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Applied Microbiology and Biotechnology (v.93, #4)

Microbial and fungal protease inhibitors—current and potential applications by Jerica Sabotič; Janko Kos (pp. 1351-1375).

Proteolytic enzymes play essential metabolic and regulatory functions in many biological processes and also offer a wide range of biotechnological applications. Because of their essential roles, their proteolytic activity needs to be tightly regulated. Therefore, small molecules and proteins that inhibit proteases can be versatile tools in the fields of medicine, agriculture and biotechnology. In medicine, protease inhibitors can be used as diagnostic or therapeutic agents for viral, bacterial, fungal and parasitic diseases as well as for treating cancer and immunological, neurodegenerative and cardiovascular diseases. They can be involved in crop protection against plant pathogens and herbivorous pests as well as against abiotic stress such as drought. Furthermore, protease inhibitors are indispensable in protein purification procedures to prevent undesired proteolysis during heterologous expression or protein extraction. They are also valuable tools for simple and effective purification of proteases, using affinity chromatography. Because there are such a large number and diversity of proteases in prokaryotes, yeasts, filamentous fungi and mushrooms, we can expect them to be a rich source of protease inhibitors as well.

Keywords: Fungi; Microorganisms; Protease; Protease inhibitor; Disease; Crop protection

Fermentative production of isobutene by Bianca N. M. van Leeuwen; Albertus M. van der Wulp; Isabelle Duijnstee; Antonius J. A. van Maris; Adrie J. J. Straathof (pp. 1377-1387).

Isobutene (2-methylpropene) is one of those chemicals for which bio-based production might replace the petrochemical production in the future. Currently, more than 10 million metric tons of isobutene are produced on a yearly basis. Even though bio-based production might also be achieved through chemocatalytic or thermochemical methods, this review focuses on fermentative routes from sugars. Although biological isobutene formation is known since the 1970s, extensive metabolic engineering is required to achieve economically viable yields and productivities. Two recent metabolic engineering developments may enable anaerobic production close to the theoretical stoichiometry of 1isobutene + 2CO₂ + 2H₂O per mol of glucose. One relies on the conversion of 3-hydroxyisovalerate to isobutene as a side activity of mevalonate diphosphate decarboxylase and the other on isobutanol dehydration as a side activity of engineered oleate hydratase. The latter resembles the fermentative production of isobutanol followed by isobutanol recovery and chemocatalytic dehydration. The advantage of a completely biological route is that not isobutanol, but instead gaseous isobutene is recovered from the fermenter together with CO₂. The low aqueous solubility of isobutene might also minimize product toxicity to the microorganisms. Although developments are at their infancy, the potential of a large scale fermentative isobutene production process is assessed. The production costs estimate is 0.9 € kg⁻¹, which is reasonably competitive. About 70% of the production costs will be due to the costs of lignocellulose hydrolysate, which seems to be a preferred feedstock.

Keywords: Bio-based isobutene; Isobutanol dehydratase; Mevalonate diphosphate decarboxylase; Gas purification

Developing fungal pigments for “painting” vascular plants by Sara C. Robinson (pp. 1389-1394).

The use of fungal pigments as color additives to wood as a method to increase forest revenue is a relatively new, but quickly developing field. Sugar maple (Acer saccharum) is currently the primary utilized hardwood for spalting and appears to be the best suited North American hardwood for such purposes. The combination of Trametes versicolor and Bjerkandera adusta has been identified in several instances as a strong fungal pairing for zone line production; however, Xylaria polymorpha is capable of creating zone lines without the antagonism of a secondary fungus. Few fungal pigments have been developed for reliable use; Scytalidium cuboideum is capable of producing a penetrating pink/red stain, as well as a blue pigment after extended incubation, and Chlorociboria sp. produces a blue/green pigment if grown on aspen (Populus tremuloides). Several opportunities exist for stimulation of fungal pigments including the use of copper sulfate and changes in wood pH.

Keywords: Bjerkandera adusta ; Chlorociboria sp.; Fungal pigments; Scytalidium cuboideum ; Trametes versicolor ; Spalting; Xylaria polymorpha

Fungal aryl-alcohol oxidase: a peroxide-producing flavoenzyme involved in lignin degradation by Aitor Hernández-Ortega; Patricia Ferreira; Angel T. Martínez (pp. 1395-1410).

Aryl-alcohol oxidase (AAO) is an extracellular flavoprotein providing the H₂O₂ required by ligninolytic peroxidases for fungal degradation of lignin, the key step for carbon recycling in land ecosystems. O₂ activation by Pleurotus eryngii AAO takes place during the redox-cycling of p-methoxylated benzylic metabolites secreted by the fungus. Only Pleurotus AAO sequences were available for years, but the number strongly increased recently due to sequencing of different basidiomycete genomes, and a comparison of 112 GMC (glucose–methanol–choline oxidase) superfamily sequences including 40 AAOs is presented. As shown by kinetic isotope effects, alcohol oxidation by AAO is produced by hydride transfer to the flavin, and hydroxyl proton transfer to a base. Moreover, site-directed mutagenesis studies showed that His502 activates the alcohol substrate by proton abstraction, and this result was extended to other GMC oxidoreductases where the nature of the base was under discussion. However, in contrast with that proposed for GMC oxidoreductases, the two transfers are not stepwise but concerted. Alcohol docking at the buried AAO active site resulted in only one catalytically relevant position for concerted transfer, with the pro-R α-hydrogen at distance for hydride abstraction. The expected hydride-transfer stereoselectivity was demonstrated, for the first time in a GMC oxidoreductase, by using the (R) and (S) enantiomers of α-deuterated p-methoxybenzyl alcohol. Other largely unexplained aspects of AAO catalysis (such as the unexpected specificity on substituted aldehydes) can also be explained in the light of the recent results. Finally, the biotechnological interest of AAO in flavor production is extended by its potential in production of chiral compounds taking advantage from the above-described stereoselectivity.

Keywords: Aryl-alcohol oxidase; Fungal enzymes; GMC oxidoreductases; Lignin biodegradation; Reaction mechanism; Stereoselectivity

Rapid detection methods for Bacillus anthracis in environmental samples: a review by Léonid M. Irenge; Jean-Luc Gala (pp. 1411-1422).

Bacillus anthracis is a Gram-positive, spore-forming bacterium, which causes anthrax, an often lethal disease of animals and humans. Although the disease has been well studied since the nineteenth century, it has witnessed a renewed interest during the past decade, due to its use as a bioterrorist agent in the fall of 2001 in the USA. A number of techniques aimed at rapidly detecting B. anthracis, in environmental samples as well as in point-of-care settings for humans suspected of exposure to the pathogen, are now available. These technologies range from culture-based methods to portable DNA amplification devices. Despite recent developments, specific identification of B. anthracis still remains difficult because of its phenotypic and genotypic similarities with other Bacillus species. Accordingly, many efforts are being made to improve the specificity of B. anthracis identification. This mini-review discusses the current challenges around B. anthracis identification, not only in reach-back laboratories but also in the field (in operational conditions).

Keywords: Bacillus anthracis ; Rapid detection; Environmental samples; Culture; Molecular; Affinity-based methods

Developing symbiotic consortia for lignocellulosic biofuel production by Trevor R. Zuroff; Wayne R. Curtis (pp. 1423-1435).

The search for petroleum alternatives has motivated intense research into biological breakdown of lignocellulose to produce liquid fuels such as ethanol. Degradation of lignocellulose for biofuel production is a difficult process which is limited by, among other factors, the recalcitrance of lignocellulose and biological toxicity of the products. Consolidated bioprocessing has been suggested as an efficient and economical method of producing low value products from lignocellulose; however, it is not clear whether this would be accomplished more efficiently with a single organism or community of organisms. This review highlights examples of mixtures of microbes in the context of conceptual models for developing symbiotic consortia for biofuel production from lignocellulose. Engineering a symbiosis within consortia is a putative means of improving both process efficiency and stability relative to monoculture. Because microbes often interact and exist attached to surfaces, quorum sensing and biofilm formation are also discussed in terms of consortia development and stability. An engineered, symbiotic culture of multiple organisms may be a means of assembling a novel combination of metabolic capabilities that can efficiently produce biofuel from lignocellulose.

Keywords: Symbiosis; Lignocellulose; Biofuel; Consortia; Consolidated bioprocessing

Improvement of specific growth rate of Pichia pastoris for effective porcine interferon-α production with an on-line model-based glycerol feeding strategy by Min-Jie Gao; Zhi-Yong Zheng; Jian-Rong Wu; Shi-Juan Dong; Zhen Li; Hu Jin; Xiao-Bei Zhan; Chi-Chung Lin (pp. 1437-1445).

Effective expression of porcine interferon-α (pIFN-α) with recombinant Pichia pastoris was conducted in a bench-scale fermentor. The influence of the glycerol feeding strategy on the specific growth rate and protein production was investigated. The traditional DO-stat feeding strategy led to very low cell growth rate resulting in low dry cell weight (DCW) of about 90 g/L during the subsequent induction phase. The previously reported Artificial Neural Network Pattern Recognition (ANNPR) model-based glycerol feeding strategy improved the cell density to 120 g DCW/L, while the specific growth rate decreased from 0.15 to 0.18 to 0.03–0.08 h⁻¹ during the last 10 h of the glycerol feeding stage leading to a variation of the porcine interferon-α production, as the glycerol feeding scheme had a significant effect on the induction phase. This problem was resolved by an improved ANNPR model-based feeding strategy to maintain the specific growth rate above 0.11 h⁻¹. With this feeding strategy, the pIFN-α concentration reached a level of 1.43 g/L, more than 1.5-fold higher than that obtained with the previously adopted feeding strategy. Our results showed that increasing the specific growth rate favored the target protein production and the glycerol feeding methods directly influenced the induction stage. Consequently, higher cell density and specific growth rate as well as effective porcine interferon-α production have been achieved by our novel glycerol feeding strategy.

Keywords: DO-stat; Fed-batch culture; Pichia pastoris ; Protein expression; Artificial neural network

Development of a novel probiotic delivery system based on microencapsulation with protectants by Song Chen; Qian Zhao; Lynnette R. Ferguson; Quan Shu; Iona Weir; Sanjay Garg (pp. 1447-1457).

The establishment of the health-promoting benefits of probiotics is challenged by the antimicrobial bio-barriers throughout the host’s gastrointestinal (GI) tract after oral administration. Although microencapsulation has been frequently utilised to enhance the delivery of probiotics, microcapsules of sub-100 μm were found to be ineffective and therefore questioned as an effective delivery vehicle for viable probiotics despite the sensory advantage. In this study, four probiotics strains were encapsulated in chitosan-coated alginate microcapsules of sub-100 μm. Only a minor protective effect was observed from this original type of microcapsule. In order to enhance the survival of these probiotics, sucrose, a metabolisable sugar, and lecithin vesicles were added to the wall material. Both of the ingredients could be readily encapsulated with the probiotics, and protected them from stresses in the simulated GI fluids. The metabolisable sugar effectively increased the survival of the probiotics in gastric acid, mainly through energizing the membrane-bound F₁F₀-ATPases. The lecithin vesicles proved to alleviate the bile salt stress, and hence notably reduced the viability loss at the elevated bile salt concentrations. Overall, three out of the total four probiotics in the reinforced sub-100 μm microencapsules could significantly survive through an 8-h sequential treatment of the simulated GI fluids, giving less than 1-log drop in viable count. The most vulnerable strain of bifidobacteria also yielded a viability increase of 3-logs from this protection. In conclusion, the sub-100 μm microcapsules can be a useful vehicle for the delivery of probiotics, as long as suitable protectants are incorporated in the wall matrix.

Keywords: Probiotic delivery system; Microencapsulation; Probiotics; Metabolisable sugar; Lecithin vesicle; Gastric pH; Bile salt

Engineering yield and rate of reductive biotransformation in Escherichia coli by partial cyclization of the pentose phosphate pathway and PTS-independent glucose transport by Solvej Siedler; Stephanie Bringer; Lars M. Blank; Michael Bott (pp. 1459-1467).

Optimization of yields and productivities in reductive whole-cell biotransformations is an important issue for the industrial application of such processes. In a recent study with Escherichia coli, we analyzed the reduction of the prochiral β-ketoester methyl acetoacetate by an R-specific alcohol dehydrogenase (ADH) to the chiral hydroxy ester (R)-methyl 3-hydroxybutyrate (MHB) using glucose as substrate for the generation of NADPH. Deletion of the phosphofructokinase gene pfkA almost doubled the yield to 4.8 mol MHB per mole of glucose, and it was assumed that this effect was due to a partial cyclization of the pentose phosphate pathway (PPP). Here, this partial cyclization was confirmed by ¹³C metabolic flux analysis, which revealed a negative net flux from glucose 6-phosphate to fructose 6-phosphate catalyzed by phosphoglucose isomerase. For further process optimization, the genes encoding the glucose facilitator (glf) and glucokinase (glk) of Zymomonas mobilis were overexpressed in recombinant E. coli strains carrying ADH and deletions of either pgi (phosphoglucose isomerase), or pfkA, or pfkA plus pfkB. In all cases, the glucose uptake rate was increased (30–47%), and for strains Δpgi and ΔpfkA also, the specific MHB production rate was increased by 15% and 20%, respectively. The yield of the latter two strains slightly dropped by 11% and 6%, but was still 73% and 132% higher compared to the reference strain with intact pgi and pfkA genes and expressing glf and glk. Thus, metabolic engineering strategies are presented for improving yield and rate of reductive redox biocatalysis by partial cyclization of the PPP and by increasing glucose uptake, respectively.

Keywords: Escherichia coli ; NADPH yield; ¹³C flux analysis; pfkA ; pfkB ; pgi ; Reductive whole-cell biotransformation; glf

Bioconversion of d-galactose to d-tagatose: continuous packed bed reaction with an immobilized thermostable l-arabinose isomerase and efficient purification by selective microbial degradation by Min Liang; Min Chen; Xinying Liu; Yafei Zhai; Xian-wei Liu; Houcheng Zhang; Min Xiao; Peng Wang (pp. 1469-1474).

The continuous enzymatic conversion of d-galactose to d-tagatose with an immobilized thermostable l-arabinose isomerase in packed-bed reactor and a novel method for d-tagatose purification were studied. l-arabinose isomerase from Thermoanaerobacter mathranii (TMAI) was recombinantly overexpressed and immobilized in calcium alginate. The effects of pH and temperature on d-tagatose production reaction catalyzed by free and immobilized TMAI were investigated. The optimal condition for free enzyme was pH 8.0, 60°C, 5 mM MnCl₂. However, that for immobilized enzyme was pH 7.5, 75°C, 5 mM MnCl₂. In addition, the catalytic activity of immobilized enzyme at high temperature and low pH was significantly improved compared with free enzyme. The optimum reaction yield with immobilized TMAI increased by four percentage points to 43.9% compared with that of free TMAI. The highest productivity of 10 g/L h was achieved with the yield of 23.3%. Continuous production was performed at 70°C; after 168 h, the reaction yield was still above 30%. The resultant syrup was then incubated with Saccharomyces cerevisiae L1 cells. The selective degradation of d-galactose was achieved, obtaining d-tagatose with the purity above 95%. The established production and separation methods further potentiate the industrial production of d-tagatose via bioconversion and biopurification processes.

Keywords: d-Tagatose; d-Galactose; l-Arabinose isomerase; Saccharomyces cerevisiae

Structure identification and fermentation characteristics of pinoresinol diglucoside produced by Phomopsis sp. isolated from Eucommia ulmoides Oliv by Junling Shi; Chao Liu; Laping Liu; Baowei Yang; Yinzhi Zhang (pp. 1475-1483).

Pinoresinol diglucoside (PDG) is the important antihypertensive compound in Eucommia ulmoides Oliv., a traditional Chinese herb medicine. The research objective was to certify the possibility of producing PDG through fermentation. PDG-producing endophytic fungi were isolated from E. ulmoides Oliv., and the highest PDG-yielding (11.65 mg/L) isolate, XP-8, was identified as Phomopsis sp. according to the morphological characteristics and the phylogenetic tree constructed on the basis of the gene sequence in the internal transcribed spacers district. The microbial PDG was isolated by using S-8 resin and purified to a purity of 98.7% using preparative high-performance liquid chromatography (HPLC). Information obtained from the UV spectrum (277 and 227 nm, in water solution), infra-red spectrum (3,428; 2,930; 2,877; 1,637; 1,600; and 1,513; 1,460; 1,421; 1,269; 1,223; 1,075; 658 cm⁻¹, in powder), molecular weight (682 Da, measured using HPLC-electrospray ionization mass spectrometry (ESI/MS) and tandem mass spectrometry), and nuclear magnetic resonance analysis show the microbial PDG is (+)-1-pinoresinol 4,4′-di-O-β-d-glucopyranoside, same as the plant-derived PDG. The microbial PDG is stable in pH range from 3 to 11 but less stable at temperature higher than 90 °C and in light exposure. During the fermentation, PDG production outside cells starts at the later stage of cell growth when the residual sugar in the medium was low. The study reveals the possibility for production of PDG by fermentation.

Keywords: Endophytic fungi; Eucommia ulmoides Oliv.; Phomopsis sp.; Pinoresinol diglucoside

Continuous butanol production with reduced byproducts formation from glycerol by a hyper producing mutant of Clostridium pasteurianum by Alok Malaviya; Yu-Sin Jang; Sang Yup Lee (pp. 1485-1494).

Butanol, a four-carbon primary alcohol (C₄H₁₀O), is an important industrial chemical and has a good potential to be used as a superior biofuel. Bio-based production of butanol from renewable feedstock is a promising and sustainable alternative to substitute petroleum-based fuels. Here, we report the development of a process for butanol production from glycerol, which is abundantly available as a byproduct of biodiesel production. First, a hyper butanol producing strain of Clostridium pasteurianum was isolated by chemical mutagenesis. The best mutant strain, C. pasteurianum MBEL_GLY2, was able to produce 10.8 g l⁻¹ butanol from 80 g l⁻¹ glycerol as compared to 7.6 g l⁻¹ butanol produced by the parent strain. Next, the process parameters were optimized to maximize butanol production from glycerol. Under the optimized batch condition, the butanol concentration, yield, and productivity of 17.8 g l⁻¹, 0.30 g g⁻¹, and 0.43 g l⁻¹ h⁻¹ could be achieved. Finally, continuous fermentation of C. pasteurianum MBEL_GLY2 with cell recycling was carried out using glycerol as a major carbon source at several different dilution rates. The continuous fermentation was run for 710 h without strain degeneration. The acetone–butanol–ethanol productivity and the butanol productivity of 8.3 and 7.8 g l⁻¹ h⁻¹, respectively, could be achieved at the dilution rate of 0.9 h⁻¹. This study reports continuous production of butanol with reduced byproducts formation from glycerol using C. pasteurianum, and thus could help design a bioprocess for the improved production of butanol.

Keywords: Anaerobic fermentation; Butanol; Clostridium pasteurianum ; Glycerol; Acetone–butanol–ethanol fermentation

Development of a glutathione production process from proteinaceous biomass resources using protease-displaying Saccharomyces cerevisiae by Kiyotaka Y. Hara; Songhee Kim; Hideyo Yoshida; Kentaro Kiriyama; Takashi Kondo; Naoko Okai; Chiaki Ogino; Hideki Fukuda; Akihiko Kondo (pp. 1495-1502).

Glutathione is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae, and supplementation of fermentation with several amino acids can increase intracellular GSH content. More recently, however, focus has been given to protein as a resource for biofuel and fine chemical production. We demonstrate that expression of a protease on the cell surface of S. cerevisiae enables the direct use of keratin and soy protein as a source of amino acids and that these substrates enhanced intracellular GSH content. Furthermore, fermentation using soy protein also enhanced cell concentration. GSH fermentation from keratin and to a greater extent from soy protein using protease-displaying yeast yielded greater GSH productivity compared to GSH fermentation with amino acid supplementation. This protease-displaying yeast is potentially applicable to a variety of processes for the bio-production of value-added chemicals from proteinaceous biomass resources.

Keywords: Glutathione; Yeast; Protease; Proteinaceous biomass; Biorefinery

A novel cold-active xylanase from the cellulolytic myxobacterium Sorangium cellulosum So9733-1: gene cloning, expression, and enzymatic characterization by Shu-Yun Wang; Wei Hu; Xiao-Yu Lin; Zhi-Hong Wu; Yue-Zhong Li (pp. 1503-1512).

The cellulolytic myxobacterium Sorangium cellulosum is able to efficiently degrade many kinds of polysaccharides, but none of the enzymes involved have been characterized. In this paper, a xylanase gene (xynA) was cloned from S. cellulosum So9733-1 using thermal asymmetric interlaced PCR. The gene is composed of 1,209 bp and has only 52.27% G + C content, which is much lower than that of most myxobacterial DNA reported (67–72%). Gene xynA encodes a 402 amino acid protein that contains a single catalytic domain belonging to the glycoside hydrolase family 10. The novel xylanase gene, xynA, was expressed in Escherichia coli BL21 (DE3) and the recombinant protein (r-XynA) was purified by Ni-affinity chromatography. The r-XynA had the optimum temperature of 30–35°C and exhibited 33.3% activity at 5°C and 13.7% activity at 0°C. Approximately 80% activity was lost after 20-min pre-incubation at 50°C. These results indicate that r-XynA is a cold-active xylanase with low thermostability. At 30°C, the K _m values of r-XynA on beechwood xylan, birchwood xylan, and oat spelt xylan were 25.77 ± 4.16, 26.52 ± 4.78, and 38.13 ± 5.35 mg/mL, respectively. The purified r-XynA displayed optimum activity at pH 7.0. The activity of r-XynA was enhanced by the presence of Ca²⁺. The r-XynA hydrolyzed beechwood xylan, birchwood xylan, and xylooligosaccharides (xylotriose, xylotetraose, and xylopentose) to produce primarily xylose and xylobiose. To our knowledge, this is the first report on the characterization of a xylanase from S. cellulosum.

Keywords: Sorangium cellulosum ; Xylanase; Cold active; Glycoside hydrolase family 10; G + C content

Discovery of nigerose phosphorylase from Clostridium phytofermentans by Takanori Nihira; Hiroyuki Nakai; Kazuhiro Chiku; Motomitsu Kitaoka (pp. 1513-1522).

A novel phosphorylase from Clostridium phytofermentans belonging to the glycoside hydrolase family (GH) 65 (Cphy1874) was characterized. The recombinant Cphy1874 protein produced in Escherichia coli showed phosphorolytic activity on nigerose in the presence of inorganic phosphate, resulting in the release of d-glucose and β-d-glucose 1-phosphate (β-G1P) with the inversion of the anomeric configuration. Kinetic parameters of the phosphorolytic activity on nigerose were k _cat = 67 s⁻¹ and K _m = 1.7 mM. This enzyme did not phosphorolyze substrates for the typical GH65 enzymes such as trehalose, maltose, and trehalose 6-phosphate except for a weak phosphorolytic activity on kojibiose. It showed the highest reverse phosphorolytic activity in the reverse reaction using d-glucose as the acceptor and β-G1P as the donor, and the product was mostly nigerose at the early stage of the reaction. The enzyme also showed reverse phosphorolytic activity, in a decreasing order, on d-xylose, 1,5-anhydro-d-glucitol, d-galactose, and methyl-α-d-glucoside. All major products were α-1,3-glucosyl disaccharides, although the reaction with d-xylose and methyl-α-d-glucoside produced significant amounts of α-1,2-glucosides as by-products. We propose 3-α-d-glucosyl-d-glucose:phosphate β-d-glucosyltransferase as the systematic name and nigerose phosphorylase as the short name for this Cphy1874 protein.

Keywords: Nigerose phosphorylase; Glycoside hydrolase family 65; Phosphorylase nigerose; Clostridium phytofermentans

Identification and characterization of DGA2, an acyltransferase of the DGAT1 acyl-CoA:diacylglycerol acyltransferase family in the oleaginous yeast Yarrowia lipolytica. New insights into the storage lipid metabolism of oleaginous yeasts by Athanasios Beopoulos; Ramdane Haddouche; Philomene Kabran; Thierry Dulermo; Thierry Chardot; Jean-Marc Nicaud (pp. 1523-1537).

Triacylglycerols (TAG) and steryl esters (SE) are the principal storage lipids in all eukaryotic cells. In yeasts, these storage lipids accumulate within special organelles known as lipid bodies (LB). In the lipid accumulation-oriented metabolism of the oleaginous yeast Yarrowia lipolytica, storage lipids are mostly found in the form of TAG, and only small amounts of SE accumulate. We report here the identification of a new DAG acyltransferase gene, DGA2, homologous to the ARE genes of Saccharomyces cerevisiae. This gene encodes a member of the type 1 acyl-CoA:diacylglycerol acyltransferase family (DGAT1), which has not previously been identified in yeasts, but is commonly found in mammals and plants. Unlike the Are proteins in S. cerevisiae, Dga2p makes a major contribution to TAG synthesis via an acyl-CoA-dependent mechanism and is not involved in SE synthesis. This enzyme appears to affect the size and morphology of LB, suggesting a direct role of storage lipid proteins in LB formation. We report that the Are1p of Y. lipolytica was essential for sterol esterification, as deletion of the encoding gene (ARE1) completely abolished SE synthesis. Unlike its homologs in yeasts, YlARE1 has no DAG acyltransferase activity. We also reconsider the role and function of all four acyltransferase enzymes involved in the final step of neutral lipid synthesis in this oleaginous yeast.

Keywords: Oleaginous yeast; Yarrowia lipolytica ; Metabolism; Biotechnology; Acyltransferases; Lipids

Influence of medium components on the expression of recombinant lipoproteins in Escherichia coli by Chi-Ling Tseng; Chih-Hsiang Leng (pp. 1539-1552).

Bacterial lipoproteins are crucial antigens for protective immunity against bacterial pathogens. Expression of exogenous lipoproteins in Escherichia coli at high levels is thought to be an extremely difficult endeavor because it frequently results in incomplete or absent lipid modification. Previously, we identified a fusion sequence (D1) from a Neisseria meningitidis lipoprotein that induced a non-lipidated protein, E3 (the domain III of the dengue virus envelope protein), to become lipidated. However, without optimizing the growth conditions, some of the D1-fusion proteins were not lipidated. Here, we report the influence of medium components on the expression of recombinant lipoproteins in E. coli. For high-level expression of mature lipoproteins in the C43 (DE3) strain, M9 medium was better than M63 and the rich medium. Furthermore, we analyzed the influence of other media factors (including nitrogen and carbon sources, phosphate, ferrous ions, calcium, magnesium, and pH) on the levels of lipoprotein expression. The results showed that excess nitrogen sources and phosphate in M9 medium could increase the amount of immature lipoproteins, and glucose was a better carbon source than glycerol for expressing mature lipoproteins. We also found that lipoproteins tended to be completely processed in the alkaline environment, even in the nutrient-rich medium. Additional constructs expressing different immunogens or lipid signal peptides as targets were also utilized, demonstrating that these targets could be expressed as completely mature lipoproteins in the M9 medium but not in the rich medium. Our results provide the useful information for expressing mature exogenous lipoproteins in E. coli.

Keywords: Bacterial lipoprotein; Recombinant lipoprotein; Minimal medium; Lipidation

Purification and characterization of heterologously expressed nitrilases from filamentous fungi by Alena Petříčková; Alicja Barbara Veselá; Ondřej Kaplan; David Kubáč; Bronislava Uhnáková; Anna Malandra; Jürgen Felsberg; Anna Rinágelová; Philip Weyrauch; Vladimír Křen; Karel Bezouška; Ludmila Martínková (pp. 1553-1561).

Nitrilases from Aspergillus niger CBS 513.88, A. niger K10, Gibberella moniliformis, Neurospora crassa OR74A, and Penicillium marneffei ATCC 18224 were expressed in Escherichia coli BL21-Gold (DE3) after IPTG induction. N. crassa nitrilase exhibited the highest yield of 69,000 U L⁻¹ culture. Co-expression of chaperones (GroEL/ES in G. moniliformis and P. marneffei; GroEL/ES and trigger factor in N. crassa and A. niger CBS 513.88) enhanced the enzyme solubility. Specific activities of strains expressing the former two enzymes increased approximately fourfold upon co-expression of GroEL/ES. The enzyme from G. moniliformis (co-purified with GroEL) preferred benzonitrile as substrate (K _m of 0.41 mM, V _max of 9.7 μmol min⁻¹ mg⁻¹ protein). The P. marneffei enzyme (unstable in its purified state) exhibited the highest V _max of 7.3 μmol min⁻¹ mg⁻¹ protein in cell-free extract, but also a high K _m of 15.4 mM, for 4-cyanopyridine. The purified nitrilases from A. niger CBS 513.88 and N. crassa acted preferentially on phenylacetonitrile (K _m of 3.4 and 2.0 mM, respectively; V _max of 10.6 and 17.5 μmol min⁻¹ mg⁻¹ protein, respectively), and hydrolyzed also (R,S)-mandelonitrile with higher K _m values. Significant amounts of amides were only formed by the G. moniliformis nitrilase from phenylacetonitrile and 4-cyanopyridine.

Keywords: Nitrilase; Chaperones; Aspergillus niger ; Gibberella moniliformis ; Neurospora crassa ; Penicillium marneffei

A novel transaminase, (R)-amine:pyruvate aminotransferase, from Arthrobacter sp. KNK168 (FERM BP-5228): purification, characterization, and gene cloning by Akira Iwasaki; Keiji Matsumoto; Junzo Hasegawa; Yoshihiko Yasohara (pp. 1563-1573).

A novel (R)-amine transaminase, which catalyzed (R)-enantioselective transamination of chiral amine, was purified to homogeneity from Arthrobacter sp. KNK168 (FERM BP-5228). The molecular mass of the enzyme was estimated to be 148 kDa by gel filtration and 37 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting a homotetrameric structure. The enzyme catalyzed transamination between amines and pyruvate stereo-specifically. The reaction on 1-methylbenzylamine was (R)-enantioselective. Pyruvate was the best amino acceptor, but the enzyme showed broad amino acceptor specificity for various ketone and aldehyde compounds. The apparent K _ms for (R)-1-methylbenzylamine and pyruvate were 2.62 and 2.29 mM, respectively. The cloned gene of the enzyme consists of an open reading frame (ORF) of 993 bp encoding a protein of 330 amino acids, with a calculated molecular weight of 36,288. The deduced amino acid sequence was found to be homologous to those of the aminotransferases belonging to fold class IV of pyridoxal-5′-phosphate-dependent enzymes, such as branched-chain amino acid aminotransferases.

Keywords: Chiral amine; Biocatalysis; Transaminase; Aminotransferase

An erythromycin process improvement using the diethyl methylmalonate-responsive (Dmr) phenotype of the Saccharopolyspora erythraea mutB strain by J. Mark Weber; William H. Cernota; Melissa C. Gonzalez; Benjamin I. Leach; Andrew R. Reeves; Roy K. Wesley (pp. 1575-1583).

The Saccharopolyspora erythraea mutB knockout strain, FL2281, having a block in the methylmalonyl-CoA mutase reaction, was found to carry a diethyl methylmalonate-responsive (Dmr) phenotype in an oil-based fermentation medium. The Dmr phenotype confers the ability to increase erythromycin A (erythromycin) production from 250–300% when the oil-based medium is supplemented with 15 mM levels of this solvent. Lower concentrations of the solvent stimulated proportionately less erythromycin production, while higher concentrations had no additional benefit. Although the mutB strain is phenotypically a low-level erythromycin producer, diethyl methylmalonate supplementation allowed it to produce up to 30% more erythromycin than the wild-type (control) strain—a strain that does not show the Dmr phenotype. The Dmr phenotype represents a new class of strain improvement phenotype. A theory to explain the biochemical mechanism for the Dmr phenotype is proposed. Other phenotypes found to be associated with the mutB knockout were a growth defect and hyper-pigmentation, both of which were restored to normal by exposure to diethyl methylmalonate. Furthermore, mutB fermentations did not significantly metabolize soybean oil in the presence of diethyl methylmalonate. Finally, a novel method is proposed for the isolation of additional mutants with the Dmr phenotype.

Keywords: Diethyl methylmalonate; Erythromycin; mutB ; Methylmalonyl-CoA mutase; Dmr; Saccharopolyspora erythraea

Isolation of a strong promoter fragment from endophytic Enterobacter cloacae and verification of its promoter activity when its host strain colonizes banana plants by Yu Guang Wang; Qi Yu Xia; Wen Liang Gu; Jian Bo Sun; He Zhang; Xue Hua Lu; Juan Lu; Ming Peng; Xin Zhang (pp. 1585-1599).

To engineer endophytic Enterobacter cloacae as a biocontrol agent against banana fusarium wilt, a promoter-probe plasmid pUCK was constructed to identify a strong promoter to express disease resistance genes. Using a kanamycin resistance gene for selection, 10 fragments with strong promoter activity were identified from the genome of the E. cloacae KKWB-10 strain. The regions of these 10 fragments that were the primary contributors to the promoter function were identified, and their promoter activities were further evaluated using green fluorescent protein (GFP) as a reporter gene. Fragment 132a″ drove the highest level of GFP activity when the bacteria bearing the fragments were cultured in Luria–Bertani and banana stem extract media. The GFP-expressing strain harboring fragment 132a″ (K-pUCK7-132a″-GT) was then inoculated into banana plantlets (about 1 × 10⁷ CFU per plant) to verify the activity of fragment 132a″ in planta. Ten days after inoculation, tissue sections of these banana plantlets were observed by laser confocal scanning microscope. Green fluorescence was observed in the tissues of banana plantlets inoculated with K-pUCK7-132a″-GT but not in uninoculated controls. These results suggest that fragment 132a″ possesses strong promoter activity when its host strain colonizes the banana plants and can be used to engineer endophytic E. cloacae KKWB-10 for biocontrol.

Keywords: Endophyte; Enterobacter cloacae ; Promoter cloning; GFP; Fluorescence observation

A homologous production system for Trichoderma reesei secreted proteins in a cellulase-free background by Fatma Uzbas; Ugur Sezerman; Lukas Hartl; Christian P. Kubicek; Bernhard Seiboth (pp. 1601-1608).

Recent demands for the production of biofuels from lignocellulose led to an increased interest in engineered cellulases from Trichoderma reesei or other fungal sources. While the methods to generate such mutant cellulases on DNA level are straightforward, there is often a bottleneck in their production since a correct posttranslational processing of these enzymes is needed to obtain highly active enzymes. Their production and subsequent enzymatic analysis in the homologous host T. reesei is, however, often disturbed by the concomitant production of other endogenous cellulases. As a useful alternative, we tested the production of cellulases in T. reesei in a genetic background where cellulase formation has been impaired by deletion of the major cellulase transcriptional activator gene xyr1. Three cellulase genes (cel7a, cel7b, and cel12a) were expressed under the promoter regions of the two highly expressed genes tef1 (encoding translation elongation factor 1-alpha) or cdna1 (encoding the hypothetical protein Trire2:110879). When cultivated on d-glucose as carbon source, the Δxyr1 strain secreted all three cellulases into the medium. Related to the introduced gene copy number, the cdna1 promoter appeared to be superior to the tef1 promoter. No signs of proteolysis were detected, and the individual cellulases could be assayed over a background essentially free of other cellulases. Hence this system can be used as a vehicle for rapid and high-throughput testing of cellulase muteins in a homologous background.

Keywords: Cellulase; Recombinant protein production; Hypocrea jecorina ; xyr1 ; cDNA1 ; tef1

N-glycans are not required for the efficient degradation of the mutant Saccharomyces cerevisiae CPY* in Schizosaccharomyces pombe by Hiroyuki Mukaiyama; Michiko Kodera; Naotaka Tanaka; Kaoru Takegawa (pp. 1609-1618).

In eukaryotic cells, aberrant proteins generated in the endoplasmic reticulum (ER) are degraded by the ER-associated degradation (ERAD) pathway. Here, we report on the ERAD pathway of the fission yeast Schizosaccharomyces pombe. We constructed and expressed Saccharomyces cerevisiae wild-type CPY (ScCPY) and CPY-G255R mutant (ScCPY*) in S. pombe. While ScCPY was glycosylated and efficiently transported to the vacuoles in S. pombe, ScCPY* was retained in the ER and was not processed to the matured form in these cells. Cycloheximide chase experiments revealed that ScCPY* was rapidly degraded in S. pombe, and its degradation depended on Hrd1p and Ubc7p homologs. We also found that Mnl1p and Yos9p, proteins that are essential for ERAD in S. cerevisiae, were not required for ScCPY* degradation in S. pombe. Moreover, the null-glycosylation mutant of ScCPY, CPY*0000, was rapidly degraded by the ERAD pathway. These results suggested that N-linked oligosaccharides are not important for the recognition of luminal proteins for ERAD in S. pombe cells.

Keywords: Schizosaccharomyces pombe ; ERAD; Carboxypeptidase Y; Protein degradation

Cloning and overexpression of ketopantoic acid reductase gene from Stenotrophomonas maltophilia and its application to stereospecific production of d-pantoic acid by Dayong Si; Nobuyuki Urano; Sakayu Shimizu; Michihiko Kataoka (pp. 1619-1625).

Ketopantoic acid (KPA) reductase catalyzes the stereospecific reduction of ketopantoic acid to d-pantoic acid. Based on the N-terminal amino acid sequence of KPA reductase from Stenotrophomonas maltophilia 845, the KPA reductase gene was cloned from S. maltophilia NBRC14161 and sequenced. This gene contains an open reading frame of 777 bp encoding 258 amino acid residues, and the deduced amino acid sequence showed high similarity to the SDR superfamily proteins. An expression vector, pETSmKPR, containing the full KPA reductase gene was constructed and introduced into Escherichia coli BL21 (DE3) to overexpress the enzyme. Bioreduction of KPA using E. coli transformant cells coexpressing KPA reductase together with cofactor regeneration enzyme gene was also performed. The conversion yield of KPA to d-pantoic acid reached over 88% with a substrate concentration up to 1.17 M.

Keywords: Ketopantoic acid reductase; Stenotrophomonas maltophilia ; d-pantoic acid; Bioreduction

Construction of a new recombinant protein expression system in the basidiomycetous yeast Cryptococcus sp. strain S-2 and enhancement of the production of a cutinase-like enzyme by Kazuo Masaki; Hiroaki Tsuchioka; Takuya Hirano; Miyoshi Kato; Hiroko Ikeda; Haruyuki Iefuji (pp. 1627-1636).

Yeast host–vector systems have been very successful in expressing recombinant proteins. However, because there are some proteins that cannot be expressed with existing systems, there is a need for new yeast expression systems. Here we describe a new host–vector system based on the basidiomycetous yeast Cryptococcus sp. strain S-2 (S-2). Two advantages of S-2 are that it naturally produces some very useful enzymes, so it would be a good system for expressing multiple copies of some of its genes, and that, it is a nonhazardous species. The orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) gene (URA5) was selected as a selectable marker for transformation in the new host–vector system. URA5 was isolated and introduced into a uracil auxotroph of S-2 by electroporation. To demonstrate the S-2 system, we selected one of its unique enzymes, a plastic-degrading cutinase-like enzyme (CLE). We were able to insert multiple copies of the CLE gene (CLE1) into the chromosomes in a high fraction of the targeted cells. Under optimal conditions, one transformant exhibited 3.5 times higher CLE activity than the wild type. Expression vectors, including an inducible promoter (the promoter for the xylanase or α-amylase gene), were constructed for recombinant protein production, and green fluorescent protein was expressed under the control of these promoters. The xylanase promoter was more tightly controlled. Furthermore, putting CLE1 under the control of the xylanase promoter, which is induced by xylose, increased CLE activity of the culture medium to approximately 15 times greater than that of the wild type.

Keywords: Recombinant protein production; Host–vector system; Basidiomycetous yeast; Inducible promoter; Cutinase

Primary metabolism in the new human cell line AGE1.HN at various substrate levels: increased metabolic efficiency and α₁-antitrypsin production at reduced pyruvate load by Jens Niklas; Christian Priesnitz; Thomas Rose; Volker Sandig; Elmar Heinzle (pp. 1637-1650).

Metabolic responses of the new neuronal human cell line AGE1.HN to various substrate levels were analyzed in this study showing that reduced substrate and especially pyruvate load improves metabolic efficiency, leading to improved growth and α₁-antitrypsin (A1AT) production. The adaptation of the metabolism to different pyruvate and glutamine concentrations was analyzed in detail using a full factorial design. The most important finding was an increasingly inefficient use of substrates as well as the reduction of cell proliferation with increasing pyruvate concentrations in the medium. Cultivations with different feeding profiles showed that the highest viable cell density and A1AT concentration (167% of batch) was reached in the culture with the lowest glucose level and without pyruvate feeding. Analysis of metabolic fluxes in the differently fed cultures revealed a more efficient metabolic phenotype in the cultures without pyruvate feeding. The measured in vitro enzyme activities of the selected enzymes involved in pyruvate metabolism were lower in AGE1.HN compared with CHO cells, which might explain the higher sensitivity and different adaptation of AGE1.HN to increased pyruvate concentrations. The results indicate on the one hand that increasing the connectivity between glycolysis and the TCA cycle might improve substrate use and, finally, the production of A1AT. On the other hand, a better balanced substrate uptake promises a reduction of energy spilling which is increased with increasing substrate levels in this cell line. Overall, the results of this study provide important insights into the regulation of primary metabolism and into the adaptation of AGE1.HN to different substrate levels, providing guidance for further optimization of production cell lines and applied process conditions.

Keywords: Mammalian cell; Human cell; Metabolic flux; Recombinant protein; CHO; Physiology

Analysis of heterologous taxadiene production in K- and B-derived Escherichia coli by Brett A. Boghigian; Daniel Salas; Parayil Kumaran Ajikumar; Gregory Stephanopoulos; Blaine A. Pfeifer (pp. 1651-1661).

Taxa-4(5),11(12)-diene is the first dedicated intermediate in the metabolic pathway responsible for synthesizing the anticancer compound Taxol. In this study, the heterologous production of taxadiene was established in and analyzed between K- and B-derived Escherichia coli strains. First, recombinant parameters associated with precursor metabolism (the upstream methylerythritol phosphate (MEP) pathway) and taxadiene biosynthesis (the downstream pathway) were varied to probe the effect different promoters and cellular backgrounds have on taxadiene production. Specifically, upstream MEP pathway genes responsible for the taxadiene precursors, dimethylallyl diphosphate and isopentenyl diphosphate, were tested with an inducible T7 promoter system within K and B E. coli strains. Whereas, inducible T7, Trc, and T5 promoters were tested with the plasmid-borne geranylgeranyl diphosphate synthase and taxadiene synthase genes responsible for the downstream pathway. The K-derivative produced taxadiene roughly 2.5-fold higher than the B-derivative. A transcriptomics study revealed significant differences in pyruvate metabolism between the K and B strains, providing insight into the differences observed in taxadiene biosynthesis and targets for future metabolic engineering efforts. Next, the effect of temperature on cell growth and taxadiene production was analyzed in these two strains, revealing similar phenotypes between the two with 22°C as the optimal production temperature. Lastly, the effect of indole on cell growth was investigated between the two strains, showing that the K-derivative demonstrated greater growth inhibition compared to the B-derivative.

Keywords: Taxol; Taxadiene; Taxadiene synthase; E. coli ; Heterologous biosynthesis; Metabolic engineering

Antibacterial property and mechanism of a novel Pu-erh tea nanofibrous membrane by Yajuan Su; Chenlu Zhang; Yan Wang; Ping Li (pp. 1663-1671).

Pu-erh tea is made via a natural fermentation process. In this study, Pu-erh tea was used as a raw material for nanomaterials preparation and as an antibacterial agent. Antibacterial activities on Escherichia coli of Pu-erh tea, Pu-erh tea powder (PTP) of different sizes, and Pu-erh tea residual powder were firstly determined, respectively. With polyvinyl alcohol as the carrier, through an electrospinning technique, different kinds of nanofibrous membranes were obtained from the extract of Pu-erh tea and nano-PTP (NPTP), and their antibacterial properties and mechanism against E. coli were evaluated. The results showed better antibacterial activity with smaller PTP particles, the nano-sized particles had the best effects, and the MIC of NPTP was 13.5 mg/mL. When NPTP was in nanofibrous membranes, the antibacterial activity decreased slightly, but increased with modification by ZnO. Pu-erh tea in nanofibrous membranes damaged the E. coli cell membranes and caused leakage of K⁺ and enzymes. What is more is that damage of the cell walls led to the leakage of fluorescent proteins from enhanced green fluorescence protein-expressing E. coli. These results indicate that the Pu-erh tea nanofibrous membranes had good antibacterial activities against E. coli, which may provide a promising application of novel antibacterial materials.

Keywords: Antibacterial; Pu-erh tea; Nanofibrous membrane

S-methyl thioesters are produced from fatty acids and branched-chain amino acids by brevibacteria: focus on l-leucine catabolic pathway and identification of acyl–CoA intermediates by Alain M. Sourabié; Henry-Eric Spinnler; Marjolaine Bourdat-Deschamps; Richard Tallon; Sophie Landaud; Pascal Bonnarme (pp. 1673-1683).

Despite their importance as potent odors that contribute to the aroma of numerous cheeses, S-methyl thioesters formation pathways have not been fully established yet. In a first part of our work, we demonstrated that Brevibacterium antiquum and Brevibacterium aurantiacum could produce S-methyl thioesters using short-chain fatty acids or branched-chain amino acids as precursors. Then, we focused our work on l-leucine catabolism using liquid chromatography tandem mass spectrometry and gas chromatography-mass spectrometry analyses coupled with tracing experiments. For the first time, several acyl–CoAs intermediates of the l-leucine to thioesters conversion pathway were identified. S-methyl thioisovalerate was produced from l-leucine, indicating that this amino acid was initially transaminated. Quite interestingly, data also showed that other S-methyl thioesters, e.g., S-methyl thioacetate or S-methyl thioisobutyrate, were produced from l-leucine. Enzymatic and tracing experiments allowed for postulating catabolic pathways leading to S-methyl thioesters biosynthesis.

Keywords: Brevibacteria; S-methyl thioesters; Acyl–CoA intermediates; Branched-chain amino acids; Short-chain fatty acids; Biosynthesis pathway

Inactivation of phosphomannose isomerase gene abolishes sporulation and antibiotic production in Streptomyces coelicolor by Thangamani Rajesh; Eunjung Song; Ji-Nu Kim; Bo-Rahm Lee; Eun-Jung Kim; Sung-Hee Park; Yun-Gon Kim; Dongwon Yoo; Hyung-Yeon Park; Yun-Hui Choi; Byung-Gee Kim; Yung-Hun Yang (pp. 1685-1693).

Phosphomannose isomerases (PMIs) in bacteria and fungi catalyze the reversible conversion of d-fructose-6-phosphate to d-mannose-6-phosphate during biosynthesis of GDP-mannose, which is the main intermediate in the mannosylation of important cell wall components, glycoproteins, and certain glycolipids. In the present study, the kinetic parameters of PMI from Streptomyces coelicolor were obtained, and its function on antibiotic production and sporulation was studied. manA (SCO3025) encoding PMI in S. coelicolor was deleted by insertional inactivation. Its mutant (S. coelicolor∆manA) was found to exhibit a bld-like phenotype. Additionally, S. coelicolor∆manA failed to produce the antibiotics actinorhodin and red tripyrolle undecylprodigiosin in liquid media. To identify the function of manA, the gene was cloned and expressed in Escherichia coli BL21 (DE3). The purified recombinant ManA exhibited PMI activity (K _cat/K _m (mM⁻¹ s⁻¹ = 0.41 for d-mannose-6-phosphate), but failed to show GDP-d-mannose pyrophosphorylase [GMP (ManC)] activity. Complementation analysis with manA from S. coelicolor or E. coli resulted in the recovery of bld-like phenotype of S. coelicolor∆manA. SCO3026, another ORF that encodes a protein with sequence similarity towards bifunctional PMI and GMP, was also tested for its ability to function as an alternate ManA. However, the purified protein of SCO3026 failed to exhibit both PMI and GMP activity. The present study shows that enzymes involved in carbohydrate metabolism could control cellular differentiation as well as the production of secondary metabolites.

Keywords: Phosphomannose isomerase; Sporulation; Pigment production; Complementation analysis; Antibiotic production

Substrate utilization by recombinant Yarrowia lipolytica growing on sucrose by Lucie Moeller; Andreas Zehnsdorf; Andreas Aurich; Thomas Bley; Beate Strehlitz (pp. 1695-1702).

We report the study of the dynamics of substrate utilization by the genetic modified strain Yarrowia lipolytica H222-S4(p67ICL1) T5. In contrast to its wild-type equivalent, this recombinant strain is able to excrete the sucrose cleaving enzyme invertase. Both the sucrose degradation rate and the glucose and fructose consumption rate have been investigated. In all experiments, satisfied amounts of invertase were produced so that all sucrose was cleaved into its monomers. While glucose and fructose as sole carbon sources were consumed with the same uptake rate, a clear preference for glucose uptake was detected in cultivations with sucrose as sole carbon source or mixed substrates when compared with fructose. Nevertheless, no real diauxie could be observed because of partly simultaneous consumption of both monosaccharides. Fructose being present in the cultivation medium at the beginning of the fermentation led to the retardation of glucose uptake. This effect was observed for various fructose starting concentrations in the range of 5–85 g/l.

Keywords: Yarrowia lipolytica ; Recombinant yeast; Substrate utilization; Sucrose

Improving the performance of an end-point PCR assay commonly used for the detection of Bacteroidales pertaining to cow feces by Rulong Liu; Cheuk F. Chan; Clare H. I. Lun; Stanley C. K. Lau (pp. 1703-1713).

Bacteroidales are normal gut flora of warm-blooded animals. Since each host species carries a different diversity of Bacteroidales, the detection of host-associated gene markers of Bacteroidales has emerged as a promising tool for the tracking of the source of fecal pollution in aquatic ecosystems. To detect cow-associated Bacteroidales, a commonly used method has been an end-point PCR assay with the 16S rRNA genes primers CF128F (cow-associated) and Bac708R (all Bacteroidales). The PCR assay has demonstrated high rates of true-positive detection (i.e., high sensitivity) in all previous studies. However, the assay also had high rates of false-positive detection to the samples of non-target hosts in some cases (i.e., low specificity). In opposite to the reason many investigators have proposed, our results suggested that false detection was not necessarily due to the presence of the target sequence of CF128F in the feces of non-target hosts. Instead, we found sequences of non-target hosts having single internal mismatches with CF128F. Those mismatches were well tolerated in PCR, partly due to the universality of Bac708R. To improve the detection performance, we designed a novel primer CF592R (targeting the same clade of sequences as CF128F) to substitute Bac708R. The use of CF529R alleviated false detection and also led to a tenfold reduction in detection limit in the samples tested, compared to the use of Bac708R. Many other end-point PCR assays that detect the 16S rRNA genes in Bacteroidales also use a host-associated primer to couple with Bac708R, and low specificity or sensitivity has been reported. Based on our findings for CF128F, we suggest that the suitability of Bac708R in those PCR assays needs to be revisited.

Keywords: Bacteroidales ; Cow feces; Microbial source tracking; Fecal pollution; 16S rRNA genes

BMP2 promotes migration and invasion of breast cancer cells via cytoskeletal reorganization and adhesion decrease: an AFM investigation by Hua Jin; Jiang Pi; Xun Huang; Feicheng Huang; Wenxiang Shao; Shengpu Li; Yong Chen; Jiye Cai (pp. 1715-1723).

Bone morphogenetic protein 2 (BMP2) has been shown to modulate the proliferation and differentiation of breast cancer cells. However, the biochemical effects and mechanisms remain unknown. In this paper, the effects of recombinant human BMP2 on the migration of MCF-7 cells—one breast cancer cell line, using transwell and wound healing experiments, as well as on the cellular morphology, cytoskeleton, cell surface adhesion, and stiffness detected at subcellular level by an atomic force microscope, were investigated. After BMP2 treatment, the untreated round-shaped MCF-7 cells transformed to a spindle-like shape with lots of specialized structures, such as lamellipodia, filopodia, membrane protrusions, and others, which are essential for cellular migration or spreading. Moreover, flow cytometry quantitatively detected the BMP2-induced changes in the expression of adhesion molecules, a significant rise of CD44, and a remarkable drop of E-cadherin. The data indicated that BMP2 promoted the migration and invasion of MCF-7 cells by regulating the reorganization of cytoskeleton and the expression of adhesion molecules in/on the cells. Thus, it is very imperative to evaluate the oncogenicity of BMP2 when used in tissue engineering.

Keywords: Bone morphogenetic protein 2; Cytoskeletal reorganization; Cell stiffness; Adhesion force; Atomic force microscopy

Molecular identification of the microbial diversity in two sequencing batch reactors with activated sludge by Martin Denecke; Sascha Eilmus; Nadine Röder; Christopher Roesch; Hermann Bothe (pp. 1725-1734).

The diversity of the microbial community was identified in two lab-scale, ideally mixed sequencing batch reactors which were run for 115 days. One of the reactors was intermittently aerated (2 h aerobically/2 h anaerobically) whereas the other was consistently aerated. The amount of biomass as dry matter, the degradation of organic carbon determined by chemical oxygen demand and nitrogen-degradation activity were followed over the operation of the two reactors and did not show significant differences between the two approaches at the end of the experiment. At this point, the composition of the microbial community was determined by a terminal restriction fragment length polymorphism approach using multiple restriction enzymes by which organisms were retrieved to the lowest taxonomic level. The microbial composition was then significantly different. The species richness was at least five-fold higher in the intermittently aerated reactor than in the permanently kept aerobic approach which is in line with the observation that ecosystem disturbances result in higher diversity.

Keywords: Activated sludge; Microbial diversity; Sequencing batch reactor (SBR); tRFLP; Intermediate disturbance hypothesis (IDH)

Surface properties and intracellular speciation revealed an original adaptive mechanism to arsenic in the acid mine drainage bio-indicator Euglena mutabilis by David Halter; Corinne Casiot; Hermann J. Heipieper; Frédéric Plewniak; Marie Marchal; Stéphane Simon; Florence Arsène-Ploetze; Philippe N. Bertin (pp. 1735-1744).

Euglena mutabilis is a protist ubiquitously found in extreme environments such as acid mine drainages which are often rich in arsenic. The response of E. mutabilis to this metalloid was compared to that of Euglena gracilis, a protist not found in such environments. Membrane fatty acid composition, cell surface properties, arsenic accumulation kinetics, and intracellular arsenic speciation were determined. The results revealed a modification in fatty acid composition leading to an increased membrane fluidity in both Euglena species under sublethal arsenic concentrations exposure. This increased membrane fluidity correlated to an induced gliding motility observed in E. mutabilis in the presence of this metalloid but did not affect the flagellar dependent motility of E. gracilis. Moreover, when compared to E. gracilis, E. mutabilis showed highly hydrophobic cell surface properties and a higher tolerance to water-soluble arsenical compounds but not to hydrophobic ones. Finally, E. mutabilis showed a lower accumulation of total arsenic in the intracellular compartment and an absence of arsenic methylated species in contrast to E. gracilis. Taken together, our results revealed the existence of a specific arsenical response of E. mutabilis that may play a role in its hypertolerance to this toxic metalloid.

Keywords: Arsenic speciation; Euglena mutabilis ; Euglena gracilis ; Hydrophobicity; Fatty acid; Unsaturation index; AMD

Endophyte-assisted promotion of biomass production and metal-uptake of energy crop sweet sorghum by plant-growth-promoting endophyte Bacillus sp. SLS18 by Shenglian Luo; Taoying Xu; Liang Chen; Jueliang Chen; Chan Rao; Xiao Xiao; Yong Wan; Guangming Zeng; Fei Long; Chengbin Liu; Yutang Liu (pp. 1745-1753).

The effects of Bacillus sp. SLS18, a plant-growth-promoting endophyte, on the biomass production and Mn/Cd uptake of sweet sorghum (Sorghum bicolor L.), Phytolacca acinosa Roxb., and Solanum nigrum L. were investigated. SLS18 displayed multiple heavy metals and antibiotics resistances. The strain also exhibited the capacity of producing indole-3-acetic acid, siderophores, and 1-aminocyclopropane-1-carboxylic acid deaminase. In pot experiments, SLS18 could not only infect plants effectively but also significantly increase the biomass of the three tested plants in the presence of Mn/Cd. The promoting effect order of SLS18 on the biomass of the tested plants was sweet sorghum > P. acinosa > S. nigrum L. In the presence of Mn (2,000 mg kg⁻¹) and Cd (50 mg kg⁻¹) in vermiculite, the total Mn/Cd uptakes in the aerial parts of sweet sorghum, P. acinosa, and S. nigrum L. were increased by 65.2%/40.0%, 55.2%/31.1%, and 18.6%/25.6%, respectively, compared to the uninoculated controls. This demonstrates that the symbiont of SLS18 and sweet sorghum has the potential of improving sweet sorghum biomass production and its total metal uptake on heavy metal-polluted marginal land. It offers the potential that heavy metal-polluted marginal land could be utilized in planting sweet sorghum as biofuel feedstock for ethanol production, which not only gives a promising phytoremediation strategy but also eases the competition for limited fertile farmland between energy crops and food crops.

Keywords: PGPE; Energy crop; Sweet sorghum; Mn/Cd; Phytoremediation

Biological activity and mechanical stability of sol–gel-based biofilters using the freeze-gelation technique for immobilization of Rhodococcus ruber by Angela Pannier; Martin Mkandawire; Ulrich Soltmann; Wolfgang Pompe; Horst Böttcher (pp. 1755-1767).

Biofilters with long lifetime and high storage stability are very important for bioremediation processes to ensure the readiness at the occurrence of sudden contaminations. By using the freeze-gelation technique, living cells can be immobilized within a mechanically and chemically stable ceramic-like matrix. Due to a freeze-drying step, the embedded microorganisms are converted into a preserved form. In that way, they can be stored under dry conditions, which comply better with storage, transport, and handling requirements. Thus, in contrast to other immobilization techniques, there is no need for storage in liquid or under humid atmosphere. The biological activity, mechanical strength, and the structure of the biologically active ceramic-like composites (biocers) produced by freeze gelation have been investigated by using the phenol-degrading bacteria Rhodococcus ruber as model organism. Samples of freeze-gelation biocers have been investigated after defined storage periods, demonstrating nearly unchanged mechanical strength of the immobilization matrix as well as good storage stability of the activity of the immobilized cells over several months of storage at 4 °C. Repeated-batch tests demonstrated further that the freeze-gelation biocers can be repeatedly used over a period of more than 12 months without losing its bioactivity. Thus, these results show that freeze-gelation biocers have high potential of being scaled up from laboratory test systems to applications in real environment because of their long bioactivity as well as mechanical stability.

Keywords: Freeze cast; Phenol degradation; Bioremediation; Cell encapsulation; Compressive strength; Tension strength

Anaerobic biodecolorization mechanism of methyl orange by Shewanella oneidensis MR-1 by Pei-Jie Cai; Xiang Xiao; Yan-Rong He; Wen-Wei Li; Jian Chu; Chao Wu; Meng-Xing He; Zhe Zhang; Guo-Ping Sheng; Michael Hon-Wah Lam; Fang Xu; Han-Qing Yu (pp. 1769-1776).

In this work, we investigated the anaerobic decolorization of methyl orange (MO), a typical azo dye, by Shewanella oneidensis MR-1, which can use various organic and inorganic substances as its electron acceptor in natural and engineered environments. S. oneidensis MR-1 was found to be able to obtain energy for growth through anaerobic respiration accompanied with dissimilatory azo-reduction of MO. Chemical analysis shows that MO reduction occurred via the cleavage of azo bond. Block of Mtr respiratory pathway, a transmembrane electron transport chain, resulted in a reduction of decolorization rate by 80%, compared to the wild type. Knockout of cymA resulted in a substantial loss of its azo-reduction ability, indicating that CymA is a key c-type cytochrome in the electron transfer chain to MO. Thus, the MtrA-MtrB-MtrC respiratory pathway is proposed to be mainly responsible for the anaerobic decolorization of azo dyes such as MO by S. oneidensis.

Keywords: Anaerobic; Degradation; Electron transfer; Methyl orange (MO); Mutant strains; Mtr respiratory pathway; Riboflavin; Shewanella oneidensis

Metabolic control of Clostridium thermocellum via inhibition of hydrogenase activity and the glucose transport rate by Hsin-Fen Li; Barbara L. Knutson; Sue E. Nokes; Bert C. Lynn; Michael D. Flythe (pp. 1777-1784).

Clostridium thermocellum has the ability to catabolize cellulosic biomass into ethanol, but acetic acid, lactic acid, carbon dioxide, and hydrogen gas (H₂) are also produced. The effect of hydrogenase inhibitors (H₂, carbon monoxide (CO), and methyl viologen) on product selectivity was investigated. The anticipated effect of these hydrogenase inhibitors was to decrease acetate production. However, shifts to ethanol and lactate production are also observed as a function of cultivation conditions. When the sparge gas of cellobiose-limited chemostat cultures was switched from N₂ to H₂, acetate declined, and ethanol production increased 350%. In resting cell suspensions, lactate increased when H₂ or CO was the inhibitor or when the cells were held at elevated hyperbaric pressure (6.8 atm). In contrast, methyl-viologen-treated resting cells produced twice as much ethanol as the other treatments. The relationship of chemostat physiology to methyl viologen inhibition was revealed by glucose transport experiments, in which methyl viologen decreased the rate of glucose transport by 90%. C. thermocellum produces NAD⁺ from NADH by H₂, lactate, and ethanol production. When the hydrogenases were inhibited, the latter two products increased. However, excess substrate availability causes fructose 1,6-diphosphate, the glycolytic intermediate that triggers lactate production, to increase. Compensatory ethanol production was observed when the chemostat fluid dilution rate or methyl viologen decreased substrate transport. This research highlights the complex effects of high concentrations of dissolved gases in fermentation, which are increasingly envisioned in microbial applications of H₂ production for the conversion of synthetic gases to chemicals.

Keywords: Chemostat; Consolidated bioprocessing; Cellulosic biomass conversion; Cellulose; Metabolic engineering; Product selectivity; Continuous culture; Paraquat

Optimization of enzymatic hydrolysis of pretreated rice straw and ethanol production by Anita Singh; Narsi R. Bishnoi (pp. 1785-1793).

Cellulase, Tween 80, and β-glucosidase loading were studied and optimized by response surface methodology to improve saccharification. Microwave alkali-pretreated rice straw used as substrate for onsite enzyme production by Aspergillus heteromorphus and Trichoderma reesei. The highest enzymatic hydrolysis (84%) was obtained from rice straw at crude enzyme loading of 10 FPU/gds of cellulase, 0.15% Tween 80, and 100 international unit/g dry solids of β-glucosidase activities. Enzymatic hydrolyzate of pretreated rice straw was used for ethanol production by Saccharomyces cerevisiae, Scheffersomyces stipitis, and by co-culture of both. The yield of ethanol was 0.50, 0.47, and 0.48 g_p/g_s by S. cerevisiae, S. stipitis, and by co-culture, respectively, using pretreated rice straw hydrolyzate. The co-culture of S. cerevisiae and S. stipitis produced 25% more ethanol than S. cerevisiae alone and 31% more ethanol than S. stipitis alone. During anaerobic fermentation 65.08, 36.45, and 50.31 μmol/ml CO₂ released by S. cerevisiae, S. stipitis, and by co-culture, respectively. The data indicated that saccharification efficiency using optimized crude enzyme cocktail was good, and enzymatic hydrolyzate could be fermented to produce ethanol.

Keywords: Rice straw; Ethanol; Fungi; Saccharomyces cerevisiae ; Scheffersomyces stipitis

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Applied Microbiology and Biotechnology (v.93, #4)

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Applied Microbiology and Biotechnology (v.93, #4)