An efficient conversion of carboxylic acids to one-carbon degraded aldehydes via 2-hydroperoxy acids

After the formation of dianions of a carboxylic acid with lithium diisopropylamide, oxygen was bubbled into the solution to produce 2-hydroperoxy acid. Then the reaction mixture was acidified with a 2 N HCl solution and subsequently elevated to 50 degrees C to afford the aldehyde with the loss of one carbon atom. Even saturated (C(10)-C(20)) and unsaturated (C(18:1)) carboxylic acids were converted into the odd aldehydes (C(9)-C(19), C(17:1)) in high yields. This conversion was found to be an efficient method for the preparation of carboxylic acids (Cn) to one-carbon degraded aldehydes (Cn-1) via 2-hydroperoxy acids.     

Lipoxygenase-mediated cleavage of fatty acids to carbonyl fragments in tomato fruits

Homogenates of tomato fruits catalysed the enzymic conversion of linoleic and linolenic acids (but not oleic acid) to C₆ aldehydes in low (3–5%) molar yield. Hexanal was formed from linoleic acid; cis-3-hexenal and smaller amounts of trans-2-hexenal were formed from linolenic acid. With the fatty acids as substrates, the major products were fatty acid hydroperoxides (50–80% yield) and the ratio of 9- to 13-hydroperoxides as isolated from an incubation with linoleic acid was at least 95:5 in favour of the 9-hydroperoxide isomer. When the 9- and 13-hydroperoxides of linoleic acid were used as substrates with tomato homogenates, the 13-hydroperoxide was readily cleaved to hexanal in high molar yield (60%) but the 9-hydroperoxide isomer was not converted to cleavage products. Properties of the hydroperoxide cleavage system are described. The results indicate that the C₆ aldehydes are formed from C₁₈ polyunsaturated fatty acids in a sequential enzyme system involving lipoxygenase (which preferentially oxygenates at the 9-position) followed by a hydroperoxide cleavage system which is, however, specific for the 13-hydroperoxy isomers.     

Accumulation of l-Glutamic Acid from Dibasic Carboxylic Acids by a Hydrocarbon Utilizing Bacterium

In order to know the substrate specificity in a hydrocarbon utilizing bacterium, the following materials were examined: n-alkanes, n-alkenes, monohydric alcohols, aldehydes, monobasic carboxylic acids, dihydric alcohols and dibasic carboxylic acids.

It was found that dibasic carboxylic acids were well utilized, and a great deal of l-glutamic acid was accumulated from them. Then suberic acid, which is C₈ dibasic carboxylic acid, was compared with n-dodecane in the effects of thiamine, penicillin, C/N ratio and substrate concentration on l-glutamic acid accumulation and cell growth.     

Inhibition of Topoisomerases by Fatty Acids

The inhibitory effects of various fatty acids on topoisomerases were examined, and their structure-activity relationships and mechanism of action were studied. Saturated fatty acids (C_6:0 to C_22:0) did not inhibit topoisomerase I, but cis-unsaturated fatty acids (C_16:1 to C_22:1) with one double bond showed strong inhibition of the enzyme. The inhibitory potency depended on the carbon chain length and the position of the double bond in the fatty acid molecule. The trans-isomer, methyl ester and hydroxyl derivative of oleic acid had no or little inhibitory effect on topoisomerases I and II. Among the compounds studied petroselinic acid and vaccenic acid (C_18:1) with a cis-double bond were the potent inhibitors. Petroselinic acid was a topoisomerase inhibitor of the cleavable complex-nonforming type and acted directly on the enzyme molecule in a noncompetitive manner without DNA intercalation.     

Cis-diastereoselectivity in pictet-spengler reactions of L-tryptophan and electronic circular dichroism studies

The diastereoselective synthesis of optically active 1,3‐disubstituted tetrahydro‐β‐carbolines using polar protic Pictet–Spengler cyclization of (S)‐tryptophan methyl ester with five aldehydes RCHO (R═CH₃, C₂H₅, C₃H₇, C₄H₉, and C₆H₅) was studied. As an alternate route, the cyclization of (S)‐tryptophan with the same aldehydes and subsequent methylation of the resulting tetrahydro‐β‐carboline carboxylic acids were also performed for comparison. ¹³C NMR and electronic circular dichroism (ECD) studies and time‐dependent density functional theory ECD calculations data established the relative 1,3 cis/trans and the absolute configuration (1S,3S/ 1R,3S) of the synthesized compounds. The solid‐state and solution ECD study of the prepared compounds, supported by ECD calculation and X‐ray data, afforded a reliable ECD method for the configurational assignment of 1,3‐disubstituted tetrahydro‐β‐carbolines and revealed the stereochemical factors that determine the characteristic ECD data. Chirality 24:789–795, 2012. © 2012 Wiley Periodicals, Inc.     

α-Oxidation of Long-chain Unsaturated Fatty Acids in the Marine Green Alga Ulva pertusa

When long-chain unsaturated fatty acids such as oleic, linoleic, and linolenic acid were incubated with crude enzymes from the marine green alga Ulva pertusa, the corresponding (R)-2-hydroperoxy acids were formed with a high enantiomeric excess (>99%).     

Aldehyde metabolism and the aroma quality of stored golden delicious apples

Aldehyde production by intact apples was monitored by reversed phase HPLC of headspace concentrates, after reaction with 2,4-dinitrophenylhydrazine. Depending on the degree of maturity and their storage history, Golden Delicious apples showed a variable headspace composition, differences being mostly of a quantitative nature. Whereas the headspace of pre-climacteric fruits was particularly rich in C₁C₆ aldehydes, that of climacteric, ripening apples was greatly reduced, and some aldehydes were only present in trace amounts. Treatment of pre-climacteric or cold stored fruits with carboxylic acid vapours had a negligible effect on the aldehyde composition. Controlled atmospheric storage, however, led to a notable increase in the aldehydes derived from the added carboxylic acids or from those shortened by β-oxidation. This confirms the presence of a reductive path of carboxylic acids into aldehydes. Further results suggest that high carbon dioxide (CA-storage) interferes with carboxylic acid metabolism and alcohol dehydrogenase activity, leading to a deterioration of the aroma quality.     

Metabolism of Chiral Ionylideneacetic Acids on the Abscisic Acid Biosynthetic Pathway in Cercospora

A Chiralcel OJ column was used to determine the absolute configuration of naturally occurring α-ionylideneacetic acid from Cercospora rosicola and γ-ionylideneacetic acid from C. cruenta as (R) enantiomers in accordance with their biosynthetic product, (S)-ABA. Both enantiomers of [1, 2-¹³C₂]-α and γ-ionylideneacetic acids were prepared and fed to C. rosicola and C. cruenta. Six combinations of feeding experiments comparatively and unequivocally demonstrated stereoselectivity in the biosynthetic conversions, including stepwise hydroxylation at C-1′ and 4′. Enzymatic isomerization from the γ to α-intermediate was suggested not to be involved in ABA biosynthesis in C. rosicola.     

CHANGES OF FATTY ACIDS IN LARVAE OF THE WHEAT BLOSSOM MIDGE,SITODIPLOSIS MOSELLANA (GEHIN) (DIPTERA: CECIDOMYIIDAE)*

Abstract The changes of fatty acids in larvae of the wheat blossom midge, Sitodiplosis mosellana (Gehin) at different periods were examined by gas choromatography. There were 10–16 kinds of fatty acids, of which the predominant ingredients were palmitic (C_16:0), oleic (C_18:1) and linoleic (C_18:2) acids which were more than 95% in total fatty acids, stearic acid (C_18:0) about 2%‐3.5% and any of the others was less than 1%. The fatty acid compositions increased from mid‐May, when larvae of the wheat blossom midge left the wheat‐ears and fallen on the ground, to April of next year before pupating and emerging. No arachidic acid (C_20.0) was discovered in over‐summering, over‐wintering as well as inactive over‐wintered larvae. The content of saturated fatty acids in over‐summering, overwintering as well as inactive over‐wintered larvae were less than those of in active over‐wintered larvae and wheat‐ear larvae. Therefore, changes of the arachidic acid and the proportions of saturated fatty acids/unsaturated fatty acids could be used as one of the biochemical criteria to determine the active state and the degree of diapause in larvae of the wheat blossom midge.     

Fatty Acids in the Species of Several Zygomycete Taxa

The composition of fatty acids synthesized de novo by thirty strains of zygomycetes from various taxa was studied. The qualitative fatty acid compositions of the fungal lipids were found to be virtually identical, but there were significant differences in the contents of individual acids. Highly active producers of essential C₁₈ fatty acids, with their content exceeding 30–40% of total fatty acids, were discovered among the fungi of the families Mucoraceae, Pilobolaceae, and Radiomycetaceae. Linoleic acid was found to predominate in the fungi of the genera Radiomyces, Mycotypha, and Circinella, and linolenic acid (identified as its -isomer by gas-liquid chromatography), in the fungi of the genera Absidia, Circinella, Pilaira, and Hesseltinella. The total yield (mg/l) of bioactive acids (C_18:3, C_18:2, C_18:1) varied from 761.4 in Pilaira anomala to 3477.9 in Syncephalastrum racemosum; the total yield of essential acids, from 520.7 in Pilaira anomala to 1154.5 in Hesseltinella vesiculosa; of linoleic acid, from 279.7 in Pilaira anomala to 836.3 in Mycotypha indica; and of linolenic acid, from 120.8 in Mycotypha indica to 708.0 in Hesseltinella vesiculosa. The data on the efficient synthesis of these acids make the actively producing strains promising for biotechnological synthesis of commercially valuable lipids. Linderina pennispora VKM F-1219, a zygomycete of the family Kickxellaceae, which was earlier singled out into the order Kickxellales, was shown to differ from zygomycetes of the order Mucorales in having a high content of cis-9-hexadecenoic (palmitoleic) acid, reaching 37.0% of the fatty acid total.     

A recombinant α-dioxygenase from rice to produce fatty aldehydes using <Emphasis Type="Italic">E. coli</Emphasis>

Fatty aldehydes are an important group of fragrance and flavor compounds that are found in different fruits and flowers. A biotechnological synthesis of fatty aldehydes based on Escherichia coli cells expressing an α-dioxygenase (αDOX) from Oryza sativa (rice) is presented. α-Dioxygenases are the initial enzymes of α-oxidation in plants and oxidize long and medium-chain C _n fatty acids to 2-hydroperoxy fatty acids. The latter are converted to C _n − 1 fatty aldehydes by spontaneous decarboxylation. Successful expression of αDOX in E. coli was proven by an in vitro luciferase assay. Using resting cells of this recombinant E. coli strain, conversion of different fatty acids to the respective fatty aldehydes shortened by one carbon atom was demonstrated. The usage of Triton X 100 improves the conversion rate up to 1 g aldehyde per liter per hour. Easy reuse of the cells was demonstrated by performing a second biotransformation without any loss of biocatalytic activity.     

Overexpression of hydroperoxide lyase,peroxygenase and epoxide hydrolase in tobacco for the biotechnological production of flavours and polymer precursors

Plants produce short‐chain aldehydes and hydroxy fatty acids, which are important industrial materials, through the lipoxygenase pathway. Based on the information that lipoxygenase activity is up‐regulated in tobacco leaves upon infection with tobacco mosaic virus (TMV), we introduced a melon hydroperoxide lyase (CmHPL) gene, a tomato peroxygenase (SlPXG) gene and a potato epoxide hydrolase (StEH) into tobacco leaves using a TMV‐based viral vector system to afford aldehyde and hydroxy fatty acid production. Ten days after infiltration, tobacco leaves infiltrated with CmHPL displayed high enzyme activities of 9‐LOX and 9‐HPL, which could efficiently transform linoleic acid into C₉ aldehydes. Protein extracts prepared from 1 g of CmHPL‐infiltrated tobacco leaves (fresh weight) in combination with protein extracts prepared from 1 g of control vector‐infiltrated tobacco leaves (as an additional 9‐LOX source) produced 758 ± 75 μg total C₉ aldehydes in 30 min. The yield of C₉ aldehydes from linoleic acid was 60%. Besides, leaves infiltrated with SlPXG and StEH showed considerable enzyme activities of 9‐LOX/PXG and 9‐LOX/EH, respectively, enabling the production of 9,12,13‐trihydroxy‐10(E)‐octadecenoic acid from linoleic acid. Protein extracts prepared from 1 g of SlPXG‐infiltrated tobacco leaves (fresh weight) in combination with protein extracts prepared from 1 g of StEH‐infiltrated tobacco leaves produced 1738 ± 27 μg total 9,12,13‐trihydroxy‐10(E)‐octadecenoic acid isomers in 30 min. The yield of trihydroxyoctadecenoic acids from linoleic acid was 58%. C₉ aldehydes and trihydroxy fatty acids could likely be produced on a larger scale using this expression system with many advantages including easy handling, time‐saving and low production cost.     

Anti‐Androgenic Activity of Fatty Acids

In this study, we show that 5α‐reductase derived from rat fresh liver was inhibited by certain aliphatic free fatty acids. The influences of chain length, unsaturation, oxidation, and esterification on the potency to inhibit 5α‐reductase activity were studied. Among the fatty acids we tested, inhibitory saturated fatty acids had C₁₂–C₁₆ chains, and the presence of a C?C bond enhanced the inhibitory activity. Esterification and hydroxy compounds were totally inactive. Finally, we tested the prostate cancer cell proliferation effect of free fatty acids. In keeping with the results of the 5α‐reductase assay, saturated fatty acids with a C₁₂ chain (lauric acid) and unsaturated fatty acids (oleic acid and α‐linolenic acid) showed a proliferation inhibitory effect on lymph‐node carcinoma of the prostate (LNCaP) cells. At the same time, the testosterone‐induced prostate‐specific antigen (PSA) mRNA expression was down‐regulated. These results suggested that fatty acids with 5α‐reductase inhibitory activity block the conversion of testosterone to 5α‐dihydrotestosterone (DHT) and then inhibit the proliferation of prostate cancer cells.     

SEASONAL CHANGES IN THE PROXIMATE AND FATTY ACID COMPOSITION OF SOME NATURALLY GROWN FRESHWATER CHLOROPHYTES1

The protein content of the filamentous Cladophora glomerata (L.) Kz., Ulothrix zonata (Web, & Mohr) Kz. and Spirogyra sp., collected from natural populations for 1 year, averaged 8.0–12.4% of the total dry weight; whereas, the corresponding levels of lipid, cellulose and ash were 11.9–16.1%, 10.0–17.8% and 14.6–24.0%, respectively. Mean values for carbohydrates, estimated by difference, ranged from 32.8 to 56.0%. The colonial Scenedesmus dimorphus (Turp.) Kz. and the unicellular Cosmarium laeve Rab., on the other hand, contained more protein, lipid and carbohydrate (estimated by difference) averaging 13–15.0%, 22.5–25.9% and 415–46.8%, respectively, and less cellulose (7.5–9.8%) and ash (8.2–9.8%). A consistent pattern of seasonal variation in the proximate composition was not normally evident for any species, reflecting the influence of several environmental parameters on the algae. Cladophora contained the greatest amount of phospholipid averaging; 10% by weight of total lipid with the smallest quantity (5%) in Scenedesmus. The predominant phospholipid fatty acid in all species was C_18:1 followed by C_18:2, C_18:3 and C_16:1 in Cladophora, Ulothrix and Spirogyra, and C_16:1, C_18:2 and C_16:0 in Scenedesmus and Cosmarium. Oleic (C_18:1) and hexadecanoic (C_16:1) acids were predominant in the neutral lipids of all the algae, followed by C_16:0, C_18:2 and C_18:3. The concentration of the different fatty acids of each Species varied considerably during the year with the proportion of C_16:0 and C_16:1, usually rising and that of C_18:1 failing during the colder months.     

Whole‐cell biocatalytic and de novo production of alkanes from free fatty acids in Saccharomyces cerevisiae

Rapid global industrialization in the past decades has led to extensive utilization of fossil fuels, which resulted in pressing environmental problems due to excessive carbon emission. This prompted increasing interest in developing advanced biofuels with higher energy density to substitute fossil fuels and bio‐alkane has gained attention as an ideal drop‐in fuel candidate. Production of alkanes in bacteria has been widely studied but studies on the utilization of the robust yeast host, Saccharomyces cerevisiae, for alkane biosynthesis have been lacking. In this proof‐of‐principle study, we present the unprecedented engineering of S. cerevisiae for conversion of free fatty acids to alkanes. A fatty acid α‐dioxygenase from Oryza sativa (rice) was expressed in S. cerevisiae to transform C_12–18 free fatty acids to C_11–17 aldehydes. Co‐expression of a cyanobacterial aldehyde deformylating oxygenase converted the aldehydes to the desired alkanes. We demonstrated the versatility of the pathway by performing whole‐cell biocatalytic conversion of exogenous free fatty acid feedstocks into alkanes as well as introducing the pathway into a free fatty acid overproducer for de novo production of alkanes from simple sugar. The results from this work are anticipated to advance the development of yeast hosts for alkane production. Biotechnol. Bioeng. 2017;114: 232–237. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Inhibition of the β-class carbonic anhydrases from Mycobacterium tuberculosis with carboxylic acids

The growth of Mycobacterium tuberculosis is strongly inhibited by weak acids although the mechanism by which these compounds act is not completely understood. A series of substituted benzoic acids, nipecotic acid, ortho- and para-coumaric acid, caffeic acid and ferulic acid were investigated as inhibitors of three β-class carbonic anhydrases (CAs, EC 4.2.1.1) from this pathogen, mtCA 1 (Rv1284), mtCA 2 (Rv3588c) and mtCA 3 (Rv3273). All three enzymes were inhibited with efficacies between the submicromolar to the micromolar one, depending on the scaffold present in the carboxylic acid. mtCA 3 was the isoform mostly inhibited by these compounds (K_Is in the range of 0.11–0.97 µM); followed by mtCA 2 (K_Is in the range of 0.59–8.10 µM), whereas against mtCA 1, these carboxylic acids showed inhibition constants in the range of 2.25–7.13 µM. This class of relatively underexplored β-CA inhibitors warrant further in vivo studies, as they may have the potential for developing antimycobacterial agents with a diverse mechanism of action compared to the clinically used drugs for which many strains exhibit multi-drug or extensive multi-drug resistance.     

Functionalized cobalt clusters. Properties of carboxylate clusters (CO)6Co2HCCCOOH and (CO)10Co4HCCCOOH as related to their structures

Reaction of propiolic acid (HCCCOOH) with dicobalt octacarbonyl (Co₂(CO)₈) and tetracobalt dodecacarbonyl (Co₄(CO)₁₂) leads to the organometallic carboxylic acids (CO)₆Co₂HCCCOOH (1) and (CO)₁₀Co₄HCCCOOH (2) in good yield. Both organometallic compounds show a cobalt carbonyl core bonded to a carboxylate function. The structure of the acetylene-carboxylic group in both clusters deviates from that of ethylene. The C(1)C(2)C(3) fragment is half way between acrylic and acetylene-carboxylic acid. The comparative acidity of the carboxylic group measured in methanol reveals that (1) is a stronger acid than (2), but less acidic than propiolic acid. Both organometallic carboxylic acids are thermally decomposed into phases with high metal content at relatively low temperatures. Fenske-Hall calculations on this series of cobaltocarbonyl cluster carboxylic acids confirm that the Co(CO)₃ donates electron density to the HCCCOOH fragment, thus decreasing the acidity of the carboxylic function. The redox properties and the electronic spectra are also well correlated to the HOMO-LUMO gap thus calculated by this non-parametric SCF method.     

Structure Determination of Fatty Acids from Tunicamycin Complex

The structures of ten fatty acids, which were obtained by the hydrolysis of tunicamycin complex, were determined. GLC-mass, ¹H NMR and IR spectra showed that the major acids were trans-α, β-unsaturated iso acids with the formula C₁₄H₂₈O₂, C₁₆H₂₈O₂, C₁₆H₃₀O₂ and C₁₇H₃₂O₂. The minor acids were α, β-unsaturated normal acids and saturated normal and iso acids.     

The distribution of fatty acids including petroselinic and tariric acids in the fruit and seed oils of the Pittosporaceae, Araliaceae, Umbelliferae, Simarubaceae and Rutaceae

The fatty acid composition of the fruit oils or seed oils of Pittosporaceae (eight genera, 10 species), Araliaceae (two species), Simarubaceae (three species), and of one umbelliferous and one rutaceous species were determined by gas chromatography, argentation TLC and ozonolysis. In the Pittosporaceae, in which the major C₁₈ fatty acid of all species was either oleic acid (18:1, 9c) or linoleic acid (18:2, 9c, 12c), large amounts of C₂₀ and C₂₂ fatty acids seem to occur regularly. Petroselinic (18:1, 6c) and tariric (18:1, 6a) acids were absent. However, petroselinic acid was the major fatty acid in the Araliaceae and Umbelliferae. In these two families only small amounts of C₂₀ and C₂₂ acids were detected and tariric acid was absent. The Rutales contained relatively high amounts of trans-octadecenoic acids (18:1, 9t). Tariric acid was the major fatty acid in the two species of Picramnia (Simarubraceae), which also contained small amounts of petroselinic acid. The major fatty acids in Ailanthus glandulosa (Simarubaceae) and Phellodendron amurense (Rutaceae) were linoleic or linolenic acid (18:3, 9c, 12c, 15c); these species contained neither tariric nor petroselinic acid and the levels of C₂₀ and C₂₂ fatty acids were low. The appearance of schizogenous resin canals and polyacetylenes and the absence of iridoids and petroselinic acid allows the Pittosporaceae to be separated from the Rutales and Araliales and to be placed in an independent order, the Pittosporales. Arguments for a rather close relationship of the Pittosporales to the Araliales and Cornales (including the Escalloniaceae) are presented.     

Systems metabolic engineering design: Fatty acid production as an emerging case study

Increasing demand for petroleum has stimulated industry to develop sustainable production of chemicals and biofuels using microbial cell factories. Fatty acids of chain lengths from C₆ to C₁₆ are propitious intermediates for the catalytic synthesis of industrial chemicals and diesel‐like biofuels. The abundance of genetic information available for Escherichia coli and specifically, fatty acid metabolism in E. coli, supports this bacterium as a promising host for engineering a biocatalyst for the microbial production of fatty acids. Recent successes rooted in different features of systems metabolic engineering in the strain design of high‐yielding medium chain fatty acid producing E. coli strains provide an emerging case study of design methods for effective strain design. Classical metabolic engineering and synthetic biology approaches enabled different and distinct design paths towards a high‐yielding strain. Here we highlight a rational strain design process in systems biology, an integrated computational and experimental approach for carboxylic acid production, as an alternative method. Additional challenges inherent in achieving an optimal strain for commercialization of medium chain‐length fatty acids will likely require a collection of strategies from systems metabolic engineering. Not only will the continued advancement in systems metabolic engineering result in these highly productive strains more quickly, this knowledge will extend more rapidly the carboxylic acid platform to the microbial production of carboxylic acids with alternate chain‐lengths and functionalities. Biotechnol. Biotechnol. Bioeng. 2014;111: 849–857. © 2014 Wiley Periodicals, Inc.