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Applied Microbiology and Biotechnology (v.91, #3)

Heme biosynthesis and its regulation: towards understanding and improvement of heme biosynthesis in filamentous fungi by Angelique C. W. Franken; B. Christien Lokman; Arthur F. J. Ram; Peter J. Punt; Cees A. M. J. J. van den Hondel; Sandra de Weert (pp. 447-460).

Heme biosynthesis in fungal host strains has acquired considerable interest in relation to the production of secreted heme-containing peroxidases. Class II peroxidase enzymes have been suggested as eco-friendly replacements of polluting chemical processes in industry. These peroxidases are naturally produced in small amounts by basidiomycetes. Filamentous fungi like Aspergillus sp. are considered as suitable hosts for protein production due to their high capacity of protein secretion. For the purpose of peroxidase production, heme is considered a putative limiting factor. However, heme addition is not appropriate in large-scale production processes due to its high hydrophobicity and cost price. The preferred situation in order to overcome the limiting effect of heme would be to increase intracellular heme levels. This requires a thorough insight into the biosynthetic pathway and its regulation. In this review, the heme biosynthetic pathway is discussed with regards to synthesis, regulation, and transport. Although the heme biosynthetic pathway is a highly conserved and tightly regulated pathway, the mode of regulation does not appear to be conserved among eukaryotes. However, common factors like feedback inhibition and regulation by heme, iron, and oxygen appear to be involved in regulation of the heme biosynthesis pathway in most organisms. Therefore, they are the initial targets to be investigated in Aspergillus niger.

Keywords: Heme biosynthesis; Heme regulation; Peroxidase; Aspergillus ; Protein production; Intracellular heme

Red mold fermented products and Alzheimer's disease: a review by Chun-Lin Lee; Tzu-Ming Pan (pp. 461-469).

Alzheimer's disease is seen mainly in individuals over the age of 65, and the morbidity rate increases with age. Regarding the health function of Monascus-fermented red mold rice (RMR), besides hypolipidemic and hypotensive effects, other health functions of RMR such as anti-oxidation, cancer prevention, anti-fatigue, and anti-obesity have also been reported. Many published studies have shown the efficacy of RMR in the prevention of Alzheimer's disease. The current article discusses and provides evidence to support the beneficial potential of RMR in the prevention of Alzheimer's disease by discussing the pathogenic factors of Alzheimer's disease and the secondary metabolites of Monascus.

Keywords: Alzheimer's disease; Red mold rice; Metabolite

Renewable energy from Cyanobacteria: energy production optimization by metabolic pathway engineering by Naira Quintana; Frank Van der Kooy; Miranda D. Van de Rhee; Gerben P. Voshol; Robert Verpoorte (pp. 471-490).

The need to develop and improve sustainable energy resources is of eminent importance due to the finite nature of our fossil fuels. This review paper deals with a third generation renewable energy resource which does not compete with our food resources, cyanobacteria. We discuss the current state of the art in developing different types of bioenergy (ethanol, biodiesel, hydrogen, etc.) from cyanobacteria. The major important biochemical pathways in cyanobacteria are highlighted, and the possibility to influence these pathways to improve the production of specific types of energy forms the major part of this review.

Keywords: Bioenergy; Biofuel; Cyanobacteria; Renewable energy; Metabolic engineering

Biological detoxification of the mycotoxin deoxynivalenol and its use in genetically engineered crops and feed additives by Petr Karlovsky (pp. 491-504).

Deoxynivalenol (DON) is the major mycotoxin produced by Fusarium fungi in grains. Food and feed contaminated with DON pose a health risk to humans and livestock. The risk can be reduced by enzymatic detoxification. Complete mineralization of DON by microbial cultures has rarely been observed and the activities turned out to be unstable. The detoxification of DON by reactions targeting its epoxide group or hydroxyl on carbon 3 is more feasible. Microbial strains that de-epoxidize DON under anaerobic conditions have been isolated from animal digestive system. Feed additives claimed to de-epoxidize trichothecenes enzymatically are on the market but their efficacy has been disputed. A new detoxification pathway leading to 3-oxo-DON and 3-epi-DON was discovered in taxonomically unrelated soil bacteria from three continents; the enzymes involved remain to be identified. Arabidopsis, tobacco, wheat, barley, and rice were engineered to acetylate DON on carbon 3. In wheat expressing DON acetylation activity, the increase in resistance against Fusarium head blight was only moderate. The Tri101 gene from Fusarium sporotrichioides was used; Fusarium graminearum enzyme which possesses higher activity towards DON would presumably be a better choice. Glycosylation of trichothecenes occurs in plants, contributing to the resistance of wheat to F. graminearum infection. Marker-assisted selection based on the trichothecene-3-O-glucosyltransferase gene can be used in breeding for resistance. Fungal acetyltransferases and plant glucosyltransferases targeting carbon 3 of trichothecenes remain promising candidates for engineering resistance against Fusarium head blight. Bacterial enzymes catalyzing oxidation, epimerization, and less likely de-epoxidation of DON may extend this list in future.

Keywords: Deoxynivalenol; Detoxification; De-epoxidation; Epimerization; Feed additive; Resistance engineering

Biocatalytic synthesis of optically active tertiary alcohols by Robert Kourist; Uwe T. Bornscheuer (pp. 505-517).

The enzymatic preparation of optically pure tertiary alcohols under sustainable conditions has received much attention. The conventional chemical synthesis of these valuable building blocks is still hampered by the use of harmful reagents such as heavy metal catalysts. Successful examples in biocatalysis used esterases, lipases, epoxide hydrolases, halohydrin dehalogenases, thiamine diphosphate-dependent enzymes, terpene cyclases, -acetylases, and -dehydratases. This mini-review provides an overview on recent developments in the discovery of new enzymes, their functional improvement by protein engineering, the design of chemoenzymatic routes leading to tertiary alcohols, and the discovery of entirely new biotransformations.

Keywords: Tertiary alcohols; Esterase; Enantioselectivity; Protein engineering; Enzyme catalysis

Regulation of trichothecene biosynthesis in Fusarium: recent advances and new insights by Jawad Merhej; Florence Richard-Forget; Christian Barreau (pp. 519-528).

Trichothecenes are toxic secondary metabolites produced by filamentous fungi mainly belonging to the Fusarium genus. Production of these mycotoxins occurs during infection of crops and is a threat to human and animal health. Although the pathway for biosynthesis of trichothecenes is well established, the regulation of the Tri genes implicated in the pathway remains poorly understood. Most of the Tri genes are gathered in a cluster which contains two transcriptional regulators controlling the expression of the other Tri genes. The regulation of secondary metabolites biosynthesis in most fungal genera has been recently shown to be controlled by various regulatory systems in response to external environment. The control of the “Tri cluster” by non-cluster regulators in Fusarium was not clearly demonstrated until recently. This review covers the recent advances concerning the regulation of trichothecene biosynthesis in Fusarium and highlights the potential implication of various general regulatory circuits. Further studies on the role of these regulatory systems in the control of trichothecene biosynthesis might be useful in designing new strategies to reduce mycotoxin accumulation.

Keywords: Fusarium ; Trichothecene; Tri cluster; Regulation

A whole cell biocatalyst for cellulosic ethanol production from dilute acid-pretreated corn stover hydrolyzates by Seunghyun Ryu; Muhammad Nazmul Karim (pp. 529-542).

In this research, a recombinant whole cell biocatalyst was developed by expressing three cellulases from Clostridium cellulolyticum—endoglucanase (Cel5A), exoglucanase (Cel9E), and β-glucosidase—on the surface of the Escherichia coli LY01. The modified strain is identified as LY01/pRE1H-AEB. The cellulases were displayed on the surface of the cell by fusing with an anchor protein, PgsA. The developed whole cell biocatalyst was used for single-step ethanol fermentation using the phosphoric acid-swollen cellulose (PASC) and the dilute acid-pretreated corn stover. Ethanol production was 3.59 ± 0.15 g/L using 10 g/L of PASC, which corresponds to a theoretical yield of 95.4 ± 0.15%. Ethanol production was 0.30 ± 0.02 g/L when 1 g/L equivalent of glucose in the cellulosic fraction of the dilute sulfuric acid-pretreated corn stover (PCS) was fermented for 84 h. A total of 0.71 ± 0.12 g/L ethanol was produced in 48 h when the PCS was fermented in the simultaneous saccharification and co-fermentation mode using the hemicellulosic (1 g/L of total soluble sugar) and as well as the cellulosic (1 g/L of glucose equivalent) parts of PCS. In a control experiment, 0.48 g/L ethanol was obtained from 1 g/L of hemicellulosic PCS. It was concluded that the whole cell biocatalyst could convert both cellulosic and hemicellulosic substrates into ethanol in a single reactor. The developed C. cellulolyticum–E. coli whole cell biocatalyst also overcame the incompatible temperature problem of the frequently reported fungal–yeast systems.

Keywords: Bioethanol; Whole cell biocatalyst; Consolidated bioprocessing; Clostridium cellulolyticum ; Escherichia coli LY01

Production and characterization of LEA29Y, a variant of cytotoxic T-lymphocyte antigen 4-immunoglobulin, in Pichia pastoris by Lin Wan; Shengyun Zhu; Yingying Li; Shan Liu; Hao Yang; Shengfu Li; Youping Li; Jingqiu Cheng; Xiaofeng Lu (pp. 543-551).

Blocking the CD28/B7 costimulatory pathway is a promising strategy in the treatment of graft rejection, graft-versus-host disease and autoimmune diseases. LEA29Y, a high-affinity variant of cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4Ig), is a more potent inhibitor of the interaction between CD28 and B7 than is CTLA4Ig. In a previous study, LEA29Y was produced in a mammalian cell system, which is time-consuming and expensive. To obtain LEA29Y more efficiently and cost effectively, we attempted to produce LEA29Y using a Pichia pastoris expression system. The gene encoding LEA29Y, with an additional 6-His tag at the N-terminus, was cloned into the yeast vector pPIC9K and expressed in the P. pastoris strain GS115. Under the optimized induction conditions for protein expression (inoculum density, OD₆₀₀ = 80; methanol concentration added daily, 1.0–3.0%; induction time point, 72–96 h; culture medium pH = 6.0), the yield of purified LEA29Y was approximately 30 mg l⁻¹ by one-step Ni-agarose affinity chromatography. PNGase F treatment showed the purified LEA29Y to be post-translational modified by N-linked glycosylation. In biological function assays, LEA29Y expressed in P. pastoris demonstrated specific binding to B7-1/B7-2-positive Raji cells and also suppressed lymphocyte proliferation in a dose-dependent manner. These results suggest that LEA29Y produced in P. pastoris is biologically active and will be useful for experimental therapy on immunotherapy for transplant rejection and autoimmune diseases.

Keywords: CTLA4Ig; Immunosuppression; Pichia pastoris

Transgenic microalgae expressing Escherichia coli AppA phytase as feed additive to reduce phytate excretion in the manure of young broiler chicks by So-Mi Yoon; So Young Kim; Kun Feng Li; Byung Hak Yoon; Senyon Choe; Mario Meng-Chiang Kuo (pp. 553-563).

Microbial phytases are widely used as feed additive to increase phytate phosphorus utilization and to reduce fecal phytates and inorganic phosphate (iP) outputs. To facilitate the process of application, we engineered an Escherichia coli appA phytase gene into the chloroplast genome of the model microalga, Chlamydomonas reinhardtii, and isolated homoplasmic plastid transformants. The catalytic activity of the recombinant E. coli AppA can be directly detected in the whole-cell lysate, termed Chlasate, prepared by freeze-drying the transgenic cell paste with liquid nitrogen. The E. coli AppA in the Chlasate has a pH and temperature optima of 4.5 and 60°C, respectively, which are similar to those described in the literature. The phytase-expressed Chlasate contains 10 phytase units per gram dry matter at pH 4.5 and 37°C. Using this transgenic Chlasate at 500 U/kg of diet for young broiler chicks, the fecal phytate excretion was reduced, and the iP was increased by 43% and 41%, respectively, as compared to those of the chicks fed with only the basal diet. The effectiveness of the Chlasate to break down the dietary phytates is compatible with the commercial Natuphos fungal phytase. Our data provide the first evidence of functional expression of microbial phytase in microalgae and demonstrate the proof of concept of using transgenic microalgae as a food additive to deliver dietary enzymes with no need of protein purification.

Keywords: Transgenic; E. coli AppA; Chlamydomonas reinhardtii ; Microalgae; Phytase

Acetone production in solventogenic Clostridium species: new insights from non-enzymatic decarboxylation of acetoacetate by Bei Han; Venkat Gopalan; Thaddeus Chukwuemeka Ezeji (pp. 565-576).

Development of a butanologenic strain with high selectivity for butanol production is often proposed as a possible route for improving the economics of biobutanol production by solventogenic Clostridium species. The acetoacetate decarboxylase (aadc) gene encoding acetoacetate decarboxylase (AADC), which catalyzes the decarboxylation of acetoacetate into acetone and CO₂, was successfully disrupted by homologous recombination in solventogenic Clostridium beijerinckii NCIMB 8052 to generate an aadc − mutant. Our fermentation studies revealed that this mutant produces a maximum acetone concentration of 3 g/L (in P2 medium), a value comparable to that produced by wild-type C. beijerinckii 8052. Therefore, we postulated that AADC-catalyzed decarboxylation of acetoacetate is not the sole means for acetone generation. Our subsequent finding that non-enzymatic decarboxylation of acetoacetate in vitro, under conditions similar to in vivo acetone–butanol–ethanol (ABE) fermentation, produces 1.3 to 5.2 g/L acetone between pH 6.5 and 4 helps rationalize why various knock-out and knock-down strategies designed to disrupt aadc in solventogenic Clostridium species did not eliminate acetone production during ABE fermentation. Based on these results, we discuss alternatives to enhance selectivity for butanol production.

Keywords: Butanol; Acetone; Coenzyme A transferase; Acetoacetate decarboxylase; Clostridium beijerinckii

Engineering Bacillus subtilis for isobutanol production by heterologous Ehrlich pathway construction and the biosynthetic 2-ketoisovalerate precursor pathway overexpression by Shanshan Li; Jianping Wen; Xiaoqiang Jia (pp. 577-589).

In the present work, Bacillus subtilis was engineered as the cell factory for isobutanol production due to its high tolerance to isobutanol. Initially, an efficient heterologous Ehrlich pathway controlled by the promoter P₄₃ was introduced into B. subtilis for the isobutanol biosynthesis. Further, investigation of acetolactate synthase of B. subtilis, ketol-acid reductoisomerase, and dihydroxy-acid dehydratase of Corynebacterium glutamicum responsible for 2-ketoisovalerate precursor biosynthesis showed that acetolactate synthase played an important role in isobutanol biosynthesis. The overexpression of acetolactate synthase led to a 2.8-fold isobutanol production compared with the control. Apart from isobutanol, alcoholic profile analysis also confirmed the existence of 1.21 g/L ethanol, 1.06 g/L 2-phenylethanol, as well as traces of 2-methyl-1-butanol and 3-methyl-1-butanol in the fermentation broth. Under microaerobic condition, the engineered B. subtilis produced up to 2.62 g/L isobutanol in shake-flask fed-batch fermentation, which was 21.3% higher than that in batch fermentation.

Keywords: Isobutanol; Bacillus subtilis ; Ehrlich pathway; Biosynthetic precursor pathway; Cell factory; Metabolic engineering

Reversible immobilization of glucoamylase onto magnetic carbon nanotubes functionalized with dendrimer by Guanghui Zhao; Yanfeng Li; Jianzhi Wang; Hao Zhu (pp. 591-601).

Magnetic carbon nanotubes (MCNTs) with necklace-like nanostructures was prepared via hydrothermal method, and hyperbranched poly(amidoamine) (PAMAM) was grafted on the surface of MCNTs on the basis of the Michael addition of methyl acrylate and the amidation of the resulting ester with a large excess of ethylenediamine (EDA), which could achieve generational growth under such uniform stepwise reactions. The terminal –NH₂ groups from the dendritic PAMAM were reacted with differently functionalized groups to form functionalized MCNTs. Subsequently, enzyme was immobilized on the functionalized MCNTs through adsorption, covalent bond, and metal-ion affinity interactions. The immobilization of glucoamylase, hereby chosen as model enzyme, onto the differently functionalized MCNTs is further demonstrated and assessed based on its activity, thermal stability, as well as reusability. Besides ease in recovery by magnetic separation, the immobilized glucoamylase on functionalized MCNTs offers superior stability and reusability, without compromising the substrate specificity of free glucoamylase. Furthermore, the results indicate that the metal-chelate dendrimer offers an efficient route to immobilize enzymes via metal-ion affinity interactions. The applicability of the regenerated supports in the current study is relevant for the conjugation of other enzymes beyond glucoamylase.

Keywords: Magnetic carbon nanotubes; Chelation; Immobilization; Regeneration; Glucoamylase

Newly generated interspecific wine yeast hybrids introduce flavour and aroma diversity to wines by Jennifer R. Bellon; Jeffery M. Eglinton; Tracey E. Siebert; Alan P. Pollnitz; Louisa Rose; Miguel de Barros Lopes; Paul J. Chambers (pp. 603-612).

Increasingly, winemakers are looking for ways to introduce aroma and flavour diversity to their wines as a means of improving style and increasing product differentiation. While currently available commercial yeast strains produce consistently sound fermentations, there are indications that sensory complexity and improved palate structure are obtained when other species of yeast are active during fermentation. In this study, we explore a strategy to increase the impact of non-Saccharomyces cerevisiae inputs without the risks associated with spontaneous fermentations, through generating interspecific hybrids between a S. cerevisiae wine strain and a second species. For our experiments, we used rare mating to produce hybrids between S. cerevisiae and other closely related yeast of the Saccharomyces sensu stricto complex. These hybrid yeast strains display desirable properties of both parents and produce wines with concentrations of aromatic fermentation products that are different to what is found in wine made using the commercial wine yeast parent. Our results demonstrate, for the first time, that the introduction of genetic material from a non-S. cerevisiae parent into a wine yeast background can impact favourably on the wine flavour and aroma profile of a commercial S. cerevisiae wine yeast.

Keywords: Saccharomyces sensu stricto; Interspecific hybrids; Metabolites; Fermentation products; Wine yeast

Highly efficient production of anti-HER2 scFv antibody variant for targeting breast cancer cells by Silvia Sommaruga; Alessio Lombardi; Agnese Salvadè; Serena Mazzucchelli; Fabio Corsi; Patrizia Galeffi; Paolo Tortora; Davide Prosperi (pp. 613-621).

The human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor overexpressed in 30% of human breast cancers. One of the mechanisms by which tumor cell proliferation can be inhibited consists in hampering HER2 dimerization by targeting its extracellular domain with specific antibodies. In recent clinical practice, a valuable alternative to entire IgGs resides in the use of smaller molecules, such as single-chain variable fragments (scFv), developed for selective molecular targeting. In this paper, we report on the production and purification of a soluble anti-HER2 scFv antibody secreted by Pichia pastoris. The gene encoding scFv800E6 with an additional 6× His-tag at the 3′-end was inserted into the expression vector pPICZα and transformed in P. pastoris. The highest expression level was obtained in presence of 0.5% methanol and 0.8% glycerol in the culture medium after 48 h of induction. The use of P. pastoris proved very valuable as an expression system, allowing the isolation of 10 mg/L of highly purified antibody, remarkably higher than previously reported data. The functionality of purified anti-HER2 scFv was assessed by cytofluorimetry and immunofluorescence on HER2-positive MCF7 breast cancer cells, showing good affinity and high selectivity for the target membrane receptor. These findings confirm that P. pastoris is a suitable host for high level expression of antibody fragments and highlight the potential role of scFv800E6 in diagnostic and therapeutic application.

Keywords: Single-chain variable fragment antibody; HER2; Pichia pastoris ; Breast cancer

Comparative characterization of endo-polygalacturonase (Pgu1) from Saccharomyces cerevisiae and Saccharomyces paradoxus under winemaking conditions by Alexis Eschstruth; Benoit Divol (pp. 623-634).

Wine strains of Saccharomyces cerevisiae have no to weak natural pectinase activity, despite their genetic ability to secrete an endo-polygalacturonase. The addition of external pectinase of fungal origin has therefore become a common step of winemaking in order to enhance the extraction of compounds located in the grape berry skins during maceration and to ease wine clarification after maturation. Recently, the strong pectinase activity of a wine strain of Saccharomyces paradoxus has been reported. In this study, the endo-polygalacturonase-encoding gene of S. paradoxus was sequenced and its activity was characterised, compared with that of S. cerevisiae and tested under winemaking conditions through overexpression of both genes individually in S. cerevisiae. A few differences in the amino acids sequences between the two proteins were revealed and the activity of the Pgu1 enzyme of S. paradoxus was shown to be weaker under winemaking conditions. Clear indicators of extracellular activity were observed in the wines made with both recombinant strains (i.e. enzyme activity in cell-free wine, higher methanol concentration and higher free-run wine), but the actual composition of the wines fermented with the mutants was only sparingly altered. Although unexpectedly found in lower concentrations in the latter wines, phenolic compounds were shown to be the most discriminatory components. Overexpressing the PGU1 gene of S. paradoxus or that of S. cerevisiae did not make much difference, showing that the higher activity of S. paradoxus strains under laboratory conditions could be due to a different regulation mechanism rather than to a different sequence of PGU1.

Keywords: Endo-polygalacturonase activity; PGU1 ; Saccharomyces paradoxus ; Wine

Isolation and characterization of two serine proteases from metagenomic libraries of the Gobi and Death Valley deserts by Julie Neveu; Christophe Regeard; Michael S. DuBow (pp. 635-644).

The screening of environmental DNA metagenome libraries for functional activities can provide an important source of new molecules and enzymes. In this study, we identified 17 potential protease-producing clones from two metagenomic libraries derived from samples of surface sand from the Gobi and Death Valley deserts. Two of the proteases, DV1 and M30, were purified and biochemically examined. These two proteases displayed a molecular mass of 41.5 kDa and 45.7 kDa, respectively, on SDS polyacrylamide gels. Alignments with known protease sequences showed less than 55% amino acid sequence identity. These two serine proteases appear to belong to the subtilisin (S8A) family and displayed several unique biochemical properties. Protease DV1 had an optimum pH of 8 and an optimal activity at 55°C, while protease M30 had an optimum pH >11 and optimal activity at 40°C. The properties of these enzymes make them potentially useful for biotechnological applications and again demonstrate that metagenomic approaches can be useful, especially when coupled with the study of novel environments such as deserts.

Keywords: Functional metagenomics; Protease; Gobi desert; Death Valley desert

Efficient cell surface display of Lip2 lipase using C-domains of glycosylphosphatidylinositol-anchored cell wall proteins of Yarrowia lipolytica by Evgeniya Y. Yuzbasheva; Tigran V. Yuzbashev; Ivan A. Laptev; Tatiana K. Konstantinova; Sergey P. Sineoky (pp. 645-654).

The cell surface display of enzymes is of great interest because of its simplified purification stage and the possibility for recycling in industrial processes. In this study, we have focused on the cell wall immobilization of Yarrowia lipolytica Lip2 protein—an enzyme that has a wide technological application. By genome analysis of Y. lipolytica in addition to already characterized Ylcwp1, we identified five putative open reading frames encoding glycosylphosphatidylinositol-anchored proteins. Lip2 translation fusion with the carboxyl termini of these proteins revealed that all proteins were capable of immobilizing lipase in active form on the cell surface. The highest level of cell-bound lipase activity was achieved using C-domains encoded by YlCWP1, YlCWP3 (YALI0D27214g) and YlCWP6 (YALI0F18282g) comprising 16,173 ± 1,800, 18,785 ± 1,130 and 17,700 ± 2,101 U/g dry cells, respectively. To the best of our knowledge, these results significantly exceed the highest cell-bound lipase activity previously reported for engineered Saccharomyces cerevisiae and Pichia pastoris strains. Furthermore, the lyophilized biomass retained the activity and was robust to collecting/resuspending procedures. Nevertheless, in most cases, a substantial amount of lipase activity was also found in the growth medium. Further work will be necessary to better understand the nature of this phenomenon.

Keywords: Yarrowia lipolytica ; Cell surface display; Lipase Lip2; Carboxyl-terminal GPI-anchored domain

Engineering of polyhydroxyalkanoate (PHA) synthase PhaC2_Ps of Pseudomonas stutzeri via site-specific mutation for efficient production of PHA copolymers by Xiao-Wen Shen; Zhen-Yu Shi; Ge Song; Zheng-Jun Li; Guo-Qiang Chen (pp. 655-665).

The site-specific mutagenesis for PHA synthase PhaC2_Ps1317 from Pseudomonas stutzeri 1317 was conducted for optimizing production of short-chain-length and medium-chain-length polyhydroxyalkanoates (scl-mcl PHA). Recombinant Ralstonia eutropha PHB-4 harboring double mutated phaC2 Ps1317 gene (phaC2 Ps QKST) produced 42 wt.% PHA content in the cell dry weight (CDW) with 93 mol% 3-hydroxybutyrate (HB) as monomer in the PHA copolymer. Compared to that of wild-type phaC2 Ps1317, the higher PHA content indicated the effectiveness of the specific point mutations for improvement on PhaC2_Ps1317 activity and PHA production. The physical characterization revealed that the PHA produced by the recombinant strain was scl-mcl PHA copolymers with molecular weights and polydispersity reasonable for practical applications. Recombinant R. eutropha PHB-4 containing mutated phaC2 Ps1317 termed phaC2 Ps QKST was demonstrated to be able to produce scl-mcl PHA copolymers consisting of even-numbered, odd-numbered, or a combination of even- and odd-numbered monomers covering the carbon chain lengths from C4 to C12 when related substrates were provided. Recombinant R. eutropha PHB-4 containing phaC2PsQKST could be used as a strain for production of copolymers consisting of dominated HB and medium-chain-length 3-hydroxyalkanoates (HA) with better application properties.

Keywords: PHB; Polyhydroxyalkanoates; PHA synthase; PhaC; Ralstonia eutropha ; Pseudomonas stutzeri

Improving the decolorization for textile dyes of a metagenome-derived alkaline laccase by directed evolution by Yu Huan Liu; Mao Ye; Yi Lu; Xia Zhang; Gang Li (pp. 667-675).

To obtain better performing laccases for textile dyes decolorization, random mutagenesis of Lac591, a metagenome-derived alkaline laccase, was carried out. After three rounds of error-prone PCR and high-throughput screening by assaying enzymatic activity toward the phenolic substrate 2,6-dimethoxyphenol (2,6-DMP), a mutant (Lac3T93) with remarkably improved enzymatic activity was obtained. Sequence analysis revealed that four amino acid substitutions (N40S, V55A, F62L, and E316V) were accumulated in the Lac3T93. Compared to the wild-type enzyme, the specific activity of Lac3T93 toward 2,6-DMP was increased to 4.8-fold (61.22 U/mg), and its optimal temperature and pH were changed to 60°C and 8.0 from 55°C and 7.5 of the wild-type enzyme, respectively. Furthermore, the degradation ability of Lac3T93 for textile dyes was investigated, and the new variant represented improved decolorization percentage for four industrial dyes with complex phenyl structure (Basic Blue 3, Methylene Blue, Bromophenol Blue, and Crystal Violet) and higher decolorization efficiency for Indigo Carmine than that of the parent enzyme. Furthermore, the decolorization percentage of Lac3T93 for five dyes in the absence of hydroxybenzotrizole (HBT) is clearly higher than those of the wild-type enzyme with 1 mM HBT, and HBT can further improve its decolorization ability.

Keywords: Laccase; Directed evolution; Enzyme properties; Textile dyes decolorization

High yield secretion of heterologous proteins in Corynebacterium glutamicum using its own Tat-type signal sequence by Hiroshi Teramoto; Keiro Watanabe; Nobuaki Suzuki; Masayuki Inui; Hideaki Yukawa (pp. 677-687).

Efficient protein secretion, the basis of large-scale production of many compounds central to the biotechnology industry, is achieved by signal peptide and propeptide optimization in addition to optimizing host factors affecting heterologous protein production. Here, we fused green fluorescent protein (GFP) to the recently identified Tat-type secretory signal peptide of CgR0949 to demonstrate a high-yield protein secretion system of Corynebacterium glutamicum. The resultant secretion vector facilitated effective secretion of active-form GFP (20 mg l⁻¹) into C. glutamicum culture medium. The expression of GFP was enhanced 2.9-fold using the Shine–Dalgarno sequence of triosephosphate isomerase in the secretion vector. Moreover, GFP drastically accumulated in the culture supernatant upon addition of calcium chloride even though Ca²⁺ addition did neither enhanced the transcription of gfp nor resulted in the accumulation of cytosolic GFP. Active-form GFP concentration reached 1.8 g l⁻¹ after 48-h incubation in a jar fermentor. Likewise, α-amylase accumulation in C. glutamicum cultures was also enhanced by Ca²⁺ addition, suggesting that Ca²⁺ may affect general protein secretion in C. glutamicum.

Keywords: Protein secretion; Corynebacterium glutamicum ; Tat-type signal sequence; PS2; Calcium

Bacterial abl-like genes: production of the archaeal osmolyte $$ {N^{varepsilon }}{ ext{ - acetyl - }}eta { ext{ - lysine}} $$ by homologous overexpression of the yodP–kamA genes in Bacillus subtilis by Stefan Müller; Tamara Hoffmann; Helena Santos; Stephan H. Saum; Erhard Bremer; Volker Müller (pp. 689-697).

$$ {N^{varepsilon }}{ ext{ - acetyl - }}eta { ext{ - lysine}} $$ is an archaeal compatible solute whose synthesis is mediated by the sequential reactions of the lysine-2,3-aminomutase AblA and the acetyltransferase AblB. α-Lysine serves as the precursor and is converted by AblA to β-lysine, and AblB then acetylates this intermediate to $$ {N^{varepsilon }}{ ext{ - acetyl - }}eta { ext{ - lysine}} $$ . The biochemical and biophysical properties of $$ {N^{varepsilon }}{ ext{ - acetyl - }}eta { ext{ - lysine}} $$ have so far not been studied intensively due to restrictions in the supply of this compound. A search for ablAB-like genes in the genomes of members of the family Bacillaceae revealed the yodP–kamA genes that encode a AblA-related lysine-2,3-aminomutase and AblB-related putative acetyltransferase. In Bacillus subtilis, the yodP–kamA genes are part of a transcriptional unit (yodT–yodS–yodR–yodQ–yodP–kamA) whose expression is upregulated during sporulation and controlled by the mother-cell-specific transcription factor SigE. $$ {N^{varepsilon }}{ ext{ - acetyl - }}eta { ext{ - lysine}} $$ was not detectable in vegetatively growing or osmotically stressed B. subtilis cells, and the deletion of the yodT–yodS–yodR–yodQ–yodP–kamA region had no noticeable effects on growth in rich or minimal media or osmotic stress resistance. However, when we expressed the yodP–kamA genes outside their natural genetic context from an isopropyl β-d-1-thiogalactopyranoside-inducible promoter on a plasmid in B. subtilis, the recombinant strain synthesized considerable amounts (0.28 μmol/mg protein) of $$ {N^{varepsilon }}{ ext{ - acetyl - }}eta { ext{ - lysine}} $$ . The data reported here thus open the bottleneck for the large-scale production of $$ {N^{varepsilon }}{ ext{ - acetyl - }}eta { ext{ - lysine}} $$ to investigate its properties as a compatible solute.

Keywords: Archaea; Methanogens; Bacteria; Salt adaptation; Compatible solutes; Lysine-2,3-aminomutase

Functional analysis of the response regulator DegU in Bacillus megaterium DSM319 and comparative secretome analysis of degSU mutants by Claudia Borgmeier; Birgit Voigt; Michael Hecker; Friedhelm Meinhardt (pp. 699-711).

We functionally analysed the two-component regulatory system DegSU (historically SacU) in Bacillus megaterium DSM319 by generating a genetic knock out as well as a sacU32 mutation. The latter—known to cause a hypersecretion phenotype in Bacillus subtilis—had no influence on extracellular protease and amylase activity in B. megaterium. Since the B. megaterium DegU complemented a Bacillus licheniformis ∆degSU mutant, functionality of the protein was proven. Expression of the sacB encoded levansucrase was found to be dependent on DegSU in B. megaterium. Consistently, the fusion of the sacB promoter to gfp revealed a strong increase in GFP-expression in the sacU32 strain. On 2 D-gels of the secretome, a large number of intracellular proteins was seen. The culture medium contained only 42 secreted proteins which can be assigned to polypeptides involved in the metabolism of the cell wall, polypeptides with proteolytic activities and those with unknown functions. Though overall protease activity matches with the wild type, two proteolytic enzymes (Vpr and YwaD) are missing in the secretome of the ∆degSU strain, while other degradative enzymes are not affected. In line with such findings, no increase of proteolytic or other degradative enzymes was seen in the sacU32 mutant. Thus, compared to B. subtilis and B. licheniformis, the number of extracellular proteins influenced by DegSU is surprisingly low in B. megaterium, a feature, probably advantageous as to the use of the sacU32 mutant for production of secreted proteins.

Keywords: Bacillus megaterium ; Two-component system; Hypersecretion; DegSU; Extracellular proteome; Degradative enzymes

Identifying genes that impact on aroma profiles produced by Saccharomyces cerevisiae and the production of higher alcohols by Gustav Styger; Dan Jacobson; Florian F. Bauer (pp. 713-730).

During alcoholic fermentation, many volatile aroma compounds are formed by Saccharomyces cerevisiae, including esters, fatty acids, and higher alcohols. While the metabolic network that leads to the formation of these compounds is reasonably well mapped, surprisingly little is known about specific enzymes involved in specific reactions, the regulation of the network, and the physiological roles of individual pathways within the network. Furthermore, different yeast strains tend to produce significantly different aroma profiles. These differences are of tremendous biotechnological interest, since producers of alcoholic beverages such as wine and beer are searching for means to diversify and improve their product range. Various factors such as the redox, energy, and nutritional balance of a cell have previously been suggested to directly or indirectly affect and regulate the network. To gain a better understanding of the regulations and physiological role of this network, we screened a subset of the EUROSCARF strain deletion library for genes that, when deleted, would impact most significantly on the aroma profile produced under fermentative conditions. The 10 genes whose deletion impacted most significantly on higher alcohol production were selected and further characterized to assess their mode of action within or on this metabolic network. This is the first description of a large-scale screening approach using aroma production as the primary selection criteria, and the data suggest that many of the identified genes indeed play central and direct roles within the aroma production network of S. cerevisiae.

Keywords: Ehrlich pathway; Branched chain amino acid metabolism; Higher alcohols; Wine aroma

Improved expression of secretory and trimeric proteins in mammalian cells via the introduction of a new trimer motif and a mutant of the tPA signal sequence by Jia-Ye Wang; Wen-Ting Song; Yan Li; Wen-Jiang Chen; Dan Yang; Guo-Cai Zhong; Hai-Zhou Zhou; Cai-Yun Ren; Hao-Tong Yu; Hong Ling (pp. 731-740).

Ideal immunogenicity in antigens is a prerequisite to eliciting a sufficiently strong immune and memory response via either DNA or protein vaccines. To improve immunogenicity, efforts have focused on high-level expression of target proteins and on maintaining their natural conformations. In the present work, two trimer motifs (MTQ and MTI) were designed and introduced into a plasmid vector with the tissue plasminogen activator signal peptide (tPA-SP). Next, we examined the efficacy and the efficiency of the two motifs as well as the introduction of tPA-SP and its mutant forms, 22P/A and 22P/G, in facilitating the secretory expression of trimeric proteins in mammalian cells. We found that both trimer motifs could produce the target protein in a trimeric form at a high level. Introduction of tPA-SP 22P/A markedly increased the secretory expression level. The combination of the trimer motif, MTQ, and the signal peptide, 22P/A, may serve as a universal mammalian vector for producing trimeric proteins in vaccine development.

Keywords: Trimer motif; Mammalian cell; Signal peptide; Tissue plasminogen activator (tPA); Protein expression

Trichoderma harzianum strain SQR-T37 and its bio-organic fertilizer could control Rhizoctonia solani damping-off disease in cucumber seedlings mainly by the mycoparasitism by Xinqi Huang; Lihua Chen; Wei Ran; Qirong Shen; Xingming Yang (pp. 741-755).

Damping-off disease is caused by Rhizoctonia solani and leads to serious loss in many crops. Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Optical micrographs, scanning electron micrographs, and the determination of hydrolytic enzymes were used to investigate the antagonism of Trichoderma harzianum SQR-T37 (SQR-T37) against R. solani. Experiments were performed in pots to assess the in vivo disease-control efficiency of SQR-T37 and bio-organic fertilizer. The results indicate that the mycoparasitism was the main mechanism accounting for the antagonistic activity of SQR-T37. In one experiment, the population of R. solani was decreased from 10⁶ internal transcribed spacer (ITS) copies per gram soil to 10⁴ ITS copies per gram soil by the presence of the antagonist. In this experiment, 45% of the control efficiency was obtained when 8 g of SQR-T37 hyphae per gram soil was applied. In a second experiment, as much as 81.82% of the control efficiency was obtained when bio-organic fertilizer (SQR-T37 fermented organic fertilizer, BIO) was applied compared to only 27.27% of the control efficiency when only 4 g of SQR-T37 hyphae per gram soil was applied. Twenty days after incubation, the population of T. harzianum was 4.12 × 10⁷ ITS copies per gram soil in the BIO treatment, which was much higher than that in the previous treatment (8.77 × 10⁵ ITS copies per gram soil), where only SQR-T37 was applied. The results indicated that SQR-T37 was a potent antagonist against R. solani in a mycoparasitic way that decreased the population of the pathogen. Applying BIO was more efficient than SQR-T37 application alone because it stabilized the population of the antagonist.

Keywords: Trichoderma harzianum SQR-T37; Rhizoctonia solani ; Mycoparasitism; Bio-organic fertilizer (BIO); Quantification real-time PCR; DGGE

Enzyme characteristics of aminotransferase FumI of Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B₁ by Doris Hartinger; Heidi Schwartz; Christian Hametner; Gerd Schatzmayr; Dietmar Haltrich; Wulf-Dieter Moll (pp. 757-768).

Fumonisins are carcinogenic mycotoxins that are frequently found as natural contaminants in maize from warm climate regions around the world. The aminotransferase FumI is encoded as part of a gene cluster of Sphingopyxis sp. MTA144, which enables this bacterial strain to degrade fumonisin B₁ and related fumonisins. FumI catalyzes the deamination of the first intermediate of the catabolic pathway, hydrolyzed fumonisin B₁. We used a preparation of purified, His-tagged FumI, produced recombinantly in Escherichia coli in soluble form, for enzyme characterization. The structure of the reaction product was studied by NMR and identified as 2-keto hydrolyzed fumonisin B₁. Pyruvate was found to be the preferred co-substrate and amino group receptor (K _M = 490 μM at 10 μM hydrolyzed fumonisin B₁) of FumI, but other α-keto acids were also accepted as co-substrates. Addition of the co-enzyme pyridoxal phosphate to the enzyme preparation enhanced activity, and saturation was already reached at the lowest tested concentration of 10 μM. The enzyme showed activity in the range of pH 6 to 10 with an optimum at pH 8.5, and in the range of 6°C to 50°C with an optimum at 35°C. The aminotransferase worked best at low salt concentration. FumI activity could be recovered after preincubation at pH 4.0 or higher, but not lower. The aminotransferase was denatured after preincubation at 60°C for 1 h, and the residual activity was also reduced after preincubation at lower temperatures. At optimum conditions, the kinetic parameters K _M = 1.1 μM and k _cat = 104/min were determined with 5 mM pyruvate as co-substrate. Based on the enzyme characteristics, a technological application of FumI, in combination with the fumonisin carboxylesterase FumD for hydrolysis of fumonisins, for deamination and detoxification of hydrolyzed fumonisins seems possible, if the enzyme properties are considered.

Keywords: Mycotoxin; Detoxification; Degradation; Kinetics; Feed; Animal

Establishment of an alternative phosphoketolase-dependent pathway for fructose catabolism in Ralstonia eutropha H16 by Christian Fleige; Jens Kroll; Alexander Steinbüchel (pp. 769-776).

The β-proteobacterium Ralstonia eutropha H16 utilizes fructose and gluconate as carbon sources for heterotrophic growth exclusively via the Entner–Doudoroff pathway with its key enzyme 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase. By deletion of the responsible gene eda, we constructed a KDPG aldolase-negative strain, which is disabled to supply pyruvate for energy metabolism from fructose or gluconate as sole carbon sources. To restore growth on fructose, an alternative pathway, similar to the fructose-6-phosphate shunt of heterofermentative bifidobacteria, was established. For this, the xfp gene from Bifidobacterium animalis, coding for a bifunctional xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp; Meile et al. in J Bacteriol 183:2929–2936, 2001), was expressed in R. eutropha H16 PHB⁻4 Δeda. This Xfp catalyzes the phosphorolytic cleavage of fructose 6-phosphate to erythrose 4-phosphate and acetylphosphate as well as of xylulose 5-phosphate to glyceralaldehyde 3-phosphate and acetylphosphate. The recombinant strain showed phosphoketolase (PKT) activity on either substrate, and was able to use fructose as sole carbon source for growth, because PKT is the only enzyme that is missing in R. eutropha H16 to establish the artificial fructose-6-phosphate shunt. The Xfp-expressing strain R. eutropha H16 PHB⁻4 Δeda (pBBR1MCS-3::xfp) should be applicable for a novel variant of a plasmid addiction system to stably maintain episomally encoded genetic information during fermentative production processes. Plasmid addiction systems are often used to ensure plasmid stability in many biotechnology relevant microorganisms and processes without the need to apply external selection pressure, like the addition of antibiotics. By episomal expression of xfp in a R. eutropha H16 mutant lacking KDPG aldolase activity and cultivation in mineral salt medium with fructose as sole carbon source, the growth of this bacterium was addicted to the constructed xfp harboring plasmid. This novel selection principle extends the applicability of R. eutropha H16 as production platform in biotechnological processes.

Keywords: Entner–Doudoroff pathway; KDPG aldolase; Phosphoketolase; Plasmid addiction system; R. eutropha ; Xfp

PCR methods for the rapid detection and identification of four pathogenic Legionella spp. and two Legionella pneumophila subspecies based on the gene amplification of gyrB by Guangpeng Zhou; Boyang Cao; Yan Dou; Yanwei Liu; Lu Feng; Lei Wang (pp. 777-787).

A total of 25 gyrB gene sequences from 20 Legionella pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were obtained and analyzed, and a multiplex PCR for the simultaneous detection of Legionella bozemanae, Legionella longbeachae, Legionella micdadei and Legioenella pneumophila, and two single PCRs for the differentiation of L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri were established. The multiplex PCR method was shown to be highly specific and reproducible when tested against 41 target strains and 17 strains of other bacteria species. The sensitivity of the multiplex PCR was also analyzed and was shown to detect levels as low as 1 ng of genomic DNA or 10 colony-forming units (CFUs) per milliliter in mock water samples. Sixty-three air conditioner condensed water samples from Shanghai City were examined, and the result was validated using 16S rRNA sequencing. The data reported here demonstrate that the multiplex PCR method described is efficient and convenient for the detection of Legionella species in water samples. Twenty L. pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were used for the validation of the two L. pneumophila subspecies-specific PCR methods, and the results indicated that the two PCR methods were both highly specific and convenient for the identification of L. pneumophila at the subspecies level.

Keywords: PCR; Detection; Legionella spp.; L. pneumophila ; Subspecies

Expression of family 3 cellulose-binding module (CBM3) as an affinity tag for recombinant proteins in yeast by Wen Wan; Dongmei Wang; Xiaolian Gao; Jiong Hong (pp. 789-798).

Easy and low-cost protein purification methods for the mass production of commonly used enzymes that play important roles in biotechnology are in high demand. In this study, we developed a fast, low-cost recombinant protein purification system in the methylotrophic yeast Pichia pastoris using the family 3 cellulose-binding module (CBM3)-based affinity tag. The codon of the cbm3 gene from Clostridium thermocellum was optimized based on the codon usage of P. pastoris. The CBM3 tag was then fused with enhanced green fluorescent protein (CBM3-EGFP) or with inulinase and expressed in P. pastoris to demonstrate its ability to function as an affinity tag in a yeast expression system. We also examined the effects of glycosylation on the secreted CBM3-tag. The secreted wild-type CBM3-EGFP was glycosylated; however, this had little influence on the adsorption of the fusion protein to the regenerated amorphous cellulose (RAC; maximum adsorption capacity of 319 mg/g). Two CBM3-EGFP mutants lacking glycosylation sites were also constructed. The three CBM3-EGFPs expressed in P. pastoris and the CBM3-EGFP expressed in Escherichia coli all had similar RAC adsorption capacity. To construct a tag-free recombinant protein purification system based on CBM3, a CBM3-intein-EGFP fusion protein was expressed in P. pastoris. This fusion protein was stably expressed and the self-cleavage of intein was efficiently induced by DTT or l-cysteine. In this study, we were able to purify the recombinant fusion protein with high efficiency using both intein and direct fusion-based strategies.

Keywords: Cellulose-binding module; Affinity tag; Yeast; Glycosylation

Microbial community succession in a bioreactor modeling a souring low-temperature oil reservoir subjected to nitrate injection by Cameron M. Callbeck; Xiaoli Dong; Indranil Chatterjee; Akhil Agrawal; Sean M. Caffrey; Christoph W. Sensen; Gerrit Voordouw (pp. 799-810).

Injection of up-flow packed-bed bioreactors with excess volatile fatty acids and limiting concentrations of nitrate and sulfate gave complete reduction of nitrate from 0 to 5.5 cm and complete or near-complete reduction of sulfate from 3.2 to 11.5 cm along the bioreactor flow path. Most of the biomass (85%) and most of the genes for nitrate reduction (narG, 96%; napA, 99%) and for sulfate reduction (dsrB, 91%) were present near the inlet (0–5.5 cm) of the 37-cm-long bioreactor. Microbial community analysis by a combination of denaturing gradient gel electrophoresis and pyrosequencing of 16S rRNA amplicons indicated that nitrate-reducing Arcobacter and Pseudomonas species were located from 0 to 3.2 cm and throughout, respectively. Desulfobulbus species were the main sulfate reducers present and acetotrophic methanogens of the genus Methanosaeta predominated at 20–37 cm. Overall, the results indicated a succession of microbial communities along the bioreactor flow path. In the absence of nitrate, the sulfate reduction zone moved nearer to the bioreactor inlet. The sulfide concentration in the bioreactor effluent was temporarily lowered after nitrate injection was re-started. Hence, the bioreactor sulfide output could be disrupted by pulsed, not by constant nitrate injection, as demonstrated also previously in a low-temperature oil field.

Keywords: Souring; Bioreactor; Nitrate; Sulfate; SRB; Pyrosequencing

Simultaneous cadmium removal and 2,4-dichlorophenol degradation from aqueous solutions by Phanerochaete chrysosporium by Anwei Chen; Guangming Zeng; Guiqiu Chen; Jiaqi Fan; Zhengjun Zou; Hui Li; Xinjiang Hu; Fei Long (pp. 811-821).

Phanerochaete chrysosporium has been recognised as an effective bioremediation agent due to its unique degradation to xenobiotic and biosorption ability to heavy metals. However, few studies have focused on the simultaneous removal of heavy metals and organic pollutants. The aim of this work was to study the feasibility of simultaneous cadmium removal and 2,4-dichlorophenol (2,4-DCP) degradation in P. chrysosporium liquid cultures. The removal efficiencies were pH dependent and the maximum removal efficiencies were observed at pH 6.5 under an initial cadmium concentration of 5 mg/L and an initial 2,4-DCP concentration of 20 mg/L. The removal efficiencies for cadmium and 2,4-DCP reached 63.62% and 83.90%, respectively, under the optimum conditions. The high production levels of lignin peroxidase (7.35 U/mL) and manganese peroxidase (8.30 U/mL) resulted in an increase in 2,4-DCP degradation. The protein content decreased with increasing cadmium concentration. The surface characteristics and functional groups of the biomass were studied by scanning electron microscopy and a Fourier-transformed infrared spectrometer. The results showed that the use of P. chrysosporium is promising for the simultaneous removal of cadmium and 2,4-DCP from liquid media.

Keywords: Biosorption; Cadmium; Degradation; 2,4-Dichlorophenol; Phanerochaete chrysosporium

Polyphasic approach for assessing changes in an autochthonous marine bacterial community in the presence of Prestige fuel oil and its biodegradation potential by Núria Jiménez; Marc Viñas; Cèlia Guiu-Aragonés; Josep M. Bayona; Joan Albaigés; Anna M. Solanas (pp. 823-834).

A laboratory experiment was conducted to identify key hydrocarbon degraders from a marine oil spill sample (Prestige fuel oil), to ascertain their role in the degradation of different hydrocarbons, and to assess their biodegradation potential for this complex heavy oil. After a 17-month enrichment in weathered fuel, the bacterial community, initially consisting mainly of Methylophaga species, underwent a major selective pressure in favor of obligate hydrocarbonoclastic microorganisms, such as Alcanivorax and Marinobacter spp. and other hydrocarbon-degrading taxa (Thalassospira and Alcaligenes), and showed strong biodegradation potential. This ranged from >99% for all low- and medium-molecular-weight alkanes (C₁₅–C₂₇) and polycyclic aromatic hydrocarbons (C₀- to C₂- naphthalene, anthracene, phenanthrene, dibenzothiophene, and carbazole), to 75–98% for higher molecular-weight alkanes (C₂₈–C₄₀) and to 55–80% for the C₃ derivatives of tricyclic and tetracyclic polycyclic aromatic hydrocarbons (PAHs) (e.g., C₃-chrysenes), in 60 days. The numbers of total heterotrophs and of n-alkane-, aliphatic-, and PAH degraders, as well as the structures of these populations, were monitored throughout the biodegradation process. The salinity of the counting medium affects the counts of PAH degraders, while the carbon source (n-hexadecane vs. a mixture of aliphatic hydrocarbons) is a key factor when counting aliphatic degraders. These limitations notwithstanding, some bacterial genera associated with hydrocarbon degradation (mainly belonging to α- and γ-Proteobacteria, including the hydrocarbonoclastic Alcanivorax and Marinobacter) were identified. We conclude that Thalassospira and Roseobacter contribute to the degradation of aliphatic hydrocarbons, whereas Mesorhizobium and Muricauda participate in the degradation of PAHs.

Keywords: Consortium; Heavy fuel oil; Hydrocarbon biodegradation; Hydrocarbonoclastic bacteria; Microbial community; Prestige oil spill

The effect of mixotrophy on microalgal growth, lipid content, and expression levels of three pathway genes in Chlorella sorokiniana by Minxi Wan; Peng Liu; Jinlan Xia; Julian N. Rosenberg; George A. Oyler; Michael J. Betenbaugh; Zhenyuan Nie; Guanzhou Qiu (pp. 835-844).

Nannochloropsis oculata CCMP 525, Dunaliella salina FACHB 435, and Chlorella sorokiniana CCTCC M209220 were compared in mixotrophic and photoautotrophic cultures in terms of growth rate, protein, and lipid content. Growth improved in glucose, and the biomass productivities of N. oculata, D. salina, and C. sorokiniana were found to be 1.4-, 2.2- and 4.2-fold that observed photoautotrophically. However, biomass and lipid production decreased at the highest glucose concentrations. Meanwhile, the content of protein and lipid were significantly augmented for mixotrophic conditions at least for some species. C. sorokiniana was found to be well suited for lipid production based on its high biomass production rate and lipid content reaching 51% during mixotrophy. Expression levels of accD (heteromeric acetyl-CoA carboxylase beta subunit), acc1 (homomeric acetyl-CoA carboxylase), rbcL (ribulose 1, 5-bisphosphate carboxylase/oxygenase large subunit) genes in C. sorokiniana were studied by real-time PCR. Increased expression levels of accD reflect the increased lipid content in stationary phase of mixotrophic growth, but expression of the acc1 gene remains low, suggesting that this gene may not be critical to lipid accumulation. Additionally, reduction of expression of the rbcL gene during mixotrophy indicated that utilization of glucose was found to reduce the role of this gene and photosynthesis.

Keywords: Mixotrophic culture; Chlorella sorokiniana ; AccD; Lipid synthesis; Gene expression; Biofuel; Algae

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Applied Microbiology and Biotechnology (v.91, #3)

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Applied Microbiology and Biotechnology (v.91, #3)