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Applied Microbiology and Biotechnology (v.69, #1)
Biotechnological production of amino acids and derivatives: current status and prospects by Wolfgang Leuchtenberger; Klaus Huthmacher; Karlheinz Drauz (pp. 1-8).
For almost 50 years now, biotechnological production processes have been used for industrial production of amino acids. Market development has been particularly dynamic for the flavor-enhancer glutamate and the animal feed amino acids l-lysine, l-threonine, and l-tryptophan, which are produced by fermentation processes using high-performance strains of Corynebacterium glutamicum and Escherichia coli from sugar sources such as molasses, sucrose, or glucose. But the market for amino acids in synthesis is also becoming increasingly important, with annual growth rates of 5–7%. The use of enzymes and whole cell biocatalysts has proven particularly valuable in production of both proteinogenic and nonproteinogenic l-amino acids, d-amino acids, and enantiomerically pure amino acid derivatives, which are of great interest as building blocks for active ingredients that are applied as pharmaceuticals, cosmetics, and agricultural products. Nutrition and health will continue to be the driving forces for exploiting the potential of microorganisms, and possibly also of suitable plants, to arrive at even more efficient processes for amino acid production.
Asymmetric synthesis of tert-butyl (3R, 5S) 6-chloro-dihydroxyhexanoate with Lactobacillus kefir by Maya Amidjojo; Ezequiel Franco-Lara; Alessandro Nowak; Hannes Link; Dirk Weuster-Botz (pp. 9-15).
An efficient whole cell biotransformation process using Lactobacillus kefir was developed for the asymmetric synthesis of tert-butyl (3R, 5S) 6-chloro-dihydroxyhexanoate, a chiral building block for the HMG-CoA reductase inhibitor. The effects of buffer concentration, temperature, pH and oxygen on the asymmetric reduction were investigated in batch reactions. Improvements in final product concentration and yields of 153% (120 mM) and 79% (0.85 mol/mol) with respect to the batch-process were achieved in an optimised fed-batch process. The pure substrate tert-butyl-6-chloro-3,5-dioxohexanoate was dispersed as microdroplets into the reaction system. This resulted in a space-time yield of 4.7 mmol l−1 h−1. A diastereomeric excess of >99% was measured for (3R, 5S) and (3S, 5S) tert-butyl 6-chloro-dihydroxyhexanoate.
The importance of aeration strategy in fuel alcohol fermentations contaminated with Dekkera/Brettanomyces yeasts by D. A. Abbott; W. M. Ingledew (pp. 16-21).
Whole corn mash fermentations infected with industrially-isolated Brettanomyces yeasts were not affected even when viable Brettanomyces yeasts out-numbered Saccharomyces yeasts tenfold at the onset of fermentation. Therefore, aeration, a parameter that is pivotal to the physiology of Dekkera/Brettanomyces yeasts, was investigated in mixed culture fermentations. Results suggest that aeration strategy plays a significant role in Dekkera/Brettanomyces-mediated inhibition of fuel alcohol fermentations. Although growth of Saccharomyces cerevisiae was not impeded, mixed culture fermentations aerated at rates of ≥20 ml air l−1 mash min−1 showed decreased ethanol yields and an accumulation of acetic acid. The importance of aeration was examined further in combination with organic acid(s). Growth of Saccharomyces occurred more rapidly than growth of Brettanomyces yeasts in all conditions. The combination of 0.075% (w/v) acetic acid and contamination with Brettanomyces TK 1404W did not negatively impact the final ethanol yield under fermentative conditions. Aeration, however, did prove to be detrimental to final ethanol yields. With the inclusion of aeration in the control condition (no organic acid stress) and in each fermentation containing organic acid(s), the final ethanol yields were decreased. It was therefore concluded that aeration strategy is the key parameter in regards to the negative effects observed in fuel alcohol fermentations infected with Dekkera/Brettanomyces yeasts.
Rat cytochrome P450-mediated transformation of dichlorodibenzo-p-dioxins by recombinant white-rot basidiomycete Coriolus hirsutus by Kanami Orihara; Takashi Yamazaki; Raku Shinkyo; Toshiyuki Sakaki; Kuniyo Inouye; Akira Tsukamoto; Jun Sugiura; Kazuo Shishido (pp. 22-28).
Rat cytochrome P450, CYP1A1, has been reported to play an important role in the metabolism of mono-trichlorodibenzo-p-dioxins (M-TriCDDs). To breed lignin (and M-TetraCDDs)-degrading basidiomycete Coriolus hirsutus strains producing rat CYP1A1, an expression cassette [C. hirsutus gpd promoter-C. hirsutus gpd 5′ portion (224-bp of 1st exon–8th base of 4th exon)-rat cyp1a1 cDNA-Lentinula edodes priA terminator] was constructed and inserted into pUCR1 carrying the C. hirsutus arg1 gene. The resulting recombinant plasmid, MIp5-(cyp1a1 + arg1) was introduced into protoplasts of C. hirsutus monokaryotic strain OJ1078 (Arg−, Leu−), obtaining three good Arg+ transformants. These transformants [ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), and ChTF5-6(CYP1A1)] were estimated to carry nine, six, and seven copies of the expression cassette on their chromosomes, respectively. Immunoblot analysis revealed that the three transformants produce similar amounts of rat CYP1A1 enzyme. ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), ChTF5-6(CYP1A1) and recipient OJ1078 were cultivated in a liquid medium containing 2,7/2,8(at a ratio of 1:1)-dichlorodibenzo-p-dioxins (2,7/2,8-DCDDs) and the amount of intra- and extracellular 2,7/2,8-DCDDs remaining was measured. The results showed that all three transformants efficiently transform 2,7/2,8-DCDDs through the action of the recombinant rat CYP1A1 enzyme.
Involvement of fengycin-type lipopeptides in the multifaceted biocontrol potential of Bacillus subtilis by Marc Ongena; Philippe Jacques; Yacine Touré; Jacqueline Destain; Abdelhamid Jabrane; Philippe Thonart (pp. 29-38).
In this work, the potential of Bacillus subtilis strain M4 at protecting plants against fungal diseases was demonstrated in different pathosystems. We provide evidence for the role of secreted lipopeptides, and more particularly of fengycins, in the protective effect afforded by the strain against damping-off of bean seedlings caused by Pythium ultimum and against gray mold of apple in post-harvest disease. This role was demonstrated by the strong biocontrol activity of lipopeptide-enriched extracts and through the detection of inhibitory quantities of fengycins in infected tissues. Beside such a direct antagonism of the pathogen, we show that root pre-inoculation with M4 enabled the host plant to react more efficiently to subsequent pathogen infection on leaves. Fengycins could also be involved in this systemic resistance-eliciting effect of strain M4, as these molecules may induce the synthesis of plant phenolics involved in or derived from the defense-related phenylpropanoid metabolism. Much remains to be discovered about the mechanisms by which Bacillus spp suppress disease. Through this study on strain M4, we reinforce the interest in B. subtilis as a pathogen antagonist and plant defense-inducing agent. The secretion of cyclic fengycin-type lipopeptides may be tightly related to the expression of these two biocontrol traits.
Expression of the chitinase gene from Trichoderma aureoviride in Saccharomyces cerevisiae by Song Jinzhu; Yang Qian; Liu Beidong; Chen Dianfu (pp. 39-43).
Chitinase gene ech42 was obtained from Trichoderma aureoviride M and amplified by PCR. The isolated DNA of ech42 was then sequenced. The results showed that the open reading frame of ech42 was 1,447 bp long, encoding 421 amino acids. Three introns were found in the sequence. The cloning vector pMD18-T and an E. coli DH5α host were used to yield clones as E. coli DH5α/ech42. The ech42 gene was integrated into the genomic DNA of pYES2 by insertion into a single site for recombination, yielding the recombinant pYES2/ech42. Chitinase expressed by pYES2/ech42 was induced by galactose (maximal activity 0.50 units ml−1) and was produced in fermentation liquid cultured for 36 h.
Branching mutants of Aspergillus oryzae with improved amylase and protease production on solid substrates by R. te Biesebeke; E. Record; N. van Biezen; M. Heerikhuisen; A. Franken; P. J. Punt; C. A. M. J. J. van den Hondel (pp. 44-50).
To study the relation between the number of hyphal tips and protein secretion during growth on a solid substrate, we have constructed two mutant strains of Aspergillus oryzae with increased hyphal branching. We have analysed hydrolytic enzyme activities during growth on wheat kernels (WK) of A. oryzae strains carrying the disrupted allele of the pclA gene encoding a secretion pathway specific (KEX2-like) endo-protease and the disrupted allele of the pg/pi-tp gene encoding a phosphatidylglycerol/phosphatidylinositol transfer protein. The biomass levels produced by the pclA and pg/pi-tp disrupted strains on wheat-based solid media were similar as found for the wild-type strain. However, the pclA disrupted strain showed much more compact colony morphology than the other two strains. Sporulation of the pclA and pg/pi-tp disrupted strains occurred, respectively, 2 days and 1 day later, compared to the wild type during fermentation on ground WK. During surface growth, microscopic analysis revealed that the hyphal growth unit length (L hgu) of the pclA and pg/pi-tp disrupted strains was, on average, 50 and 74% of that of the wild-type strain. This implies that in both mutant strains, a higher branching frequency occurs than in the wild-type strain. Compared to the wild-type strain, the pclA and pg/pi-tp disrupted strains produced at least 50% more amylase, at least 100% more glucoamylase and at least 90% more protease activity levels after growth on WK. These results support the hypothesis that branching mutants with an increased branching frequency can improve the solid state fermentation process.
Cloning and expression of a gene for an alpha-glucosidase from Saccharomycopsis fibuligera homologous to family GH31 of yeast glucoamylases by Eva Hostinová; Adriana Solovicová; Juraj Gašperík (pp. 51-56).
Cloning of cDNA encoding an α-glucosidase from the dimorphous yeast Saccharomycopsis fibuligera and characterization of the gene product were performed. The cDNA of the putative α-glucosidase gene consists of 2,886 bp, which includes an open reading frame encoding a 19 amino acid signal peptide at the N-terminal end and a 944 amino acid mature protein with a predicted molecular mass of 105.4 kDa and pI value of 4.52. The deduced amino acid sequence shows a high degree of identity (70%) with two yeast glucoamylases, namely, the extracellular glucoamylase Gam from Schwanniomyces occidentalis and the cell surface glucoamylase Gca from Candida albicans. The recombinant product, synthesized in Saccharomyces cerevisiae, is localized on the cell surface and hydrolyses maltooligosaccharides exclusively without the ability to digest soluble starch, which is consistent with the specificity characteristic of α-glucosidase, EC. 3.2.1.20.
The effects of ftsZ mutation on the production of recombinant protein in Bacillus subtilis by In-Suk Park; June-Hyung Kim; Byung-Gee Kim (pp. 57-64).
In this paper, the possibility of using a mutation of ftsZ as a pseudo-spore mutant is investigated. ftsZ, which is essential for cell division and sporulation of Bacillus subtilis, was placed under the spac promoter, which is inducible with isopropyl thiogalactose (IPTG). Cell growth of the ftsZ mutant and its β-galactosidase activity under the aprE promoter were compared with the wild type. In the presence of 1 mM IPTG, cell growth of the ftsZ mutant was almost the same as that of the wild type and its sporulation frequency was slightly lower than that of the wild type. However, under uninduced conditions, cell growth of ftsZ mutant was severely impaired. When induced with 0.2 mM IPTG, the ftsZ mutant showed about 13 times higher β-galactosidase activity than the wild type. When the ftsZ mutant was used for secretory production of subtilisin, only three times higher extracellular subtilisin activity was measured, compared with the wild type. By real-time PCR investigation, it was revealed that the ftsZ mutant intracellular mRNA level for subtilisin was more than 16 times higher, compared with the wild type. However, it appears that the secretion pathway is somewhat damaged in the ftsZ mutant. These results suggest that the cell division mutant can also be used like a sporulation mutant to produce recombinant proteins, with a precise control of cell growth and induction.
A versatile and general splitting technology for generating targeted YAC subclones by YeonHee Kim; Minetaka Sugiyama; Kazuo Yamagishi; Yoshinobu Kaneko; Kiichi Fukui; Akio Kobayashi; Satoshi Harashima (pp. 65-70).
Yeast artificial chromosomes (YAC) splitting technology was developed as a means to subclone any desired region of eukaryotic chromosomes from one YAC into new YACs. In the present study, the conventional YAC splitting technology was improved by incorporating PCR-mediated chromosome splitting technique and by adding autonomously replicating sequence (ARS) to the system. To demonstrate the performance of the improved method, a 60-kb region from within a 590-kb YAC (clone CIC9e2 from Arabidopsis thaliana chromosome 5) that could not be subcloned using the original method was split to convert into a replicating YAC. Two template plasmids, pSK-KCA and pSKCLY, were used to generate two splitting fragments by PCR. Two splitting fragments consisted of telomeric (C4A2)6 repeats, 400-bp target region, CEN4, H4ARS and Kmr (selective marker for plant transformants), or CgLEU2. These splitting fragments were introduced into Saccharomyces cerevisiae harboring the 100-kb split YAC generated by splitting of the 590-kb YAC and containing the 60-kb region. Among 12 Leu+ transformants, four exhibited the expected karyotype in which two newly split 40- and 60-kb chromosomes were generated. These results demonstrate that the improved method can convert a targeted region of a eukaryotic chromosome within a YAC into a replicating YAC.
Xylanase production by fungal strains on spent sulphite liquor by Zawadi A. Chipeta; James C. du Preez; George Szakacs; Lew Christopher (pp. 71-78).
Xylanase production by seven fungal strains was investigated using concentrated spent sulphite liquor (SSLc), xylan and d-xylose as carbon substrates. An SSLc-based medium induced xylanase production at varying levels in all of these strains, with Aspergillus oryzae NRRL 3485 and Aspergillus phoenicis ATCC 13157 yielding activities of 164 and 146 U ml−1, respectively; these values were higher than those obtained on xylan or d-xylose with the same fungal strains. The highest xylanase activity of 322 U ml−1 was obtained with Aspergillus foetidus ATCC 14916 on xylan. Electrophoretic and zymogram analysis indicated three xylanases from A. oryzae with molecular weights of approximately 32, 22 and 19 kDa, whereas A. phoenicis produced two xylanases with molecular weights of about 25 and 21 kDa. Crude xylanase preparations from these A. oryzae and A. phoenicis strains exhibited optimal activities at pH 6.5 and 5.0 and at 65 and 55°C, respectively. The A. oryzae xylanolytic activity was stable at 50°C over the pH range 4.5–10. The crude xylanase preparations from these A. oryzae and A. phoenicis strains had negligible cellulase activity, and their application in the biobleaching of hardwood pulp reduced chlorine dioxide consumption by 20–30% without sacrificing brightness.
Citrate catabolism and production of acetate and succinate by Lactobacillus helveticus ATCC 15807 by M. I. Torino; M. P. Taranto; G. Font de Valdez (pp. 79-85).
The citrate metabolism of Lactobacillus helveticus ATCC 15807 was studied under controlled-pH fermentations at pH 4.5 and pH 6.2. The micro-organism was able to co-metabolize citrate and lactose at both pH from the beginning of growth, which enhanced the rate of lactose consumption and lactic acid production, compared with cultures without citrate. The effect of citrate on cell growth was dependent on the balance between the ratio of dissociated to non-dissociated forms of the acetic acid produced and the extra ATP gained by the cells, both facts related to the citrate metabolism. The citrate catabolism determined a change in the fermentation pattern of L. helveticus ATCC 15807 from homolactic to a mixed-acid profile, regardless of the external pH. Within this new fermentation pattern, acetate was the major product formed (13–20 mM), followed by succinate (2.4–3.7 mM), while acetoine, dyacetile or butanediol were not detected. The mixed-acid profile displayed by L. helveticus ATCC 15807 was linked to NADH2 oxidase activity rather than the acetate kinase enzyme.
Characterization of Rat8 localization and mRNA export in Saccharomyces cerevisiae during the brewing of Japanese sake by Shingo Izawa; Reiko Takemura; Kayo Ikeda; Kiyoshi Fukuda; Yoshinori Wakai; Yoshiharu Inoue (pp. 86-91).
Ethanol affects the nuclear export of mRNA in a similar way to heat shock in Saccharomyces cerevisiae. We recently reported that the nuclear accumulation of Rat8 caused by ethanol stress correlates well with blocking of the export of bulk poly(A)+ mRNA. Here, we characterize the localization of Rat8 and bulk poly(A)+ mRNA in sake (Japanese rice wine) yeast during the brewing of sake. In wine must and synthetic dextrose medium, sake yeast showed the same responses to ethanol regarding changes in the localization of Rat8 as wine yeast and a laboratory strain: i.e., cells began the nuclear accumulation of Rat8 at an ethanol concentration of 6% and completed it at 9%. In contrast, during the sake-brewing process, sake yeast showed unique phenomena: i.e., cells did not start the nuclear accumulation of Rat8 until the ethanol concentration of the sake mash reached around 12% and they showed a normal localization of Rat8 around the nuclear envelope at the late stage of fermentation. These results provide new information about the transport of mRNA in yeast cells during actual alcoholic fermentation.
Antimicrobial activity of conditioned medium fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni Hi5 insect cells by Ingrid Svensson; Karin Calles; Eva Lindskog; Hongbin Henriksson; Ulrika Eriksson; Lena Häggström (pp. 92-98).
Concentrated conditioned medium (CM) fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni cells, eluting from a gel filtration column at around 10 kDa, were found to exhibit strong antibacterial activity against Bacillus megaterium and Escherichia coli. The B. megaterium cells incubated in the CM fraction from Sf9 cells rapidly lost viability: after 8 min the viability had decreased to 0.7%, as compared with the control. Addition of the CM fraction to E. coli cells resulted in a less drastic drop in viability: 65% viability was lost after 60 min of incubation. Further, exposure to the CM fraction caused a substantial leakage of intracellular proteins, as demonstrated by SDS-PAGE analysis. Cell lysis was confirmed by optical density measurements, microscopic investigations and flow cytometry. B. megaterium exposed to a CM fraction from T. ni cells lost 97% of their viability in about 40 min. Ubiquitin, thioredoxin and cyclophilin were identified in the antibacterial fraction from Sf9 cells by mass spectrometry and N-terminal amino acid sequencing. Other proteins in the fraction gave no matches in a database search. Since ubiquitin was shown not to cause the antimicrobial effect and thioredoxin and cyclophilin were likely not involved, the responsible agent may be an unknown protein, not yet registered in databases. The antimicrobial effect of the CM fraction from T. ni cells most probably comes from a lysozyme precursor protein.
DGGE analysis of 16S rDNA of ammonia-oxidizing bacteria in chemical–biological flocculation and chemical coagulation systems by Siqing Xia; Yan Shi; Yigang Fu; Xingmao Ma (pp. 99-105).
Microbial community DNA was extracted from activated sludge samples taken from a chemical bioflocculation process and a chemical coagulation process in Shanghai, China. 16S rDNA of ammonia-oxidizing bacteria (AOB)was amplified by nested polymerase chain reaction and fingerprinted by denaturing gradient gel electrophoresis for microbial structure analysis. The Shannon diversity index of each sample was determined. The results indicated that the microbial structure of AOB in chemical bioflocculation process was comparable at two operational conditions. The ammonia-oxidizing bacterial communities were similar in three channels of the chemical bioflocculation process and in three serial tanks in the chemical coagulation process at the same condition. The diversity of microbial structures in the chemical bioflocculation process was higher than in the chemical coagulation process, in which the microbial structure was similar to that in the influent. Although the microbial study provides insights to the nitrification removal, higher microbial diversity of AOB does not necessarily mean higher ammonia oxidization. Molecular analysis should be combined with chemical assays to optimize operational conditions.
Antibiotic resistance of bacteria in raw and biologically treated sewage and in groundwater below leaking sewers by C. Gallert; K. Fund; J. Winter (pp. 106-112).
More than 750 isolates of faecal coliforms (>200 strains), enterococci (>200 strains) and pseudomonads (>340 strains) from three wastewater treatment plants (WTPs) and from four groundwater wells in the vicinity of leaking sewers were tested for resistance against 14 antibiotics. Most, or at least some, strains of the three bacterial groups, isolated from raw or treated sewage of the three WTPs, were resistant against penicillin G, ampicillin, vancomycin, erythromycin, triple sulfa and trimethoprim/sulfamethoxazole (SXT). Only a few strains of pseudomonads or faecal coliforms were resistant against some of the other tested antibiotics. The antibiotic resistances of pseudomonads, faecal coliforms and enterococci from groundwater varied to a higher extent. In contrast to the faecal coliforms and enterococci, most pseudomonads from all groundwater samples, including those from non-polluted groundwater, were additionally resistant against chloramphenicol and SXT. Pseudomonads from sewage and groundwater had more multiple antibiotic resistances than the faecal coliforms or the enterococci, and many pseudomonads from groundwater were resistant to more antibiotics than those from sewage. The pseudomonads from non-polluted groundwater were the most resistant isolates of all. The few surviving faecal coliforms in groundwater seemed to gain multiple antibiotic resistances, whereas the enterococci lost antibiotic resistances. Pseudomonads, and presumably, other autochthonous soil or groundwater bacteria, such as antibiotic-producing Actinomyces sp., seem to contribute significantly to the gene pool for acquisition of resistances against antibiotics in these environments.