The accD3 gene for mycolic acid biosynthesis as a target for improving fatty acid production by fatty acid-producing Corynebacterium glutamicum strains

We have recently developed Corynebacterium glutamicum strains that produce free fatty acids in culture supernatant due to enhanced fatty acid biosynthesis. Of these producing strains, the basic producer PAS-15 has a defect in the gene for a fatty acid biosynthesis repressor protein, and the advanced producer PCC-6 has two additional mutations to augment the production by strain PAS-15. The aim of the present study was to obtain novel genetic traits for improving fatty acid production by these producers. A new mutant with increased production derived from strain PAS-15 had a missense mutation in the accD3 gene (mutation accD3^A433T), which is involved in the biosynthesis of mycolic acids that are cell envelope lipids of C. glutamicum, as the causal mutation. Mutation accD3^A433T was verified to reduce the AccD3 enzymatic activity and increase fatty acid production in strain PAS-15 by 1.8-fold. Deletion of the accD3 gene in strain PAS-15, which was motivated by the characteristic of mutation accD3^A433T, increased fatty acid production by 3.2-fold. Susceptibility of strain PAS-15 to vancomycin was significantly increased by accD3 gene deletion and by mutation accD3^A433T to the intermediate level, suggesting that the cell envelope permeability barrier by mycolic acids is weakened by this engineering. Furthermore, mutation accD3^A433T also increased fatty acid production in strain PCC-6 by 1.3-fold. These increased production levels were suggested to be involved not only in the redirection of carbon flux from mycolic acid biosynthesis to fatty acid production but also in the permeability of the cell envelope.

     

Etude “in vitro” des acides gras synthétisés dans les microsomes de cerveaux de souris normales et “quaking”

Radiogas chromatographic studies of the products of fatty acid biosynthesis in mice brain microsomes confirm the existence of a «de novo system from acetyl-CoA and malonyl-CoA and of a least two elongating systems for long chain fatty acids, involving malonyl-CoA. The possibility of an intermediary system leading from C₁₈ to C₂₀ fatty acids has been evoked.Comparison between non mutant and quaking mice indicates that all the microsomal fatty acid biosynthetic systems are depressed. The biosynthetic system elongating fatty acids from C₁₈ is the one which is the most modified quantitatively and qualitatively in quaking. Microsomal and soluble «de novo systems are qualitatively intact.     

Effets physiologiques de ľ atrazine à doses sublétales sur Lemna minor, VII. Incorporation ďacétate-l,2-[14C] dans les groupes de lipides et leurs acides gras

Physiological effects of sublethal doses of atrazine on Lemna minor. VII. 1,2-[¹⁴C] acetate incorporation into the groups of lipids and their fatty acids. The lipids and the fatty acids of ten-day old duckweed (Lemna minor L.), cultivated aseptically in mineral solution containing sublethal concentrations of 0,10 and 0,50 ppm (0.46 and 2.3 μM, respectively) of atrazine, were analyzed by thin-layer chromatography and gas-liquid radiochromatography after 1,2-[¹⁴C] acetate feeding. Sublethal concentrations of atrazine increased the incorporation of radioactivity in total lipids, diacylgalactosylglycerol (DGG), diacyldigalactosylglycerol (DDG), sul-folipids (SL), phosphatidylglycerol (PG), diacylglycerol (DAG) and triacylglycerol + steroll esters (TAG+SE). The incorporation of acetate-1,2-[¹⁴C] decreased in phos-phatidylcholine (PC) and in phosphatidylethanolamine (PE) in the presence of atrazine. The radioactivity increased in total Transic-hexadecenoic, linoleic and α -linolenic acids while it decreased in the other fatty acids. This indicates that the sublethal concentrations of atrazine stimulate the desaturation of fatty acids of L. minor. The radioactivity was strongly incorporated in the α -linolenic acid of DGG in the presence of atrazine. The specific radioactivity of α-linolenic acid was greater in DAG than in PG > TAG + SE > PC > PE > DGG > SL > DDG and it increased in all groupd of lipids analyzed under the influence of sublethal doses of atrazine. The labelling of Translchexadecenoic acid of PG and its specific radioactivity increased in the presence of atrazine. These changes suggest that the sublethal concentrations of atrazine stimulate especially the lipid metabolism of the chloroplasts of L. minor and they could explain the increase in the number of grana per chloroplast in treated L. minor. The results are discussed in relation to the biosynthesis of galactolipids.     

THE LIPID COMPOSITION OF THORACOSPHAERA HEIMII: EVIDENCE FOR INCLUSION IN THE DINOPHYCEAE1

The fatty acid, sterol and chlorophyll composition of the calcified, unicellular alga Thoracosphaera heimii (Lohmann) Kamptner are reported. The presence of 4,23,24-termethyl-5α-cholest-22E-en-3β-ol (dinosterol), 4,23,24-trimethyl-5α-cholest-22E-en-3-one (dinosterone) and the predominance of C₁₈, C₂₀ and C₂₂ unsaturated fatty acids, including the acid 18:5ω3, indicates that T. heimii is a dinoflagellate. The fatty acid: sterol ratio (1.3), is typical of dinoflagellates. The geochemical significance of dinosterone, the high relative concentration of 4-desmethyl-5α-stanols and the role of 23-methyl-5α-cholest-22E-en-3β-ol in the biosynthesis of dinosterol in T. heimii are also discussed.     

The effect of cadmium on lipid and fatty acid biosynthesis in tomato leaves

This research aims to examine the effect of cadmium uptake on lipid composition and fatty acid biosynthesis, in young leaves of tomato treated seedlings (Lycopersicon esculentum cv. Ibiza F1). Results in membrane lipids investigations revealed that high cadmium concentrations affect the main lipid classes, leading to strong changes in their composition and fatty acid content. Thus, the exposure of tomato plants to cadmium caused a concentration-related decrease in the unsaturated fatty acid content, resulting in a lower degree of fatty acid unsaturation. The level of lipid peroxides was significantly enhanced at high Cd concentrations. Studies of the lipid metabolism using radioactive labelling with [1-¹⁴C]acetate as a major precursor of lipid biosynthesis, showed that levels of radioactivity incorporation in total lipids as well as in all lipid classes were lowered by Cd doses. In total lipid fatty acids, [1-¹⁴C]acetate incorporation was reduced in tri-unsaturated fatty acids (C16:3 and C18:3); While it was enhanced in the palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0) and linoleic (C18:2) acids. [1-¹⁴C]acetate incorporation into C16:3 and C18:3 of galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] and some phospholipids [phosphatidylcholine (PC) and phosphatidylglycerol (PG)] was inhibited by Cd stress. Our results showed that in tomato plants, cadmium stress provoked an inhibition of polar lipid biosynthesis and reduced fatty acid desaturation process.     

Glucose stimulates a decrease of the fatty acid saturation degree in Acinetobacter calcoaceticus

The fatty acid composition of Acinetobacter calcoaceticus 69-V was determined under various growth conditions. Saturated, unsaturated, and hydroxy fatty acids with chain lengths of 12–18 carbon atoms predominated in the fatty acid profile. With acetate or propanol as growth substrates, the ratio of saturated to unsaturated fatty acids varied with changes in the temperature. This was the only adaptive mechanism detected that compensated for the physical effects of temperature alterations on the cell membranes. The fatty acid composition of A. calcoaceticus grown at 40 °C had a saturation degree of approximately 50%; after growth at 20 °C it was approximately 35%. In the presence of a carbon and energy source, A. calcoaceticus was able to respond to temperature reductions under oxic conditions regardless of whether fatty acid biosynthesis was inhibited or not. This suggests an aerobic mechanism of fatty acid biosynthesis and the involvement of a fatty acid desaturase system. Addition of the non-growth substrate, glucose, helped the organism to adapt to lower temperature. The molecular mechanism of the aid is not really understood. The oxidation of glucose could provide the desaturase either with electrons directly via a pyrrolo-quinoline-quinone-linked glucose dehydrogenase or with NADH after fatty acid degradation has been initiated by ATP generated by the oxidation of glucose. Received: 19 June 1998 / Accepted: 28 December 1998     

The Biogenesis of the Photosynthetic Apparatus and the Activity of Chlorophyll Biosynthesis in a Plastome Mutant of Sunflower

It was demonstrated that, in the phenotypically colorless leaves of a sunflower (Helianthus annuusL.) plastome mutant with a heavily reduced level of chlorophyll, all pigment–protein complexes of the photosynthetic apparatus typical for the wild type were present. However, the ratio between them was changed. During aging of the mutant leaves, pigment–protein complexes of photosystem I were destroyed first followed by those of photosystem II. Chlorophyll a/b-containing light-harvesting complex II turned out to be the most stable. This conforms to an increased content of lutein and violaxanthin in mutant leaves. A synchrony of the decreases in the chlorophyll and 5-aminolevulinic acid (ALA) contents throughout all ontogenetic stages of the colorless mutant leaves made it possible to suggest that a decrease in the synthesis and resynthesis of chlorophyll during the formation and development of such leaves is caused by the inhibition of an initial stage of this process, namely, the biosynthesis of ALA molecules. The activity of the enzymes converting ALA into protochlorophyllide did not limit chlorophyll biosynthesis. Possible mechanisms controlling the synthesis of ALA destined for chlorophyll formation are discussed.     

Developmental specific expression and organelle targeting of the Escherichia coli fabD gene,encoding malonyl coenzyme A-acyl carrier protein transacylase in transgenic rape and tobacco seeds

In both plants and bacteria, de novo fatty acid biosynthesis is catalysed by a type II fatty acid synthetase (FAS) system which consists of a group of eight discrete enzyme components. The introduction of heterologous, i.e. bacterial, FAS genes in plants could provide an alternative way of modifying the plant lipid composition. In this study the Escherichia coli fabD gene, encoding malonyl CoA-ACP transacylase (MCAT), was used as a model gene to investigate the effects of over-producing a bacterial FAS component in the seeds of transgenic plants. Chimeric genes were designed, so as not to interfere with the household activities of fatty acid biosynthesis in the earlier stages of seed development, and introduced into tobacco and rapeseed using the Agrobacterium tumefaciens binary vector system. A napin promoter was used to express the E. coli MCAT in a seed-specific and developmentally specific manner. The rapeseed enoyl-ACP reductase transit peptide was used successfully, as confirmed by immunogold labelling studies, for plastid targeting of the bacterial protein. The activity of the bacterial enzyme reached its maximum (up to 55 times the maximum endogenous MCAT activity) at the end of seed development, and remained stable in mature transgenic seeds. Significant changes in fatty acid profiles of storage lipids and total seed lipid content of the transgenic plants were not found. These results are in support of the notion that MCAT does not catalyse a rate-limiting step in plant fatty acid biosynthesis.     

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.     

Control of tuberisation in potato by gibberellins and phytochrome B

Phytochrome B-deficient plants exhibit increased gibberellin (GA) levels or responsiveness, which may contribute to their elongated growth and reduced chlorophyll levels. We have investigated the effects of applications of gibberellic acid and an inhibitor of gibberellin biosynthesis, ancymidol, on wild-type and phytochrome B-antisense potato (Solanum tuberosum ssp. andigena) plants. The results showed that some phenotypes of the phytochrome B-antisense plants, i.e. increased stem length and reduced chlorophyll, can be mimicked by treating wild-type plants with gibberellic acid. However, another phenotype, i.e. tuberisation response in long days, is mimicked by application of a GA biosynthesis inhibitor ancymidol, thus appearing to be the result of a reduction in the gibberellin levels. A simple increase in gibberellin levels or sensitivity is, therefore, not sufficient to explain the phenotype of the antisense plants.     

Biosynthese de l'acide linolenique dans la feuille de pois

During the development of pea seedlings in complete darkness or under a short-day photoperiod, the capacity of linolenic acid biosynthesis reaches a maximum about 7 days after germination. At all stages of development the light markedly and specifically increases the incorporation of 1-¹⁴C-acetate into the linolenic acid, causing a 20-fold increase in the labelling at the maximum as compared with dark incubation. The evolution of the capacity of linolenic acid biosynthesis in leaves follows strictly the ability to produce chlorophyll under light. First analysis shows that the linolenic acid biosynthesis observed occurs specifically into the galactolipids.     

Cadmium response and redoxin targets in Chlamydomonas reinhardtii: a proteomic approach

A proteomic approach including two-dimensional electrophoresis and MALDI-TOF analysis has been developed to identify the soluble proteins of the unicellular photosynthetic algae Chlamydomonas reinhardtii. We first described the partial 2D-picture of soluble proteome obtained from whole cells grown on acetate. Then we studied the effects of the exposure of these cells to 150 μM cadmium (Cd). The most drastic effect was the decrease in abundance of both large and small subunits of the ribulose-1,5-bisphosphate carboxylase/oxygenase, in correlation with several other enzymes involved in photosynthesis, Calvin cycle and chlorophyll biosynthesis. Other down-regulated processes were fatty acid biosynthesis, aminoacid and protein biosynthesis. On the other hand, proteins involved in glutathione synthesis, ATP metabolism, response to oxidative stress and protein folding were up-regulated in the presence of cadmium. In addition, we observed that most of the cadmium-sensitive proteins were also regulated via two major cellular thiol redox systems, thioredoxin and glutaredoxin.     

Functions of the Clostridium acetobutylicium FabF and FabZ proteins in unsaturated fatty acid biosynthesis

Background  

The original anaerobic unsaturated fatty acid biosynthesis pathway proposed by Goldfine and Bloch was based on in vivo labeling studies in Clostridium butyricum ATCC 6015 (now C. beijerinckii) but to date no dedicated unsaturated fatty acid biosynthetic enzyme has been identified in Clostridia. C. acetobutylicium synthesizes the same species of unsaturated fatty acids as E. coli, but lacks all of the known unsaturated fatty acid synthetic genes identified in E. coli and other bacteria. A possible explanation was that two enzymes of saturated fatty acid synthesis of C. acetobutylicium, FabZ and FabF might also function in the unsaturated arm of the pathway (a FabZ homologue is known to be an unsaturated fatty acid synthetic enzyme in enterococci).     

Polyploid genome of <Emphasis Type="Italic">Camelina sativa</Emphasis>revealed by isolation of fatty acid synthesis genes

Background  

Camelina sativa, an oilseed crop in the Brassicaceae family, has inspired renewed interest due to its potential for biofuels applications. Little is understood of the nature of the C. sativa genome, however. A study was undertaken to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase (FAD) 2 and fatty acid elongase (FAE) 1, which revealed unexpected complexity in the C. sativa genome.     

Investigation of <i>GhFAT</i> Genes Related to Seed Oil Content and Fatty Acid Composition in <i>Gossypium hirsutum</i> L.

Fatty Acyl-ACP thioesterase (FAT) is a key enzyme controlling oil biosynthesis in plant seeds. FATs can be divided into two subfamilies, FATA and FATB according to their amino acid sequences and substrate specificity. The Upland cotton genome contains 20 GhFAT genes, amongst which 6 genes were of the GhFATA subfamily and 14 of the GhFATB subfamily. The 20 GhFAT genes are unevenly distributed on 14 chromosomes. The GhFATA genes have 5 or 7 exons and the GhFATB genes have 6 or 7 exons. All GhFAT proteins have the conserved Acyl-ACP_TE domain and PLN02370 super family, the typical characteristics of plant thioesterases. Analyses of the expression level of GhFATs and the compositions of fatty acid in 5–60 days-post-anthesis seeds showed that the ratio of saturated fatty acids to unsaturated fatty acids was consistent with the expression profile of GhFATB12, GhFATB3, and GhFATB10; the ratio of monounsaturated fatty acid to polyunsaturated fatty acids was consistent with the expression profile of GhFATA3. The oil contents of mature cottonseeds were positively correlated with the contents of palmitic acid and linolenic acid as well as seed vigor. These results provide essential information for further exploring the role(s) of the specific GhFATs in determining oil biosynthesis and cottonseed compositions.     

Small Cab-like proteins regulating tetrapyrrole biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803

In the cyanobacterium Synechocystis sp. PCC 6803 five open reading frames (scpA–scpE) have been identified that code for single-helix proteins resembling helices I and III of chlorophyll a/b-binding (Cab) antenna proteins from higher plants. They have been named SCPs (small Cab-like proteins). Deletion of a single scp gene in a wild-type or in a photosystem I-less (PS I-less) strain has little effect. However, the effects of functional deletion of scpB or scpE were remarkable under conditions where chlorophyll availability was limited. When cells of a strain lacking PS I and chlL (coding for a polypeptide needed for light-independent protochlorophyllide reduction) were grown in darkness, the phycobilin and protochlorophyllide levels decreased upon deletion of scpB or scpE and the protoheme level was reduced in the strain lacking scpE. Addition of -aminolevulinic acid (ALA) in darkness drastically increased the level of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester in the PS I-less/chlL ^–/scpE ^– strain, whereas PChlide accumulated in the PS I-less/chlL ^–/scpB ^– strain. In the PS I-less/chlL ^– control strain ALA supplementation did not lead to large changes in the levels of tetrapyrrole biosynthesis intermediates. We propose that ScpE and ScpB regulate tetrapyrrole biosynthesis as a function of pigment availability. This regulation occurs primarily at an early step of tetrapyrrole biosynthesis, prior to ALA. In view of the conserved nature of chlorophyll-binding sites in these proteins, it seems likely that regulation by SCPs occurs as a function of chlorophyll availability, with SCPs activating chlorophyll biosynthesis steps when they do not have pigments bound.