scholarly journals Metabolic Engineering of Acinetobacter baylyi ADP1 for Improved Growth on Gluconate and Glucose

2014 ◽  
Vol 80 (22) ◽  
pp. 7021-7027 ◽  
Author(s):  
Matti Kannisto ◽  
Tommi Aho ◽  
Matti Karp ◽  
Ville Santala
Keyword(s):  
Growth Rate ◽  
Metabolic Engineering ◽  
Carbon Source ◽  
Pyruvate Kinase ◽  
Wax Ester ◽  
Similar Amount ◽  
Acinetobacter Baylyi ◽  
Kinase Gene ◽  
Content Type ◽  
Storage Lipids

ABSTRACTA high growth rate in bacterial cultures is usually achieved by optimizing growth conditions, but metabolism of the bacterium limits the maximal growth rate attainable on the carbon source used. This limitation can be circumvented by engineering the metabolism of the bacterium.Acinetobacter baylyihas become a model organism for studies of bacterial metabolism and metabolic engineering due to its wide substrate spectrum and easy-to-engineer genome. It produces naturally storage lipids, such as wax esters, and has a unique gluconate catabolism as it lacks a gene for pyruvate kinase. We engineered the central metabolism ofA. baylyiADP1 more favorable for gluconate catabolism by expressing the pyruvate kinase gene (pykF) ofEscherichia coli. This modification increased growth rate when cultivated on gluconate or glucose as a sole carbon source in a batch cultivation. The engineered cells reached stationary phase on these carbon sources approximately twice as fast as control cells carrying an empty plasmid and produced similar amount of biomass. Furthermore, when grown on either gluconate or glucose,pykFexpression did not lead to significant accumulation of overflow metabolites and consumption of the substrate remained unaltered. Increased growth rate on glucose was not accompanied with decreased wax ester production, and thepykF-expressing cells accumulated significantly more of these storage lipids with respect to cultivation time.

scholarly journals Wax ester production in nitrogen-rich conditions by metabolically engineered Acinetobacter baylyi ADP1

2019 ◽  
Author(s):  
Jin Luo ◽  
Elena Efimova ◽  
Pauli Losoi ◽  
Ville Santala ◽  
Suvi Santala
Keyword(s):  
Metabolic Engineering ◽  
Isocitrate Lyase ◽  
Fatty Acyl ◽  
Wax Esters ◽  
Wax Ester ◽  
High Carbon ◽  
Acinetobacter Baylyi ◽  
Storage Lipids ◽  
Carbon Nitrogen Ratio ◽  
Nitrogen Ratio

AbstractMetabolic engineering can be used as a powerful tool to redirect cell resources towards product synthesis, also in conditions that are not optimal. An example of a synthesis pathway strongly dependent on external conditions is the production of storage lipids, which typically requires high carbon/nitrogen ratio. Acinetobacter baylyi ADP1 is known for its ability to produce industrially interesting storage lipids, namely wax esters (WEs). Here, we engineered the central carbon metabolism of A. baylyi ADP1 by deletion of the gene aceA encoding for isocitrate lyase in order to allow redirection of carbon towards WEs. The production was further enhanced by overexpression of fatty acyl-CoA reductase Acr1 in the wax ester production pathway. This strategy led to 3-fold improvement in yield (0.075 g/g glucose) and 3.15-fold improvement in titer (1.82 g/L) and productivity (0.038 g/L/h) by a simple one-stage batch cultivation with glucose as carbon source. The engineered strain accumulated up to 27% WEs of cell dry weight. The titer and cellular WE content are the highest reported to date among microbes. We further showed that the engineering strategy alleviated the inherent requirement for high carbon/nitrogen ratio and demonstrated the production of wax esters using nitrogen-rich substrates including casamino acids, yeast extract and baker’s yeast hydrolysate, which support biomass production but not WE production in wild-type cells. The study demonstrates the power of metabolic engineering in overcoming natural limitations in the production of storage lipids.

Identification of an upstream activating sequence and an upstream repressible sequence of the pyruvate kinase gene of the yeast Saccharomyces cerevisiae

1989 ◽  
Vol 9 (2) ◽  
pp. 442-451
Author(s):  
M Nishizawa ◽  
R Araki ◽  
Y Teranishi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Pyruvate Kinase ◽  
Transcriptional Activation ◽  
Regulatory Elements ◽  
Noncoding Region ◽  
Yeast Cells ◽  
Kinase Gene ◽  
Yeast Saccharomyces Cerevisiae ◽  
Upstream Activating Sequence

To clarify carbon source-dependent control of the glycolytic pathway in the yeast Saccharomyces cerevisiae, we have initiated a study of transcriptional regulation of the pyruvate kinase gene (PYK). By deletion analysis of the 5'-noncoding region of the PYK gene, we have identified an upstream activating sequence (UASPYK1) located between 634 and 653 nucleotides upstream of the initiating ATG codon. The promoter activity of the PYK 5'-noncoding region was abolished when the sequence containing the UASPYK1 was deleted from the region. Synthetic UASPYK1 (26mer), in either orientation, was able to restore the transcriptional activity of UAS-depleted mutants when placed upstream of the TATA sequence located at -199 (ATG as +1). While the UASPYK1 was required for basal to intermediate levels of transcriptional activation, a sequence between -714 and -811 was found to be necessary for full activation. On the other hand, a sequence between -344 and -468 was found to be responsible for transcriptional repression of the PYK gene when yeast cells were grown on nonfermentable carbon sources. This upstream repressible sequence also repressed transcription, although to a lesser extent, when glucose was present in the medium. The possible mechanism for carbon source-dependent regulation of PYK expression through these cis-acting regulatory elements is discussed.

scholarly journals Multiple Pathways for Triacylglycerol Biosynthesis in Streptomyces coelicolor

2008 ◽  
Vol 74 (9) ◽  
pp. 2573-2582 ◽  
Author(s):  
Ana Arabolaza ◽  
Eduardo Rodriguez ◽  
Silvia Altabe ◽  
Hector Alvarez ◽  
Hugo Gramajo
Keyword(s):  
Mutant Strain ◽  
De Novo ◽  
Streptomyces Coelicolor ◽  
Lipid Production ◽  
Wax Ester ◽  
Acinetobacter Baylyi ◽  
Triple Mutant ◽  
Storage Lipids ◽  
Tag Biosynthesis

ABSTRACT The terminal reaction in triacylglyceride (TAG) biosynthesis is the esterification of diacylglycerol (DAG) with a fatty acid molecule. To study this reaction in Streptomyces coelicolor, we analyzed three candidate genes (sco0958, sco1280, and sco0123) whose products significantly resemble the recently identified wax ester synthase/acyl-coenzyme A (CoA):DAG acyltransferase (DGAT) from Acinetobacter baylyi. The deletion of either sco0123 or sco1280 resulted in no detectable decrease in TAG accumulation. In contrast, the deletion of sco0958 produced a dramatic reduction in neutral lipid production, whereas the overexpression of this gene yielded a significant increase in de novo TAG biosynthesis. In vitro activity assays showed that Sco0958 mediates the esterification of DAG using long-chain acyl-CoAs (C14 to C18) as acyl donors. The Km and V max values of this enzyme for myristoyl-CoA were 45 μM and 822 nmol mg−1 min−1, respectively. Significantly, the triple mutant strain was not completely devoid of storage lipids, indicating the existence of alternative TAG-biosynthetic routes. We present strong evidence demonstrating that the residual production of TAG in this mutant strain is mediated, at least in part, by an acyl-CoA-dependent pathway, since the triple mutant still exhibited DGAT activity. More importantly, there was substantial phospholipid:DGAT (PDAT) activity in the wild type and in the triple mutant. This is the first time that a PDAT activity has been reported for bacteria, highlighting the extreme metabolic diversity of this industrially important soil microorganism.

scholarly journals Identification of an upstream activating sequence and an upstream repressible sequence of the pyruvate kinase gene of the yeast Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (2) ◽  
pp. 442-451 ◽  
Author(s):  
M Nishizawa ◽  
R Araki ◽  
Y Teranishi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Pyruvate Kinase ◽  
Transcriptional Activation ◽  
Regulatory Elements ◽  
Noncoding Region ◽  
Yeast Cells ◽  
Kinase Gene ◽  
Yeast Saccharomyces Cerevisiae ◽  
Upstream Activating Sequence

To clarify carbon source-dependent control of the glycolytic pathway in the yeast Saccharomyces cerevisiae, we have initiated a study of transcriptional regulation of the pyruvate kinase gene (PYK). By deletion analysis of the 5'-noncoding region of the PYK gene, we have identified an upstream activating sequence (UASPYK1) located between 634 and 653 nucleotides upstream of the initiating ATG codon. The promoter activity of the PYK 5'-noncoding region was abolished when the sequence containing the UASPYK1 was deleted from the region. Synthetic UASPYK1 (26mer), in either orientation, was able to restore the transcriptional activity of UAS-depleted mutants when placed upstream of the TATA sequence located at -199 (ATG as +1). While the UASPYK1 was required for basal to intermediate levels of transcriptional activation, a sequence between -714 and -811 was found to be necessary for full activation. On the other hand, a sequence between -344 and -468 was found to be responsible for transcriptional repression of the PYK gene when yeast cells were grown on nonfermentable carbon sources. This upstream repressible sequence also repressed transcription, although to a lesser extent, when glucose was present in the medium. The possible mechanism for carbon source-dependent regulation of PYK expression through these cis-acting regulatory elements is discussed.

Evaluation of thioesterases from Acinetobacter baylyi for production of free fatty acids

2017 ◽  
Vol 63 (4) ◽  
pp. 321-329 ◽  
Author(s):  
Rahul Ukey ◽  
William E. Holmes ◽  
Rakesh Bajpai ◽  
Andrei Y. Chistoserdov
Keyword(s):  
Fatty Acids ◽  
Free Fatty Acids ◽  
Vaccenic Acid ◽  
Fold Increase ◽  
Coli Strain ◽  
Wax Ester ◽  
Acinetobacter Baylyi ◽  
E Coli ◽  
Storage Lipids ◽  
Ester Biosynthesis

Acinetobacter baylyi is one of few Gram-negative bacteria capable of accumulating storage lipids in the form of triacylglycerides and wax esters, which makes it an attractive candidate for production of lipophilic products, including biofuel precursors. Thioesterases play a significant dual role in the triacylglyceride and wax ester biosynthesis by either providing or removing acyl-CoA from this pathway. Therefore, 4 different thioesterase genes were cloned from Acinetobacter baylyi ADP1 and expressed in Escherichia coli to investigate their contribution to free fatty acids (FFAs) accumulation. Overexpression of the genes tesA′ (a leaderless form of the gene tesA) and tesC resulted in increased accumulation of FFAs when compared with the host E. coli strain. Overexpression of tesA′ showed a 1.87-fold increase in production of long-chain fatty acids (C16 to C18) over the host strain. Unlike TesC and the other investigated thioesterases, the TesA′ thioesterase also produced shorter chain FFAs (e.g., myristic acid) and unsaturated FFAs (e.g., cis-vaccenic acid (18:1Δ11)). A comparison of the remaining 3 A. baylyi ADP1 thioesterases (encoded by the tesB, tesC, and tesD genes) revealed that only the strain containing the tesC gene produced statistically higher levels of FFAs over the control, suggesting that it possesses the acyl-ACP thioesterase activity. Both E. coli strains containing the tesB and tesD genes produced levels of FFAs similar to those of the plasmid-free control E. coli strain, which indicates that TesB and TesD lack the acyl-ACP thioesterase activity.

scholarly journals Bacterial Evolution in High-Osmolarity Environments

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Spencer Cesar ◽  
Maya Anjur-Dietrich ◽  
Brian Yu ◽  
Ethan Li ◽  
Enrique Rojas ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Carbon Source ◽  
Cell Size ◽  
Metagenomic Sequencing ◽  
Evolutionary Mechanisms ◽  
Mean Cell Volume ◽  
Content Type ◽  
High Osmolarity ◽  
Trade Offs

ABSTRACT Bacteria must maintain a cytosolic osmolarity higher than that of their environment in order to take up water. High-osmolarity environments therefore present formidable stress to bacteria. To explore the evolutionary mechanisms by which bacteria adapt to high-osmolarity environments, we selected Escherichia coli in media with a variety of osmolytes and concentrations for 250 generations. Adaptation was osmolyte dependent, with sorbitol stress generally resulting in increased fitness under conditions with higher osmolarity, while selection in high concentrations of proline resulted in increased fitness specifically on proline. Consistent with these phenotypes, sequencing of the evolved populations showed that passaging in proline resulted in specific mutations in an associated metabolic pathway that increased the ability to utilize proline for growth, while evolution in sorbitol resulted in mutations in many different genes that generally resulted in improved growth under high-osmolarity conditions at the expense of growth at low osmolarity. High osmolarity decreased the growth rate but increased the mean cell volume compared with growth on proline as the sole carbon source, demonstrating that osmolarity-induced changes in growth rate and cell size follow an orthogonal relationship from the classical Growth Law relating cell size and nutrient quality. Isolates from a sorbitol-evolved population that captured the likely temporal sequence of mutations revealed by metagenomic sequencing demonstrated a trade-off between growth at high osmolarity and growth at low osmolarity. Our report highlights the utility of experimental evolution for dissecting complex cellular networks and environmental interactions, particularly in the case of behaviors that can involve both specific and general metabolic stressors. IMPORTANCE For bacteria, maintaining higher internal solute concentrations than those present in the environment allows cells to take up water. As a result, survival is challenging in high-osmolarity environments. To investigate how bacteria adapt to high-osmolarity environments, we maintained Escherichia coli in a variety of high-osmolarity solutions for hundreds of generations. We found that the evolved populations adopted different strategies to improve their growth rates depending on the osmotic passaging condition, either generally adapting to high-osmolarity conditions or better metabolizing the osmolyte as a carbon source. Single-cell imaging demonstrated that enhanced fitness was coupled to faster growth, and metagenomic sequencing revealed mutations that reflected growth trade-offs across osmolarities. Our study demonstrated the utility of long-term evolution experiments for probing adaptation occurring during environmental stress.

scholarly journals Differences in Substrate Specificities of Five Bacterial Wax Ester Synthases

2012 ◽  
Vol 78 (16) ◽  
pp. 5734-5745 ◽  
Author(s):  
Brett M. Barney ◽  
Bradley D. Wahlen ◽  
EmmaLee Garner ◽  
Jiashi Wei ◽  
Lance C. Seefeldt
Keyword(s):  
Kinetic Studies ◽  
Fatty Acyl ◽  
Biofuel Production ◽  
Wax Ester ◽  
Acinetobacter Baylyi ◽  
Content Type ◽  
Rapid Purification ◽  
Range Characterization ◽  
Acyl Coenzyme A

ABSTRACTWax esters are produced in certain bacteria as a potential carbon and energy storage compound. The final enzyme in the biosynthetic pathway responsible for wax ester production is the bifunctional wax ester synthase/acyl-coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT), which utilizes a range of fatty alcohols and fatty acyl-CoAs to synthesize the corresponding wax ester. We report here the isolation and substrate range characterization for five WS/DGAT enzymes from four different bacteria:Marinobacter aquaeoleiVT8,Acinetobacter baylyi,Rhodococcus jostiiRHA1, andPsychrobacter cryohalolentisK5. The results from kinetic studies of isolated enzymes reveal a differential activity based on the order of substrate addition and reveal subtle differences between the substrate selectivity of the different enzymes. Thesein vitroresults are compared to the wax ester and triacylglyceride product profiles obtained from each organism grown under neutral lipid accumulating conditions, providing potential insights into the role that the WS/DGAT enzyme plays in determining the final wax ester products that are produced under conditions of nutrient stress in each of these bacteria. Further, the analysis revealed that one enzyme in particular fromM. aquaeoleiVT8 showed the greatest potential for future study based on rapid purification and significantly higher activity than was found for the other isolated WS/DGAT enzymes. The results provide a framework to test prospective differences between these enzymes for potential biotechnological applications such as high-value petrochemicals and biofuel production.

scholarly journals Identification of a Residue Affecting Fatty Alcohol Selectivity in Wax Ester Synthase

2012 ◽  
Vol 79 (1) ◽  
pp. 396-399 ◽  
Author(s):  
Brett M. Barney ◽  
Rachel L. Mann ◽  
Janet M. Ohlert
Keyword(s):  
Kinetic Parameters ◽  
Fatty Alcohol ◽  
Biosynthetic Pathway ◽  
Fatty Acyl ◽  
Wax Ester ◽  
Acinetobacter Baylyi ◽  
Content Type ◽  
Marinobacter Aquaeolei ◽  
Acyl Coenzyme A ◽  
Wax Ester Synthase

ABSTRACT The terminal enzyme in the bacterial wax ester biosynthetic pathway is the bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT), which utilizes a fatty alcohol and a fatty acyl-coenzyme A (CoA) to synthesize the corresponding wax ester. In this report, we identify a specific residue in WS/DGAT enzymes obtained from Marinobacter aquaeolei VT8 and Acinetobacter baylyi that alters fatty alcohol selectivity and kinetic parameters when modified to alternative residues.

scholarly journals Charting the Metabolic Landscape of the Facultative Methylotroph Bacillus methanolicus

mSystems ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Baudoin Delépine ◽  
Marina Gil López ◽  
Marc Carnicer ◽  
Cláudia M. Vicente ◽  
Volker F. Wendisch ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Carbon Source ◽  
Industrial Chemistry ◽  
Content Type ◽  
Facultative Methylotroph ◽  
Cell Factories ◽  
Bacillus Methanolicus ◽  
Methanol Economy ◽  
Natural Strains ◽  
Potential Carbon

Methanol is inexpensive, is easy to transport, and can be produced both from renewable and from fossil resources without mobilizing arable lands. As such, it is regarded as a potential carbon source to transition toward a greener industrial chemistry. Metabolic engineering of bacteria and yeast able to efficiently consume methanol is expected to provide cell factories that will transform methanol into higher-value chemicals in the so-called methanol economy. Toward that goal, the study of natural methylotrophs such as Bacillus methanolicus is critical to understand the origin of their efficient methylotrophy. This knowledge will then be leveraged to transform such natural strains into new cell factories or to design methylotrophic capability in other strains already used by the industry.

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