Effect of heterologous expression of phaG [(R)-3-hydroxyacyl-ACP-CoA transferase] on polyhydroxyalkanoate accumulation from the aromatic hydrocarbon phenylacetic acid in Pseudomonas species

Five Pseudomonas strains capable of growth with the aromatic carboxylic acid phenylacetic acid were investigated with a view to improving PHA accumulation. The overexpression of (R)-3-hydroxyacyl-ACP-CoA transferase (PhaG) from Pseudomonas putida CA-3 increased PHA accumulation in only one of the five strains tested, namely Pseudomonas jessenii C8. Recombinant P. jessenii C8 harbouring the phaG gene showed a 4.1-fold increase (9.6-39% cell dry weight) in PHA accumulation when grown on phenylacetic acid (15 mM) compared with the wild-type strain. This is the highest reported level of PHA accumulation from phenylacetic acid. This is also the first time the heterologous expression of phaG has resulted in improved PHA accumulation from an aromatic carbon source. The growth patterns of the wild type and recombinant strains were very similar, with no significant differences observed in carbon and nitrogen utilization.     

The production of polyhydroxyalkanoate by Bacillus licheniformis using sequential mutagenesis and optimization

The purpose of this study was to enhance the production of polyhydroxyalkanoate (PHA) by sequential mutation of Bacillus licheniformis PHAs-007, using UV and N-methyl-N′-nitro-N-nitrosoguanidine (NTG). In addition, the effect of nutrient additions and environmental conditions were optimized to increase the production of PHA. Bacillus licheniformis PHAs-007 produced high amounts of PHA (64.09 ～ 68.80% of DCW) under both synthetic and renewable substrates. After mutagenesis treatment, mutant M2-12 was selected from 380 strains, based on its high biomass and PHA concentration. The mutant M2-12 gave the highest value of specific growth rate (0.09/h), biomass (22.24 g/L) and PHA content (19.55 g/L) under optimal conditions, consisting of 3% palm oil mill effluent, with no additional trace elements, at 45oC and pH 7. The mutant strain showed higher resistance to substrate concentrations, as well as pH and temperature, than the wild type. The accumulation of PHA was increased by 3.18-fold compared to the wild type, and the production of PHA by the mutant M2-12 was constantly retained over 12 times of cultivation. The mutation and optimization strategy appear to be suitable for producing high density PHA, reducing the medium cost and consequently lowering the production cost. Interestingly, the mutant strain could synthesize the novel PHA copolymers such as 3-hydroxyvalerate and 3-hydroxyhexanoate, which were not produced by the wild type.     

Polyhydroxyalkanoates are essential for maintenance of redox state in the Antarctic bacterium Pseudomonas sp. 14-3 during low temperature adaptation

Polyhydroxyalkanoates (PHAs) are highly reduced bacterial storage compounds that increase fitness in changing environments. We have previously shown that phaRBAC genes from the Antarctic bacterium Pseudomonas sp. 14-3 are located in a genomic island containing other genes probably related with its adaptability to cold environments. In this paper, Pseudomonas sp. 14-3 and its PHA synthase-minus mutant (phaC) were used to asses the effect of PHA accumulation on the adaptability to cold conditions. The phaC mutant was unable to grow at 10°C and was more susceptible to freezing than its parent strain. PHA was necessary for the development of the oxidative stress response induced by cold treatment. Addition of reduced compounds cystine and gluthathione suppressed the cold sensitive phenotype of the phaC mutant. Cold shock produced very rapid degradation of PHA in the wild type strain. The NADH/NAD ratio and NADPH content, estimated by diamide sensitivity, decreased strongly in the mutant after cold shock while only minor changes were observed in the wild type. Accordingly, the level of lipid peroxidation in the mutant strain was 25-fold higher after temperature downshift. We propose that PHA metabolism modulates the availability of reducing equivalents, contributing to alleviate the oxidative stress produced by low temperature.     

Inactivation of Isocitrate Lyase Leads to Increased Production of Medium-Chain-Length Poly(3-Hydroxyalkanoates) in Pseudomonas putida

Medium-chain-length (mcl) poly(3-hydroxyalkanoates) (PHAs) are storage polymers that are produced from various substrates and accumulate in Pseudomonas strains belonging to rRNA homology group I. In experiments aimed at increasing PHA production in Pseudomonas strains, we generated an mcl PHA-overproducing mutant of Pseudomonas putida KT2442 by transposon mutagenesis, in which the aceA gene was knocked out. This mutation inactivated the glyoxylate shunt and reduced the in vitro activity of isocitrate dehydrogenase, a rate-limiting enzyme of the citric acid cycle. The genotype of the mutant was confirmed by DNA sequencing, and the phenotype was confirmed by biochemical experiments. The aceA mutant was not able to grow on acetate as a sole carbon source due to disruption of the glyoxylate bypass and exhibited two- to fivefold lower isocitrate dehydrogenase activity than the wild type. During growth on gluconate, the difference between the mean PHA accumulation in the mutant and the mean PHA accumulation in the wild-type strain was 52%, which resulted in a significant increase in the amount of mcl PHA at the end of the exponential phase in the mutant P. putida KT217. On the basis of a stoichiometric flux analysis we predicted that knockout of the glyoxylate pathway in addition to reduced flux through isocitrate dehydrogenase should lead to increased flux into the fatty acid synthesis pathway. Therefore, enhanced carbon flow towards the fatty acid synthesis pathway increased the amount of mcl PHA that could be accumulated by the mutant.     

Inactivation of isocitrate lyase leads to increased production of medium-chain-length poly(3-hydroxyalkanoates) in Pseudomonas putida

Medium-chain-length (mcl) poly(3-hydroxyalkanoates) (PHAs) are storage polymers that are produced from various substrates and accumulate in Pseudomonas strains belonging to rRNA homology group I. In experiments aimed at increasing PHA production in Pseudomonas strains, we generated an mcl PHA-overproducing mutant of Pseudomonas putida KT2442 by transposon mutagenesis, in which the aceA gene was knocked out. This mutation inactivated the glyoxylate shunt and reduced the in vitro activity of isocitrate dehydrogenase, a rate-limiting enzyme of the citric acid cycle. The genotype of the mutant was confirmed by DNA sequencing, and the phenotype was confirmed by biochemical experiments. The aceA mutant was not able to grow on acetate as a sole carbon source due to disruption of the glyoxylate bypass and exhibited two- to fivefold lower isocitrate dehydrogenase activity than the wild type. During growth on gluconate, the difference between the mean PHA accumulation in the mutant and the mean PHA accumulation in the wild-type strain was 52%, which resulted in a significant increase in the amount of mcl PHA at the end of the exponential phase in the mutant P. putida KT217. On the basis of a stoichiometric flux analysis we predicted that knockout of the glyoxylate pathway in addition to reduced flux through isocitrate dehydrogenase should lead to increased flux into the fatty acid synthesis pathway. Therefore, enhanced carbon flow towards the fatty acid synthesis pathway increased the amount of mcl PHA that could be accumulated by the mutant.     

Synthesis of poly(3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains

We have studied the accumulation kinetics and physical characteristics of the poly(3-hydroxyalkanoates) (PHAs) formed by several Pseudomonas strains, mutants and recombinants. Although PHA synthesis generally begins only after an essential nutrient such as N, P, S or Mg becomes limiting, we have identified at least one strain (P. putida KT2442) that begins producing PHA during the exponential growth phase. This PHA is chemically and physically identical to that produced by P. oleovorans GPol, the strain in which we first identified PHA. Analysis of the PHA formed by a mutant strain defective in PHA degradation (P. oleovorans GPo500) revealed that the molecular mass (M_w), the monomer composition and thermal characteristics were similar to that of the PHA of the wild-type parent strain P. oleovorans GPo1. The pha locus of P. oleovorans encodes enzymes that are involved in PHA biosynthesis and degradation. It has been subcloned to study the two PHA polymerases separately in a PHA^– mutant (GPp104) derived from P. putida KT2442. The recombinant strains accumulated lower PHA levels than the wild-type strains, and the M_w of these polymers were lower than those produced by the wild-type P. oleovorans and parent strain. The monomer composition of the two PHAs formed by the two PHA polymerases differed, indicating that the PHA polymerases have different substrate specificities for the incorporation of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers into PHA. Despite these differences, the PHAs formed were essentially indistinguishable from wild-type PHAs with respect to their thermal characteristics.Correspondence to: B. Witholt     

In vivo evolution of the Aeromonas punctata polyhydroxyalkanoate (PHA) synthase: isolation and characterization of modified PHA synthases with enhanced activity

In vivo random mutagenesis of the polyhydroxyalkanoate (PHA) synthase gene from Aeromonas punctata was performed employing the mutator strain Escherichia coli XL1-Red. About 200,000 mutants were screened on Nile red-containing medium and five mutants with enhanced fluorescence were selected. Four of these mutants exhibited enhanced in vivo and in vitro PHA synthase activity. Mutant M1, which carried the single mutation F518I, showed a five-fold increase in specific PHA synthase activity, whereas the corresponding mediated PHA accumulation increased by 20%, as compared with the wild-type PHA synthase. Mutant M2, which carried the single mutation V214G, showed a two-fold increase in specific PHA synthase activity and PHA accumulation only increased by 7%. Overall, the in vitro activities of the overproducing mutants ranged from 1.1- to 5-fold more than the wild-type activity, whereas the amounts of accumulated PHA ranged over 107–126% of that of the wild type. Moreover, all mutants mediated synthesis of PHAs with an increased weight average molar mass, but the molar fractions of 3-hydroxybutyrate and 3-hydroxyhexanoate remained almost constant. In vivo random mutagenesis proved to be a versatile tool to isolate mutants exerting improved properties with respect to PHA biosynthesis. Electronic Publication     

Fusarium graminearum Tri12p influences virulence to wheat and trichothecene accumulation

The gene Tri12 encodes a predicted major facilitator superfamily protein suggested to play a role in export of trichothecene mycotoxins produced by Fusarium spp. It is unclear, however, how the Tri12 protein (Tri12p) may influence trichothecene sensitivity and virulence of the wheat pathogen Fusarium graminearum. In this study, we establish a role for Tri12 in toxin accumulation and sensitivity as well as in pathogenicity toward wheat. Tri12 deletion mutants (tri12) are reduced in virulence and result in decreased trichothecene accumulation when inoculated on wheat compared with the wild-type strain or an ectopic mutant. Reduced radial growth of tri12 mutants on trichothecene biosynthesis induction medium was observed relative to the wild type and the ectopic strains. Diminished trichothecene accumulation was observed in liquid medium cultures inoculated with tri12 mutants. Wild-type fungal cells grown under conditions that induce trichothecene biosynthesis develop distinct subapical swelling and form large vacuoles. A strain expressing Tri12p linked to green fluorescent protein shows localization of the protein consistent with the plasma membrane. Our results indicate Tri12 plays a role in self-protection and influences toxin production and virulence of the fungus in planta.     

Effect of hydroxylated carotenoid deficiency on ABA accumulation in Arabidopsis

Abscisic acid (ABA) is a sesquiterpene compound (C₁₅) derived from C₄₀ carotenoids. The immediate carotenoid precursors for ABA biosynthesis, 9- cis -violaxanthin and 9'- cis -neoxanthin, are produced from β -carotene by a series of hydroxylation, epoxidation, and isomerization reactions. Carotenoid hydroxylase deficient mutants contain severely reduced levels of violaxanthin and neoxanthin ( < 20% of wild type level) and provide a unique system to correlate carotenoid substrate availability and ABA production in photosynthetic tissues under non-stressed conditions. Quantitative measurements indicated that ABA levels in the carotenoid hydroxylase mutants are reduced nearly 50% compared to the wild type plants under non-stressed conditions. When drought-stressed, wild type plants showed up to a 17-fold increase in ABA levels, while ABA levels in the carotenoid hydroxylase mutants were only increased 6- to 7-fold (25% of wild type drought-stressed ABA levels). Expression of AtNCED3 ( Arabidopsis thaliana nine- cis -epoxycarotenoid dioxygenase 3, the rate-limiting activity for ABA biosynthesis) was induced in the carotenoid hydroxylase mutants, but to a lesser extent than the 40-fold increase in wild type plants. Therefore, the reduced ABA accumulation in response to drought-stress is at least partially due to the attenuated increase in AtNCED3 gene expression in the carotenoid hydroxylase mutants. The remaining violaxanthin and neoxanthin in the carotenoid hydroxylase mutants can not be converted into ABA, indicating that there is probably a separate pool of violaxanthin and neoxanthin that is not accessible to the cleavage enzymes, because it is sequestered in the light-harvesting complexes.     

Effect of reduced arginine decarboxylase activity on salt tolerance and on polyamine formation during salt stress in Arabidopsis thaliana

Polyamines have been suggested to play an important role in stress protection. However, attempts to determine the function of polyamines have been complicated by the fact that, dependent on the conditions, polyamine contents increase or decrease during stress. To determine the importance of polyamine formation during salt stress, we analysed polyamine contents and salt tolerance in two Arabidopsis thaliana mutants, spe1-1 and spe2-1 (Watson et al. Plant J 13: 231–239, 1998), with reduced activity of arginine decarboxylase (EC 4.1.1.19), an important enzyme in polyamine synthesis. Polyamines accumulated in wild-type plants (Col-0 and Ler-0) that were pre-treated with 100 m M NaCl before transfer to 125 m M NaCl, but not in plants that were directly transferred to 125 m M NaCl without prior treatment with 100 m M NaCl. This shows that polyamine accumulation depends on acclimation to salinity. The salt treatment that induced polyamine accumulation in wild-type plants did not lead to polyamine accumulation in the spe1-1 and spe2-1 mutants. Decreased fresh weight, chlorophyll content and photosynthetic efficiency indicated that the spe1-1 mutant was more severely affected by salt stress than its wild type, Col-0. In the spe2-1 mutant decreased salt tolerance compared to its wild type, Ler-0, became apparent as bleaching under severe salt stress. The present results demonstrate that decreased polyamine formation due to lower arginine decarboxylase activity leads to reduced salt tolerance.     

细菌群体感应参与铜绿假单胞菌胞内聚羟基脂肪酸酯合成的调控

群体感应是细菌根据细胞密度变化调控基因表达的一种调节机制。铜绿假单胞菌中QS系统由lasI和rhlI合成的信号分子3OC12-HSL和C4-HSL以及各自的受体蛋白LasR、RhlR组成,它们以级联方式调控多个基因表达。【目的】研究细菌群体感应(QS)对聚羟基脂肪酸酯合成的调控。【方法】利用铜绿假单胞菌PAO1及其QS突变株为材料通过气相色谱、荧光定量PCR在生理和分子水平上研究QS对聚羟基脂肪酸酯合成的调控。【结果】QS信号分子合成抑制剂阿奇霉素处理铜绿假单胞菌PAO1和QS突变株导致胞内PHA积累量显著减少;铜绿假单胞菌PAO1中C4-HSL合成酶基因rhlI缺失突变株PAO210胞内PHA积累量与野生型无差别;而3OC12-HSL合成酶基因lasI缺失突变株PAO55、3OC12-HSL受体合成酶基因lasR缺失突变株PAO56以及lasI/lasR双缺失突变株PAO57胞内PHA含量与野生型相比明显减少;lasI和lasR的突变株体内PHA合成酶基因phaC1的表达量显著降低,信号分子3OC12-HSL回补实验使phaC1的表达量可恢复到野生株水平,但只可部分恢复lasI缺失导致的胞内PHA合成。【结论】由此推测,铜绿假单胞菌群体感应系统中lasI/lasR系统参与胞内聚羟基脂肪酸酯合成的调控。     

A metal-accumulator mutant of Arabidopsis thaliana.

A mutation designated man1 (for manganese accumulator) was found to cause Arabidopsis thaliana seedlings to accumulate a range of metals. The man1 mutation segregated as a single recessive locus located on chromosome 3. When grown on soil, mutant seedlings accumulated Mn (7.5 times greater than wild type), Cu (4.6 times greater than wild type), Zn (2.8 times greater than wild type), and Mg (1.8 times greater than wild type) in leaves. In addition to these metals, the man1 mutant accumulated 2.7-fold more S in leaves, primarily in the oxidized form, than wild-type seedlings. Analysis of seedlings grown by hydroponic culture showed a similar accumulation of metals in leaves of man1 mutants. Roots of man1 mutants also accumulated metals, but unlike leaves they accumulated 10-fold more total Fe (symplasmic and apoplasmic combined) than wild-type roots. Roots of man1 mutants possessed greater (from 1.8- to 20-fold) ferric-chelate reductase activity than wild-type seedings, and this activity was not responsive to changes of Mn nutrition in either genotype. Taken together, these results suggest that the man1 mutation disrupts the regulation of metal-ion uptake or homeostasis in Arabidopsis.     

Mutants of Streptococcus gordonii Challis over-producing glucosyltransferase

Two mutants of Streptococcus gordonii which over-produced extracellular polysaccharide when grown on sucrose-containing medium were isolated after mutagenesis of strain Challis with ethyl methanesulphonate. The mutants, designated strains OB20 and OB30, expressed 2.6-fold and 4.7-fold respectively more glucosyltransferase (GTF) activities than the wild-type strain. Transformation experiments suggested that the two mutants carried different mutations, denoted gtf-20 and gtf-30. A double mutant (gtf-20 gtf-30) was constructed and this strain produced 6.4-fold more GTF. Enzymes from wild-type and mutant strains were biochemically indistinguishable and they synthesized structurally identical glucans. Increasing the Na+ concentration of the bacterial growth medium reduced GTF production in all strains by about 60%. Tween 80 also inhibited enzyme production and more specifically reduced GTF synthesis by the mutants. The mutations gtf-20 and gtf-30 appear to define separate genetic loci involved in regulating expression of GTF activity in S. gordonii.     

Effect of water activity on invertase production in solid state fermentation by improved diploid strains of Aspergillus niger

Invertase production under solid state fermentation (SSF) was determined using two overproducing mutants (Aw96-3 and Aw96-4) isolated previously from the wild type strain Aspergillus niger C28B25, as well as one diploid (DAR1) and two autodiploid strains (AD96-3 and AD96-4) constructed by parasexual crossings among these mutants. Using polyurethane foam (PUF) as an inert carrier, two initial water activity (Aw) values were evaluated (0.99 and 0.96). At Aw=0.99, maximal activity was reached by diploid AD96-4 (48.91 IU/ml) representing 30- and 13-fold increases with respect to maximal values achieved by the wild type and the haploid parental mutant (Aw96-4), respectively. Similar levels were achieved by this strain at Aw=0.96. However, diploid DAR1 only produced high levels of invertase at Aw=0.96 (43.90 IU/ml), whereas strain AD96-3 reached its highest production (31.10 IU/ml) at Aw=0.99. Both productivity and yields were also analysed for every strain at each Aw value.     

Genetically modified strains of Ralstonia eutropha H16 with β-ketothiolase gene deletions for production of copolyesters with defined 3-hydroxyvaleric acid contents

β-Ketothiolases catalyze the first step of poly(3-hydroxybutyrate) [poly(3HB)] biosynthesis in bacteria by condensation of two acetyl coenzyme A (acetyl-CoA) molecules to acetoacetyl-CoA and also take part in the degradation of fatty acids. During growth on propionate or valerate, Ralstonia eutropha H16 produces the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)]. In R. eutropha, 15 β-ketothiolase homologues exist. The synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) could be significantly reduced in an 8-fold mutant (Lindenkamp et al., Appl. Environ. Microbiol. 76:5373-5382, 2010). In this study, a 9-fold mutant deficient in nine β-ketothiolase gene homologues (phaA, bktB, H16_A1713, H16_B1771, H16_A1528, H16_B0381, H16_B1369, H16_A0170, and pcaF) was generated. In order to examine the polyhydroxyalkanoate production capacity when short- or long-chain and even- or odd-chain-length fatty acids were provided as carbon sources, the growth and storage behavior of several mutants from the previous study and the newly generated 9-fold mutant were analyzed. Propionate, valerate, octanoate, undecanoic acid, or oleate was chosen as the sole carbon source. On octanoate, no significant differences in growth or storage behavior were observed between wild-type R. eutropha and the mutants. In contrast, during the growth on oleate of a multiple mutant lacking phaA, bktB, and H16_A0170, diminished poly(3HB) accumulation occurred. Surprisingly, the amount of accumulated poly(3HB) in the multiple mutants grown on gluconate differed; it was much lower than that on oleate. The β-ketothiolase activity toward acetoacetyl-CoA in H16ΔphaA and all the multiple mutants remained 10-fold lower than the activity of the wild type, regardless of which carbon source, oleate or gluconate, was employed. During growth on valerate as a sole carbon source, the 9-fold mutant accumulated almost a poly(3-hydroxyvalerate) [poly(3HV)] homopolyester with 99 mol% 3HV constituents.     

Succinate:quinol oxidoreductases in the cyanobacterium synechocystis sp. strain PCC 6803: presence and function in metabolism and electron transport

The open reading frames sll1625 and sll0823, which have significant sequence similarity to genes coding for the FeS subunits of succinate dehydrogenase and fumarate reductase, were deleted singly and in combination in the cyanobacterium Synechocystis sp. strain PCC 6803. When the organic acid content in the Deltasll1625 and Deltasll0823 strains was analyzed, a 100-fold decrease in succinate and fumarate concentrations was observed relative to the wild type. A similar analysis for the Deltasll1625 Deltasll0823 strain revealed that 17% of the wild-type succinate levels remained, while only 1 to 2% of the wild-type fumarate levels were present. Addition of 2-oxoglutarate to the growth media of the double mutant strain prior to analysis of organic acids in cells caused succinate to accumulate. This indicates that succinate dehydrogenase activity had been blocked by the deletions and that 2-oxoglutarate can be converted to succinate in vivo in this organism, even though a traditional 2-oxoglutarate dehydrogenase is lacking. In addition, reduction of the thylakoid plastoquinone pool in darkness in the presence of KCN was up to fivefold slower in the mutants than in the wild type. Moreover, in vitro succinate dehydrogenase activity observed in wild-type membranes is absent from those isolated from the double mutant and reduced in those from the single mutants, further indicating that the sll1625 and sll0823 open reading frames encode subunits of succinate dehydrogenase complexes that are active in the thylakoid membrane of the cyanobacterium.