18 research outputs found
Identification and physiological characterization of phosphatidic acid phosphatase enzymes involved in triacylglycerol biosynthesis in Streptomyces coelicolor
BACKGROUND: Phosphatidic acid phosphatase (PAP, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. Despite the importance of PAP activity in TAG producing bacteria, studies to establish its role in lipid metabolism have been so far restricted only to eukaryotes. Considering the increasing interest of bacterial TAG as a potential source of raw material for biofuel production, we have focused our studies on the identification and physiological characterization of the putative PAP present in the TAG producing bacterium Streptomyces coelicolor. RESULTS: We have identified two S. coelicolor genes, named lppα (SCO1102) and lppβ (SCO1753), encoding for functional PAP proteins. Both enzymes mediate, at least in part, the formation of DAG for neutral lipid biosynthesis. Heterologous expression of lppα and lppβ genes in E. coli resulted in enhanced PAP activity in the membrane fractions of the recombinant strains and concomitantly in higher levels of DAG. In addition, the expression of these genes in yeast complemented the temperature-sensitive growth phenotype of the PAP deficient strain GHY58 (dpp1lpp1pah1). In S. coelicolor, disruption of either lppα or lppβ had no effect on TAG accumulation; however, the simultaneous mutation of both genes provoked a drastic reduction in de novo TAG biosynthesis as well as in total TAG content. Consistently, overexpression of Lppα and Lppβ in the wild type strain of S. coelicolor led to a significant increase in TAG production. CONCLUSIONS: The present study describes the identification of PAP enzymes in bacteria and provides further insights on the genetic basis for prokaryotic oiliness. Furthermore, this finding completes the whole set of enzymes required for de novo TAG biosynthesis pathway in S. coelicolor. Remarkably, the overexpression of these PAPs in Streptomyces bacteria contributes to a higher productivity of this single cell oil. Altogether, these results provide new elements and tools for future cell engineering for next-generation biofuels productionFil: Comba, Santiago. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Rosario. Instituto de BiologĂa Molecular y Celular de Rosario; ArgentinaFil: Menendez Bravo, SimĂłn MatĂas. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Rosario. Instituto de BiologĂa Molecular y Celular de Rosario; ArgentinaFil: Arabolaza, Ana Lorena. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Rosario. Instituto de BiologĂa Molecular y Celular de Rosario; ArgentinaFil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Rosario. Instituto de BiologĂa Molecular y Celular de Rosario; Argentin
Expanding the chemical diversity of natural esters by engineering a polyketide-derived pathway into Escherichia coli
Microbial fatty acid (FA)-derived molecules have emerged as promising alternatives to petroleum-based chemicals for reducing dependence on fossil hydrocarbons. However, native FA biosynthetic pathways often yield limited structural diversity, and therefore restricted physicochemical properties, of the end products by providing only a limited variety of usually linear hydrocarbons. Here we have engineered into Escherichia coli a mycocerosic polyketide synthase-based biosynthetic pathway from Mycobacterium tuberculosis and redefined its biological role towards the production of multi-methyl-branched-esters (MBEs) with novel chemical structures. Expression of FadD28, Mas and PapA5 enzymes enabled the biosynthesis of multi-methyl-branched-FA and their further esterification to an alcohol. The high substrate tolerance of these enzymes towards different FA and alcohol moieties resulted in the biosynthesis of a broad range of MBE. Further metabolic engineering of the MBE producer strain coupled this system to long-chain-alcohol biosynthetic pathways resulting in de novo production of branched wax esters following addition of only propionate.Fil: Menendez-Bravo, SimĂłn MatĂas. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de BiologĂa Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas; ArgentinaFil: Comba, Santiago. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de BiologĂa Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas; ArgentinaFil: Sabatini, MartĂn. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de BiologĂa Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario; ArgentinaFil: Arabolaza, Ana Lorena. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de BiologĂa Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario; ArgentinaFil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de BiologĂa Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas; Argentin
Genetic Alterations in Gliomas Remodel the Tumor Immune Microenvironment and Impact Immune-Mediated Therapies
High grade gliomas are malignant brain tumors that arise in the central nervous system, in patients of all ages. Currently, the standard of care, entailing surgery and chemo radiation, exhibits a survival rate of 14-17 months. Thus, there is an urgent need to develop new therapeutic strategies for these malignant brain tumors. Currently, immunotherapies represent an appealing approach to treat malignant gliomas, as the pre-clinical data has been encouraging. However, the translation of the discoveries from the bench to the bedside has not been as successful as with other types of cancer, and no long-lasting clinical benefits have been observed for glioma patients treated with immune-mediated therapies so far. This review aims to discuss our current knowledge about gliomas, their molecular particularities and the impact on the tumor immune microenvironment. Also, we discuss several murine models used to study these therapies pre-clinically and how the model selection can impact the outcomes of the approaches to be tested. Finally, we present different immunotherapy strategies being employed in clinical trials for glioma and the newest developments intended to harness the immune system against these incurable brain tumors.Fil: Garcia Fabiani, Maria Belen. University of Michigan; Estados Unidos. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; ArgentinaFil: Haase, Santiago. University of Michigan; Estados Unidos. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; ArgentinaFil: Comba, Andrea. University of Michigan; Estados Unidos. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; ArgentinaFil: Carney, Stephen. University of Michigan; Estados UnidosFil: McClellan, Brandon. University of Michigan; Estados UnidosFil: Banerjee, Kaushik. University of Michigan; Estados UnidosFil: Alghamri, Mahmoud S.. University of Michigan; Estados UnidosFil: Syed, Faisal. University of Michigan; Estados UnidosFil: Kadiyala, Padma. University of Michigan; Estados UnidosFil: Nuñez, Felipe. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Parque Centenario. Instituto de Investigaciones BioquĂmicas de Buenos Aires. FundaciĂłn Instituto Leloir. Instituto de Investigaciones BioquĂmicas de Buenos Aires; ArgentinaFil: Candolfi, Marianela. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de Investigaciones BiomĂ©dicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones BiomĂ©dicas; ArgentinaFil: Asad, Antonela SofĂa. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de Investigaciones BiomĂ©dicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones BiomĂ©dicas; ArgentinaFil: González, Nazareno. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de Investigaciones BiomĂ©dicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones BiomĂ©dicas; ArgentinaFil: Aikins, Marisa E.. University of Michigan; Estados UnidosFil: Schwendeman, Anna. University of Michigan; Estados UnidosFil: Moon, James J.. University of Michigan; Estados UnidosFil: Lowenstein, Pedro R.. University of Michigan; Estados UnidosFil: Castro, Maria G.. University of Michigan; Estados Unido
Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study
Summary
Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally.
Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies
have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of
the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income
countries globally, and identified factors associated with mortality.
Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to
hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis,
exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a
minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical
status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary
intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause,
in-hospital mortality for all conditions combined and each condition individually, stratified by country income status.
We did a complete case analysis.
Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital
diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal
malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome
countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male.
Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3).
Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income
countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups).
Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome
countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries;
p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients
combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11],
p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20
[1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention
(ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety
checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed
(ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of
parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65
[0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality.
Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome,
middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will
be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger
than 5 years by 2030
RegulaciĂłn del metabolismo lipĂdico en Streptomyces coelicolor
Streptomyces es un gĂ©nero bacteriano caracterizado por poseer un complejo ciclo de vida que involucra varios pasos de diferenciaciĂłn fisiolĂłgica y morfolĂłgica. Durante su desarrollo, las bacterias de este gĂ©nero suelen producir una amplia gama de metabolitos con actividad biolĂłgica, que han sido tradicionalmente utilizados como antibiĂłticos, antiparasitarios, antifĂşngicos, anticancerĂgenos, entre otros. Sin embargo, los Streptomyces son capaces tambiĂ©n de sintetizar y acumular triglicĂ©ridos (TAG) como reserva de energĂa y carbono. Esta caracterĂstica, si bien es inusual en bacterias, es compartida con otros gĂ©neros del orden de los Actinomicetales como Rhodoccocus, Nocardia y Mycobacterium.
A pesar de su diversificado metabolismo lipĂdico, los estudios en el gĂ©nero Streptomyces se han concentrado generalmente en el área de la producciĂłn de metabolitos secundarios, y muy poco se conoce acerca de cĂłmo este organismo sintetiza sus ácidos grasos y regula su flujo hacia fosfolĂpidos o TAG. Además, en S. coelicolor la sĂntesis de ácidos grasos y del metabolito secundario actinorrodina comparten un precursor clave, el malonil-CoA, lo que plantea un complejo escenario regulatorio para la sĂntesis de estos compuestos carbonados en este microorganismo.
AsĂ, como objetivo general de este trabajo de tesis nos propusimos identificar y caracterizar los mecanismos genĂ©ticos y bioquĂmicos que regulan la sĂntesis de ácidos grasos y lĂpidos de reserva en Streptomyces coelicolor. Para ello, en una primera etapa nos centramos en la caracterizaciĂłn de un regulador transcripcional de la sĂntesis de ácidos grasos en esta bacteria, FasR, con el fin de develar su rol fisiolĂłgico y entender de quĂ© manera lleva a cabo su funciĂłn regulatoria. Demostramos, mediante experimentos genĂ©ticos y bioquĂmicos, que FasR es un activador transcripcional del operĂłn fab (fabD-fabH-acpP-fabF), el cual codifica para las proteĂnas esenciales del sistema de sintasa de ácidos grasos de tipo II de este organismo. FasR lleva a cabo su funciĂłn uniĂ©ndose directamente a la regiĂłn corriente arriba del operĂłn fab, y su actividad estarĂa modulada por algĂşn metabolito relacionado con el metabolismo de lĂpidos.
A continuaciĂłn nos planteamos identificar el paso enzimático que genera el diacilglicerol (DAG), un sustrato clave para la sĂntesis de TAG. A pesar del rol que juegan estas molĂ©culas en las diferentes especies como reserva de energĂa y carbono, la ruta de abastecimiento de DAG para su sĂntesis es completamente desconocida en bacterias. En este apartado trabajamos bajo la hipĂłtesis de que enzimas fosfatidato fosfatasas de tipo 2 (PAP2) serĂan las responsables de defosforilar al intermediario ácido fosfatĂdico y generar asĂ el DAG necesario para la sĂntesis de TAG. A travĂ©s de análisis informáticos fuimos capaces de identificar tres probables proteĂnas con actividad PAP. El posterior estudio de las mismas revelĂł que sĂłlo dos de ellas, Lppα y Lppβ, eran capaces de defosforilar fosfatidato in vitro y tenĂan además un rol en el metabolismo lipĂdico de S. coelicolor. Una cepa mutante en ambos genes presentĂł niveles reducidos de TAG a tiempos cortos de crecimiento, equiparándose con los niveles de la cepa salvaje a tiempos mayores. Estas evidencias indican que si bien tanto Lppα como Lppβ estarĂan proveyendo DAG para la sĂntesis de TAG en S. coelicolor, existen en este organismo rutas alternativas de generaciĂłn de DAG que prevalecen a tiempos largos de cultivo.
Por Ăşltimo, con la informaciĂłn obtenida a partir de estos estudios básicos se reconstituyĂł la vĂa de sĂntesis de TAG en la bacteria no oleaginosa E. coli como prueba de concepto acerca de la funcionalidad de los genes caracterizados en la vĂa de sĂntesis de TAG en S. coelicolor. Para ello se utilizĂł como hospedador una cepa de E. coli ΔdgkA, que posee una deleciĂłn en el gen que codifica para una DAG quinasa capaz de reciclar el DAG hacia la sĂntesis de fosfolĂpidos. En este contexto se expresaron en forma heterĂłloga las enzimas fosfatidato fosfatasa Lppβ y DAG aciltransferasa Sco0958, que catalizan los dos Ăşltimos pasos de la sĂntesis de TAG. Si bien la sola expresiĂłn de Sco0958 demostrĂł ser suficiente para la sĂntesis de TAG en este contexto genĂ©tico, la expresiĂłn adicional de la fosfatasa Lppβ tuvo un efecto positivo en el contenido de TAG, confirmando su capacidad de generar el sustrato DAG limitante para la sĂntesis de TAG. Luego de una optimizaciĂłn de la cepa de E. coli productora de TAG, y de su cultivo en sistemas de alta densidad, fuimos capaces de obtener una producciĂłn de 722,1 mg TAG/L, lo que constituye el mayor tĂtulo de TAG reportado para E. coli recombinantes en la literatura. En conjunto, estos resultados muestran cĂłmo las evidencias que surgen a partir de estudios básicos pueden volcarse en el campo aplicado para resolver una necesidad particular. Sin embargo, a pesar de lograr el mayor tĂtulo de TAG reportado hasta el momento para E. coli, es necesario aĂşn incrementar la productividad volumĂ©trica de TAG en este sistema para lograr el establecimiento de este huĂ©sped heterĂłlogo como futura plataforma de biosĂntesis de lĂpidos neutros aptos para la producciĂłn de, por ejemplo, biocombustibles.Fil: Comba, Santiago. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario (IBR-CONICET); Argentina
EMERGING ENGINEERING PRINCIPLES FOR YIELD IMPROVEMENT IN MICROBIAL CELL DESIGN
Metabolic Engineering has undertaken a rapid transformation in the last ten years making real progress towards the production of a wide range of molecules and fine chemicals using a designed cellular host. However, the maximization of product yields through pathway optimization is a constant and central challenge of this field. Traditional methods used to improve the production of target compounds from engineered biosynthetic pathways in non-native hosts include: codon usage optimization, elimination of the accumulation of toxic intermediates or byproducts, enhanced production of rate-limiting enzymes, selection of appropriate promoter and ribosome binding sites, application of directed evolution of enzymes, and chassis re-circuit. Overall, these approaches tend to be specific for each engineering project rather than a systematic practice based on a more generalizable strategy. In this mini-review, we highlight some novel and extensive approaches and tools intended to address the improvement of a target product formation, founded in sophisticated principles such as dynamic control, pathway genes modularization, and flux modeling
Metabolic engineering of microorganisms for the production of structurally diverse esters
Conventional petroleum-based chemical industry, although economically still thriving, is now facing great socio-political challenges due to the increasing concerns on climate change and limited availability of fossil resources. In this context, microbial production of fuels and commodity oleochemicals from renewable biomass is being considered a promising sustainable alternative. The increasing understanding of cellular systems has enabled the redesign of microbial metabolism for the production of compounds present in many daily consumer products such as esters, waxes, fatty acids (FA) and fatty alcohols. Small aliphatic esters are important flavour and fragrance elements while long-chain esters, composed of FA esterified to fatty alcohols, are widely used in lubricant formulas, paints, coatings and cosmetics. Here, we review recent advances in the biosynthesis of these types of mono alkyl esters in vivo. We focus on the critical ester bond-forming enzymes and the latest metabolic engineering strategies employed for the biosynthesis of a wide range of products ranging from low-molecular-weight esters to waxy compounds.Fil: Menendez-Bravo, SimĂłn MatĂas. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de BiologĂa Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario; ArgentinaFil: Comba, Santiago. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de BiologĂa Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario; ArgentinaFil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de BiologĂa Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario; ArgentinaFil: Arabolaza, Ana Lorena. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de BiologĂa Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario; Argentin
Overexpression of a phosphatidic acid phosphatase type 2 leads to an increase in triacylglycerol production in oleaginous Rhodococcus strains
Oleaginous Rhodococcus strains are able to accumulate large amounts of triacylglycerol (TAG). Phosphatidic acid phosphatase (PAP) enzyme catalyzes the dephosphorylation of phosphatidic acid (PA) to yield diacylglycerol (DAG), a key precursor for TAG biosynthesis. Studies to establish its role in lipid metabolism have been mainly focused in eukaryotes but not in bacteria. In this work, we identified and characterized a putative PAP type 2 (PAP2) encoded by the ro00075 gene in Rhodococcus jostii RHA1. Heterologous expression of ro00075 in Escherichia coli resulted in a fourfold increase in PAP activity and twofold in DAG content. The conditional deletion of ro00075 in RHA1 led to a decrease in the content of DAG and TAG, whereas its overexpression in both RHA1 and Rhodococcus opacus PD630 promoted an increase up to 10 to 15% by cellular dry weight in TAG content. On the other hand, expression of ro00075 in the nonoleaginous strain Rhodococcus fascians F7 promoted an increase in total fatty acid content up to 7% at the expense of free fatty acid (FFA), DAG, and TAG fractions. Moreover, coexpression of ro00075 / atf 2 genes resulted in a fourfold increase in total fatty acid content by a further increase of the FFA and TAG fractions. The results of this study suggest that ro00075 encodes for a PAP2 enzyme actively involved in TAG biosynthesis. Overexpression of this gene, as single one or with an atf gene, provides an alternative approach to increase the biosynthesis and accumulation of bacterial oils as a potential source of raw material for biofuel production.Fil: Hernández, MartĂn Alejandro. Universidad Nacional de la Patagonia; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; ArgentinaFil: Comba, Santiago. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de BiologĂa Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario; ArgentinaFil: Arabolaza, Ana Lorena. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de BiologĂa Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario; ArgentinaFil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de BiologĂa Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario; ArgentinaFil: Alvarez, Hector Manuel. Universidad Nacional de la Patagonia; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; Argentin