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Applied Microbiology and Biotechnology (v.83, #4)

Understanding the industrial application potential of lactic acid bacteria through genomics by Yan Zhu; Yanping Zhang; Yin Li (pp. 597-610).

Lactic acid bacteria (LAB) are a heterogeneous group of bacteria contributing to various industrial applications, ranging from food and beverage fermentation, bulk and fine chemicals production to pharmaceuticals manufacturing. Genome sequencing is booming; hitherto, 25 genomes of LAB have been published and many more are in progress. Based on genomic content of LAB, this review highlights some findings related to applications revealed by genomics and functional genomics analyses. Finally, this review summarizes mathematical modeling strategies of LAB in the context of genomics, to further our understanding of industrial related features.

Keywords: Genomics; Lactic acid bacteria; Application

The RNA interference—virus interplay: tools of nature for gene modulation, morphogenesis, evolution and a possible mean for aflatoxin control by F. R. Schmidt (pp. 611-615).

This article points out, that viruses, in an interplay with RNA interference and as vehicles for intergenic and interspecies gene transfer, may work as agents for intracellular gene modulation, for steering of individual morphogenesis and as a driving force of evolution in the toolbox of nature. This is illustrated in particular in the light of a fungal double-stranded RNA virus that may be employed as a suitable agent for a biological control of aflatoxins, the most carcinogenic natural substances occurring in food and feedstuff.

Keywords: Interplay virus; RNA interference; Gene modulation; Evolution; Biological aflatoxin control

Stereoselective synthesis of (R)-3-quinuclidinol through asymmetric reduction of 3-quinuclidinone with 3-quinuclidinone reductase of Rhodotorula rubra by Atsuko Uzura; Fumiki Nomoto; Akiko Sakoda; Yukifumi Nishimoto; Michihiko Kataoka; Sakayu Shimizu (pp. 617-626).

A novel nicotinamide adenine dinucleotide phosphate-dependent carbonyl reductase, 3-quinuclidinone reductase, was isolated from Rhodotorula rubra JCM3782. The enzyme catalyzes the asymmetric reduction of 3-quinuclidinone to (R)-3-quinuclidinol. The gene encoding the enzyme was also cloned and sequenced. A 819-bp nucleotide fragment was confirmed to be the gene encoding the 3-quinuclidinone reductase by agreement of the internal amino acid sequences of the purified enzyme. The gene encodes a total of 272 amino acid residues, and the deduced amino acid sequence shows similarity to those of several short-chain dehydrogenase/reductase family proteins. An expression vector, pWKLQ, which contains the full length 3-quinuclidinone reductase gene was constructed. Using Escherichia coli cells coexpressing the 3-quinuclidinone reductase and glucose dehydrogenase (cofactor regeneration enzyme) genes, 618 mM 3-quinuclidinone was almost stiochiometrically converted to (R)-3-quinuclidinol with an >99.9% enantiomeric excess within 21 h of reaction.

Keywords: 3-Quinuclidinone reductase; Rhodotorula rubra ; Asymmetric reduction; (R)-3-Quinuclidinol

Optimization of temperature, sugar concentration, and inoculum size to maximize ethanol production without significant decrease in yeast cell viability by Cecilia Laluce; João Olimpio Tognolli; Karen Fernanda de Oliveira; Crisla Serra Souza; Meline Rezende Morais (pp. 627-637).

Aiming to obtain rapid fermentations with high ethanol yields and a retention of high final viabilities (responses), a 2³ full-factorial central composite design combined with response surface methodology was employed using inoculum size, sucrose concentration, and temperature as independent variables. From this statistical treatment, two well-fitted regression equations having coefficients significant at the 5% level were obtained to predict the viability and ethanol production responses. Three-dimensional response surfaces showed that increasing temperatures had greater negative effects on viability than on ethanol production. Increasing sucrose concentrations improved both ethanol production and viability. The interactions between the inoculum size and the sucrose concentrations had no significant effect on viability. Thus, the lowering of the process temperature is recommended in order to minimize cell mortality and maintain high levels of ethanol production when the temperature is on the increase in the industrial reactor. Optimized conditions (200 g/l initial sucrose, 40 g/l of dry cell mass, 30 °C) were experimentally confirmed and the optimal responses are 80.8 ± 2.0 g/l of maximal ethanol plus a viability retention of 99.0 ± 3.0% for a 4-h fermentation period. During consecutive fermentations with cell reuse, the yeast cell viability has to be kept at a high level in order to prevent the collapse of the process.

Keywords: RSM; Viability; Ethanol production; Temperature; Sugar concentration; Inoculum size

Development of serum-free medium supplemented with hydrolysates for the production of therapeutic antibodies in CHO cell cultures using design of experiments by Sung Hyun Kim; Gyun Min Lee (pp. 639-648).

An efficient method of formulating serum-free medium (SFM) for production of therapeutic antibodies by recombinant CHO (rCHO) cells was developed using two rCHO cell lines producing a therapeutic antibody. In this method, ten kinds of SFM were prepared by supplementing the basal SFM with statistically designed mixtures (total 5 g L⁻¹) of three non-animal-derived hydrolysates: yeastolate, soy hydrolysate, and wheat gluten hydrolysate. When the two rCHO cell lines were cultivated, the mixtures of soy hydrolysate and wheat gluten hydrolysate showed a positive effect on cell growth. On the other hand, the mixtures including a high portion of yeastolate significantly enhanced specific antibody productivity. To reconstitute the mixture ratios of the three hydrolysates for high growth and antibody production, the effect of each medium was analyzed by the statistical program Design-Expert®. The resulting medium gave a 1.9–3.3-fold increase in the maximum antibody concentration, compared to the basal SFM. Taken together, the supplementation of hydrolysates to the basal SFM with the help of statistical analysis is an efficient means of developing SFM for therapeutic antibody production by rCHO cells.

Keywords: Antibody; CHO cells; Hydrolysates; Serum-free medium; Statistical design

Directed evolution of endoglucanase III (Cel12A) from Trichoderma reesei by Hikaru Nakazawa; Katsunori Okada; Tomoko Onodera; Wataru Ogasawara; Hirofumi Okada; Yasushi Morikawa (pp. 649-657).

The stability and specific activity of endo-β-1,4-glucanase III from Trichoderma reesei QM9414 was enhanced, and the expression efficiency of its encoding gene, egl3, was optimized by directed evolution using error-prone PCR and activity screening in Escherichia coli RosettaBlue (DE3) pLacI as a host. Relationship between increase in yield of active enzyme in the clones and improvement in its stability was observed among the mutants obtained in the present study. The clone harboring the best mutant 2R4 (G41E/T110P/K173M/Y195F/P201S/N218I) selected in via second-round mutagenesis after optimal recombinating of first-round mutations produced 130-fold higher amount of mutant enzyme than the transformant with wild-type EG III. Mutant 2R4 produced by the clone showed broad pH stability (4.4–8.8) and thermotolerance (entirely active at 55°C for 30 min) compared with those of the wild-type EG III (pH stability, 4.4–5.2; thermostability, inactive at 55°C for 30 min). k _cat of 2R4 against carboxymethyl-cellulose was about 1.4-fold higher than that of the wild type, though the K _m became twice of that of the wild type.

Keywords: Directed evolution; Endoglucanase; Trichoderma reesei ; Error-prone PCR

An extracellular polyhydroxybutyrate depolymerase in Thermus thermophilus HB8 by Christos P. Papaneophytou; Anastasia A. Pantazaki; Dimitrios A. Kyriakidis (pp. 659-668).

The thermophilic bacterium Thermus thermophilus HB8 has been characterized as a polyhydroxybutyrate (PHB)-degrading microorganism since it grows efficiently and forms clear zones on agar plates containing PHB as sole carbon source. T. thermophilus extracellular PHB depolymerase was purified to homogeneity using an affinity chromatography protocol. The purified enzyme was estimated to have an apparent molecular mass of 42 kDa. The extracellular PHB depolymerase gene was identified as the TTHA0199 gene product of T. thermophilus HB8. The amino acid sequence of the TTHA0199 gene product shared significant homologies to other carboxylesterases. A catalytic triad was identified consisting of S₁₈₃, E₃₁₀, and H₄₀₅. A pentapeptide sequence (GX₁SX₂G) exists within the molecule, characteristic for PHB depolymerases (lipase box) and for other serine hydrolases. Purified extracellular PHB depolymerase was stable at high temperatures with an optimum activity at pH 8.0. The apparent Km value of the purified enzyme for PHB was 53 μg/ml. As the main product of the enzymic hydrolysis of PHB, the monomer 3-hydroxybutyrate was identified, suggesting that the enzyme acts principally as an exo-type hydrolase.

Keywords: Poly-hydroxybutyrate (PHB); PHB-depolymerase; Thermus thermophilus

Paraffin oil as a “methane vector” for rapid and high cell density cultivation of Methylosinus trichosporium OB3b by Bing Han; Tao Su; Hao Wu; Zhongxuan Gou; Xin-Hui Xing; Hao Jiang; Yin Chen; Xin Li; J. Colin Murrell (pp. 669-677).

Slow growth and relatively low cell densities of methanotrophs have limited their uses in industrial applications. In this study, a novel method for rapid cultivation of Methylosinus trichosporium OB3b was studied by adding a water-immiscible organic solvent in the medium. Paraffin oil was the most effective at enhancing cell growth and final cell density. This is at least partially due to the increase of methane gas transfer between gas and medium phases since methane solubility is higher in paraffin than in water/nitrate minimal salt medium. During cultivation with paraffin oil at 5% (v/v) in the medium, M. trichosporium OB3b cells also showed higher concentrations of the intermediary metabolites, such as formic acid and pyruvic acid, and consumed more methane compared with the control. Paraffin as methane vector to improve methanotroph growth was further studied in a 5-L fermentor at three concentrations (i.e., 2.5%, 5%, and 10%). Cell density reached about 14 g dry weight per liter with 5% paraffin, around seven times higher than that of the control (without paraffin). Cells cultivated with paraffin tended to accumulate around the interface between oil droplets and the water phase and could exist in oil phase in the case of 10% (v/v) paraffin. These results indicated that paraffin could enhance methanotroph growth, which is potentially useful in cultivation of methanotrophs in large scale in industry.

Keywords: Methanotrophs; Cultivation; Methane Vector; Methylosinus trichosporium OB3b

Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis by Edwin van Bloois; Remko T. Winter; Dick B. Janssen; Marco W. Fraaije (pp. 679-687).

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.

Keywords: Carbohydrate oxidase; Whole cell biocatalysis; Flavo enzyme; Periplasmic transport; Surface display

Characterization of two distinct feruloyl esterases, AoFaeB and AoFaeC, from Aspergillus oryzae by Takuya Koseki; Akane Hori; Shouji Seki; Tetsuya Murayama; Yoshihito Shiono (pp. 689-696).

Two hypothetical proteins XP_001818628 and XP_001819091 (designated AoFaeB and AoFaeC, respectively), showing sequence identity with known type-C feruloyl esterases, have been found in the genomic sequence of Aspergillus oryzae. We cloned the putative A. oryzae feruloyl esterase-encoding genes and expressed them in Pichia pastoris. Both purified recombinant AoFaeB (rAoFaeB) and AoFaeC (rAoFaeC) had apparent relative molecular masses of 61,000 and 75,000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After N-deglycosylation, both proteins had a relative molecular mass of 55,000. The optimum pH for rAoFaeB was 6.0, although it was stable at pH values ranging from 3.0 to 9.0; rAoFaeC had an optimum pH of 6.0 and was stable in the pH range of 7.0–10.0. Thermostability of rAoFaeC was greater than that of rAoFaeB. Whereas rAoFaeC displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, methyl ferulate, and methyl sinapate, rAoFaeB displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, and methyl ferulate but not toward methyl sinapate. Substrate specificity profiling of rAoFaeB and rAoFaeC revealed type-B and type-C feruloyl esterases, respectively. Ferulic acid was efficiently released from wheat arabinoxylan when both esterases were applied with xylanase from Thermomyces lanuginosus. Both recombinant proteins also exhibited hydrolytic activity toward chlorogenic acid.

Keywords: Feruloyl esterase; Plant cell wall; Ferulic acid; Aspergillus oryzae

Improvement of compactin (ML-236B) production by genetic engineering in compactin high-producing Penicillium citrinum by S. Baba; Y. Abe; T. Suzuki; C. Ono; K. Iwamoto; T. Nihira; M. Hosobuchi (pp. 697-704).

An increase in compactin (ML-236B) production was achieved by introducing a whole compactin biosynthetic gene cluster or the regulatory gene mlcR into compactin high-producing Penicillium citrinum. In the previous report, we introduced mlcR encoding the positive regulator of compactin biosynthetic genes into compactin high-producing strain no. 41520, and most of the transformants produced higher amounts of compactin. Here, we characterize one of the resulting high producers (strain TIR-35, which produced 50% more compactin) and reveal that TIR-35 contained five copies of mlcR and that early, enhanced expression of mlcR caused compactin overproduction. Similarly, the introduction of mlcR into strain T48.19, which was created previously from strain no. 41520 by introducing a partial compactin biosynthetic gene cluster, enhanced compactin production further. Our results indicated that genetic engineering is an effective tool to improve compactin production, even in compactin high producers.

Keywords: Penicillium citrinum ; Secondary metabolite production; Compactin (ML-236B); Genetic engineering

C₁ compounds as auxiliary substrate for engineered Pseudomonas putida S12 by Frank W. Koopman; Johannes H. de Winde; Harald J. Ruijssenaars (pp. 705-713).

The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to efficiently utilize the C₁ compounds methanol and formaldehyde as auxiliary substrate. The hps and phi genes of Bacillus brevis, encoding two key steps of the ribulose monophosphate (RuMP) pathway, were introduced to construct a pathway for the metabolism of the toxic methanol oxidation intermediate formaldehyde. This approach resulted in a remarkably increased biomass yield on the primary substrate glucose when cultured in C-limited chemostats fed with a mixture of glucose and formaldehyde. With increasing relative formaldehyde feed concentrations, the biomass yield increased from 35% (C-mol biomass/C-mol glucose) without formaldehyde to 91% at 60% relative formaldehyde concentration. The RuMP-pathway expressing strain was also capable of growing to higher relative formaldehyde concentrations than the control strain. The presence of an endogenous methanol oxidizing enzyme activity in P. putida S12 allowed the replacement of formaldehyde with the less toxic methanol, resulting in an 84% (C-mol/C-mol) biomass yield. Thus, by introducing two enzymes of the RuMP pathway, co-utilization of the cheap and renewable substrate methanol was achieved, making an important contribution to the efficient use of P. putida S12 as a bioconversion platform host.

Keywords: Pseudomonas putida ; Auxiliary substrate; C₁ compounds

Novel gene clusters involved in arsenite oxidation and resistance in two arsenite oxidizers: Achromobacter sp. SY8 and Pseudomonas sp. TS44 by Lin Cai; Christopher Rensing; Xiangyang Li; Gejiao Wang (pp. 715-725).

This study describes three gene clusters involved in arsenic redox transformation of two arsenite oxidizers: Achromobacter sp. SY8 and Pseudomonas sp. TS44. A 17.5-kb sequence containing the arsenite oxidase (aox) gene cluster (aoxX-aoxS-aoxR and aoxA-aoxB-aoxC-aoxD) was isolated from SY8 using a fosmid library approach. Similarly, a 14.6-kb sequence including the aox cluster (arsD-arsA-aoxA-aoxB) and the arsenic resistance (ars) gene cluster (arsC1-arsR-arsC2-ACR3-arsH-dual specificity phosphatase (DSP)-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-major facilitator superfamily (MFS)) was obtained from TS44 by inverse polymerase chain reaction (PCR). According to reverse transcription (RT) PCR experiments, SY8 aoxXSR and aoxABCD transcribed as two different transcripts in opposite directions, and TS44 aox and ars clusters transcribed as a single transcript in their respective cluster. All of these genes were found to be upregulated by the addition of arsenite [As(III)], arsenate [As(V)], and antimonite [Sb(III)], except that TS44 arsC1-arsR appeared to be expressed constitutively. The SY8 aox cluster was predicted to be regulated by a two-component signal transduction system and a potential regulatory model was proposed. The TS44 aox cluster is unusual since it contains structural genes only and arsDA in its upstream. The TS44 ars cluster includes several genes previously identified not associated with arsenic resistance or transformation. This study showed novel structures and arrangements of arsenic gene clusters associated with bacterial As(III) oxidation and As(V) reduction.

Keywords: Arsenic; Arsenite oxidizer; aox cluster; ars cluster

Effects of indole-3-acetic acid on Sinorhizobium meliloti survival and on symbiotic nitrogen fixation and stem dry weight production by Esther Imperlini; Carmelina Bianco; Enza Lonardo; Serena Camerini; Michele Cermola; Giancarlo Moschetti; Roberto Defez (pp. 727-738).

We evaluated the effects of the main auxin phytohormone, indole-3-acetic acid (IAA), on the central metabolism of Sinorhizobium meliloti 1021. We either treated S. meliloti 1021 wild-type cells with 0.5 mM IAA, 1021+, or use a derivative, RD64, of the same strain harboring an additional pathway for IAA biosynthesis (converting tryptophan into IAA via indoleacetamide). We assayed the activity of tricarboxylic acid cycle (TCA) key enzymes and found that activity of citrate synthase and α-ketoglutarate dehydrogenase were increased in both 1021+ and RD64 as compared to the wild-type strain. We also showed that the intracellular acetyl-CoA content was enhanced in both RD64 and 1021+ strains when compared to the control strain. The activity of key enzymes, utilizing acetyl-CoA for poly-β-hydroxybutyrate (PHB) biosynthesis, was also induced. The PHB level measured in these cells were significantly higher than that found in control cells. Moreover, 4-week-long survival experiments showed that 80% of 1021 cells died, whereas 50% of RD64 cells were viable. Medicago truncatula plants nodulated by RD64 (Mt-RD64) showed an induction of both acetylene reduction activity and stem dry weight production.

Keywords: Cell survival; PHB; TCA; Nitrogen fixation; Stem dry weight; Starch

Growth of Pseudomonas chloritidismutans AW-1^T on n-alkanes with chlorate as electron acceptor by Farrakh Mehboob; Howard Junca; Gosse Schraa; Alfons J. M. Stams (pp. 739-747).

Microbial (per)chlorate reduction is a unique process in which molecular oxygen is formed during the dismutation of chlorite. The oxygen thus formed may be used to degrade hydrocarbons by means of oxygenases under seemingly anoxic conditions. Up to now, no bacterium has been described that grows on aliphatic hydrocarbons with chlorate. Here, we report that Pseudomonas chloritidismutans AW-1^T grows on n-alkanes (ranging from C7 until C12) with chlorate as electron acceptor. Strain AW-1^T also grows on the intermediates of the presumed n-alkane degradation pathway. The specific growth rates on n-decane and chlorate and n-decane and oxygen were 0.5 ± 0.1 and 0.4 ± 0.02 day⁻¹, respectively. The key enzymes chlorate reductase and chlorite dismutase were assayed and found to be present. The oxygen-dependent alkane oxidation was demonstrated in whole-cell suspensions. The strain degrades n-alkanes with oxygen and chlorate but not with nitrate, thus suggesting that the strain employs oxygenase-dependent pathways for the breakdown of n-alkanes.

Keywords: n-Alkane oxidation; Chlorate reduction; Pseudomonas chloritidismutans AW-1^T

Regulation of glycerol metabolism in Enterobacter aerogenes NBRC12010 under electrochemical conditions by Kouta Hatayama; Tatsuo Yagishita (pp. 749-756).

Enterobacter aerogenes NBRC12010 was able to ferment glycerol to ethanol and hydrogen gas. Fermentation of glycerol ceased in the stationary phase of growth, and it was activated by electrochemical reactions using thionine as an electron transfer mediator from bacterial cells to an electrode. Using resting cells of E. aerogenes NBRC12010 in only citrate buffer solution, the cells did not consume glycerol at all, but they could metabolize glucose. These results suggest that the regulation of glycerol metabolism occurred at enzymatic steps before glycolysis. In E. aerogenes NBRC12010, glycerol was metabolized via glycerol dehydrogenase (GDH) and then dehydroxyacetone kinase. The GDH-catalyzed reaction mainly depended on the ratio of NAD⁺/NADH. At a NAD⁺/NADH ratio of nearly 1 or less, it was substantially suppressed and glycerol metabolism stopped. When the ratio was higher than 1, GDH was activated and glycerol was metabolized. Thus, the reaction of glycerol metabolism depended on the balance of cellular NAD⁺/NADH. Exogenous NADH was oxidized to NAD⁺ by electrochemical reactions with thionine. We proposed the activation mechanism of glycerol metabolism under electrochemical conditions.

Keywords: Enterobacter aerogenes ; Glycerol; Electrochemical reaction; Glycerol dehydrogenase; Metabolism

Characterization and application of a native lactic acid bacterium isolated from tannery fleshings for fermentative bioconversion of tannery fleshings by Amit Kumar Rai; N. Bhaskar; Prakash M. Halami; K. Indirani; P. V. Suresh; N. S. Mahendrakar (pp. 757-766).

Lactic acid bacteria (LAB) species isolated from limed and delimed tannery fleshings (TF) were evaluated for their fermentation efficiency and antibacterial property. The native LAB isolates efficiently fermented TF and resulted in a fermented mass with antioxidant properties, indicating their potential for effective eco-friendly bioconversion of TF. From among the LAB isolated, a proteolytic isolate showing better antimicrobial spectrum and reasonably good fermentation efficiency was identified as Enterococcus faecium HAB01 based on various biochemical and molecular tests. This isolate afforded a better degree of hydrolysis (81.36%) of TF than Pediococcus acidilactici (54.64%) that was previously reported by us. The bacteriocin produced by E. faecium was found to be antagonistic to several human pathogens including Listeria, Aeromonas, Staphylococcus and Salmonella. Further, E. faecium HAB01 bacteriocin was thermostable and had a molecular weight of around 5 kDa, apart from being stable at both acidic and alkaline conditions. The bacteriocin was unstable against proteases.

Keywords: Tannery fleshings; Lactic acid bacteria; Antioxidant activity; Bacteriocin; Fermentation

Fermentation and growth kinetic study of Aeromonas caviae under anaerobic conditions by Changsoo Lee; Jaai Kim; Kwanghyun Hwang; Seokhwan Hwang (pp. 767-773).

Although Aeromonas caviae is pathogenic to a broad range of invertebrates including human, frequent in aquatic environments, and potentially vital for acidogenesis in anaerobic digestion, virtually no biokinetic information on its anaerobic growth is at hand. Therefore, this study focused on evaluating its anaerobic growth kinetics on glucose. To provide a set of relevant biokinetic coefficients for modeling, a combination of curve fitting and numerical modeling was used. Microcultivations were performed at eight different initial glucose concentrations of 0.1 to 2.5 g l⁻¹ to establish a function of specific growth rate versus substrate concentration. A batch anaerobic bioreactor was then operated to collect a data set for the numerical analysis. Kinetic coefficients were estimated from three different biomass growth profiles monitored by optical density, volatile suspended solids (VSS), or DNA measurement, and applied for simulating continuous operations at various hydraulic retention times (HRTs). Assuming the influent glucose concentration is 5,000 mg l⁻¹, the substrate utilization efficiency predicted to be 77.2% to 92.0% at 17 to 36 h HRTs. For the VSS-model-based simulation, the washout HRT was estimated to be 16.6 h, and similar for the other models. Overall, the anaerobic biokinetic coefficients of A. caviae grown on glucose were successfully estimated and found to follow a substrate inhibition model.

Keywords: Acidogenesis; Aeromonas caviae ; Biokinetic modeling; Fermentation; Substrate inhibition

Functional characterization of the trigger factor protein PceT of tetrachloroethene-dechlorinating Desulfitobacterium hafniense Y51 by Yasuyuki Morita; Taiki Futagami; Masatoshi Goto; Kensuke Furukawa (pp. 775-781).

Desulfitobacterium hafniense strain Y51 dechlorinates tetrachloroethene to cis-1,2-dichloroethene (cis-DCE) via trichloroethene by the action of the PceA reductive dehalogenase encoded by pceA. The pceA gene constitutes a gene cluster with pceB, pceC, and pceT. However, the gene components, except for pceA, still remained to be characterized. In the present study, we characterized the function of PceT. PceT of strain Y51 showed a sequence homology with trigger factor proteins, although it is evolutionally distant from the well-characterized trigger factor protein of Escherichia coli. The PceT protein tagged with 6x histidine was expressed as a soluble form in E. coli. The recombinant PceT fusion protein exhibited peptidyl-proryl cis–trans isomerase activity toward the chromogenic peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The PceT fusion protein also exhibited chaperon activity towards the chemically denatured citrate synthase. Immunoprecipitation analysis using antibodies raised against PceA and PceT demonstrated that PceT specifically binds to the precursor form of PceA with an N-terminal twin-arginine translocation (TAT) signal sequence. On the other hand, PceT failed to bind the mature form of PceA that lost the TAT signal sequence. This is the first report in dehalorespiring bacteria, indicating that PceT is responsible for the correct folding of the precursor PceA.

Keywords: Desulfitobacterium hafniense ; Dehalorespiring bacteria; Tetrachloroethene; PCE genes; Chaperon; Trigger factor

Marker-disruptive gene integration and URA3 recycling for multiple gene manipulation in Saccharomyces cerevisiae by Shohei Kaneko; Tsutomu Tanaka; Hideo Noda; Hideki Fukuda; Rinji Akada; Akihiko Kondo (pp. 783-789).

The introduction of several kinds of genes into the yeast chromosome is a powerful tool in many fields from fundamental study to industrial application. Here, we describe a general strategy for one-step gene integration and a marker recycling method. Forty base pairs of a short sequence derived from a region adjacent to the HIS3 locus were placed between cell surface displaying β-glucosidase (BGL) and URA3 marker genes. HIS3 deletion and BGL–URA3 fragment integration were achieved via a PCR fragment consisting of the BGL–URA3 fragment attached to homology sequences flanked by the HIS3 targeting locus. The obtained his3::URA3 disruptants were plated on a 5-FOA plate to select for the URA3 deletion due to repeated sequences at both sides of URA3 gene. In all selected colonies, BGL genes were integrated at the targeted HIS3 locus and URA3 was completely deleted. In addition, introduced BGL was efficiently expressed, and the transformants fermented cellobiose to ethanol effectively. As our strategy creates next transformation markers continuously together with gene integration, this method can serve as a simple and powerful tool for multiple genetic manipulations in yeast engineering.

Keywords: Marker recycling; Gene integration; β-Glucosidase; Ethanol fermentation

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Applied Microbiology and Biotechnology (v.83, #4)

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Applied Microbiology and Biotechnology (v.83, #4)