scholarly journals PccD Regulates Branched-Chain Amino Acid Degradation and Exerts a Negative Effect on Erythromycin Production inSaccharopolyspora erythraea

2018 ◽  
Vol 84 (8) ◽  
pp. e00049-18 ◽  
Author(s):  
Zhen Xu ◽  
Yong Liu ◽  
Bang-Ce Ye
Keyword(s):  
Growth Rate ◽  
Amino Acid ◽  
Transcriptional Regulator ◽  
Fermentation Medium ◽  
Industrial Fermentation ◽  
Content Type ◽  
Branched Chain Amino Acid ◽  
Negative Effect ◽  
Branched Chain ◽  
Erythromycin Production

ABSTRACTBranched-chain amino acid (BCAA) degradation is a major source of propionyl coenzyme A (propionyl-CoA), a key precursor of erythromycin biosynthesis inSaccharopolyspora erythraea. In this study, we found that thebkdoperon, responsible for BCAA degradation, was regulated directly by PccD, a transcriptional regulator of propionyl-CoA carboxylase genes. The transcriptional level of thebkdoperon was upregulated 5-fold in apccDgene deletion strain (ΔpccDstrain) and decreased 3-fold in apccDoverexpression strain (WT/pIB-pccD), demonstrating that PccD was a negative transcriptional regulator of the operon. The deletion ofpccDsignificantly improved the ΔpccDstrain's growth rate, whereaspccDoverexpression repressed WT/pIB-pccDgrowth rate, in basic Evans medium with 30 mM valine as the sole carbon and nitrogen source. The deletion ofgdhA1and the BcdhE1 gene (genes in thebkdoperon) resulted in lower growth rates of ΔgdhA1and ΔBcdhE1 strains, respectively, on 30 mM valine, further suggesting that thebkdoperon is involved in BCAA degradation. Bothbkdoverexpression (WT/pIB-bkd) andpccDinactivation (ΔpccDstrain) improve erythromycin production (38% and 64%, respectively), whereas the erythromycin production of strain WT/pIB-pccDwas decreased by 48%. Lastly, we explored the applications of engineeringpccDandbkdin an industrial high-erythromycin-producing strain.pccDdeletion in industrial strainS. erythraeaE3 (E3pccD) improved erythromycin production by 20%, and the overexpression ofbkdin E3ΔpccD(E3ΔpccD/pIB-bkd) increased erythromycin production by 39% compared withS. erythraeaE3 in an industrial fermentation medium. Addition of 30 mM valine to industrial fermentation medium further improved the erythromycin production by 23%, a 72% increase from the initial strainS. erythraeaE3.IMPORTANCEWe describe abkdoperon involved in BCAA degradation inS. erythraea. The genes of the operon are repressed by a TetR regulator, PccD. The results demonstrated that PccD controlled the supply of precursors for biosynthesis of erythromycin via regulating the BCAA degradation and propionyl-CoA assimilation and exerted a negative effect on erythromycin production. The findings reveal a regulatory mechanism in feeder pathways and provide new strategies for designing metabolic engineering to increase erythromycin yield.

scholarly journals Role of Branched-Chain Amino Acid Transport in Bacillus subtilis CodY Activity

2015 ◽  
Vol 197 (8) ◽  
pp. 1330-1338 ◽  
Author(s):  
Boris R. Belitsky
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Growth Medium ◽  
Amino Acid Transport ◽  
Transcriptional Regulator ◽  
Acid Transport ◽  
Content Type ◽  
Branched Chain Amino Acid ◽  
Branched Chain

ABSTRACTCodY is a branched-chain amino acid-responsive transcriptional regulator that controls the expression of several dozen transcription units inBacillus subtilis. The presence of isoleucine, valine, and leucine in the growth medium is essential for achieving high activity of CodY and for efficient regulation of the target genes. We identified three permeases—BcaP, BraB, and BrnQ—that are responsible for the bulk of isoleucine and valine uptake and are also involved in leucine uptake. At least one more permease is capable of efficient leucine uptake, as well as low-affinity transport of isoleucine and valine. The lack of the first three permeases strongly reduced activity of CodY in an amino acid-containing growth medium. BcaP appears to be the most efficient isoleucine and valine permease responsible for their utilization as nitrogen sources. The previously described strong CodY-mediated repression of BcaP provides a mechanism for fine-tuning CodY activity by reducing the availability of amino acids and for delaying the utilization of isoleucine and valine as nitrogen and carbon sources under conditions of nutrient excess.IMPORTANCEBacillus subtilisCodY is a global transcriptional regulator that is activated by branched-chain amino acids (BCAA). Since the level of BCAA achieved by intracellular synthesis is insufficient to fully activate CodY, transport of BCAA from the environment is critical for CodY activation, but the permeases needed for such activation have not been previously identified. This study identifies three such permeases, reports their amino acid transport specificity, and reveals their impact on CodY activation.

scholarly journals Acid Stress-Mediated Metabolic Shift in Lactobacillus sanfranciscensis LSCE1

2011 ◽  
Vol 77 (8) ◽  
pp. 2656-2666 ◽  
Author(s):  
Diana I. Serrazanetti ◽  
Maurice Ndagijimana ◽  
Sylvain L. Sado-Kamdem ◽  
Aldo Corsetti ◽  
Rudi F. Vogel ◽  
...  
Keyword(s):  
Amino Acid ◽  
Solid Phase ◽  
Acid Stress ◽  
Redox Balance ◽  
Gas Chromatography Mass Spectrometry ◽  
Metabolic Shift ◽  
Content Type ◽  
Lactobacillus Sanfranciscensis ◽  
Branched Chain Amino Acid ◽  
Branched Chain

ABSTRACTLactobacillus sanfranciscensisLSCE1 was selected as a target organism originating from recurrently refreshed sourdough to study the metabolic rerouting associated with the acid stress exposure during sourdough fermentation. In particular, the acid stress induced a metabolic shift toward overproduction of 3-methylbutanoic and 2-methylbutanoic acids accompanied by reduced sugar consumption and primary carbohydrate metabolite production. The fate of labeled leucine, the role of different nutrients and precursors, and the expression of the genes involved in branched-chain amino acid (BCAA) catabolism were evaluated at pH 3.6 and 5.8. The novel application of the program XCMS to the solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) data allowed accurate separation and quantification of 2-methylbutanoic and 3-methylbutanoic acids, generally reported as a cumulative datum. The metabolites coming from BCAA catabolism increased up to seven times under acid stress. The gene expression analysis confirmed that some genes associated with BCAA catabolism were overexpressed under acid conditions. The experiment with labeled leucine showed that 2-methylbutanoic acid originated also from leucine. While the overproduction of 3-methylbutanoic acid under acid stress can be attributed to the need to maintain redox balance, the rationale for the production of 2-methylbutanoic acid from leucine can be found in a newly proposed biosynthesis pathway leading to 2-methylbutanoic acid and 3 mol of ATP per mol of leucine. Leucine catabolism to 3-methylbutanoic and 2-methylbutanoic acids suggests that the switch from sugar to amino acid catabolism supports growth inL. sanfranciscensisin restricted environments such as sourdough characterized by acid stress and recurrent carbon starvation.

scholarly journals Genetically encoded biosensors for branched-chain amino acid metabolism to monitor mitochondrial and cytosolic production of isobutanol and isopentanol in yeast

2020 ◽  
Author(s):  
Yanfei Zhang ◽  
Sarah K. Hammer ◽  
Cesar Carrasco-Lopez ◽  
Sergio A. Garcia Echauri ◽  
José L. Avalos
Keyword(s):  
Amino Acid ◽  
Amino Acid Metabolism ◽  
Metabolic Flux ◽  
Acid Metabolism ◽  
Transcriptional Regulator ◽  
Higher Alcohol ◽  
Alcohol Production ◽  
Branched Chain Amino Acid ◽  
Branched Chain ◽  
Genetically Encoded Biosensor

AbstractBranched-chain amino acid (BCAA) metabolism can be harnessed to produce many valuable chemicals. Among these, isobutanol, which is derived from valine degradation, has received substantial attention due to its promise as an advanced biofuel. While Saccharomyces cerevisiae is the preferred organism for isobutanol production, the lack of isobutanol biosensors in this organism has limited the ability to screen strains at high throughput. Here, we use a transcriptional regulator of BCAA biosynthesis, Leu3p, to develop the first genetically encoded biosensor for isobutanol production in yeast. Small modifications allowed us to redeploy Leu3p in a second biosensor for isopentanol, another BCAA-derived product of interest. Each biosensor is highly specific to isobutanol or isopentanol, respectively, and was used to engineer metabolic enzymes to increase titers. The isobutanol biosensor was additionally employed to isolate high-producing strains, and guide the construction and enhancement of mitochondrial and cytosolic isobutanol biosynthetic pathways, including in combination with optogenetic actuators to enhance metabolic flux. These biosensors promise to accelerate the development of enzymes and strains for branched-chain higher alcohol production, and offer a blueprint to develop biosensors for other products derived from BCAA metabolism.

scholarly journals Importance of Branched-Chain Amino Acid Utilization in Francisella Intracellular Adaptation

2014 ◽  
Vol 83 (1) ◽  
pp. 173-183 ◽  
Author(s):  
Gael Gesbert ◽  
Elodie Ramond ◽  
Fabiola Tros ◽  
Julien Dairou ◽  
Eric Frapy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Critical Role ◽  
Cell Types ◽  
Host Cells ◽  
Metabolic Adaptation ◽  
Amino Acid Transporters ◽  
Loss Of Function ◽  
Content Type ◽  
Branched Chain Amino Acid ◽  
Branched Chain

Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication ofFrancisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation ofFrancisella. Inactivation of IleP severely impaired intracellularF. tularensissubsp.novicidamultiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of theilePgene inF. tularensispathogenesis, we constructed a chromosomal deletion mutant ofileP(ΔFTL_1803) in theF. tularensissubsp.holarcticalive vaccine strain (LVS). Inactivation of IleP in theF. tularensisLVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication ofFrancisellaand suggest that virulentF. tularensissubspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens.

scholarly journals Biosensor for branched-chain amino acid metabolism in yeast and applications in isobutanol and isopentanol production

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Yanfei Zhang ◽  
Jeremy D. Cortez ◽  
Sarah K. Hammer ◽  
César Carrasco-López ◽  
Sergio Á. García Echauri ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Metabolism ◽  
Acid Metabolism ◽  
Transcriptional Regulator ◽  
Strain Engineering ◽  
Higher Alcohol ◽  
Metabolic Studies ◽  
Branched Chain Amino Acid ◽  
Branched Chain ◽  
High Throughput Screens

AbstractBranched-chain amino acid (BCAA) metabolism fulfills numerous physiological roles and can be harnessed to produce valuable chemicals. However, the lack of eukaryotic biosensors specific for BCAA-derived products has limited the ability to develop high-throughput screens for strain engineering and metabolic studies. Here, we harness the transcriptional regulator Leu3p from Saccharomyces cerevisiae to develop a genetically encoded biosensor for BCAA metabolism. In one configuration, we use the biosensor to monitor yeast production of isobutanol, an alcohol derived from valine degradation. Small modifications allow us to redeploy Leu3p in another biosensor configuration that monitors production of the leucine-derived alcohol, isopentanol. These biosensor configurations are effective at isolating high-producing strains and identifying enzymes with enhanced activity from screens for branched-chain higher alcohol (BCHA) biosynthesis in mitochondria as well as cytosol. Furthermore, this biosensor has the potential to assist in metabolic studies involving BCAA pathways, and offers a blueprint to develop biosensors for other products derived from BCAA metabolism.

scholarly journals The Intermediates in Branched-Chain Amino Acid Biosynthesis Are Indispensable for Conidial Germination of the Insect-Pathogenic Fungus Metarhizium robertsii

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
Feifei Luo ◽  
Hongxia Zhou ◽  
Xue Zhou ◽  
Xiangyun Xie ◽  
You Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Pathogenic Fungus ◽  
Pathogenic Fungi ◽  
Conidial Germination ◽  
Keto Acid ◽  
Metarhizium Robertsii ◽  
Content Type ◽  
Branched Chain Amino Acid ◽  
Key Enzyme ◽  
Branched Chain

ABSTRACT Metarhizium spp. are well-known biocontrol agents used worldwide to control different insect pests. Keto-acid reductoisomerase (ILVC) is a key enzyme for branched-chain amino acid (BCAA) biosynthesis, and it regulates many physiological activities. However, its functions in insect-pathogenic fungi are poorly understood. In this work, we identified MrilvC in M. robertsii and dissected its roles in fungal growth, conidiation, germination, destruxin biosynthesis, environmental stress response, and insecticidal virulence. BCAA metabolism affects conidial yields and germination. However, BCAAs cannot recover the conidial germination of an MrilvC-deficient strain. Further feeding assays with intermediates showed that some conidia of the ΔMrilvC mutant start to germinate. Therefore, it is the germination defect that causes the complete failures of conidial penetration and pathogenicity in the ΔMrilvC mutant. In conclusion, we found intermediates in BCAA biosynthesis are indispensable for Metarhizium robertsii conidial germination. This study will advance our understanding of the fungal germination mechanism. IMPORTANCE Branched-chain amino acid (BCAA) metabolism plays a significant role in many biological activities beyond protein synthesis. Spore germination initiates the first stage of vegetative growth, which is critical for the virulence of pathogenic fungi. In this study, we demonstrated that the keto-acid reductoisomerase MrILVC, a key enzyme for BCAA biosynthesis, from the insect-pathogenic fungus Metarhizium robertsii is associated with conidial germination and fungal pathogenicity. Surprisingly, the germination of the ΔMrilvC mutant was restored when supplemented with the intermediates of BCAA metabolism rather than three BCAAs. The result was significantly different from that of plant-pathogenic fungi. Therefore, this report highlights that the intermediates in BCAA biosynthesis are indispensable for conidial germination of M. robertsii.

scholarly journals Analysis of the LIV System of Campylobacter jejuni Reveals Alternative Roles for LivJ and LivK in Commensalism beyond Branched-Chain Amino Acid Transport

2011 ◽  
Vol 193 (22) ◽  
pp. 6233-6243 ◽  
Author(s):  
Deborah A. Ribardo ◽  
David R. Hendrixson
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Campylobacter Jejuni ◽  
Transport System ◽  
Amino Acid Transport ◽  
Branched Chain Amino Acids ◽  
Acid Transport ◽  
Content Type ◽  
Branched Chain Amino Acid ◽  
Branched Chain

Campylobacter jejuniis a leading cause of diarrheal disease in humans and an intestinal commensal in poultry and other agriculturally important animals. These zoonotic infections result in significant amounts ofC. jejunipresent in the food supply to contribute to disease in humans. We previously found that a transposon insertion inCjj81176_1038, encoding a homolog of theEscherichia coliLivJ periplasmic binding protein of the leucine, isoleucine, and valine (LIV) branched-chain amino acid transport system, reduced the commensal colonization capacity ofC. jejuni81-176 in chicks.Cjj81176_1038is the first gene of a six-gene locus that encodes homologous components of theE. coliLIV system. By analyzing mutants with in-frame deletions of individual genes or pairs of genes, we found that this system constitutes a LIV transport system inC. jejuniresponsible for a high level of leucine acquisition and, to a lesser extent, isoleucine and valine acquisition. Despite each LIV protein being required for branched-chain amino acid transport, only the LivJ and LivK periplasmic binding proteins were required for wild-type levels of commensal colonization of chicks. All LIV permease and ATPase components were dispensable forin vivogrowth. These results suggest that the biological functions of LivJ and LivK for colonization are more complex than previously hypothesized and extend beyond a role for binding and acquiring branched-chain amino acids during commensalism. In contrast to other studies indicating a requirement and utilization of other specific amino acids for colonization, acquisition of branched-chain amino acids does not appear to be a determinant forC. jejuniduring commensalism.

Sign in / Sign up

Export Citation Format

Share Document