Incorporation of extracellular fatty acids by a fatty acid kinase‐dependent pathway in Staphylococcus aureus

Acyl‐CoA and acyl‐acyl carrier protein (ACP) synthetases activate exogenous fatty acids for incorporation into phospholipids in Gram‐negative bacteria. However, Gram‐positive bacteria utilize an acyltransferase pathway for the biogenesis of phosphatidic acid that begins with the acylation of sn‐glycerol‐3‐phosphate by PlsY using an acyl‐phosphate (acyl‐PO₄) intermediate. PlsX generates acyl‐PO₄ from the acyl‐ACP end‐products of fatty acid synthesis. The plsX gene of Staphylococcus aureus was inactivated and the resulting strain was both a fatty acid auxotroph and required de novo fatty acid synthesis for growth. Exogenous fatty acids were only incorporated into the 1‐position and endogenous acyl groups were channeled into the 2‐position of the phospholipids in strain PDJ39 (ΔplsX). Extracellular fatty acids were not elongated. Removal of the exogenous fatty acid supplement led to the rapid accumulation of intracellular acyl‐ACP and the abrupt cessation of fatty acid synthesis. Extracts from the ΔplsX strain exhibited an ATP‐dependent fatty acid kinase activity, and the acyl‐PO₄ was converted to acyl‐ACP when purified PlsX is added. These data reveal the existence of a novel fatty acid kinase pathway for the incorporation of exogenous fatty acids into S. aureus phospholipids.     

Xanthomonas campestris RpfB is a fatty Acyl‐CoA ligase required to counteract the thioesterase activity of the RpfF diffusible signal factor (DSF) synthase

In Xanthomonas campestris pv. campestris (Xcc), the proteins encoded by the rpf (regulator of pathogenicity factor) gene cluster produce and sense a fatty acid signal molecule called diffusible signalling factor (DSF, 2(Z)‐11‐methyldodecenoic acid). RpfB was reported to be involved in DSF processing and was predicted to encode an acyl‐CoA ligase. We report that RpfB activates a wide range of fatty acids to their CoA esters in vitro. Moreover, RpfB can functionally replace the paradigm bacterial acyl‐CoA ligase, Escherichia coli FadD, in the E. coli ß‐oxidation pathway and deletion of RpfB from the Xcc genome results in a strain unable to utilize fatty acids as carbon sources. An essential RpfB function in the pathogenicity factor pathway was demonstrated by the properties of a strain deleted for both the rpfB and rpfC genes. The ΔrpfB ΔrpfC strain grew poorly and lysed upon entering stationary phase. Deletion of rpfF, the gene encoding the DSF synthetic enzyme, restored normal growth to this strain. RpfF is a dual function enzyme that synthesizes DSF by dehydration of a 3‐hydroxyacyl‐acyl carrier protein (ACP) fatty acid synthetic intermediate and also cleaves the thioester bond linking DSF to ACP. However, the RpfF thioesterase activity is of broad specificity and upon elimination of its RpfC inhibitor RpfF attains maximal activity and its thioesterase activity proceeds to block membrane lipid synthesis by cleavage of acyl‐ACP intermediates. This resulted in release of the nascent acyl chains to the medium as free fatty acids. This lack of acyl chains for phospholipid synthesis results in cell lysis unless RpfB is present to counteract the RpfF thioesterase activity by catalysing uptake and activation of the free fatty acids to give acyl‐CoAs that can be utilized to restore membrane lipid synthesis. Heterologous expression of a different fatty acid activating enzyme, the Vibrio harveyi acyl‐ACP synthetase, replaced RpfB in counteracting the effects of high level RpfF thioesterase activity indicating that the essential role of RpfB is uptake and activation of free fatty acids.     

Biosynthesis and function of simple amides in Xenorhabdus doucetiae

Xenorhabdus doucetiae, the bacterial symbiont of the entomopathogenic nematode Steinernema diaprepesi produces several different fatty acid amides. Their biosynthesis has been studied using a combination of analysis of gene deletions and promoter exchanges in X. doucetiae and heterologous expression of candidate genes in E. coli. While a decarboxylase is required for the formation of all observed phenylethylamides and tryptamides, the acyltransferase XrdE encoded in the xenorhabdin biosynthesis gene cluster is responsible for the formation of short chain acyl amides. Additionally, new, long‐chain and cytotoxic acyl amides were identified in X. doucetiae infected insects and when X. doucetiae was grown in Galleria Instant Broth (GIB). When the bioactivity of selected amides was tested, a quorum sensing modulating activity was observed for the short chain acyl amides against the two different quorum sensing systems from Chromobacterium and Janthinobacterium.     

Fatty Acid Specificity and Selectivity of the Chloroplast sn-Glycerol 3-Phosphate Acyltransferase of the Chilling Sensitive Plant, Amaranthus lividus

Chilling sensitivity of plants is strongly correlated with the presence of high levels of a species of chloroplast phosphatidylglycerol that contains two saturated fatty acids. The most straightforward synthetic pathway for this lipid would require the primary acylation of sn-glycerol 3-phosphate (G3P) with a saturated fatty acid (palmitic acid) rather than with oleic acid, an unsaturated acid. This selective incorporation would differ markedly from the reported properties of the chloroplast G3P acyltransferases of pea and spinach, two chilling resistant plants and thus we have studied the chloroplast G3P acyltransferase of Amaranthus lividus, a chilling sensitive plant. In contrast to our results and those of others (M. Frentzen et al. 1983 Eur J Biochem 129: 629-636 and previous work) with the pea and spinach enzymes, the amaranthus chloroplast G3P acyltranferase did not select oleic acid donors from a mixture of oleic and palmitic acid donors (either coenzyme A or acyl carrier protein thioesters). Instead the fatty acid composition of the synthesized 1-acyl G3P faithfully reflected the composition of the acyl donor mixture. However, the amaranthus enzyme did strongly select against incorporation of stearic acid. The properties of the amaranthus G3P acyltransferase are consistent with this enzyme having the major role in synthesis of the disaturated phosphatidylglycerol species.     

The role of Δ6-desaturase acyl-carrier specificity in the efficient synthesis of long-chain polyunsaturated fatty acids in transgenic plants

The role of acyl‐CoA‐dependent Δ6‐desaturation in the heterologous synthesis of omega‐3 long‐chain polyunsaturated fatty acids was systematically evaluated in transgenic yeast and Arabidopsis thaliana. The acyl‐CoA Δ6‐desaturase from the picoalga Ostreococcus tauri and orthologous activities from mouse (Mus musculus) and salmon (Salmo salar) were shown to generate substantial levels of Δ6‐desaturated acyl‐CoAs, in contrast to the phospholipid‐dependent Δ6‐desaturases from higher plants that failed to modify this metabolic pool. Transgenic plants expressing the acyl‐CoA Δ6‐desaturases from either O. tauri or salmon, in conjunction with the two additional activities required for the synthesis of C20 polyunsaturated fatty acids, contained higher levels of eicosapentaenoic acid compared with plants expressing the borage phospholipid‐dependent Δ6‐desaturase. The use of acyl‐CoA‐dependent Δ6‐desaturases almost completely abolished the accumulation of unwanted biosynthetic intermediates such as γ‐linolenic acid in total seed lipids. Expression of acyl‐CoA Δ6‐desaturases resulted in increased distribution of long‐chain polyunsaturated fatty acids in the polar lipids of transgenic plants, reflecting the larger substrate pool available for acylation by enzymes of the Kennedy pathway. Expression of the O. tauriΔ6‐desaturase in transgenic Camelina sativa plants also resulted in the accumulation of high levels of Δ6‐desaturated fatty acids. This study provides evidence for the efficacy of using acyl‐CoA‐dependent Δ6‐desaturases in the efficient metabolic engineering of transgenic plants with high value traits such as the synthesis of omega‐3 LC‐PUFAs.     

A Functional Homolog of Escherichia coli NhaR in Vibrio cholerae

Escherichia coli NhaR controls expression of a sodium/proton (Na⁺/H⁺) antiporter, NhaA. The Vibrio cholerae NhaR protein shows over 60% identity to those of Escherichia coli and Salmonella enteritidis. V. cholerae NhaR complements an E. coli nhaR mutant for growth in 100 mM LiCl–33 mM NaCl, pH 7.6, and enhances the Na⁺-dependent induction of an E. coli chromosomal nhaA::lacZ fusion. These findings indicate functional homology to E. coli NhaR. Two V. cholerae nhaR mutants were constructed by using kanamycin resistance cartridge insertion at different sites to disrupt the gene. Both mutants showed sensitivity to growth in 120 mM LiCl, pH 9.2, compared with the wild-type strain and could be complemented by the introduction of V. cholerae nhaR on a low-copy-number plasmid. An nhaR mutation had no detectable effect on the virulence of the V. cholerae strain in the infant mouse model, suggesting that the antiporter system involved is not required in vivo, at least in this animal model.     

Metabolic engineering of Escherichia coli for the production of polylactic acid and its copolymers

Polylactic acid (PLA) is a promising biomass‐derived polymer, but is currently synthesized by a two‐step process: fermentative production of lactic acid followed by chemical polymerization. Here we report production of PLA homopolymer and its copolymer, poly(3‐hydroxybutyrate‐co‐lactate), P(3HB‐co‐LA), by direct fermentation of metabolically engineered Escherichia coli. As shown in an accompanying paper, introduction of the heterologous metabolic pathways involving engineered propionate CoA‐transferase and polyhydroxyalkanoate (PHA) synthase for the efficient generation of lactyl‐CoA and incorporation of lactyl‐CoA into the polymer, respectively, allowed synthesis of PLA and P(3HB‐co‐LA) in E. coli, but at relatively low efficiency. In this study, the metabolic pathways of E. coli were further engineered by knocking out the ackA, ppc, and adhE genes and by replacing the promoters of the ldhA and acs genes with the trc promoter based on in silico genome‐scale metabolic flux analysis in addition to rational approach. Using this engineered strain, PLA homopolymer could be produced up to 11 wt% from glucose. Also, P(3HB‐co‐LA) copolymers containing 55–86 mol% lactate could be produced up to 56 wt% from glucose and 3HB. P(3HB‐co‐LA) copolymers containing up to 70 mol% lactate could be produced to 46 wt% from glucose alone by introducing the Cupriavidus necator β‐ketothiolase and acetoacetyl‐CoA reductase genes. Thus, the strategy of combined metabolic engineering and enzyme engineering allowed efficient bio‐based one‐step production of PLA and its copolymers. This strategy should be generally useful for developing other engineered organisms capable of producing various unnatural polymers by direct fermentation from renewable resources. Biotechnol. Bioeng. 2010; 105: 161–171. © 2009 Wiley Periodicals, Inc.     

Identification of a broad family of lipid A late acyltransferases with non‐canonical substrate specificity

Most Gram‐negative organisms produce lipopolysaccharide (LPS), a complex macromolecule anchored to the bacterial membrane by the lipid A moiety. Lipid A is synthesized via the Raetz pathway, a conserved nine‐step enzymatic process first characterized in Escherichia coli. The Epsilonproteobacterium Helicobacter pylori uses the Raetz pathway to synthesize lipid A; however, only eight of nine enzymes in the pathway have been identified in this organism. Here, we identify the missing acyltransferase, Jhp0255, which transfers a secondary acyl chain to the 3′‐linked primary acyl chain of lipid A, an activity similar to that of E. coli LpxM. This enzyme, reannotated as LpxJ due to limited sequence similarity with LpxM, catalyses addition of a C12:0 or C14:0 acyl chain to the 3′‐linked primary acyl chain of lipid A, complementing an E. coli LpxM mutant. Enzymatic assays demonstrate that LpxJ and homologues in Campylobacter jejuni and Wolinella succinogenes can act before the 2′ secondary acyltransferase, LpxL, as well as the 3‐deoxy‐d ‐manno‐octulosonic acid (Kdo) transferase, KdtA. Ultimately, LpxJ is one member of a large class of acyltransferases found in a diverse range of organisms that lack an E. coli LpxM homologue, suggesting that LpxJ participates in lipid A biosynthesis in place of an LpxM homologue.     

Vernonia DGATs increase accumulation of epoxy fatty acids in oil

Vernolic acid (cis‐12‐epoxy‐octadeca‐cis‐9‐enoic acid) is valuable as a renewable chemical feedstock. This fatty acid can accumulate to high levels in the seed oil of some plant species such as Vernonia galamensis and Stokesia laevis which are unsuitable for large‐scale production. A cost‐effective alternative for production of epoxy fatty acids is to genetically engineer its biosynthesis in commercial oilseeds. An epoxygenase cDNA (SlEPX) responsible for vernolic acid synthesis and two acyl‐CoA : diacylglycerol acyltransferase cDNAs (VgDGAT1 and VgDGAT2) catalysing triacylglycerol (TAG) formation were cloned from developing seeds of S. laevis and V. galamensis. Co‐expression of SlEPX and VgDGAT1 or VgDGAT2 greatly increases accumulation of vernolic acid both in petunia leaves and soybean somatic embryos. Seed‐specific expression of VgDGAT1 and VgDGAT2 in SlEPX mature soybean seeds results in vernolic acid levels of ～15% and 26%. Both DGAT1 and DGAT2 increase epoxy fatty acid accumulation with DGAT2 having much greater impact.     

Enhancement of extraplastidic oil synthesis in Chlamydomonas reinhardtii using a type‐2 diacylglycerol acyltransferase with a phosphorus starvation–inducible promoter

When cultivated under stress conditions, many plants and algae accumulate oil. The unicellular green microalga Chlamydomonas reinhardtii accumulates neutral lipids (triacylglycerols; TAGs) during nutrient stress conditions. Temporal changes in TAG levels in nitrogen (N)‐ and phosphorus (P)‐starved cells were examined to compare the effects of nutrient depletion on TAG accumulation in C. reinhardtii. TAG accumulation and fatty acid composition were substantially changed depending on the cultivation stage before nutrient starvation. Profiles of TAG accumulation also differed between N and P starvation. Logarithmic‐growth‐phase cells diluted into fresh medium showed substantial TAG accumulation with both N and P deprivation. N deprivation induced formation of oil droplets concomitant with the breakdown of thylakoid membranes. In contrast, P deprivation substantially induced accumulation of oil droplets in the cytosol and maintaining thylakoid membranes. As a consequence, P limitation accumulated more TAG both per cell and per culture medium under these conditions. To enhance oil accumulation under P deprivation, we constructed a P deprivation‐dependent overexpressor of a Chlamydomonas type‐2 diacylglycerol acyl‐CoA acyltransferase (DGTT4) using a sulphoquinovosyldiacylglycerol 2 (SQD2) promoter, which was up‐regulated during P starvation. The transformant strongly enhanced TAG accumulation with a slight increase in 18 : 1 content, which is a preferred substrate of DGTT4. These results demonstrated enhanced TAG accumulation using a P starvation–inducible promoter.     

Cloning and characterization of the Vibrio cholerae genes encoding the utilization of iron from haemin and haemoglobin

Vibrio cholerae can utilize haemin or haemoglobin as its sole source of iron. Four haem utilization mutants of a classical strain of V. cholerae were isolated. These mutations were complemented with pHUT1, a cosmid clone isolated from a library of wild-type CA401 DNA. Two independent Tn5 insertions into the cloned sequence disrupted function in all of the complemented mutants. Escherichia coli 1017 transformed with pHUT1 failed to utilize haemin as an iron source; a second plasmid containing a different cloned fragment of V. cholerae DNA (pHUT3) was required in addition to pHUT1 to reconstitute the system in E. coli. Minicell analysis and SDS-PAGE of protein fractions indicate that pHUT10 (a subclone of p>HUT1) encodes a 26 kDa inner membrane protein, and pHUT3 encodes a 77 kDa outer membrane protein. Loss of either protein by Tn5 mutagenesis abolishes haem utilization in E. coli. An E. coli hemA mutant that cannot synthesize porphyrins was transformed with the recombinant plasmids to determine whether the plasmids encoded the ability to transport the porphyrin as well as the iron. The transformants grew aerobically in media containing haemin, whereas the parental strain was unable to grow under these conditions. This indicates that V. cholerae haem-iron utilization genes allow transport of the entire haem moiety into the cell.     

Secondary Acylation of Vibrio cholerae Lipopolysaccharide Requires Phosphorylation of Kdo

The lipopolysaccharide of Vibrio cholerae has been reported to contain a single 3-deoxy-d-manno-octulosonic acid (Kdo) residue that is phosphorylated. The phosphorylated Kdo sugar further links the hexa-acylated V. cholerae lipid A domain to the core oliogosaccharide and O-antigen. In this report, we confirm that V. cholerae possesses the enzymatic machinery to synthesize a phosphorylated Kdo residue. Further, we have determined that the presence of the phosphate group on the Kdo residue is necessary for secondary acylation in V. cholerae. The requirement for a secondary substituent on the Kdo residue (either an additional Kdo sugar or a phosphate group) was also found to be critical for secondary acylation catalyzed by LpxL proteins from Bordetella pertussis, Escherichia coli, and Haemophilus influenzae. Although three putative late acyltransferase orthologs have been identified in the V. cholerae genome (Vc0212, Vc0213, and Vc1577), only Vc0213 appears to be functional. Vc0213 functions as a myristoyl transferase acylating lipid A at the 2′-position of the glucosamine disaccharide. Generally acyl-ACPs serve as fatty acyl donors for the acyltransferases required for lipopolysaccharide biosynthesis; however, in vitro assays indicate that Vc0213 preferentially utilizes myristoyl-CoA as an acyl donor. This is the first report to biochemically characterize enzymes involved in the biosynthesis of the V. cholerae Kdo-lipid A domain.Lipopolysaccharide (LPS),² the major surface molecule in the outer membrane of Gram-negative bacteria, is composed of three domains: lipid A, core oligosaccharide, and O-antigen (1). The core oligosaccharide is further divided into two distinct regions: inner and outer core. The inner core consists of the Kdo sugars, which are responsible for linking the core region to the lipid A moiety of LPS. Lipid A is the hydrophobic anchor of LPS and is the only portion of LPS required for activating the host innate immune response by interacting with Toll-like receptor 4 and the accessory molecule, MD2.Kdo-lipid A biosynthesis is a well conserved and ordered process among Gram-negative bacteria; however, not all Gram-negative bacteria produce similar lipid A structures (2). In Escherichia coli, the biosynthesis of the Kdo-lipid A domain occurs via a nine-step process, resulting in the production of a hexa-acylated lipid A structure known as Kdo₂-lipid A. Kdo₂-lipid A has long been thought to be essential for the viability of E. coli; however, viable suppressor strains have been isolated that lack the Kdo sugar (3). The late steps of the biosynthetic pathway involve the addition of the Kdo sugars and the secondary or “late” acyl chains. The enzyme responsible for the addition of the Kdo sugars is the Kdo transferase (WaaA). In E. coli, this enzyme is bifunctional, transferring two Kdo sugars to the lipid A precursor, lipid IV_A (4); however, other Gram-negative bacteria have been shown to possess a monofunctional or trifunctional WaaA, as is the case in Haemophilus influenzae (5) or Chlamydia trachomatis (6), respectively.Previous reports have shown that in E. coli, the addition of the Kdo sugars is critical for the functionality of the secondary acyltransferases (LpxL, LpxM, and LpxP). The E. coli late acyltransferase LpxL catalyzes the transfer of laurate (C12:0) to the acyl chain linked at the 2′-position of Kdo₂-lipid IV_A (7). LpxM then catalyzes the addition of a myristate (C14:0) to the 3′-linked acyl chain of the penta-acylated lipid A precursor (8). When E. coli experience cold shock conditions (temperatures below 20 °C), the late acyltransferase LpxP transfers a palmitoleate (C16:1) to the 2′-position of Kdo₂-lipid IV_A, replacing the C12:0 acyl chain transferred by LpxL (9). Lipid A secondary acyltransferases have been shown to primarily utilize acyl-acyl carrier proteins (acyl-ACPs) as their acyl chain donor; however, a recent report by Six et al. (10) has shown that purified E. coli LpxL is capable of utilizing acyl-coenzyme A (acyl-CoA) as an alternative acyl donor at a lesser rate.The Gram-negative bacteria Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. Cholera is transmitted via the fecal-oral route by ingestion of contaminated drinking water or food. The World Health Organization reported ∼130,000 cases of cholera in 2005 with the majority occurring in Africa. There are two serogroups of V. cholerae capable of epidemic and pandemic disease: O1 and O139 (11). Previous structural analyses have revealed that these serogroups possess very different lipid A structures. The V. cholerae O1 lipid A structure was reported as hexa-acylated, bearing secondary acyl chains at the 2- and 2′-positions of phosphorylated Kdo-lipid A (11–13); however, V. cholerae O139 was reported as having an octa-acylated lipid A (see Fig. 1) (11, 14).Open in a separate windowFIGURE 1.Comparison of E. coli K12 lipid A species to V. cholerae O1 and V. cholerae O139 lipid A species. The covalent modifications of lipid A are indicated with dashed bonds, and the lengths of the acyl chains are indicated below each structure. The lipid A of E. coli K12 is a hexa-acylated structure, bearing two secondary acyl chains at the 2′- and 3′-positions. The E. coli lipid A structure is glycosylated at the 6′-position with two Kdo moieties and is phosphorylated at the 1- and 4′-positions of the disaccharide backbone. Similar to E. coli, the lipid A species of V. cholerae serogroup O1 is hexa-acylated, but with a symmetrical acyl chain distribution. The proposed lipid A structure of V. cholerae O139 is the octa-acylated structure. Both V. cholerae serogroups O1 and O139 reported lipid A species have a single Kdo sugar that is phosphorylated (red) and a phosphoethanolamine (magenta) attached to the 1-phosphate.Our report focuses on V. cholerae O1 El Tor, which is the predominant disease-causing strain worldwide. Because little attention has been given to the Kdo-lipid A domain of V. cholerae, we investigated the assembly of the inner core structure of V. cholerae O1 LPS and the late acylation steps. This report demonstrates the importance of a secondary negative charge on the primary Kdo sugar of lipid A for late acyltransferase activity in V. cholerae and in other Gram-negative bacteria. Also, we have identified the putative V. cholerae late acyltransferase, Vc0213 as the LpxL homolog, transferring a myristate (C14:0) to the 2′-position of V. cholerae lipid A. These initial findings provide us with the groundwork needed to investigate the modifications of the V. cholerae Kdo-lipid A structure, which may serve as attractive vaccine targets in future research.     

Coupled biosynthesis and esterification of 1,2,4‐butanetriol to simplify its separation from fermentation broth

1,2,4‐Butanetriol (BT) is a valuable chemical with versatile applications in many fields and can be produced through biosynthetic pathways. As a trihydric alcohol, BT possesses good water solubility and is very difficult to separate from fermentation broth, which does complicate the production process and increase the cost. To develop a novel method for BT separation, a biosynthetic pathway for 1,2,4‐butanetriol esters with poor water solubility was constructed. Wax ester synthase/acyl‐coenzyme A: diacylglycerol acyltransferase (Atf) from Acinetobacter baylyi, Mycobacterium smegmatis, and Escherichia coli were screened, and the acyltransferase from A. baylyi (AtfA) was found to have higher capability. The BT producing strain with AtfA overexpression produced 49.5 mg/L BT oleate in flask cultivation. Through enhancement of acyl‐CoA production by overexpression of the acyl‐CoA synthetase gene fadD and deleting the acyl coenzyme A dehydrogenase gene fadE, the production was improved to 64.4 mg/L. Under fed‐batch fermentation, the resulting strain produced up to 1.1 g/L BT oleate. This is the first time showed that engineered E. coli strains can successfully produce BT esters from xylose and free fatty acids.