Inactivation of Yarrowia lipolytica YIACL2 gene coding subunit of ATP citrate lyase using CRISPR/Cas9 system

2020 ◽  
Vol 36 (1) ◽  
pp. 16-24
Author(s):  
T.V. Yuzbashev ◽  
E.B. Vinogradova ◽  
I.M. Kosikhina ◽  
M.O. Taratynova ◽  
D.A. Dementev ◽  
...  
Keyword(s):  
Carbon Source ◽  
Yarrowia Lipolytica ◽  
Traditional Approach ◽  
Atp Citrate Lyase ◽  
Alternative Source ◽  
Gene Coding ◽  
Citrate Lyase ◽  
Mutant Strains ◽  
The Media ◽  
Single Carbon Source

In this study, Y1ACL2 was inactivated by two methods: traditional approach based on homologous recombination and uracil marker and markerless system using CRISPR/Cas9. The efficiency of YIACL2 inactivation using traditional approach was 4% (one ΔYlacl2 strain out of 24 tested transformants) whereas knockout efficiency using CRISPR/Cas9 system was 75% (18 ΔYlacl2 strains out of 24 tested transformants). YIACL2 null mutant strains were not able to utilize citrate as a single carbon source. Growth kinetics was investigated in the media with glucose and acetate as a single carbon source. The fact that ΔYlacl2 is able to grow in the minimal medium with glucose as a single carbon source provides evidence that there is an alternative source of acetyl-CoA on carbohydrate substrates in Y. lipolytica. Yarrowia lipolytica, CRISPR/Cas9 system, ATP citrate lyase, YIACL2. The work was carried out using the equipment of the Unique Scientific Facility of BRC VKPM with technical support of the Centre for Collective Use of NRC «Kurchatov Institute» -GosNIIgenetika and with financial support of Russian Federation (state task No. 595-00003-19 PR) as well as partially supported by grant No. MK-2241.2019.7.

Inactivation of Yarrowia lipolytica YlACL2 gene Coding Subunit of ATP Citrate Lyase Using CRISPR/Cas9 System

2020 ◽  
Vol 56 (9) ◽  
pp. 885-892
Author(s):  
E. Y. Yuzbasheva ◽  
T. V. Yuzbashev ◽  
E. B. Vinogradova ◽  
I. M. Kosikhina ◽  
M. O. Taratynova ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Atp Citrate Lyase ◽  
Gene Coding ◽  
Citrate Lyase

scholarly journals ATP-Citrate lyase fuels axonal transport across species

2020 ◽  
Author(s):  
Aviel Even ◽  
Giovanni Morelli ◽  
Romain Le Bail ◽  
Michal Shilian ◽  
Silvia Turchetto ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Molecular Motors ◽  
Recessive Mutation ◽  
Projection Neurons ◽  
Atp Citrate Lyase ◽  
Tubulin Acetylation ◽  
Gene Coding ◽  
Citrate Lyase ◽  
Autosomal Recessive Mutation ◽  
Dependent Transport

AbstractMicrotubule (MT)-based transport is an evolutionary conserved processed finely tuned by posttranslational modifications. Among them, α-tubulin acetylation, which is catalyzed by the α-tubulin N-acetyltransferase 1, Atat1, promotes the recruitment and processivity of molecular motors along MT tracks. However, the mechanisms that controls Atat1 activity remains poorly understood. Here, we show that a pool of vesicular ATP-citrate lyase Acly acts as a rate limiting enzyme to modulate Atat1 activity by controlling availability of Acetyl-Coenzyme-A (Acetyl-CoA). In addition, we showed that Acly expression is reduced upon loss of Elongator activity, further connecting Elongator to Atat1 in the pathway regulating α-tubulin acetylation and MT-dependent transport in projection neurons, across species. Remarkably, comparable defects occur in fibroblasts from Familial Dysautonomia (FD) patients bearing an autosomal recessive mutation in the gene coding for the Elongator subunit ELP1. Our data may thus shine new light on the pathophysiological mechanisms underlying FD.

Analysis of the Yarrowia lipolytica proteome reveals subtle variations in expression levels between lipogenic and non-lipogenic conditions

2021 ◽  
Vol 21 (2) ◽  
Author(s):  
Ryan Sestric ◽  
Vic Spicer ◽  
Oleg V Krokhin ◽  
Richard Sparling ◽  
David B Levin
Keyword(s):  
Yarrowia Lipolytica ◽  
Carbon Flux ◽  
Neutral Lipids ◽  
Pyruvate Decarboxylase ◽  
Atp Citrate Lyase ◽  
Expression Levels ◽  
Rich Media ◽  
Citrate Lyase ◽  
The Relationship

ABSTRACT Oleaginous yeasts have the ability to store greater than 20% of their mass as neutral lipids, in the form of triacylglycerides. The ATP citrate lyase is thought to play a key role in triacylglyceride synthesis, but the relationship between expression levels of this and other related enzymes is not well understood in the role of total lipid accumulation conferring the oleaginous phenotype. We conducted comparative proteomic analyses with the oleaginous yeast, Yarrowia lipolytica, grown in either nitrogen-sufficient rich media or nitrogen-limited minimal media. Total proteins extracted from cells collected during logarithmic and late stationary growth phases were analyzed by 1D liquid chromatography, followed by mass spectroscopy. The ATP citrate lyase enzyme was expressed at similar concentrations in both conditions, in both logarithmic and stationary phase, but many upstream and downstream enzymes showed drastically different expression levels. In non-lipogenic conditions, several pyruvate enzymes were expressed at higher concentration. These enzymes, especially the pyruvate decarboxylase and pyruvate dehydrogenase, may be regulating carbon flux away from central metabolism and reducing the amount of citrate being produced in the mitochondria. While crucial for the oleaginous phenotype, the constitutively expressed ATP citrate lyase appears to cleave citrate in response to carbon flux upstream from other enzymes creating the oleaginous phenotype.

scholarly journals ATP-citrate lyase promotes axonal transport across species

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Aviel Even ◽  
Giovanni Morelli ◽  
Silvia Turchetto ◽  
Michal Shilian ◽  
Romain Le Bail ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Molecular Motors ◽  
Recessive Mutation ◽  
Projection Neurons ◽  
Atp Citrate Lyase ◽  
Tubulin Acetylation ◽  
Gene Coding ◽  
Citrate Lyase ◽  
Autosomal Recessive Mutation ◽  
Dependent Transport

AbstractMicrotubule (MT)-based transport is an evolutionary conserved process finely tuned by posttranslational modifications. Among them, α-tubulin acetylation, primarily catalyzed by a vesicular pool of α-tubulin N-acetyltransferase 1 (Atat1), promotes the recruitment and processivity of molecular motors along MT tracks. However, the mechanism that controls Atat1 activity remains poorly understood. Here, we show that ATP-citrate lyase (Acly) is enriched in vesicles and provide Acetyl-Coenzyme-A (Acetyl-CoA) to Atat1. In addition, we showed that Acly expression is reduced upon loss of Elongator activity, further connecting Elongator to Atat1 in a pathway regulating α-tubulin acetylation and MT-dependent transport in projection neurons, across species. Remarkably, comparable defects occur in fibroblasts from Familial Dysautonomia (FD) patients bearing an autosomal recessive mutation in the gene coding for the Elongator subunit ELP1. Our data may thus shine light on the pathophysiological mechanisms underlying FD.

Cloning and Expression Analysis of ATP-Citrate Lyase Genes from Sugarcane

2013 ◽  
Vol 38 (11) ◽  
pp. 2024-2033 ◽  
Author(s):  
Chang-Ning LI ◽  
Qian NONG ◽  
Qin-Liang TAN ◽  
SRIVASTAVA Manoj Kumar ◽  
Li-Tao YANG ◽  
...  
Keyword(s):  
Expression Analysis ◽  
Cloning And Expression ◽  
Atp Citrate Lyase ◽  
Citrate Lyase

Acetyl-CoA is produced by the citrate synthase homology module of ATP-citrate lyase

2021 ◽  
Author(s):  
Kenneth Verstraete ◽  
Koen H. G. Verschueren ◽  
Ann Dansercoer ◽  
Savvas N. Savvides
Keyword(s):  
Citrate Synthase ◽  
Atp Citrate Lyase ◽  
Acetyl Coa ◽  
Citrate Lyase ◽  
Homology Module

scholarly journals IGF1-mediated HOXA13 overexpression promotes colorectal cancer metastasis through upregulating ACLY and IGF1R

2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Chenyang Qiao ◽  
Wenjie Huang ◽  
Jie Chen ◽  
Weibo Feng ◽  
Tongyue Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Growth Factor ◽  
Cancer Metastasis ◽  
Combined Treatment ◽  
Insulin Like Growth Factor ◽  
Atp Citrate Lyase ◽  
Citrate Lyase ◽  
Elevated Expression ◽  
Igf1r Inhibitor ◽  
Colorectal Cancer Metastasis

AbstractMetastasis is the major reason for the high mortality of colorectal cancer (CRC) patients and its molecular mechanism remains unclear. Here, we report a novel role of Homeobox A13 (HOXA13), a member of the Homeobox (HOX) family, in promoting CRC metastasis. The elevated expression of HOXA13 was positively correlated with distant metastasis, higher AJCC stage, and poor prognosis in two independent CRC cohorts. Overexpression of HOXA13 promoted CRC metastasis whereas downregulation of HOXA13 suppressed CRC metastasis. Mechanistically, HOXA13 facilitated CRC metastasis by transactivating ATP-citrate lyase (ACLY) and insulin-like growth factor 1 receptor (IGF1R). Knockdown of ACLY and IGFIR inhibited HOXA13-medicated CRC metastasis, whereas ectopic overexpression of ACLY and IGFIR rescued the decreased CRC metastasis induced by HOXA13 knockdown. Furthermore, Insulin-like growth factor 1 (IGF1), the ligand of IGF1R, upregulated HOXA13 expression through the PI3K/AKT/HIF1α pathway. Knockdown of HOXA13 decreased IGF1-mediated CRC metastasis. In addition, the combined treatment of ACLY inhibitor ETC-1002 and IGF1R inhibitor Linsitinib dramatically suppressed HOXA13-mediated CRC metastasis. In conclusion, HOXA13 is a prognostic biomarker in CRC patients. Targeting the IGF1-HOXA13-IGF1R positive feedback loop may provide a potential therapeutic strategy for the treatment of HOXA13-driven CRC metastasis.

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