Modulation of Lipid Metabolism and Spiramycin Biosynthesis in Streptomyces ambofaciens Unstable Mutants

Streptomyces ambofaciens is prone to genetic instability involving genomic rearrangements at the extremities of the chromosomal DNA. An amplified DNA sequence (ADS205), including an open reading frame (orfPS), is responsible for the reversible loss of spiramycin production in the mutant strain NSA205 (ADS205⁺ Spi⁻). The product of orfPS is homologous to polyketide synthase systems (PKSs) involved in the biosynthesis of erythromycin and rapamycin and is overexpressed in strain NSA205 compared with the parental strain RP181110. As PKSs and fatty acid synthase systems have the same precursors, we tested the possibility that overexpression of orfPS also affects lipid metabolism in strain NSA205. This report focuses on comparative analysis of lipids in strain RP181110, the mutant strain NSA205, and a derivative, NSA228 (ADS205⁻ Spi⁺). NSA205 showed a dramatically depressed lipid content consisting predominantly of phospholipids and triacylglycerols. This lipid content was globally restored in strain NSA228, which had lost ADS205. Furthermore, strains RP181110 and NSA205 presented similar phospholipid and triacylglycerol compositions. No abnormal fatty acids were detected in NSA205.     

Large genomic rearrangements of the unstable region in Streptomyces ambofaciens are associated with major changes in global gene expression

Global gene expression is dramatically altered by genomic rearrangements in Streptomyces ambofaciens RP181110. Partial genome mapping of two derivatives of strain RP181110 (strains NSA205 and NSA228) revealed rearrangements located in the unstable region of the genome (deletion in strain NSA228; deletion and amplification in strain NSA205). Computerized comparisons of pulse-labelled proteins separated by two-dimensional electrophoresis have revealed numerous differences in gene expression among the three strains during both exponential and stationary phases of growth: 31 proteins were absent in both mutant strains, 16 were absent only in strain NSA228, 17 were absent only in strain NSA205 and 9 were found to be present or over expressed in strain NSA205. Thus, in spite of the scarcity of genetic markers in the unstable region and its dispensability for growth under laboratory conditions, these results suggest that it includes genes which are actively expressed. Spontaneous gene amplifications, which occur frequently in this region of the chromosome, can further activate their expression.     

集胞藻PCC6803膜脂循环关键基因酶学性质和生理功能

集胞藻PCC6803野生型和其脂酰ACP合酶敲除突变株的自由脂肪酸含量和组成表明膜脂的重构和降解是细胞内自由脂肪酸的来源之一。在这一过程中脂肪酶起到关键性作用。通过基因组数据库检索,发现集胞藻PCC6803基因组中只有一个脂肪酶编码基因sll1969,但是还没有其功能相关的生化证据。为了确定该基因的功能及其在脂肪酸代谢途径中的作用,加深对集胞藻PCC6803脂肪酸代谢途径的了解,文中将sll1969基因在大肠杆菌中过表达和体外纯化,得到重组蛋白Sll1969,并对其酶学性质进行初步分析。在30℃条件下,测得Sll1969以对硝基苯丁酸酯作为底物时的Km和kcat值分别为(1.16±0.01)mmol/L和(332.8±10.0)/min;该脂肪酶的最适反应温度为55℃。通过比较分析sll1969突变株中脂肪酸含量和组成变化,发现sll1969的表达量与细胞自由脂肪酸的产量呈正相关,但Sll1969不是细胞中唯一的脂肪酶。     

Reconstitution of the FK228 biosynthetic pathway reveals cross talk between modular polyketide synthases and fatty acid synthase

Functional cross talk between fatty acid biosynthesis and secondary metabolism has been discovered in several cases in microorganisms; none of them, however, involves a modular biosynthetic enzyme. Previously, we reported a hybrid modular nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) pathway for the biosynthesis of FK228 anticancer depsipeptide in Chromobacterium violaceum strain 968. This pathway contains two PKS modules on the DepBC enzymes that lack a functional acyltransferase (AT) domain, and no apparent AT-encoding gene exists within the gene cluster or its vicinity. We report here that, through reconstitution of the FK228 biosynthetic pathway in Escherichia coli cells, two essential genes, fabD1 and fabD2, both encoding a putative malonyl coenzyme A (CoA) acyltransferase component of the fatty acid synthase complex, are positively identified to be involved in FK228 biosynthesis. Either gene product appears sufficient to complement the AT-less PKS modules on DepBC for polyketide chain elongation. Concurrently, a gene (sfp) encoding a putative Sfp-type phosphopantetheinyltransferase was identified to be necessary for FK228 biosynthesis as well. Most interestingly, engineered E. coli strains carrying variable genetic components produced significant levels of FK228 under both aerobic and anaerobic cultivation conditions. Discovery of the trans complementation of modular PKSs by housekeeping ATs reveals natural product biosynthesis diversity. Moreover, demonstration of anaerobic production of FK228 by an engineered facultative bacterial strain validates our effort toward the engineering of novel tumor-targeting bioagents.     

Temperature-sensitive Saccharomyces cerevisiae mutant defective in lipid biosynthesis.

A temperature-sensitive mutant of Saccharomyces cerevisiae (DAM303) is described that exhibits an early defect in lipid biosynthesis at the restrictive growth temperature, 37 degrees C. This strain rapidly lost viability after 1 h of incubation at 37 degrees C, and this was accompanied by a significantly reduced incorporation of 32Pi into cellular lipid and an accumulation of [1-14C]acetate into the free fatty acid fraction. The temperature-sensitive DAM303 mutation failed to complement the sec13 mutation described by Novick et al. (Cell 21:205-215, 1980), and from analysis of invertase secretion in the temperature-sensitive DAM303 strain, it is clear that the loss of invertase secretion in the mutant occurs after the loss of phospholipid synthesis. Although the precise nature of the temperature-sensitive lesion in the DAM303 strain has still to be identified, the results from the study of this mutant indicate that a defect in lipid biosynthesis can be correlated with subsequent alterations in extracellular protein secretion and loss of other macromolecular functions including DNA, RNA, and protein syntheses. From studies of this mutant, two procedures of enriching for other temperature-sensitive mutants with defects in lipid biosynthesis have emerged: inositol overproduction and screening for increased buoyant densities.     

Lipid peroxidation as a source of oxidative damage in Helicobacter pylori: protective roles of peroxiredoxins

Oxidative stress conditions lead to enzymatic and non-enzymatic unsaturated fatty acid-initiated lipid peroxidation reactions. One exacerbating product is lipid hydroperoxide (LOOH) which itself promotes formation of several additional peroxyl radicals. Helicobacter pylori mutant strains with disruptions in genes encoding the peroxiredoxins, alkyl hydroperoxide reductase (ahpC) and the bacterioferritin comigratory protein (bcp), were more sensitive than the parent strain to oxidizing agents. These mutant strains were particularly sensitive, compared to the wild type, to killing by the unsaturated fatty acid linolenic acid but were not sensitive to the saturated fatty acid palmitic acid. A double mutant strain (ahpC bcp) accumulated more than 3-fold more lipid peroxides than the parent strain, indicating these peroxiredoxins together play a role in detoxifying lipid peroxides. The level of free iron accumulation, a signature of oxidative stress damage, was correlated specifically to organic peroxide-mediated stress by both in vivo and in vitro approaches. Free iron accumulation and concomitant destruction of [Fe-S] cluster-containing proteins (hydrogenase and aconitase) was correlated to damage mediated by exogenous t-butyl peroxide, or separately to intracellular accumulation of lipid peroxides in mutant strains. A major macromolecular target of accumulating lipid peroxides in H. pylori is DNA, as mutant analysis approaches combined with quantitative DNA fragmentation studies and specific DNA damage assessment (i.e. 8-oxoguanine formation) were used to demonstrate that such damage was especially associated with ahpC and ahpC bcp strains.     

Development of microalga Scenedesmus dimorphus mutant with higher lipid content by radiation breeding

In this study, a high lipid-accumulating mutant strain of the microalgae Scenedesmus dimorphus was developed via radiation breeding. To induce mutant strain, S. dimorphus was gamma-irradiated at doses from 100 to 800 Gy, and then a mutant (Sd-Pm210) with 25 % increased lipid content was selected using Nile red staining methodology. Sd-Pm210 showed morphological changes and had higher growth rate compared to the wild type. From random amplified polymorphic DNA analysis, partial genetic modifications were also observed in Sd-Pm210. In comparisons of lipid content between wild type and Sd-Pm210 using thin-layer chromatography, the content of triacylglycerol was markedly higher in the Sd-Pm210 strain. The total peak area of fatty acid methyl ester was shown to have about 1.4-fold increase in Sd-Pm210, and major fatty acids were identified as palmitic acid, oleic acid, linoleic acid, and linolenic acid. To define the metabolic changes in the mutant strain, 2-dimensional electrophoresis was conducted. Several proteins related to lipid synthesis and energy metabolisms were overexpressed in the mutant strain. These results showed that radiation breeding can be utilized for the development of efficient microalgae strains for biofuel production.     

Effects of Mutations and Growth Conditions on Lipid Synthesis in Neurospora crassa

A morphological mutant (col-2) of Neurospora, which is partially deficient in glucose-6-phosphate dehydrogenase (G-6-PD) activity and has lower levels of reduced nicotinamide adenine dinucleotide phosphate (NADPH), accumulated three-fold more triglycerides during log-phase growth than the wild-type strain. Increased lipid deposition was not found in other strains that included slow-growing morphological mutants, NADPH-deficient strains, G-6-PD-deficient mutants, wild-type revertants from col-2, and a cel, col-2 double mutant. The cel, col-2 strain was supplemented with an exogenous source of fatty acids because it cannot synthesize these lipid moieties. The observed normal lipid content of this strain suggests that the lipid deposition in col-2 on glucose is due to an overstimulation of fatty acid synthesis and not a deficiency in fatty acid breakdown. The neutral lipid levels in both wild type and col-2 were decreased to identical levels when grown on glutamate as a carbon source. This effect was not due to changes in glutamic dehydrogenase levels. The omission of citrate from the glutamate medium reduced wild-type neutral lipid levels even further, but had no effect on col-2. The variations with time in the neutral lipid levels of col-2 upon changes in these carbon sources are presented, as well as a discussion of the possible types of regulatory effects unique to the col-2 mutation which might affect fatty acid synthesis.     

Improving of lipid productivity of the biodiesel promising green microalga <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis> via low-energy ion implantation

High lipid content in microalgae is an essential parameter for adopting of microalgal biomass as a feedstock for biodiesel. Mutation is one approach to obtain desired algal strain with high lipid production. In this study, a mutant strain of Chlorella pyrenoidosa was isolated using 1.5?×?10¹⁵ ions cm^?2 s^?1 of N⁺ ion beam implantation technique, which has been widely used in mutagenesis of agricultural crops. N⁺ implantation slightly improved the growth of the mutant over the corresponding wild strain with significant increase in lipid content (32.4 % higher than the wild strain), which resulted in significant increase in lipid productivity by 35 %. In addition, ion implantation mutagenesis of C. pyrenoidosa resulted in 21.4 % decrease in total saturated fatty acids (SFAs) compared to the wild type, with a noticeable increase in polyunsaturated fatty acids (PUFAs). The increase in PUFAs was due mainly to stimulation of hexadecadienoic acid (C16:2) and octadecadienoic acid (C18:2) production. However, the SFA content of wild and mutant strains was 31.7 and 24.9 % of total fatty acids, respectively, highlighting the oxidative stability of biodiesel produced by both strains according to the European standards. Cultivation of C. pyrenoidosa mutant in selenite enrichment medium for five successive cultivation experiments showed insignificant changes in biomass productivity, lipid content, and lipid productivity alongside the study period, which confirms the genetic stability of the produced mutant. The present study confirmed the feasibility of generation of microalgae mutants with significant high lipid production using ion beam implantation.     

Overproduction of beta-ketoacyl-acyl carrier protein synthase I imparts thiolactomycin resistance to Escherichia coli K-12.

Thiolactomycin [(4S)(2E,5E)-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene- 4-thiolide] (TLM) is a unique antibiotic structure that inhibits dissociated type II fatty acid synthase systems but not the multifunctional type I fatty acid synthases found in mammals. We screened an Escherichia coli genomic library for recombinant plasmids that impart TLM resistance to a TLM-sensitive strain of E. coli K-12. Nine independent plasmids were isolated, and all possessed a functional beta-ketoacyl-acyl carrier protein synthase I gene (fabB) based on their restriction enzyme maps and complementation of the temperature-sensitive growth of a fabB15(Ts) mutant. A plasmid (pJTB3) was constructed that contained only the fabB open reading frame. This plasmid conferred TLM resistance, complemented the fabB(Ts) mutation, and directed the overproduction of synthase I activity. TLM selectively inhibited unsaturated fatty acid synthesis in vivo; however, synthase I was not the only TLM target, since supplementation with oleate to circumvent the cellular requirement for an active synthase I did not confer TLM resistance. Overproduction of the FabB protein resulted in TLM-resistant fatty acid biosynthesis in vivo and in vitro. These data show that beta-ketoacyl-acyl carrier protein synthase I is a major target for TLM and that increased expression of this condensing enzyme is one mechanism for acquiring TLM resistance. However, extracts from a TLM-resistant mutant (strain CDM5) contained normal levels of TLM-sensitive synthase I activity, illustrating that there are other mechanisms of TLM resistance.     

Fatty acid and complex lipid composition of a Saccharomyces cerevisiae mutant unable to synthesize delta-aminolevulinic acid.

The lipid composition of a Saccharomyces cerevisiae mutant (GL 1–38) lacking δ-aminolevulinic acid synthase (EC 2.3.1.37) was investigated. This mutant is unable to synthesize heme compounds and, as a consequence, cannot make unsaturated fatty acids or ergosterol. The mutant cells were grown (i) in medium supplemented with δ-aminolevulinic acid or (ii) in medium supplemented with Tween 80 (as a source of oleate) and ergosterol. After growth in the presence of δ-aminolevulinic acid, the fatty acid composition of total lipids and mitochondrial lipids was the same as that of the corresponding wild-type strain. After growth in the presence of Tween 80 and ergosterol, the mutant cells contained increased levels of oleate and greatly decreased levels of palmitoleate. The ratio of unsaturated to saturated fatty acids in these cells was still close to that of the wild type but much lower than that of the medium. The sphingolipids accounted for 5.2% of the lipid phosphate in the wild type and, after growth in Tween 80 and ergosterol, for 12.7% in the mutant. Changes in other phospholipids were too small to be considered significant.     

Acyl‐lipid desaturase 1 primes cold acclimation response in Arabidopsis

Membrane fluidity change has long been suggested as the primary mechanism by which, plants adapt to cold stress, but the underlying molecular mechanisms are not completely established. In this study, we found that a knockout of acyl‐lipid/CoA desaturase 1 gene (ADS1; EC 1.14.99) enhances freezing tolerance after cold acclimation (CA). Fatty acid composition analysis demonstrated that 18:1 content in ads1 mutant plants was 20% lower than in wild‐type (WT) grown at 23°C. Lipidomics revealed that 34C‐species of monogalactosyl diacylglycerol (MGDG) content in ads1 mutants were 3.3–14.9% lower than in WT. Lipid positional analysis identified 10% lower 18:1 fatty acid content at the sn‐2 position of MGDG. The cytosolic calcium content in ads1 mutant plants was also approximately two‐times higher than that of WT in response to cold shock. Each of these biochemical differences between WT and ads1 mutant disappeared after CA. Subcellular localization of C‐ and N‐terminal enhanced‐fluorescence‐fusion proteins indicated that ADS1 localized exclusively to chloroplasts. These observations suggest that ADS1‐mediated alteration of chloroplast membrane fluidity is required to prime a CA response, and is the upstream event of cytosolic calcium signaling.     

Beta-ketoacyl-acyl carrier protein synthase III (FabH) is essential for bacterial fatty acid synthesis

beta-Ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, also called acetoacetyl-ACP synthase) encoded by the fabH gene is thought to catalyze the first elongation reaction (Claisen condensation) of type II fatty acid synthesis in bacteria and plant plastids. However, direct in vivo evidence that KAS III catalyzes an essential reaction is lacking, because no mutant organism deficient in this activity has been isolated. We report the first bacterial strain lacking KAS III, a fabH mutant constructed in the Gram-positive bacterium Lactococcus lactis subspecies lactis IL1403. The mutant strain carries an in-frame deletion of the KAS III active site region and was isolated by gene replacement using a medium supplemented with a source of saturated and unsaturated long-chain fatty acids. The mutant strain is devoid of KAS III activity and fails to grow in the absence of supplementation with exogenous long-chain fatty acids demonstrating that KAS III plays an essential role in cellular metabolism. However, the L. lactis fabH deletion mutant requires only long-chain unsaturated fatty acids for growth, a source of long-chain saturated fatty acids is not required. Because both saturated and unsaturated fatty acids are required for growth when fatty acid synthesis is blocked by biotin starvation (which prevents the synthesis of malonyl-CoA), another pathway for saturated fatty acid synthesis must remain in the fabH deletion strain. Indeed, incorporation of [1-14C]acetate into fatty acids in vivo showed that the fabH mutant retained about 10% of the fatty acid synthetic ability of the wild-type strain and that this residual synthetic capacity was preferentially diverted to the saturated branch of the pathway. Moreover, mass spectrometry showed that the fabH mutant retained low levels of palmitic acid upon fatty acid starvation. Derivatives of the fabH deletion mutant strain were isolated that were octanoic acid auxotrophs consistent with biochemical studies indicating that the major role of FabH is production of short-chain fatty acid primers. We also confirmed the essentiality of FabH in Escherichia coli by use of a plasmid-based gene insertion/deletion system. Together these results provide the first genetic evidence demonstrating that FabH conducts the major condensation reaction in the initiation of type II fatty acid biosynthesis in both Gram-positive and Gram-negative bacteria.     

Identification and characterization of a new enoyl coenzyme A hydratase involved in biosynthesis of medium-chain-length polyhydroxyalkanoates in recombinant Escherichia coli

The biosynthetic pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) from fatty acids has been established in fadB mutant Escherichia coli strain by expressing the MCL-PHA synthase gene. However, the enzymes that are responsible for the generation of (R)-3-hydroxyacyl coenzyme A (R3HA-CoAs), the substrates for PHA synthase, have not been thoroughly elucidated. Escherichia coli MaoC, which is homologous to Pseudomonas aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1), was identified and found to be important for PHA biosynthesis in a fadB mutant E. coli strain. When the MCL-PHA synthase gene was introduced, the fadB maoC double-mutant E. coli WB108, which is a derivative of E. coli W3110, accumulated 43% less amount of MCL-PHA from fatty acid compared with the fadB mutant E. coli WB101. The PHA biosynthetic capacity could be restored by plasmid-based expression of the maoCEc gene in E. coli WB108. Also, E. coli W3110 possessing fully functional beta-oxidation pathway could produce MCL-PHA from fatty acid by the coexpression of the maoCEc gene and the MCL-PHA synthase gene. For the enzymatic analysis, MaoC fused with His6-Tag at its C-terminal was expressed in E. coli and purified. Enzymatic analysis of tagged MaoC showed that MaoC has enoyl-CoA hydratase activity toward crotonyl-CoA. These results suggest that MaoC is a new enoyl-CoA hydratase involved in supplying (R)-3-hydroxyacyl-CoA from the beta-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E. coli strain.     

Physcomitrella PpORS,Basal to Plant Type III Polyketide Synthases in Phylogenetic Trees,Is a Very Long Chain 2′-Oxoalkylresorcinol Synthase

The plant type III polyketide synthases (PKSs), which produce diverse secondary metabolites with different biological activities, have successfully co-evolved with land plants. To gain insight into the roles that ancestral type III PKSs played during the early evolution of land plants, we cloned and characterized PpORS from the moss Physcomitrella. PpORS has been proposed to closely resemble the most recent common ancestor of the plant type III PKSs. PpORS condenses a very long chain fatty acyl-CoA with four molecules of malonyl-CoA and catalyzes decarboxylative aldol cyclization to yield the pentaketide 2′-oxoalkylresorcinol. Therefore, PpORS is a 2′-oxoalkylresorcinol synthase. Structure modeling and sequence alignments identified a unique set of amino acid residues (Gln²¹⁸, Val²⁷⁷, and Ala²⁸⁶) at the putative PpORS active site. Substitution of the Ala²⁸⁶ to Phe apparently constricted the active site cavity, and the A286F mutant instead produced triketide alkylpyrones from fatty acyl-CoA substrates with shorter chain lengths. Phylogenetic analysis and comparison of the active sites of PpORS and alkylresorcinol synthases from sorghum and rice suggested that the gramineous enzymes evolved independently from PpORS to have similar functions but with distinct active site architecture. Microarray analysis revealed that PpORS is exclusively expressed in nonprotonemal moss cells. The in planta function of PpORS, therefore, is probably related to a nonprotonemal structure, such as the cuticle.     

利用转座标签mTn-lacZ/leu2插入突变发酵性丝孢酵母2.1368-Leu?筛选高效产油突变株

为提高微生物油脂产率,降低其生产成本,以转座标签mTn-lacZ/leu2插入突变发酵性丝孢酵母2.1368-Leu?筛选高效产油突变株。利用LacZ显色反应、脂肪酸合成酶抑制剂Cerulenin和磷酸香草醛反应,最终在玉米秸秆糖化液中筛选出一株高效产油突变株2.1368-Leu?-7。结果表明其油脂含量为38.30％,比对照的29.33％高了8.97％,而其产油率为8.35％,比对照的6.92％提高了20.63％;在玉米秸秆糖化液中的糖利用率为77％,每100 g玉米秸秆可转化油脂8.32 g。可为未来生物柴油产业提供了廉价原料。     

A polyketide synthase gene required for biosynthesis of fumonisin mycotoxins in Gibberella fujikuroi mating population A.

Fumonisins are toxins associated with several mycotoxicoses and are produced by the maize pathogen Gibberella fujikuroi mating population A (MP-A). Biochemical analyses indicate that fumonisins are a product of either polyketide or fatty acid biosynthesis. To isolate a putative polyketide synthase (PKS) gene involved in fumonisin biosynthesis, we employed PCR with degenerate PKS primers and a cDNA template prepared from a fumonisin-producing culture of G. fujikuroi. Sequence analysis of the single PCR product and its flanking DNA revealed a gene (FUM5) with a 7.8-kb coding region. The predicted FUM5 translation product was highly similar to bacterial and fungal Type I PKSs. Transformation of a cosmid clone carrying FUM5 into G. fujikuroi enhanced production in three strains and restored wild-type production in a fumonisin nonproducing mutant. Disruption of FUM5 reduced fumonisin production by over 99% in G. fujikuroi MP-A. Together, these results indicate that FUM5 is a PKS gene required for fumonisin biosynthesis.