Mannuronate C-5 epimerases and their use in alginate modification.

Alginate is a polysaccharide consisting of ß-D-mannuronate (M) and α-L-guluronate (G) produced by brown algae and some bacterial species. Alginate has a wide range of industrial and pharmaceutical applications, owing mainly to its gelling and viscosifying properties. Alginates with high G content are considered more valuable since the G residues can form hydrogels with divalent cations. Alginates are modified by lyases, acetylases, and epimerases. Alginate lyases are produced by alginate-producing organisms and by organisms that use alginate as a carbon source. Acetylation protects alginate from lyases and epimerases. Following biosynthesis, alginate C-5 epimerases convert M to G residues at the polymer level. Alginate epimerases have been found in brown algae and alginate-producing bacteria, predominantly Azotobacter and Pseudomonas species. The best characterised epimerases are the extracellular family of AlgE1-7 from Azotobacter vinelandii(Av). AlgE1-7 all consist of combinations of one or two catalytic A-modules and one to seven regulatory R-modules, but even though they are sequentially and structurally similar, they create different epimerisation patterns. This makes the AlgE enzymes promising for tailoring of alginates to have the desired properties. The present review describes the current state of knowledge regarding alginate-active enzymes with focus on epimerases, characterisation of the epimerase reaction, and how alginate epimerases can be used in alginate production.

Improved site-specific mutagenesis in Rhodococcus opacus using a novel conditional suicide plasmid.

Rhodococcus opacus PD630 is a biotechnologically important bacterium with metabolic capability for bioremediation, metal recovery, and storage of triacylglycerols. Genome editing by homologous recombination in R. opacus is hampered by a very low combined frequency of DNA transfer and recombination. To improve recombination in the species, a conjugative, conditional suicide plasmid based on the replicon derived from the Corynebacterium glutamicum plasmid pGA1 was constructed and evaluated in R. opacus. The replication of this plasmid is controlled by a dual inducible and repressible promoter system originally developed for Mycobacterium spp. Next, we demonstrated that a derivative of this plasmid containing sacB as a counterselection marker and homologous regions of R. opacus could be used for homologous recombination, and that the problem of obtaining recombinants had been solved. Like for other Corynebacteriales, the cell wall of Rhodococcus spp. contains mycolic acids which form a hydrophobic and impermeable outer layer. Mycolic acids are essential for Mycobacterium smegmatis, but not for Corynebacterium glutamicum, and the new vector was used to study if mycolic acid is essential for R. opacus. We found that accD3 that is necessary for mycolic acid synthesis could only be deleted from the chromosome in strains containing a plasmid-encoded copy of accD3. This indicates that mycolic acid is important for R. opacus viability. The conditional suicide vector should be useful for homologous recombination or for delivering gene products like recombinases or Cas proteins and gRNA to Rhodococcus and related genera, while the approach should be applicable for any plasmid needing a plasmid-encoded protein for replication. KEY POINTS: • Improved vector for homologous recombination in R. opacus. • Mycolic acid is important for survival of R. opacus like it is for Mycobacterium. • Similar conditional suicide plasmids may be constructed for other bacteria.

Strain Construction and Process Development for Efficient Recombinant Production of Mannuronan C-5 Epimerases in Hansenula polymorpha.

Alginates are linear polysaccharides produced by brown algae and some bacteria and are composed of ß-D-mannuronic acid (M) and α-L-guluronic acid (G). Alginate has numerous present and potential future applications within industrial, medical and pharmaceutical areas and G rich alginates are traditionally most valuable and frequently used due to their gelling and viscosifying properties. Mannuronan C-5 epimerases are enzymes converting M to G at the polymer level during the biosynthesis of alginate. The Azotobacter vinelandii epimerases AlgE1-AlgE7 share a common structure, containing one or two catalytic A-modules (A), and one to seven regulatory R-modules (R). Despite the structural similarity of the epimerases, they create different M-G patterns in the alginate; AlgE4 (AR) creates strictly alternating MG structures whereas AlgE1 (ARRRAR) and AlgE6 (ARRR) create predominantly G-blocks. These enzymes are therefore promising tools for producing in vitro tailor-made alginates. Efficient in vitro epimerization of alginates requires availability of recombinantly produced alginate epimerases, and for this purpose the methylotrophic yeast Hansenula polymorpha is an attractive host organism. The present study investigates whether H. polymorpha is a suitable expression system for future large-scale production of AlgE1, AlgE4, and AlgE6. H. polymorpha expression strains were constructed using synthetic genes with reduced repetitive sequences as well as optimized codon usage. High cell density cultivations revealed that the largest epimerases AlgE1 (147 kDa) and AlgE6 (90 kDa) are subject to proteolytic degradation by proteases secreted by the yeast cells. However, degradation could be controlled to a large extent either by co-expression of chaperones or by adjusting cultivation conditions. The smaller AlgE4 (58 kDa) was stable under all tested conditions. The results obtained thus point toward a future potential for using H. polymorpha in industrial production of mannuronan C-5 epimerases for in vitro tailoring of alginates.

Overexpression of Two New Acyl-CoA:Diacylglycerol Acyltransferase 2-Like Acyl-CoA:Sterol Acyltransferases Enhanced Squalene Accumulation in Aurantiochytrium limacinum.

Thraustochytrids are heterotrophic marine eukaryotes known to accumulate large amounts of triacylglycerols, and they also synthesize terpenoids like carotenoids and squalene, which all have an increasing market demand. However, a more extensive knowledge of the lipid metabolism is needed to develop thraustochytrids for profitable biomanufacturing. In this study, two putative type-2 Acyl-CoA:diacylglycerol acyltransferases (DGAT2) genes of Aurantiochytrium sp. T66, T66ASATa, and T66ASATb, and their homologs in Aurantiochytrium limacinum SR21, AlASATa and AlASATb, were characterized. In A. limacinum SR21, genomic knockout of AlASATb reduced the amount of the steryl esters of palmitic acid, SE (16:0), and docosahexaenoic acid, SE (22:6). The double mutant of AlASATa and AlASATb produced even less of these steryl esters. The expression and overexpression of T66ASATb and AlASATb, respectively, enhanced SE (16:0) and SE (22:6) production more significantly than those of T66ASATa and AlASATa. In contrast, these mutations did not significantly change the level of triacylglycerols or other lipid classes. The results suggest that the four genes encoded proteins possessing acyl-CoA:sterol acyltransferase (ASAT) activity synthesizing both SE (16:0) and SE (22:6), but with the contribution from AlASATb and T66ASATb being more important than that of AlASATa and T66ASATa. Furthermore, the expression and overexpression of T66ASATb and AlASATb enhanced squalene accumulation in SR21 by up to 88%. The discovery highlights the functional diversity of DGAT2-like proteins and provides valuable information on steryl ester and squalene synthesis in thraustochytrids, paving the way to enhance squalene production through metabolic engineering.

Mechanistic Basis for Understanding the Dual Activities of the Bifunctional Azotobacter vinelandii Mannuronan C-5-Epimerase and Alginate Lyase AlgE7.

The structure and functional properties of alginates are dictated by the monomer composition and molecular weight distribution. Mannuronan C-5-epimerases determine the monomer composition by catalyzing the epimerization of ß-d-mannuronic acid (M) residues into α-l-guluronic acid (G) residues. The molecular weight is affected by alginate lyases, which catalyze a ß-elimination mechanism that cleaves alginate chains. The reaction mechanisms for the epimerization and lyase reactions are similar, and some enzymes can perform both reactions. These dualistic enzymes share high sequence identity with mannuronan C-5-epimerases without lyase activity. The mechanism behind their activity and the amino acid residues responsible for it are still unknown. We investigate mechanistic determinants involved in the bifunctional epimerase and lyase activity of AlgE7 from Azotobacter vinelandii. Based on sequence analyses, a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by nuclear magnetic resonance (NMR) spectroscopy. Our results show that calcium promotes lyase activity, whereas NaCl reduces the lyase activity of AlgE7. By using defined polymannuronan (polyM) and polyalternating alginate (polyMG) substrates, the preferred cleavage sites of AlgE7 were found to be M|XM and G|XM, where X can be either M or G. From the study of AlgE7 mutants, R148 was identified as an important residue for the lyase activity, and the point mutant R148G resulted in an enzyme with only epimerase activity. Based on the results obtained in the present study, we suggest a unified catalytic reaction mechanism for both epimerase and lyase activities where H154 functions as the catalytic base and Y149 functions as the catalytic acid. IMPORTANCE Postharvest valorization and upgrading of algal constituents are promising strategies in the development of a sustainable bioeconomy based on algal biomass. In this respect, alginate epimerases and lyases are valuable enzymes for tailoring the functional properties of alginate, a polysaccharide extracted from brown seaweed with numerous applications in food, medicine, and material industries. By providing a better understanding of the catalytic mechanism and of how the two enzyme actions can be altered by changes in reaction conditions, this study opens further applications of bacterial epimerases and lyases in the enzymatic tailoring of alginate polymers.

Method Development Progress in Genetic Engineering of Thraustochytrids.

Thraustochytrids are unicellular, heterotrophic marine eukaryotes. Some species are known to store surplus carbon as intracellular lipids, and these also contain the long-chain polyunsaturated fatty acid docosahexaenoic acid (DHA). Most vertebrates are unable to synthesize sufficient amounts of DHA, and this fatty acid is essential for, e.g., marine fish, domesticated animals, and humans. Thraustochytrids may also produce other commercially valuable fatty acids and isoprenoids. Due to the great potential of thraustochytrids as producers of DHA and other lipid-related molecules, a need for more knowledge on this group of organisms is needed. This necessitates the ability to do genetic manipulation of the different strains. Thus far, this has been obtained for a few strains, while it has failed for other strains. Here, we systematically review the genetic transformation methods used for different thraustochytrid strains, with the aim of aiding studies on strains not yet successfully transformed. The designs of transformation cassettes are also described and compared. Moreover, the potential problems when trying to establish transformation protocols in new thraustochytrid species/strains are discussed, along with suggestions utilized in other organisms to overcome similar challenges. The approaches discussed in this review could be a starting point when designing protocols for other non-model organisms.

A non-canonical Δ9-desaturase synthesizing palmitoleic acid identified in the thraustochytrid Aurantiochytrium sp. T66.

Thraustochytrids are oleaginous marine eukaryotic microbes currently used to produce the essential omega-3 fatty acid docosahexaenoic acid (DHA, C22:6 n-3). To improve the production of this essential fatty acid by strain engineering, it is important to deeply understand how thraustochytrids synthesize fatty acids. While DHA is synthesized by a dedicated enzyme complex, other fatty acids are probably synthesized by the fatty acid synthase, followed by desaturases and elongases. Which unsaturated fatty acids are produced differs between different thraustochytrid genera and species; for example, Aurantiochytrium sp. T66, but not Aurantiochytrium limacinum SR21, synthesizes palmitoleic acid (C16:1 n-7) and vaccenic acid (C18:1 n-7). How strain T66 can produce these fatty acids has not been known, because BLAST analyses suggest that strain T66 does not encode any Δ9-desaturase-like enzyme. However, it does encode one Δ12-desaturase-like enzyme. In this study, the latter enzyme was expressed in A. limacinum SR21, and both C16:1 n-7 and C18:1 n-7 could be detected in the transgenic cells. Our results show that this desaturase, annotated T66Des9, is a Δ9-desaturase accepting C16:0 as a substrate. Phylogenetic studies indicate that the corresponding gene probably has evolved from a Δ12-desaturase-encoding gene. This possibility has not been reported earlier and is important to consider when one tries to deduce the potential a given organism has for producing unsaturated fatty acids based on its genome sequence alone. KEY POINTS: • In thraustochytrids, automatic gene annotation does not always explain the fatty acids produced. • T66Des9 is shown to synthesize palmitoleic acid (C16:1 n-7). • T66des9 has probably evolved from Δ12-desaturase-encoding genes.

Combined Metabolome and Lipidome Analyses for In-Depth Characterization of Lipid Accumulation in the DHA Producing Aurantiochytrium sp. T66.

Thraustochytrids are marine heterotrophic microorganisms known for their potential to accumulate docosahexaenoic acid (DHA)-enriched lipids. There have been many attempts to improve thraustochytrid DHA bioprocesses, especially through traditional optimization of cultivation and media conditions. Nevertheless, thraustochytrid-based bioprocesses are still not commercially competitive for high volume-low cost production of DHA. Thus, it is realized that genetic and metabolic engineering strategies are needed for the development of commercially competitive thraustochytrid DHA cell factories. Here, we present an analytical workflow for high resolution phenotyping at metabolite and lipid levels to generate deeper insight into the thraustochytrid physiology, with particular focus on central carbon and redox metabolism. We use time-series sampling during unlimited growth and nitrogen depleted triggering of DHA synthesis and lipid accumulation (LA) to show-case our methodology. The mass spectrometric absolute quantitative metabolite profiling covered glycolytic, pentose phosphate pathway (PPP) and tricarboxylic acid cycle (TCA) metabolites, amino acids, complete (deoxy)nucleoside phosphate pools, CoA and NAD metabolites, while semiquantitative high-resolution supercritical fluid chromatography MS/MS was applied for the lipid profiling. Interestingly, trace amounts of a triacylglycerols (TG) with DHA incorporated in all three acyl positions was detected, while TGs 16:0_16:0_22:6 and 16:0_22:6_22:6 were among the dominant lipid species. The metabolite profiling data indicated that lipid accumulation is not limited by availability of the acyl chain carbon precursor acetyl-CoA nor reducing power (NADPH) but rather points to the TG head group precursor glycerol-3-phosphate as the potential cause at the metabolite level for the gradual decline in lipid production throughout the cultivation. This high-resolution phenotyping provides new knowledge of changes in the central metabolism during growth and LA in thraustochytrids and will guide target selection for metabolic engineering needed for further improvements of this DHA cell factory.

Functional characterization of three Azotobacter chroococcum alginate-modifying enzymes related to the Azotobacter vinelandii AlgE mannuronan C-5-epimerase family.

Bacterial alginate initially consists of 1-4-linked ß-D-mannuronic acid residues (M) which can be later epimerized to α-L-guluronic acid (G). The family of AlgE mannuronan C-5-epimerases from Azotobacter vinelandii has been extensively studied, and three genes putatively encoding AlgE-type epimerases have recently been identified in the genome of Azotobacter chroococcum. The three A. chroococcum genes, here designated AcalgE1, AcalgE2 and AcalgE3, were recombinantly expressed in Escherichia coli and the gene products were partially purified. The catalytic activities of the enzymes were stimulated by the addition of calcium ions in vitro. AcAlgE1 displayed epimerase activity and was able to introduce long G-blocks in the alginate substrate, preferentially by attacking M residues next to pre-existing G residues. AcAlgE2 and AcAlgE3 were found to display lyase activities with a substrate preference toward M-alginate. AcAlgE2 solely accepted M residues in the positions - 1 and + 2 relative to the cleavage site, while AcAlgE3 could accept either M or G residues in these two positions. Both AcAlgE2 and AcAlgE3 were bifunctional and could also catalyze epimerization of M to G. Together, we demonstrate that A. chroococcum encodes three different AlgE-like alginate-modifying enzymes and the biotechnological and biological impact of these findings are discussed.

Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coli.

BACKGROUND: Advantages of translocation of recombinant proteins to the periplasm in Escherichia coli include simplified downstream processing, and improved folding and in vivo activity of the target protein. There are, however, problems encountered in the periplasmic production that can be associated with the incorrect formation of disulfide bonds, incomplete cleavage of the signal peptide, and proteolytic degradation. A common strategy used to overcome these difficulties involves manipulating the cellular levels of proteases and periplasmic folding assistants like chaperones, signal peptide peptidases or thiol-disulfide oxidoreductases. To date, this has been achieved by plasmid-based over-expression or knockouts of the relevant genes. RESULTS: We changed the translation efficiencies of five native E. coli proteins, DsbA, DsbB, Skp, SppA, and DegP, by modifying the strength of their ribosome binding sites (RBS). The genomic RBS sequences were replaced with synthetic ones that provided a predicted translation initiation rate. Single- and double-gene mutant strains were created and tested for production of two pharmaceutically relevant proteins, PelB-scFv173-2-5-AP and OmpA-GM-CSF. Almost all the single-gene mutant strains showed improved periplasmic production of at least one of the recombinant proteins. No further positive effects were observed when the mutations were combined. CONCLUSIONS: Our findings confirm that our strain engineering approach involving translational regulation of endogenous proteins, in addition to plasmid-based methods, can be used to manipulate the cellular levels of periplasmic folding assistants and proteases to improve the yields of translocated recombinant proteins. The positive effects of SppA overexpression should be further investigated in E. coli.

Lipid and DHA-production in Aurantiochytrium sp. - Responses to nitrogen starvation and oxygen limitation revealed by analyses of production kinetics and global transcriptomes.

Thraustochytrids of the genera Schizochytrium and Aurantiochytrium accumulate oils rich in the essential, marine n3 fatty acid docosahexaenoic acid (DHA). DHA production in Aurantiochytrium sp T66 was studied with the aim to provide more knowledge about factors that affect the DHA-productivities and the contributions of the two enzyme systems used for fatty acid synthesis in thraustochytrids, fatty acid synthetase (FAS) and PUFA-synthase. Fermentations with nitrogen starvation, which is well-known to initiate lipid accumulation in oleaginous organisms, were compared to fermentations with nitrogen in excess, obtained by oxygen limitation. The specific productivities of fatty acids originating from FAS were considerably higher under nitrogen starvation than with nitrogen in excess, while the specific productivities of DHA were the same at both conditions. Global transcriptome analysis showed significant up-regulation of FAS under N-deficient conditions, while the PUFA-synthase genes were only marginally upregulated. Neither of them was upregulated under O2-limitation where nitrogen was in excess, suggesting that N-starvation mainly affects the FAS and may be less important for the PUFA-synthase. The transcriptome analysis also revealed responses likely to be related to the generation of reducing power (NADPH) for fatty acid synthesis.

Construction and characterization of broad-host-range reporter plasmid suitable for on-line analysis of bacterial host responses related to recombinant protein production.

BACKGROUND: Bacteria are widely used as hosts for recombinant protein production due to their rapid growth, simple media requirement and ability to produce high yields of correctly folded proteins. Overproduction of recombinant proteins may impose metabolic burden to host cells, triggering various stress responses, and the ability of the cells to cope with such stresses is an important factor affecting both cell growth and product yield. RESULTS: Here, we present a versatile plasmid-based reporter system for efficient analysis of metabolic responses associated with availability of cellular resources utilized for recombinant protein production and host capacity to synthesize correctly folded proteins. The reporter plasmid is based on the broad-host range RK2 minimal replicon and harbors the strong and inducible XylS/Pm regulator/promoter system, the ppGpp-regulated ribosomal protein promoter PrpsJ, and the σ32-dependent synthetic tandem promoter Pibpfxs, each controlling expression of one distinguishable fluorescent protein. We characterized the responsiveness of all three reporters in Escherichia coli by quantitative fluorescence measurements in cell cultures cultivated under different growth and stress conditions. We also validated the broad-host range application potential of the reporter plasmid by using Pseudomonas putida and Azotobacter vinelandii as hosts. CONCLUSIONS: The plasmid-based reporter system can be used for analysis of the total inducible recombinant protein production, the translational capacity measured as transcription level of ribosomal protein genes and the heat shock-like response revealing aberrant protein folding in all studied Gram-negative bacterial strains.

Identification of Regulatory Genes and Metabolic Processes Important for Alginate Biosynthesis in Azotobacter vinelandii by Screening of a Transposon Insertion Mutant Library.

Azotobacter vinelandii produces the biopolymer alginate, which has a wide range of industrial and pharmaceutical applications. A random transposon insertion mutant library was constructed from A. vinelandii ATCC12518Tc in order to identify genes and pathways affecting alginate biosynthesis, and about 4,000 mutant strains were screened for altered alginate production. One mutant, containing a mucA disruption, displayed an elevated alginate production level, and several mutants with decreased or abolished alginate production were identified. The regulatory proteins AlgW and AmrZ seem to be required for alginate production in A. vinelandii, similarly to Pseudomonas aeruginosa. An algB mutation did however not affect alginate yield in A. vinelandii although its P. aeruginosa homolog is needed for full alginate production. Inactivation of the fructose phosphoenolpyruvate phosphotransferase system protein FruA resulted in a mutant that did not produce alginate when cultivated in media containing various carbon sources, indicating that this system could have a role in regulation of alginate biosynthesis. Furthermore, impaired or abolished alginate production was observed for strains with disruptions of genes involved in peptidoglycan biosynthesis/recycling and biosynthesis of purines, isoprenoids, TCA cycle intermediates, and various vitamins, suggesting that sufficient access to some of these compounds is important for alginate production. This hypothesis was verified by showing that addition of thiamine, succinate or a mixture of lysine, methionine and diaminopimelate increases alginate yield in the non-mutagenized strain. These results might be used in development of optimized alginate production media or in genetic engineering of A. vinelandii strains for alginate bioproduction.

Identification of genes affecting alginate biosynthesis in Pseudomonas fluorescens by screening a transposon insertion library.

BACKGROUND: Polysaccharides often are necessary components of bacterial biofilms and capsules. Production of these biopolymers constitutes a drain on key components in the central carbon metabolism, but so far little is known concerning if and how the cells divide their resources between cell growth and production of exopolysaccharides. Alginate is an industrially important linear polysaccharide synthesized from fructose 6-phosphate by several bacterial species. The aim of this study was to identify genes that are necessary for obtaining a normal level of alginate production in alginate-producing Pseudomonas fluorescens. RESULTS: Polysaccharide biosynthesis is costly, since it utilizes nucleotide sugars and sequesters carbon. Consequently, transcription of the genes necessary for polysaccharide biosynthesis is usually tightly regulated. In this study we used an engineered P. fluorescens SBW25 derivative where all genes encoding the proteins needed for biosynthesis of alginate from fructose 6-phosphate and export of the polymer are expressed from inducible Pm promoters. In this way we would avoid identification of genes merely involved in regulating the expression of the alginate biosynthetic genes. The engineered strain was subjected to random transposon mutagenesis and a library of about 11500 mutants was screened for strains with altered alginate production. Identified inactivated genes were mainly found to encode proteins involved in metabolic pathways related to uptake and utilization of carbon, nitrogen and phosphor sources, biosynthesis of purine and tryptophan and peptidoglycan recycling. CONCLUSIONS: The majority of the identified mutants resulted in diminished alginate biosynthesis while cell yield in most cases were less affected. In some cases, however, a higher final cell yield were measured. The data indicate that when the supplies of fructose 6-phosphate or GTP are diminished, less alginate is produced. This should be taken into account when bacterial strains are designed for industrial polysaccharide production.

New insights into Pseudomonas fluorescens alginate biosynthesis relevant for the establishment of an efficient production process for microbial alginates.

Alginate denotes a family of linear polysaccharides with a wide range of industrial and pharmaceutical applications. Presently, all commercially available alginates are manufactured from brown algae. However, bacterial alginates have advantages with regard to compositional homogeneity and reproducibility. In order to be able to design bacterial strains that are better suited for industrial alginate production, defining limiting factors for alginate biosynthesis is of vital importance. Our group has been studying alginate biosynthesis in Pseudomonas fluorescens using several complementary approaches. Alginate is synthesised and transported out of the cell by a multiprotein complex spanning from the inner to the outer membrane. We have developed an immunogold labelling procedure in which the porin AlgE, as a part of this alginate factory, could be detected by transmission electron microscopy. No time-dependent correlation between the number of such factories on the cell surface and alginate production level was found in alginate-producing strains. Alginate biosynthesis competes with the central carbon metabolism for the key metabolite fructose 6-phosphate. In P. fluorescens, glucose, fructose and glycerol, are metabolised via the Entner-Doudoroff and pentose phosphate pathways. Mutational analysis revealed that disruption of the glucose 6-phosphate dehydrogenase gene zwf-1 resulted in increased alginate production when glycerol was used as carbon source. Furthermore, alginate-producing P. fluorescens strains cultivated on glucose experience acid stress due to the simultaneous production of alginate and gluconate. The combined results from our studies strongly indicate that the availability of fructose 6-phosphate and energy requires more attention in further research aimed at the development of an optimised alginate production process.

Identification of a New Phosphatase Enzyme Potentially Involved in the Sugar Phosphate Stress Response in Pseudomonas fluorescens.

The alginate-producing bacterium Pseudomonas fluorescens utilizes the Entner-Doudoroff (ED) and pentose phosphate (PP) pathways to metabolize fructose, since the upper part of its Embden-Meyerhof-Parnas pathway is defective. Our previous study indicated that perturbation of the central carbon metabolism by diminishing glucose-6-phosphate dehydrogenase activity could lead to sugar phosphate stress when P. fluorescens was cultivated on fructose. In the present study, we demonstrate that PFLU2693, annotated as a haloacid dehalogenase-like enzyme, is a new sugar phosphate phosphatase, now designated Spp, which is able to dephosphorylate a range of phosphate substrates, including glucose 6-phosphate and fructose 6-phosphate, in vitro The effect of spp overexpression on growth and alginate production was investigated using both the wild type and several mutant strains. The results obtained suggested that sugar phosphate accumulation caused diminished growth in some of the mutant strains, since this was partially relieved by spp overexpression. On the other hand, overexpression of spp in fructose-grown alginate-producing strains negatively affected both growth and alginate production. The latter implies that Spp dephosphorylates the sugar phosphates, thus depleting the pool of these important metabolites. Deletion of the spp gene did not affect growth of the wild-type strain on fructose, but the gene could not be deleted in the alginate-producing strain. This indicates that Spp is essential for relieving the cells of sugar phosphate stress in P. fluorescens actively producing alginate. IMPORTANCE: In enteric bacteria, the sugar phosphate phosphatase YigL is known to play an important role in combating stress caused by sugar phosphate accumulation. In this study, we identified a sugar phosphate phosphatase, designated Spp, in Pseudomonas fluorescens Spp utilizes glucose 6-phosphate, fructose 6-phosphate, and ribose 5-phosphate as substrates, and overexpression of the gene had a positive effect on growth in P. fluorescens mutants experiencing sugar phosphate stress. The gene was localized downstream of gnd and zwf-2, which encode enzymes involved in the pentose phosphate and Entner-Doudoroff pathways. Genes encoding Spp homologues were identified in similar genetic contexts in some bacteria belonging to several phylogenetically diverse families, suggesting similar functions.

Guluronic acid content as a factor affecting turbidity removal potential of alginate.

Alginates are natural polymers composed of mannuronic and guluronic acid residues. They are currently extracted from brown algae; however, alginate can also be synthesized by some species of Azotobacter and Pseudomonas. Alginates with different proportion of mannuronic and guluronic acids are known to have different characteristics and form gels at different extents in the presence of calcium ions. The aim of this work was to investigate the usefulness of alginate as a non-toxic coagulant used in purification of drinking water. This study utilized alginates from Azotobacter vinelandii having different guluronic acid levels. These were obtained partly by changing the cultivation parameters, partly by epimerizing a purified alginate sample in vitro using the A. vinelandii mannuronan C-5 epimerase AlgE1. The different alginates were then used for coagulation together with calcium. The results showed that turbidity removal capability was dependent on the content of guluronic acid residues. For the best performing samples, the turbidity decreased from 10 NTU to 1 NTU by the use of only 2 mg/L of alginate and 1.5 mM of calcium chloride.

Draft genome sequence of the docosahexaenoic acid producing thraustochytrid Aurantiochytrium sp. T66.

Thraustochytrids are unicellular, marine protists, and there is a growing industrial interest in these organisms, particularly because some species, including strains belonging to the genus Aurantiochytrium, accumulate high levels of docosahexaenoic acid (DHA). Here, we report the draft genome sequence of Aurantiochytrium sp. T66 (ATCC PRA-276), with a size of 43 Mbp, and 11,683 predicted protein-coding sequences. The data has been deposited at DDBJ/EMBL/Genbank under the accession LNGJ00000000. The genome sequence will contribute new insight into DHA biosynthesis and regulation, providing a basis for metabolic engineering of thraustochytrids.

Thraustochytrids as production organisms for docosahexaenoic acid (DHA), squalene, and carotenoids.

Thraustochytrids have been applied for industrial production of the omega-3 fatty acid docosahexaenoic (DHA) since the 1990s. During more than 20 years of research on this group of marine, heterotrophic microorganisms, considerable increases in DHA productivities have been obtained by process and medium optimization. Strains of thraustochytrids also produce high levels of squalene and carotenoids, two other commercially interesting compounds with a rapidly growing market potential, but where yet few studies on process optimization have been reported. Thraustochytrids use two pathways for fatty acid synthesis. The saturated fatty acids are produced by the standard fatty acid synthesis, while DHA is synthesized by a polyketide synthase. However, fundamental knowledge about the relationship between the two pathways is still lacking. In the present review, we extract main findings from the high number of reports on process optimization for DHA production and interpret these in the light of the current knowledge of DHA synthesis in thraustochytrids and lipid accumulation in oleaginous microorganisms in general. We also summarize published reports on squalene and carotenoid production and review the current status on strain improvement, which has been hampered by the yet very few published genome sequences and the lack of tools for gene transfer to the organisms. As more sequences now are becoming available, targets for strain improvement can be identified and open for a system-level metabolic engineering for improved productivities.