transcript to the wild-type strain and displayed NasG protein and NADH–nitrate reductase activity with all N-sources tested, except with ammonium. Ammonium repression of nasABGHC was dependent on the Ntr system. The ntrBC and ntrYX genes were expressed at low levels regardless of the nitrogen source supporting growth. Mutational analysis of the ntrBCYX genes indicated that while ntrBC genes are required for nitrate assimilation, ntrYX genes can only partially restore growth on nitrate in the absence of ntrBC genes. The existence of a regulation mechanism for nitrate assimilation in P. denitrificans, by which nitrate induction operates at both transcriptional and translational levels, is proposed.

scholarly journals Biochemical Characterization of Molybdenum Cofactor-free Nitrate Reductase from Neurospora crassa

2013 ◽  
Vol 288 (20) ◽  
pp. 14657-14671 ◽  
Author(s):  
Phillip Ringel ◽  
Joern Krausze ◽  
Joop van den Heuvel ◽  
Ute Curth ◽  
Antonio J. Pierik ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Neurospora Crassa ◽  
Biochemical Characterization ◽  
Nitrate Assimilation ◽  
Molybdenum Cofactor ◽  
Site Formation ◽  
Epr Spectrum ◽  
Redox Active

Nitrate reductase (NR) is a complex molybdenum cofactor (Moco)-dependent homodimeric metalloenzyme that is vitally important for autotrophic organism as it catalyzes the first and rate-limiting step of nitrate assimilation. Beside Moco, eukaryotic NR also binds FAD and heme as additional redox active cofactors, and these are involved in electron transfer from NAD(P)H to the enzyme molybdenum center where reduction of nitrate to nitrite takes place. We report the first biochemical characterization of a Moco-free eukaryotic NR from the fungus Neurospora crassa, documenting that Moco is necessary and sufficient to induce dimer formation. The molybdenum center of NR reconstituted in vitro from apo-NR and Moco showed an EPR spectrum identical to holo-NR. Analysis of mutants unable to bind heme or FAD revealed that insertion of Moco into NR occurs independent from the insertion of any other NR redox cofactor. Furthermore, we showed that at least in vitro the active site formation of NR is an autonomous process.

scholarly journals A Novel Gene (narM) Required for Expression of Nitrate Reductase Activity in the Cyanobacterium Synechococcus elongatus Strain PCC7942

2004 ◽  
Vol 186 (7) ◽  
pp. 2107-2114 ◽  
Author(s):  
Shin-ichi Maeda ◽  
Tatsuo Omata
Keyword(s):  
Nitrate Reductase ◽  
Structural Gene ◽  
Nitrate Reductase Activity ◽  
Insertional Mutagenesis ◽  
Reductase Activity ◽  
Nitrate Assimilation ◽  
Gene Cassette ◽  
Synechococcus Elongatus ◽  
Prosthetic Group ◽  
Functional Link

ABSTRACT A new class of mutants deficient in nitrate assimilation was obtained from the cyanobacterium Synechococcus elongatus strain PCC7942 by means of random insertional mutagenesis. A 0.5-kb genomic region had been replaced by a kanamycin resistance gene cassette in the mutant, resulting in inactivation of two genes, one of which was homologous to the recently characterized cnaT gene of Anabaena sp. strain PCC7120 (J. E. Frías, A. Herrero, and E. Flores, J. Bacteriol. 185:5037-5044, 2003). While insertional mutation of the cnaT homolog did not affect expression of the nitrate assimilation operon or the activity of the nitrate assimilation enzymes in S. elongatus, inactivation of the other gene, designated narM, resulted in specific loss of the cellular nitrate reductase activity. The deduced NarM protein is a hydrophilic protein consisting of 161 amino acids. narM was expressed constitutively at a low level. The narM gene has its homolog only in the cyanobacterial strains that are capable of nitrate assimilation. In most of the cyanobacterial strains, narM is located downstream of narB, the structural gene of the cyanobacterial nitrate reductase, suggesting the functional link between the two genes. NarM is clearly not the structural component of the cyanobacterial nitrate reductase. The narM insertional mutant normally expressed narB, indicating that narM is not the transcriptional regulator of the structural gene of nitrate reductase. These results suggested that narM is required for either synthesis of the prosthetic group of nitrate reductase or assembly of the prosthetic groups to the NarB polypeptide to form functional nitrate reductase in cyanobacteria.

scholarly journals Isoform-Specific NO Synthesis by Arabidopsis thaliana Nitrate Reductase

Plants ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 67 ◽  
Author(s):  
Marie Mohn ◽  
Besarta Thaqi ◽  
Katrin Fischer-Schrader
Keyword(s):  
Arabidopsis Thaliana ◽  
Nitrate Reductase ◽  
Biochemical Characterization ◽  
Special Functions ◽  
Reductase Activity ◽  
Nitrate Assimilation ◽  
Product Formation ◽  
Electron Transfer Pathway ◽  
Electron Reduction ◽  
Reduced Nicotinamide Adenine Dinucleotide

Nitrate reductase (NR) is important for higher land plants, as it catalyzes the rate-limiting step in the nitrate assimilation pathway, the two-electron reduction of nitrate to nitrite. Furthermore, it is considered to be a major enzymatic source of the important signaling molecule nitric oxide (NO), that is produced in a one-electron reduction of nitrite. Like many other plants, the model plant Arabidopsis thaliana expresses two isoforms of NR (NIA1 and NIA2). Up to now, only NIA2 has been the focus of detailed biochemical studies, while NIA1 awaits biochemical characterization. In this study, we have expressed and purified functional fragments of NIA1 and subjected them to various biochemical assays for comparison with the corresponding NIA2-fragments. We analyzed the kinetic parameters in multiple steady-state assays using nitrate or nitrite as substrate and measured either substrate consumption (nitrate or nitrite) or product formation (NO). Our results show that NIA1 is the more efficient nitrite reductase while NIA2 exhibits higher nitrate reductase activity, which supports the hypothesis that the isoforms have special functions in the plant. Furthermore, we successfully restored the physiological electron transfer pathway of NR using reduced nicotinamide adenine dinucleotide (NADH) and nitrate or nitrite as substrates by mixing the N-and C-terminal fragments of NR, thus, opening up new possibilities to study NR activity, regulation and structure.

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