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 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 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 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.

On the nutrition and metabolism of zooplankton XI. Lipids in Calanus helgolandicus grazing Biddulphia sinensis

1977 ◽  
Vol 57 (2) ◽  
pp. 525-533 ◽  
Author(s):  
J. R. Sargent ◽  
R. R. Gatten ◽  
E. D. S. Corner ◽  
C. C. Kilvington
Keyword(s):  
Brood Size ◽  
Developmental Stages ◽  
Dry Weight ◽  
Wax Esters ◽  
Wax Ester ◽  
Calanoid Copepods ◽  
Lipid Levels ◽  
History Of ◽  
Northern Pacific

Many of the calanoid copepods contain large amounts of neutral lipid that is predominantly wax esters (Lee, Hirota & Barnett, 1971; Lee & Hirota, 1973; Sargent & Garten, 1976). The role of these compounds in the life-history of copepods is particularly well documented for the large Northern Pacific calanoid Euchaeta japonica Marukawa. These animals have their highest levels of total lipid (50% of the dry weight) at stage V, up to 80% being wax esters (Lee, Nevenzel & Lewis, 1974). Wax ester levels are lower in adult females but are high in the eggs, the contents of which are subsequently used during the naupliar stages (Lee et al. 1974). Thus, wax esters accumulated during the later developmental stages provide a fuel for the developing eggs and embryos; from which it follows that the amounts of these lipids present in mature animals are likely to be critical in determining the brood size and survival of the ensuing generation. So far, however, the factors controlling lipid levels in copepods have been poorly understood.

The lipid, fatty acid and fatty alcohol composition of the myctophid fish Electrona antarctica: high level of wax esters and food-chain implications

1997 ◽  
Vol 9 (3) ◽  
pp. 258-265 ◽  
Author(s):  
Charles F. Phleger ◽  
Peter D. Nichols ◽  
Patti Virtue
Keyword(s):  
Fatty Acids ◽  
Total Lipid ◽  
Dry Weight ◽  
Monounsaturated Fatty Acids ◽  
Wax Esters ◽  
Wax Ester ◽  
Lipid Fatty Acid ◽  
Myctophid Fish ◽  
High Level ◽  
Elephant Island

The myctophid, Electrona antarctica, was collected by trawl from the Elephant Island region of the Antarctic Peninsula, and from East Antarctica near 61°S and 93°W. Total lipid was higher in Elephant Island E. antarctica (whole fish, 466–585 mg g−1 dry weight) than those from Eastern Antarctica (394–459 mg g−1). Wax esters comprised 86.2–90.5% of total lipid in E. antarctica flesh. There were no significant differences between Eastern Antarctica and Elephant Island in total wax ester levels, or in levels of wax esters between different tissues analysed. Oily bones (up to 326 mg g−1 in the neurocranium) characterized E. antarctica from both locations, with wax esters as the major skeletal lipid class (67.0–87.9%, percent of lipid). The wax esters may have a buoyancy role in E. antarctica. The only substantial amount of triacylglycerols (29.4%) were found in the viscera of Elephant Island fish. The principal fatty acids of all fish analysed included the monounsaturated fatty acids 18:1(n-9) and 16:1(n-7), with lower levels of 16:0 and 14:0 saturated acids. Fatty alcohols were dominated by the saturated 16:0 and 14:0 (37.8–47.8%) and the monounsaturated 18:1(n-9) and 18:1(n-7) (38.3–59.2%). The low ratio of 22:1/20:1 alcohols observed for E. antarctica is consistent with a diet of amphipods, copepods and other items low in 22:1 alcohols.

Wax Esters in the Marine Environment: Origin and Composition of the Wax from Bute Inlet, British Columbia

1979 ◽  
Vol 36 (12) ◽  
pp. 1519-1523 ◽  
Author(s):  
Richard F. Lee ◽  
Judd C. Nevenzel
Keyword(s):  
British Columbia ◽  
Marine Pollution ◽  
Major Change ◽  
Dry Weight ◽  
Wax Esters ◽  
Wax Ester ◽  
Winter Temperatures ◽  
The World ◽  
Major Hydrocarbon ◽  
Fatty Acid Compositions

During severe winters large amounts of a solid lipid accumulate on the water surface and on the shores of Bute Inlet, a deep fjord on the mainland of British Columbia. Chromatographic analyses show that this wax consists chiefly of wax esters (48%), with minor amounts of hydrocarbons (4%) and triacylglycerols (6%). Dominant among the zooplankton in this inlet is the copepod Calanus plumchrus, which also is rich in wax esters (40–50% of dry weight). Except for the lack of polyunsaturation in Bute Inlet wax there is a close similarity between the wax ester, alcohol, and fatty acid compositions of C. plumchrus and Bute Inlet wax. The major hydrocarbon of both is pristane. We hypothesize that Bute Inlet wax is produced by some major change in water conditions which cause a massive kill of copepods. At winter temperatures this lipid is a solid and as such may accumulate on the beach. Copepods are the likely source for the wax esters in slicks and sediments reported from oceanic areas throughout the world. Key words: marine pollution, biological pollutant, lipid, hydrocarbon, wax, zooplankton, Copepoda

scholarly journals Do minke whales (Balaenoptera acutorostrata) digest wax esters?

1995 ◽  
Vol 74 (5) ◽  
pp. 717-722 ◽  
Author(s):  
Erling S. Nordøy
Keyword(s):  
Energy Density ◽  
Dry Matter ◽  
General Pattern ◽  
Dry Mass ◽  
Wax Esters ◽  
Gut Contents ◽  
Wax Ester ◽  
Balaenoptera Acutorostrata ◽  
Minke Whales

Mammals are known to utilize wax esters with an efficiency of less than 50%. The purpose of the present study was to examine whether or not minke whales (Balaenoptera acutorostrata), which at times may eat considerable amounts of wax-ester-rich krill, represent an exception to this general pattern. Samples of fresh undigested forestomach, as well as colon, contents were obtained from minke whales (n5) that had been feeding on krill (Thysanoessa inermis) for some time. The samples were analysed for dry mass, energy density, lipid content and the major lipid classes, including wax esters. The concentrations of wax esters were compared with previous estimates of dry-matter disappearance of the same type of prey using anin vitrotechnique, to calculate the dry-matter digestibility of wax esters (DMDwax). Wax esters contributed 21% of the energy and 47% of total lipids in the krill diet. The energy density of gut contents decreased by 50% after their passage from forestomach to the end of the colon. The DMDwaxwas 94·1 (SD 2·8)% (n5). This high DMDwaxand the occurrence of fatty alcohols, one of the products of wax-ester hydrolysis, in faeces show that minke whales are very efficient digesters of wax esters and absorb most of the energy-rich products of this process.

scholarly journals Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

2010 ◽  
Vol 76 (9) ◽  
pp. 2884-2894 ◽  
Author(s):  
Efraín Manilla-Pérez ◽  
Alvin Brian Lange ◽  
Stephan Hetzler ◽  
Marc Wältermann ◽  
Rainer Kalscheuer ◽  
...  
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Marine Bacterium ◽  
Neutral Lipids ◽  
Nile Red ◽  
Dry Weight ◽  
Wax Ester ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Key Enzyme

ABSTRACT In many microorganisms, the key enzyme responsible for catalyzing the last step in triacylglycerol (TAG) and wax ester (WE) biosynthesis is an unspecific acyltransferase which is also referred to as wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT; AtfA). The importance and function of two AtfA homologues (AtfA1 and AtfA2) in the biosynthesis of TAGs and WEs in the hydrocarbon-degrading marine bacterium Alcanivorax borkumensis SK2 have been described recently. However, after the disruption of both the AtfA1 and AtfA2 genes, reduced but substantial accumulation of TAGs was still observed, indicating the existence of an alternative TAG biosynthesis pathway. In this study, transposon-induced mutagenesis was applied to an atfA1 atfA2 double mutant to screen for A. borkumensis mutants totally defective in biosynthesis of neutral lipids in order to identify additional enzymes involved in the biosynthesis of these lipids. At the same time, we have searched for a totally TAG-negative mutant in order to study the function of TAGs in A. borkumensis. Thirteen fluorescence-negative mutants were identified on Nile red ONR7a agar plates and analyzed for their abilities to synthesize lipids. Among these, mutant 2 M131 was no longer able to synthesize and accumulate TAGs if pyruvate was used as the sole carbon source. The transposon insertion was localized in a gene encoding a putative cytochrome c family protein (ABO_1185). Growth and TAG accumulation experiments showed that the disruption of this gene resulted in the absence of TAGs in 2 M131 but that growth was not affected. In cells of A. borkumensis SK2 grown on pyruvate as the sole carbon source, TAGs represented about 11% of the dry weight of the cells, while in the mutant 2 M131, TAGs were not detected by thin-layer and gas chromatography analyses. Starvation and lipid mobilization experiments revealed that the lipids play an important role in the survival of the cells. The function of neutral lipids in A. borkumensis SK2 is discussed.

CHANGES IN ENZYMES OF CARBOHYDRATE METABOLISM IN RAT UTERUS DURING EARLY PREGNANCY

1971 ◽  
Vol 68 (4) ◽  
pp. 805-816 ◽  
Author(s):  
M. A. H. Surani ◽  
P. J. Heald
Keyword(s):  
Energy Requirement ◽  
Lipid Synthesis ◽  
Dry Weight ◽  
Glycolytic Enzymes ◽  
Rat Uterus ◽  
Malic Dehydrogenase ◽  
Phosphogluconate Dehydrogenase ◽  
Tissue Changes ◽  
Implantation Sites ◽  
Glucose 6 Phosphate Dehydrogenase

ABSTRACT The enzymes phosphofructokinase (PFK), pyruvate kinase (PK), isocitric dehydrogenase (ICDH), malic dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G-6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) have been measured in rat uterus during the first 9 days of pregnancy. It was found that after implantation on day 6, the activities of PFK and PK (the key glycolytic enzymes) increased in terms of dry weight — and in terms of protein in the implantation sites, but decreased in non-implanted tissue. The pentose shunt enzymes changed similarly to those of the glycolytic enzymes. ICDH activity increased in the non-implanted tissue and decreased in the implanted tissue. Changes in malic dehydrogenase were extremely variable and did not show a consistent pattern. Administration of Actinomycin D on day 6 of pregnancy abolished the increase in PK and PFK in the implantation sites and indeed led to a major decrease in activity. This implies that the increased PK and PFK in the implantation sites, arise from a DNA dependent RNA directed synthesis of new enzyme protein. The results are discussed in relation to the energy requirement of the decidualising tissue and the need for increased pentose for RNA synthesis. It is suggested that the extra NADPH resulting from the pentose shunt is involved in increased lipid synthesis.

Metabolism of Food Reserves in Germinating Velvetleaf

Weed Science ◽  
1970 ◽  
Vol 18 (5) ◽  
pp. 565-571
Author(s):  
J. A. Mulliken ◽  
C. A. Kust ◽  
L. E. Schrader
Keyword(s):  
Hydrogen Cyanide ◽  
Isocitrate Lyase ◽  
Dry Weight ◽  
Maximal Activity ◽  
Radicle Growth ◽  
Fat Mobilization ◽  
Lyase Activity ◽  
Radicle Emergence ◽  
Weight Protein ◽  
Food Reserves

Endosperm dry weight, protein, and fat losses accompanied rapid radicle growth of velvetleaf (Abutilon theophrasti Medic.) between 12 and 36 hr of germination at 31 C. Cotyledonary reserves were mobilized after 36 hr. Isocitrate lyase activity sedimented with a particulate fraction in varying degrees, but maximal activity developed at times coincident with fat mobilization. Respiration of excised endosperms reached maximal rates shortly after radicle emergence. The actions of hydrogen cyanide, carbon monoxide, and 2,4-dinitrolphenol indicated that respiration of endosperms excised from imbibed and germinated seed was due to cytochrome oxidase activity, and was coupled to phosphorylation.

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