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.

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 Dynamic decoupling of biomass and lipid biosynthesis by autonomously regulated switch

2018 ◽  
Author(s):  
Suvi Santala ◽  
Elena Efimova ◽  
Ville Santala
Keyword(s):  
Isocitrate Lyase ◽  
Glucose Dehydrogenase ◽  
Lipid Synthesis ◽  
Dry Weight ◽  
Carbon Flow ◽  
Wax Esters ◽  
Wax Ester ◽  
Acinetobacter Baylyi ◽  
Metabolic Switch ◽  
Engineered Strain

For improving the microbial production of fuels and chemicals, gene knock-outs and overexpression are routinely applied to intensify the carbon flow from substrate to product. However, their possibilities in dynamic control of the flux between the biomass and product synthesis are limited, whereas dynamic metabolic switches can be used for optimizing the distribution of carbon and resources. The production of single cell oils is especially challenging, as the synthesis is strongly regulated, competes directly with biomass, and requires defined conditions, such as nitrogen limitation. Here, we engineered a metabolic switch for redirecting carbon flow from biomass to wax ester production in Acinetobacter baylyi ADP1 using acetate as a carbon source. Isocitrate lyase, an essential enzyme for growth on acetate, was expressed under an arabinose inducible promoter. The autonomous downregulation of the expression is based on the gradual oxidation of the arabinose inducer by a glucose dehydrogenase gcd. The depletion of the inducer, occurring simultaneously to acetate consumption, switches the cells from a biomass mode to a lipid synthesis mode, enabling the efficient channelling of carbon to wax esters in a simple batch culture. In the engineered strain, the yield and titer of wax esters were improved by 3.8 and 3.1 folds, respectively, over the control strain. In addition, the engineered strain accumulated wax esters 19% of cell dry weight, being the highest reported among microbes. The study provides important insights into the dynamic engineering of the biomass-dependent synthesis pathways for the improved production of biocompounds from low-cost, sustainable substrates.

scholarly journals Fatty Alcohols for Wax Esters in Marinobacter aquaeolei VT8: Two Optional Routes in the Wax Biosynthesis Pathway

2013 ◽  
Vol 79 (22) ◽  
pp. 7055-7062 ◽  
Author(s):  
Eric M. Lenneman ◽  
Janet M. Ohlert ◽  
Nagendra P. Palani ◽  
Brett M. Barney
Keyword(s):  
Fatty Alcohol ◽  
Fatty Acyl ◽  
Wax Esters ◽  
Transcriptional Analysis ◽  
Wax Ester ◽  
Fatty Aldehyde ◽  
Marinobacter Aquaeolei

ABSTRACTThe biosynthesis of wax esters in bacteria is accomplished by a unique pathway that combines a fatty alcohol and a fatty acyl coenzyme A substrate. Previousin vitroenzymatic studies indicated that two different enzymes could be involved in the synthesis of the required fatty alcohol inMarinobacter aquaeoleiVT8. In this study, we demonstrate through a series of gene deletions and transcriptional analysis that either enzyme is capable of fulfilling the role of providing the fatty alcohol required for wax ester biosynthesisin vivo, but evolution has clearly selected one of these, a previously characterized fatty aldehyde reductase, as the preferred enzyme to perform this reaction under typical wax ester-accumulating conditions. These results complement previousin vitrostudies and provide the first glimpse into the role of each enzymein vivoin the native organism.

scholarly journals A Fatty Acyl Coenzyme A Reductase Promotes Wax Ester Accumulation in Rhodococcus jostii RHA1

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
James Round ◽  
Raphael Roccor ◽  
Shu-Nan Li ◽  
Lindsay D. Eltis
Keyword(s):  
Specific Activity ◽  
Dry Weight ◽  
Fatty Acyl ◽  
Wax Esters ◽  
Wax Ester ◽  
Wild Type ◽  
Considerable Potential ◽  
Commodity Chemicals ◽  
Acyl Coenzyme A ◽  
Rhodococcus Jostii

ABSTRACT Many rhodococci are oleaginous and, as such, have considerable potential for the sustainable production of lipid-based commodity chemicals. Herein, we demonstrated that Rhodococcus jostii RHA1, a soil bacterium that catabolizes a wide range of organic compounds, produced wax esters (WEs) up to 0.0002% of its cellular dry weight during exponential growth on glucose. These WEs were fully saturated and contained primarily 31 to 34 carbon atoms. Moreover, they were present at higher levels during exponential growth than under lipid-accumulating conditions. Bioinformatics analyses revealed that RHA1 contains a gene encoding a putative fatty acyl coenzyme A (acyl-CoA) reductase (FcrA). The purified enzyme catalyzed the NADPH-dependent transformation of stearoyl-CoA to stearyl alcohol with a specific activity of 45 ± 3 nmol/mg · min and dodecanal to dodecanol with a specific activity of 5,300 ± 300 nmol/mg · min. Deletion of fcrA did not affect WE accumulation when grown in either carbon- or nitrogen-limited medium. However, the ΔfcrA mutant accumulated less than 20% of the amount of WEs as the wild-type strain under conditions of nitric oxide stress. A strain of RHA1 overproducing FcrA accumulated WEs to ∼13% cellular dry weight under lipid-accumulating conditions, and their acyl moieties had longer average chain lengths than those in wild-type cells (C17 versus C16). The results provide insight into the biosynthesis of WEs in rhodococci and facilitate the development of this genus for the production of high-value neutral lipids. IMPORTANCE Among the best-studied oleaginous bacteria, rhodococci have considerable potential for the sustainable production of lipid-based commodity chemicals, such as wax esters. However, many aspects of lipid synthesis in these bacteria are poorly understood. The current study identifies a key enzyme in wax ester synthesis in rhodococci and exploits it to significantly improve the yield of wax esters in bacteria. In so doing, this work contributes to the development of novel bioprocesses for an important class of oleochemicals that may ultimately allow us to phase out their unsustainable production from sources such as petroleum and palm oil.

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 Bio-Floc Technology: Prospects & Challenges in Fish Farming of Nepal

2020 ◽  
Vol 8 (2) ◽  
pp. 140-145
Author(s):  
Prajina Neupane ◽  
Madhusudhan Adhikari ◽  
Manita Kumari Thapa ◽  
Astha Kiran Pandeya
Keyword(s):  
Water Quality ◽  
Carbon Source ◽  
Nutrient Recycling ◽  
Bacterial Biomass ◽  
Fish Farming ◽  
High Carbon ◽  
Carbon Nitrogen Ratio ◽  
Nitrogen Ratio ◽  
Technical Manpower ◽  
Blue Revolution

Bio-floc technology is the blue revolution in aquaculture & new technique of enhancing water quality & utilizing feed wastes in the aquaculture system. It follows the concept of conversion of ammonium in addition to organic nitrogenous wastes into bacterial biomass in where heterotrophic bacterial growth is stimulated & nitrogen uptake through the production of microbial proteins is promoted by the addition of carbohydrates to the pond. Nitrogen generated by uneaten feed and excreta of cultured organisms is converted into proteinaceous feed available for those same organisms. This technique recycles nutrients & nitrogenous wastes by maintaining a high carbon: nitrogen ratio and provides essential & higher quality nutrition to the shrimps & fishes in achieving fast growth, lesser FCR & possibility to prevent diseases. Water requirement in BFT is extremely less & it is advantageous than the conventional system where there is continuous water & nutrient recycling, lower FCR. On the other hand, many challenges are existing in practicing bio-floc in Nepal as it requires frequent pond monitoring by the technical manpower. The choice of carbon source should be made wisely and correctly as the performance of fish and water quality in the bio-floc ponds depend highly upon carbon source. Further, vitamins required for fish may not be produced by microbes thus needed to identify them and supply through the feed. However, the practice of bio-floc technology will be proven worth for farmers in Nepal. Int. J. Appl. Sci. Biotechnol. Vol 8(2): 140-145  

scholarly journals Alkane and wax ester production from lignin derived molecules

2018 ◽  
Author(s):  
Milla Salmela ◽  
Tapio Lehtinen ◽  
Elena Efimova ◽  
Suvi Santala ◽  
Ville Santala
Keyword(s):  
Value Added ◽  
Product Formation ◽  
Wax Esters ◽  
Wax Ester ◽  
Acinetobacter Baylyi ◽  
Bacterial Host ◽  
Synthetic Pathway ◽  
Growth Product ◽  
Lignin Depolymerization ◽  
Value Added Products

AbstractLignin has potential as a sustainable feedstock for microbial production of industrially relevant molecules. However, the required lignin depolymerization yields a heterogenic mixture of aromatic monomers that are challenging substrates for the microorganisms commonly used in industry. Here, we investigated the properties of lignin-derived molecules (LDMs), namely coumarate, ferulate, and caffeate, in the synthesis of biomass and products in a LDM-utilizing bacterial hostAcinetobacter baylyiADP1. The biosynthesis products, wax esters and alkanes, are relevant compounds for the chemical and fuel industries. InA. baylyiADP1, wax esters are produced by a native pathway, whereas alkanes are produced by a synthetic pathway introduced to the host. Using individual LDMs as substrates, the growth, product formation, and toxicity to cells were monitored with internal biosensors. Of the tested LDMs, coumarate was the most propitious in terms of product synthesis. Wax esters were produced from coumarate with a yield and titer of 40 mg /gcoumarateand 221 mg/L, whereas alkanes were produced with a yield of 62.3 μg /gcoumarateand titer of 152 μg/L. This study demonstrates the microbial preference for certain LDMs, and highlights the potential ofA. baylyiADP1 as a convenient host for LDM upgrading to value-added products.

scholarly journals Plastidial Wax Ester Biosynthesis As A Tool To Synthesize Shorter And More Saturated Wax Esters

2021 ◽  
Author(s):  
Katharina Vollheyde ◽  
Ellen Hornung ◽  
Cornelia Herrfurth ◽  
Till Ischebeck ◽  
Ivo Feussner
Keyword(s):  
Transgenic Plants ◽  
Fossil Fuel ◽  
Neutral Lipids ◽  
Fatty Acyl ◽  
Wax Esters ◽  
Wax Ester ◽  
In Planta ◽  
Alternative Source ◽  
Transit Peptides ◽  
Ester Biosynthesis

Abstract Background: Wax esters (WE) are neutral lipids that consist of a fatty alcohol esterified to a fatty acid. WE are valuable feedstocks in industry for producing lubricants, coatings and cosmetics. They can be produced chemically from fossil fuel or plant derived triacylglycerol. As fossil fuel resources are finite, the synthesis of WE in transgenic plants may serve as an alternative source. As chain length and desaturation degree of the alcohol and acyl moieties determine the physicochemical properties of WE and their field of application, a tightly controlled tailor-made WE synthesis in plants is aimed for. Here, we report the generation of ten combinations of WE producing transgenes expressed in Arabidopsis thaliana. In order to study their suitability for WE production in planta, we analyzed WE amount and synthesized WE species in the transgenic plants.Results: The transgenes consisted of different combinations of a fatty acyl-CoA/ACP reductase (FAR) and two wax synthases/acyl-CoA:diacylglycerol O-acyltransferases (WSD) namely WSD2 and WSD5 from the bacterium Marinobacter aquaeoleoi. We generated constructs with and without plastidial transit peptides to access diverse alcohol and acyl substrate pools within A. thaliana. We observed WE formation with plastid and cytosol-localized FAR and WSD in seeds. A comparative WE analysis revealed the production of shorter and more saturated WE by plastid-localized WE biosynthesis compared to cytosolic WE synthesis.Conclusions: A shift of WE formation into seed plastids is a suitable approach for tailor-made WE production and can be used to synthesize WE mainly derived from mid and long chain saturated and monounsaturated substrates.

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 Triacylglycerol and phytyl ester synthesis inSynechocystissp. PCC6803

2020 ◽  
Vol 117 (11) ◽  
pp. 6216-6222 ◽  
Author(s):  
Mohammed Aizouq ◽  
Helga Peisker ◽  
Katharina Gutbrod ◽  
Michael Melzer ◽  
Georg Hölzl ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Acyl Carrier Protein ◽  
Sequence Similarity ◽  
Wax Esters ◽  
Oxygenic Photosynthesis ◽  
Wax Ester ◽  
Ester Synthesis ◽  
Acyltransferase Activity ◽  
Storage Lipids ◽  
Triacylglycerol Synthesis

Cyanobacteria are unicellular prokaryotic algae that perform oxygenic photosynthesis, similar to plants. The cells harbor thylakoid membranes composed of lipids related to those of chloroplasts in plants to accommodate the complexes of photosynthesis. The occurrence of storage lipids, including triacylglycerol or wax esters, which are found in plants, animals, and some bacteria, nevertheless remained unclear in cyanobacteria. We show here that the cyanobacteriumSynechocystissp. PCC6803 accumulates both triacylglycerol and wax esters (fatty acid phytyl esters). Phytyl esters accumulate in higher levels under abiotic stress conditions. The analysis of an insertional mutant revealed that the acyltransferase slr2103, with sequence similarity to plant esterase/lipase/thioesterase (ELT) proteins, is essential for triacylglycerol and phytyl ester synthesis inSynechocystis. The recombinant slr2103 enzyme showed acyltransferase activity with phytol and diacylglycerol, thus producing phytyl esters and triacylglycerol. Acyl-CoA thioesters were the preferred acyl donors, while acyl-ACP (acyl carrier protein), free fatty acids, or galactolipid-bound fatty acids were poor substrates. The slr2103 protein sequence is unrelated to acyltransferases from bacteria (AtfA) or plants (DGAT1, DGAT2, PDAT), and therefore establishes an independent group of bacterial acyltransferases involved in triacylglycerol and wax ester synthesis. The identification of the geneslr2103responsible for triacylglycerol synthesis in cyanobacteria opens the possibility of using prokaryotic photosynthetic cells in biotechnological applications.

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