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Applied Microbiology and Biotechnology (v.96, #5)

The ends and means of artificially induced targeted protein degradation by C. Ratna Prabha; Soumya Mukherjee; Renuka Raman; Swapnali Kulkarni (pp. 1111-1123).

Studies on knockout mutants and conditional mutants are invaluable to biological research and have been used extensively to probe the intricacies of biological systems through loss of function associated with attenuation of a particular protein. Besides, RNAi technology has been developed in recent years to further aid the process of scientific inquiry. Even though, the methods, dealing with DNA and RNA have met with great success, are not without their shortcomings. In order to overcome the inadequacies of existing methods, a host of new techniques, aimed at knockdowns at the protein rather than the nucleic acid level, have been devised. Essentially, these methods can achieve rapid degradation of cellular pools of a target protein in response to an inducible signal coupled with dose-dependent modulation and exquisite temporal control, features which are absent from techniques involving manipulations at the DNA or RNA level. This review aims to provide a broad overview of a gamut of these methods, while highlighting the strengths and weaknesses of each one. Last two decades of advances presented here in the field of targeted protein degradation serve as a beacon to further research and are likely to find applications in the areas of medicine and allied fields of biology.

Keywords: Targeted protein degradation; Ubiquitin; Proteasome; Antizyme; Ornithine decarboxylase; PROTACs

An Indian scenario on renewable and sustainable energy sources with emphasis on algae by S. Hemaiswarya; Rathinam Raja; Isabel S. Carvalho; R. Ravikumar; Vasudeo Zambare; Debmalya Barh (pp. 1125-1135).

India is the fifth largest primary energy consumer and fourth largest petroleum consumer after USA, China, and Japan. Despite the global economic crisis, India’s economy is expected to grow at 6 to 8 %/year. There is an extreme dependence on petroleum products with considerable risks and environmental issues. Petroleum-derived transport fuels are of limited availability and contribute to global warming, making renewable biofuel as the best alternative. The focus on biogas and biomass-based energy, such as bioethanol and biohydrogen, will enhance cost-effectiveness and provide an opportunity for the rural community. Among all energy sources, microalgae have received, so far, more attention due to their facile adaptability to grow in the photobioreactors or open ponds, high yields, and multiple applications. Microalgae can produce a substantial amount of triacylglycerols as a storage lipid under photooxidative stress or other adverse environmental conditions. In addition to renewable biofuels, they can provide different types of high-value bioproducts added to their advantages, such as higher photosynthetic efficiency, higher biomass production, and faster growth compared to any other energy crops. The viability of first-generation biofuels production is, however, questionable because of the conflict with food supply. In the future, biofuels should ideally create the environmental, economic, and social benefits to the communities and reflect energy efficiency so as to plan a road map for the industry to produce third-generation biofuels.

Keywords: Renewable and sustainable energy; Biofuel; Biomass; Microalgae

Microbial rescue to plant under habitat-imposed abiotic and biotic stresses by D. K. Choudhary (pp. 1137-1155).

Habitat-imposed abiotic and biotic stress is a serious condition and is also a land-degradation problem in arid and semi-arid regions, causing major problem for crop productivity. Most of the cultivable and a least half of irrigated lands around the world are severely affected by environmental stresses. However, in these conditions, there are plant populations successfully adapted and evolutionarily different in their strategy of stress tolerance. Vascular plants do not function as autonomous individuals, but house diverse communities of symbiotic microbes. The role of these microbes can no longer be ignored. Microbial interactions are critical not only for host but also for fungal survival in stressed environments. Plants benefit extensively by harboring these associated microbes; they promote plant growth and confer enhanced resistance to various pathogens by producing antibiotics. To date, improvements in plant quality, production, abiotic and biotic stress resistance, nutrient, and water use have relied largely on manipulating plant genomes by breeding and genetic modification. Increasing evidence indicates that the function of symbiotic microbes seems to parallel more than one of these characteristics.

Keywords: Habitat-imposed stresses; Stress tolerance; Bacterial volatiles; Induced systemic resistance

Peptidoglycan hydrolases-potential weapons against Staphylococcus aureus by Piotr Szweda; Marta Schielmann; Roman Kotlowski; Grzegorz Gorczyca; Magdalena Zalewska; Slawomir Milewski (pp. 1157-1174).

Bacteria of the genus Staphylococcus are common pathogens responsible for a broad spectrum of human and animal infections and belong to the most important etiological factors causing food poisoning. Because of rapid increase in the prevalence of isolation of staphylococci resistant to many antibiotics, there is an urgent need for the development of new alternative chemotherapeutics. A number of studies have recently demonstrated the strong potential of peptidoglycan hydrolases (PHs) to control and treat infections caused by this group of bacteria. PHs cause rapid lysis and death of bacterial cells. The review concentrates on enzymes hydrolyzing peptidoglycan of staphylococci. Usually, they are characterized by high specificity to only Staphylococcus aureus cell wall components; however, some of them are also able to lyse cells of other staphylococci, e.g., Staphylococcus epidermidis-human pathogen of growing importance and also other groups of bacteria. Some PHs strengthen the bactericidal or bacteriostatic activity of common antibiotics, and as a result, they should be considered as component of combined therapy which could definitely reduced the development of bacterial resistance to both enzymes and antibiotics. The preliminary research revealed that most of these enzymes can be produced using heterologous, especially Escherichia coli expression systems; however, still much effort is required to develop more efficient and large-scale production technologies. This review discusses current state on knowledge with emphasis on the possibilities of application of PHs in the context of therapeutics for infections caused by staphylococci.

Keywords: Staphylococcus ; Staphylococcus aureus ; Antibiotic resistance; Peptidoglycan hydrolases

Evolution of atrazine-degrading capabilities in the environment by Nikolina Udiković-Kolić; Colin Scott; Fabrice Martin-Laurent (pp. 1175-1189).

Since their first introduction in the mid 1950s, man-made s-triazine herbicides such as atrazine have extensively been used in agriculture to control broadleaf weed growth in different crops, and thus contributed to improving crop yield and quality. Atrazine is the most widely used s-triazine herbicide for the control of weeds in crops such as corn and sorghum. Although atrazine was initially found to be slowly and partially biodegradable, predominantly by nonspecific P450 monoxygenases which do not sustain microbial growth, microorganisms gradually evolved as a result of repeated exposure, started using it as a growth substrate and eventually succeeded in mineralizing it. Within three decades, an entirely new hydrolase-dependent pathway for atrazine mineralization emerged and rapidly spread worldwide among genetically different bacteria. This review focuses on the enzymes involved in atrazine mineralization and their evolutionary histories, the genetic composition of microbial populations involved in atrazine degradation and the biotechnologies that have been developed, based on these systems, for the bioremediation of atrazine contamination in the environment.

Keywords: Atrazine; Evolution; Biodegradation; Atrazine-degrading enzymes; atz ; trz genes; Bioremediation

Sugar transport systems in Corynebacterium glutamicum: features and applications to strain development by Masato Ikeda (pp. 1191-1200).

Corynebacterium glutamicum uses the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) to take up and phosphorylate glucose, fructose, and sucrose, the major sugars from agricultural crops that are used as the primary feedstocks for industrial amino acid fermentation. This means that worldwide amino acid production using this organism has depended exclusively on the PTS. Recently, a better understanding not only of PTS-mediated sugar uptake but also of global regulation associated with the PTS has permitted the correction of certain negative aspects of this sugar transport system for amino acid production. In addition, the recent identification of different glucose uptake systems in this organism has led to a strategy for the generation of C. glutamicum strains that express non-PTS routes instead of the original PTS. The potential practical advantages of the development of such strains are discussed.

Keywords: Sugar transport systems; PTS; Amino acid production; Strain improvement; Corynebacterium glutamicum

Nanocrystal Cu₂O-loaded TiO₂ nanotube array films as high-performance visible-light bactericidal photocatalyst by Shengsen Zhang; Chang Liu; Xiaolu Liu; Haimin Zhang; Porun Liu; Shanqing Zhang; Feng Peng; Huijun Zhao (pp. 1201-1207).

In this work, we report the use of a non-toxic nanocrystal Cu₂O-loaded TiO₂ nanotube array (Cu₂O/TNTs) film as high-performance visible-light bactericidal photocatalyst. The samples were characterized by field-emission scanning electron microscopy, X-ray photoelectron spectroscopy, and ultraviolet–visible diffusion reflection spectroscopy. This Cu₂O/TNTs film photocatalyst is capable of complete inactivation of Escherichia coli in 5 × 10⁷ colony-forming units/mL within a record short disinfection time of 20 min under visible-light irradiation. The average bactericidal percentage of the Cu₂O/TNTs for E. coli under visible-light irradiation are 20 times and 6.6 times higher than those of TNTs under the same conditions and Cu₂O/TNTs without light, respectively. This superior bactericidal performance is mainly attributed to the high ability to produce OH radicals by both photogenerated electron and hole of the prepared photocatalyst under visible light. The Cu₂O/TNTs film photocatalyst makes it applicable to broad fields including drinking water disinfection.

Keywords: Cu₂O; TiO₂ nanotube; Visible light; Bactericidal photocatalyst

Microbial production of itaconic acid: developing a stable platform for high product concentrations by Anja Kuenz; Yvonne Gallenmüller; Thomas Willke; Klaus-Dieter Vorlop (pp. 1209-1216).

Biotechnologically produced itaconic acid (IA) is a promising organic acid with a wide range of applications and the potential to open up new application fields in the area of polymer chemistry, pharmacy, and agriculture. In this study, a systematic process optimization was performed with an own isolated strain of Aspergillus terreus and transferred from a 250-mL to a 15-L scale. An IA concentration of 86.2 g/L was achieved within 7 days with an overall productivity of 0.51 g/(L h), a maximum productivity of 1.2 g/(L h), and a yield of 86 mol%. A cultivation of other well-known A. terreus strains with the developed process showed no significant differences. Based on this, a process is developed providing a high final IA concentration independent of the used strain combined with high reproducibility.

Keywords: Aspergillus terreus ; Itaconic acid; Optimization; Submerse cultivation; Scale up

Streptomyces sp. JS520 produces exceptionally high quantities of undecylprodigiosin with antibacterial, antioxidative, and UV-protective properties by Nada Stankovic; Vanja Radulovic; Milos Petkovic; Ivan Vuckovic; Milka Jadranin; Branka Vasiljevic; Jasmina Nikodinovic-Runic (pp. 1217-1231).

A Gram-positive, red-pigment-producing bacterial strain, designated JS520 was isolated from the pristine sediment from the cave on mountain Miroc in Serbia. Strain was confirmed to belong to Streptomyces genus based on phenotypic and genetic analysis. Streptomyces sp. JS520 has the ability to produce exceptionally high amounts of deep red pigment into both solid and liquid media. Liquid chromatography and mass spectroscopy of the purified pigments revealed the major component to be undecylprodigiosin (93 %) with minor component being oxidatively cyclized derivative. The pigment production was affected by medium composition, temperature, pH, and the aeration rate. By medium optimization, yields of undecylprodigiosin of 138 mg l⁻¹ were achieved, what is the highest level of undecylprodigiosin production reported for the members of Gram-positive Streptomyces genus. Purified pigment had antimicrobial properties against bacterial Bacillus and Micrococcus species (50 μg ml⁻¹) and against Candida albicans species (100–200 μg ml⁻¹ range). The ability to affect auto-oxidation of the linoleic acid was demonstrated for the purified undecylprodigiosin, suggesting antioxidative properties of this pigment. Multiple ecophysiological roles of the pigment were revealed by comparing cultures grown under pigment-producing and pigment-nonproducing conditions. Cells grown under undecylprodigiosin-producing conditions could tolerate presence of hydrogen peroxide exhibiting three times smaller zones of inhibition at 100 mM H₂O₂. Undecylprodigiosin-producing cells were also less susceptible to tetracycline, kanamycin, chloramphenicol, and 8-hydroxyquinoline. While the growth of the cells not producing pigment was completely inhibited by 15 min of exposure to ultraviolet light (254 nm), cells producing undecylprodigiosin and cells supplied with purified pigment in vitro showed survival rates at 22 and 8 %, respectively.

Keywords: Pigment; Undecylprodigiosin; Antioxidative; UV protection; Streptomyces; Medium optimization

Construction and development of a mammalian cell-based full-length antibody display library for targeting hepatocellular carcinoma by Feng Li; Yan-Hong Liu; Yan-Wen Li; Yue-Hui Li; Ping-Li Xie; Qiang Ju; Lin Chen; Guan-Cheng Li (pp. 1233-1241).

We present a detailed method for constructing a mammalian cell-based full-length antibody display library for targeting hepatocellular carcinoma. Two novel mammalian library vectors pcDNA3-CHm and pcDNA3-CLm were constructed that contained restriction enzyme sites NheI, ClaI and antibody constant domain. Mammalian expression vector pcDNA3-CHm contains IgG heavy-chain (HC) constant region and glycosylphosphatidylinositol anchor (GPI) that could be anchored full-length antibodies on the surface of mammalian cells. GOLPH2 prokaryotic expression vector was carried out in Escherichia coli and purified by immobilized metal affinity chromatography. Variable domain of heavy-chain and variable domain of light-chain genes were respectively inserted into the vector pcDNA3-CHm and pcDNA3-CLm by ligation, and antibody libraries are displayed as whole IgG molecules on the cell surface by co-transfecting this HC-GPI with a light chain. By screening the cell library using magnetic beads and cell ELISA, the cell clone that displayed GOLPH2-specific antibodies on cell surfaces was identified. The mammalian cell-based antibody display library is a great potential application for displaying full-length functional antibodies of targeting hepatocellular carcinoma on the surface of mammalian cells. Anti-GOLPH2 display antibody was successfully isolated from the library.

Keywords: Antibody library; Mammalian cell display; Hepatocellular carcinoma; GOLPH2; Antibody engineering

Asymmetric synthesis of d-glyceric acid by an alditol oxidase and directed evolution for enhanced oxidative activity towards glycerol by Sandra Gerstenbruch; Hauke Wulf; Nina Mußmann; Timothy O’Connell; Karl-Heinz Maurer; Uwe T. Bornscheuer (pp. 1243-1252).

Glycerol as a by-product of biodiesel production is an attractive precursor for producing d-glyceric acid. Here, we demonstrate the successful production of d-glyceric acid based on glycerol via glyceraldehyde in a two-step enzyme reaction with the FAD-dependent alditol oxidase from Streptomyces coelicolor A3(2). The hydrogen peroxide generated in the reaction can be used in detergent, food, and paper industry. In order to apply the alditol oxidase in industry, the enzyme was subjected to protein engineering. Different strategies were used to enhance the substrate specificity towards glycerol. Initial attempts based on rational protein design in the active site region were found unsuccessful to increase activity. However, through directed evolution, an alditol oxidase double mutant (V125M/A244T) with 1.5-fold improved activity for glycerol was found by screening 8,000 clones. Further improvement of activity was achieved by combinatorial experiments, which led to a quadruple mutant (V125M/A244T/V133M/G399R) with 2.4-fold higher specific activity towards glycerol compared to the wild-type enzyme. Through studying the effects of mutations created, we were able to understand the importance of certain amino acids in the structure of alditol oxidase, not only for conferring enzymatic structural stability but also with respect to their influence on oxidative activity.

Keywords: Oxidase; Protein engineering; Directed evolution; Rational protein design; Glycerol; Enzyme catalysis

Optimization of the production of gurmarin, a sweet-taste-suppressing protein, secreted by the methylotrophic yeast Pichia pastoris by Maud Sigoillot; Anne Brockhoff; Ewen Lescop; Nicolas Poirier; Wolfgang Meyerhof; Loïc Briand (pp. 1253-1263).

Gurmarin, a 35-residue polypeptide, is known to selectively inhibit responses to sweet substances in rodents without affecting responses to other basic taste stimuli, such as NaCl, HCl, and quinine. Here, we report the heterologous expression of gurmarin using the methylotrophic yeast Pichia pastoris. Gurmarin was secreted into the buffered minimal medium using the α-factor preprosequence without the EAEA spacer peptide of Saccharomyces cerevisiae and was under the control of the methanol-inducible alcohol oxidase promoter. We found that gurmarin accumulated in the yeast culture medium reaching 5 mg per liter of culture over an expression period of 4 days. To compare the production level and the signal peptide processing, the N-terminal amino acid of gurmarin was substituted by a glutamic acid residue. This construct resulted in a 6-fold increase in the level of gurmarin secretion leading to 30 mg of purified protein per liter of culture. Purified recombinant gurmarin resulting from both constructs was characterized using mass spectrometry. Circular dichroism and NMR spectroscopy revealed that recombinant gurmarin was properly folded and had secondary and tertiary structures. We also confirmed its capability to inhibit the rat heterodimeric sweet taste T1R2/T1R3 receptor by functional expression in human embryonic kidney HEK293T cells. The high level of fully active gurmarin obtained in P. pastoris makes this expression system attractive for fermentor growth and pharmacological investigations of taste receptor and gurmarin functions.

Keywords: Gurmarin; Sweetness-suppressing peptide; Taste receptor; Gymnema sylvestre ; Yeast; Pichia pastoris

Comparison of alkyl hydroperoxide reductase and two water-forming NADH oxidases from Bacillus cereus ATCC 14579 by Liang Wang; Huiqing Chong; Rongrong Jiang (pp. 1265-1273).

Bacillus cereus (B. cereus) is an ubiquitous facultative anaerobic bacterium, and its growth in aerobic environment correlates to the functions of its oxygen defense system. Water-forming NADH oxidase (nox-2) can catalyze the conversion of oxygen to water with concomitant NADH oxidation in anaerobic microorganisms. Here, we report the cloning and characterization of two annotated nox-2 s (nox-2(444) and nox-2(554)) from B. cereus ATCC 14579 and their comparison with another oxidative stress defense system alkyl hydroperoxide reductase (AhpR) from this microbe, which composed of two enzymes—hydrogen peroxide-forming NADH oxidase (nox-1) and peroxidase. Both nox-2 and AhpR catalyze the same reaction in the presence of oxygen. With the stimulation of exogenously added FAD, the maximum activity of nox-1, nox-2(444), and nox-2(554) could reach 27.7 U/mg, 22.9 U/mg, and 2.4 U/mg, respectively, at pH 7.0, 30 °C. Different from nox-1, both nox-2 s were thermotolerant enzymes and could maintain above 87% of their optimum activity at 80 °C, which was not found in other nox-2 s. As for operational stability, all are turnover-limited. Exogenously added reductive reagent dithiothreitol could dramatically increase the total turnover number of nox-2(444) and nox-2(554) by twofold and threefold, respectively, but had no effect on AhpR or nox-1.

Keywords: NADH oxidase; Alkyl hydroperoxide reductase; Oxygen scavenger; Anaerobic bacterium

Transcripts of a heterologous gene encoding mite allergen Der f 7 are stabilized by codon optimization in Aspergillus oryzae by Mizuki Tanaka; Masafumi Tokuoka; Takahiro Shintani; Katsuya Gomi (pp. 1275-1282).

We have previously demonstrated that transcripts of an AT-biased heterologous gene encoding mite allergen Der f 7 from Dermatophagoides farinae were polyadenylated prematurely within the coding region when native cDNA was expressed in Aspergillus oryzae, and that this premature polyadenylation was prevented by the codon optimization of the Der f 7 gene, resulting in increased steady-state mRNA levels. In this study, we tested the stability of transcription products derived from expression constructs of the native and codon-optimized Der f 7 gene in A. oryzae using 1,10-phenanthroline as a transcription inhibitor. Transcription products of native Der f 7 cDNA fused to the A. oryzae glucoamylase gene (glaA) were rapidly degraded; the half-life of the mRNA was approximately 13 min. However, the half-life of codon-optimized Der f 7 mRNA fused to glaA was approximately 43 min, which was highly similar to that of endogenous glaA mRNA. These results indicate that Der f 7 mRNA is significantly stabilized by codon optimization. In addition, Der f 7 mRNA was stabilized by the codon optimization of only the 3′-half region, where premature polyadenylation sites were exclusively situated; the half-life of the chimeric Der f 7 mRNA was approximately 39 min. This suggested that destabilization of native Der f 7 mRNA is mainly triggered by premature polyadenylation within the coding region. To the best of our knowledge, this is the first report to provide experimental evidence that heterologous mRNA is significantly stabilized by codon optimization in eukaryotic cells.

Keywords: mRNA stability; Aspergillus oryzae ; Codon optimization; Heterologous gene expression; 1,10-Phenanthroline; Nonstop mRNA decay

Recombinant production of mGLP-1 by coupling of refolding and intein-mediated self-cleavage (CRIS) by Mingming Gao; Yue Tong; Hong Tian; Xiangdong Gao; Wenbing Yao (pp. 1283-1290).

Glucagon-like peptide-1 as an endogenous glucose-lowering peptide is a promising candidate for anti-diabetic drug development. Here, we developed a convenient method by coupling of refolding and intein-mediated self-cleavage (CRIS) to improve the recombinant production of a mutated glucagon-like peptide-1 (mGLP-1). Bacterial cell culture employing auto-induction was performed at 37 °C to avoid the intracellular self-cleavage of the intein fusion protein. The impacts of urea, pH, and temperature on the efficiency of CRIS were tested, and then, the optimized CRIS was established. Using the optimized method, we obtained the purified mGLP-1 with a yield of 3.41 mg peptide/g bacterial cells which was 5.6-fold higher than before. After that, using chromatography, peptide electrophoresis, and mass spectrometry, we determined the purity and molecular weight of the purified peptide and then confirmed its glucose-lowering activity by performing glucose tolerance test in mice. These results suggest that CRIS is a relatively simple and efficacious method for the recombinant production of mGLP-1, and as a general method, it can also be used for the recombinant preparation of some other proteins and peptides.

Keywords: Glucagon-like peptide-1; Incretin; Auto-induction; Refolding; Intein

Laboratory metabolic evolution improves acetate tolerance and growth on acetate of ethanologenic Escherichia coli under non-aerated conditions in glucose-mineral medium by M. T. Fernández-Sandoval; G. Huerta-Beristain; B. Trujillo-Martinez; P. Bustos; V. González; F. Bolivar; G. Gosset; A. Martinez (pp. 1291-1300).

In this work, Escherichia coli MG1655 was engineered to produce ethanol and evolved in a laboratory process to obtain an acetate tolerant strain called MS04 (E. coli MG1655: ΔpflB, ΔadhE, ΔfrdA, ΔxylFGH, ΔldhA, PpflB::pdc Zm -adhB Zm, evolved). The growth and ethanol production kinetics of strain MS04 were determined in mineral medium, mainly under non-aerated conditions, supplemented with glucose in the presence of different concentrations of sodium acetate at pH 7.0 and at different values of acid pH and a constant concentration of sodium acetate (2 g/l). Results revealed an increase in the specific growth rate, cell mass formation, and ethanol volumetric productivity at moderate concentrations of sodium acetate (2–10 g/l), in addition to a high tolerance to acetate because it was able to grow and produce a high yield of ethanol in the presence of up to 40 g/l of sodium acetate. Genomic analysis of the ΔpflB evolved strain identified that a chromosomal deletion of 27.3 kb generates the improved growth and acetate tolerance in MG1655 ΔpflB derivative strains. This deletion comprises genes related to the respiration of nitrate, repair of alkylated DNA and synthesis of the ompC gene coding for porin C, cytochromes C, thiamine, and colonic acid. Strain MS04 is advantageous for the production of ethanol from hemicellulosic hydrolysates that contain acetate.

Keywords: Escherichia coli ; Metabolic engineering; Metabolic evolution; Fuel ethanol; Glucose; Acetate

Analysis of two distinct mycelial populations in liquid-grown Streptomyces cultures using a flow cytometry-based proteomics approach by G. Jerre van Veluw; Marloes L. C. Petrus; Jacob Gubbens; Richard de Graaf; Inez P. de Jong; Gilles P. van Wezel; Han A. B. Wösten; Dennis Claessen (pp. 1301-1312).

Streptomycetes are proficient producers of enzymes and antibiotics. When grown in bioreactors, these filamentous microorganisms form mycelial pellets that consist of interconnected hyphae. We here employed a flow cytometry approach designed for large particles (COPAS) and demonstrate that liquid-grown Streptomyces cultures consist of two distinct populations of pellets. One population consists of mycelia with a constant mean diameter of approximately 260 μm, whereas the other population contains larger mycelia whose diameter depends on the strain, the age of the culture, and medium composition. Quantitative proteomics analysis revealed that 37 proteins differed in abundance between the two populations of pellets. Stress-related proteins and biosynthetic proteins for production of the calcium-dependent antibiotic were more abundant in the population of large mycelia, while proteins involved in DNA topology, modification, or degradation were overrepresented in the population of small mycelia. Deletion of genes for the cellulose synthase-like protein CslA and the chaplins affected the average size of the population of large pellets but not that of small pellets. Considering the fact that the production of enzymes and metabolites depends on pellet size, these results provide new leads toward rational strain design of Streptomyces strains tailored for industrial fermentations.

Keywords: Heterogeneity; Fermentation; COPAS; Cell wall; Proteomics

Formation of ethyl acetate by Kluyveromyces marxianus on whey during aerobic batch cultivation at specific trace element limitation by Thanet Urit; Anton Stukert; Thomas Bley; Christian Löser (pp. 1313-1323).

Kluyveromyces marxianus is able to transform lactose into ethyl acetate as a bulk product which offers a chance for an economical reuse of whey-borne sugar. Ethyl acetate is highly volatile and allows its process-integrated recovery by stripping from the aerated bioreactor. Extensive formation of ethyl acetate by K. marxianus DSM 5422 required restriction of yeast growth by a lack of trace elements. Several aerobic batch processes were done in a 1-L stirred reactor using whey-borne culture medium supplemented with an individual trace element solution excluding Mn, Mo, Fe, Cu, or Zn for identifying the trace element(s) crucial for the observed ester synthesis. Only a lack of Fe, Cu, or Zn restricted yeast growth while exclusion of Mn and Mo did not exhibit any effect due to a higher amount of the latter in the used whey. Limitation of growth by Fe or Cu caused significant production of ethyl acetate while limitation by Zn resulted in formation of ethanol. A lack of Fe or Cu obviously makes the respiratory chain inefficient resulting in an increased mitochondrial NADH level followed by a reduced metabolic flux of acetyl-SCoA into the citrate cycle. Synthesis of ethyl acetate from acetyl-SCoA and ethanol by alcoholysis is thus interpreted as an overflow metabolism.

Keywords: Kluyveromyces marxianus ; Whey; Ethyl acetate; Trace elements; Iron; Copper

New insights into the butyric acid metabolism of Clostridium acetobutylicum by Dörte Lehmann; Nadine Radomski; Tina Lütke-Eversloh (pp. 1325-1339).

Biosynthesis of acetone and n-butanol is naturally restricted to the group of solventogenic clostridia with Clostridium acetobutylicum being the model organism for acetone-butanol-ethanol (ABE) fermentation. According to limited genetic tools, only a few rational metabolic engineering approaches were conducted in the past to improve the production of butanol, an advanced biofuel. In this study, a phosphotransbutyrylase-(Ptb) negative mutant, C. acetobutylicum ptb::int(87), was generated using the ClosTron methodology for targeted gene knock-out and resulted in a distinct butyrate-negative phenotype. The major end products of fermentation experiments without pH control were acetate (3.2 g/l), lactate (4.0 g/l), and butanol (3.4 g/l). The product pattern of the ptb mutant was altered to high ethanol (12.1 g/l) and butanol (8.0 g/l) titers in pH ≥ 5.0-regulated fermentations. Glucose fed-batch cultivation elevated the ethanol concentration to 32.4 g/l, yielding a more than fourfold increased alcohol to acetone ratio as compared to the wildtype. Although butyrate was never detected in cultures of C. acetobutylicum ptb::int(87), the mutant was still capable to take up butyrate when externally added during the late exponential growth phase. These findings suggest that alternative pathways of butyrate re-assimilation exist in C. acetobutylicum, supposably mediated by acetoacetyl-CoA:acyl-CoA transferase and acetoacetate decarboxylase, as well as reverse reactions of butyrate kinase and Ptb with respect to previous studies.

Keywords: Biofuels; Butanol; Ethanol; Butyrate; ABE fermentation; Metabolic engineering

Microplate-based assays for the evaluation of antibacterial effects of photocatalytic coatings by Urška Žvab; Martina Bergant Marušič; Urška Lavrenčič Štangar (pp. 1341-1351).

Three microplate-based viability assays for assessing the antibacterial effects of photocatalytic coatings were compared to the conventional colony count method. In the experimental design, cultured Escherichia coli were exposed to photocatalysis on various TiO₂ films in the presence of either UVA or visible light. The photocatalytic effects on the bacterial physiology were determined by real-time measurements of metabolic activity (XTT assay), biomass formation in the liquid medium (growth assay), and by assessing membrane integrity (with propidium iodide and SYTO 9 fluorescent nucleic acid binding dyes—BacLight assay). All three methods proved to be more sensitive and reproducible than colony count for the evaluation of the bactericidal effect of photocatalysis, XTT, and growth assay succeeded in detecting differences in both UVA and visible light-activated photocatalytic coatings. BacLight could efficiently detect the visible light-dependent photocatalytic effect on bacteria and identify membrane damage, but resulted inadequate for evaluating the UVA-dependent antibacterial effects. The described microplate-based evaluation methods proved being more effective and rapid than the colony count assay for assessing the antibacterial effect of various photocatalytic coatings.

Keywords: Microplate-based assay; Photocatalytic coating; Titanium dioxide; Antibacterial photo-oxidation; Escherichia coli

Fast and efficient generation of recombinant baculoviruses by in vitro transposition by Jae Young Choi; Yang-Su Kim; Yong Wang; Xue Ying Tao; Qin Liu; Jong Yul Roh; Soo Dong Woo; Byung Rae Jin; Yeon Ho Je (pp. 1353-1360).

A novel recombinant bacmid, bEasyBac, that enables the easy and fast generation of pure recombinant baculovirus without any purification step was constructed. In bEasyBac, attR recombination sites were introduced to facilitate the generation of a recombinant viral genome by in vitro transposition. Moreover, the extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of the Cotesia plutellae bracovirus early promoter to negatively select against the non-recombinant background. The bEasyBac bacmid could only replicate in host insect cells when the barnase gene was replaced with the gene of interest by in vitro transposition. When bEasyBac was transposed with pDualBac-EGFP, the resulting recombinant virus, AcEasy-EGFP, showed comparable levels of EGFP expression efficiency to the plaque-purified recombinant virus AcEGFP, which was constructed using the bAcGOZA system. In addition, no non-recombinant backgrounds were detected in unpurified AcEasy-EGFP stocks. Based on these results, a high-throughput system for the generation of multiple recombinant viruses at a time was established.

Keywords: Baculovirus expression system; EasyBac; In vitro transposition; Barnase; High-throughput

Intestinal microbiota associated with differential feed conversion efficiency in chickens by Dragana Stanley; Stuart E. Denman; Robert J. Hughes; Mark S. Geier; Tamsyn M. Crowley; Honglei Chen; Volker R. Haring; Robert J. Moore (pp. 1361-1369).

Analysis of model systems, for example in mice, has shown that the microbiota in the gastrointestinal tract can play an important role in the efficiency of energy extraction from diets. The study reported here aimed to determine whether there are correlations between gastrointestinal tract microbiota population structure and energy use in chickens. Efficiency in converting food into muscle mass has a significant impact on the intensive animal production industries, where feed represents the major portion of production costs. Despite extensive breeding and selection efforts, there are still large differences in the growth performance of animals fed identical diets and reared under the same conditions. Variability in growth performance presents management difficulties and causes economic loss. An understanding of possible microbiota drivers of these differences has potentially important benefits for industry. In this study, differences in cecal and jejunal microbiota between broiler chickens with extreme feed conversion capabilities were analysed in order to identify candidate bacteria that may influence growth performance. The jejunal microbiota was largely dominated by lactobacilli (over 99% of jejunal sequences) and showed no difference between the birds with high and low feed conversion ratios. The cecal microbial community displayed higher diversity, and 24 unclassified bacterial species were found to be significantly (<0.05) differentially abundant between high and low performing birds. Such differentially abundant bacteria represent target populations that could potentially be modified with prebiotics and probiotics in order to improve animal growth performance.

Keywords: 16S rRNA gene; Chicken; Feed conversion ratio; Gut; Microbiota

Degradation of the novel herbicide ZJ0273 by Amycolatopsis sp. M3-1 isolated from soil by Zhiqiang Cai; Qiaoli Chen; Haiyan Wang; Yucai He; Wei Wang; Xiyue Zhao; Qingfu Ye (pp. 1371-1379).

ZJ0273, propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoate, is a novel and broad-spectrum herbicide. In this study, 15 bacteria capable of utilizing ZJ0273 as the sole carbon source were isolated from soil. One of the isolates belonged to the family Amycolatopsis and was designated to Amycolatopsis sp. M3-1; at 30°C and pH 7.0, degradation rate of ZJ0273 could reach at 59.3% and 68.5% in 25 days and 60 days, respectively. Furthermore, six metabolites (M1–M6) during the degradation of ZJ0273 by Amycolatopsis sp. M3-1 were identified by a combination with multi-position ¹⁴C-labeled compounds (B-ZJ0273 and C-ZJ0273), chromatography, liquid scintillation spectrometer, and LC–MS, a novel pathway of ZJ0273 degradation by Amycolatopsis sp. M3-1 was proposed based on the identified metabolites and their biodegradation courses. ZJ0273 was initially hydrolyzed into M1 (4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoic acid), then further oxidized into M3 (2-(4,6-dimethoxypyrimidin-2-yloxy) benzoic acid). M1 also could undergo a carbonylation into M2 (4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzamido) benzoic acid), and then its C–N and C–O bonds were cleaved to yield M3 (2-(4,6-dimethoxypyrimidin-2-yloxy) benzoic acid) and M4 (4,6-dimethoxypyrimidin-2-ol), respectively. Moreover, another two new metabolites, M5 (2-(4-hydroxy, 6-methoxypyrimidin-2-yloxy) benzoic acid) and M6 (2, 4-dihydroxy-pyrimidine) were found. M5 was formed through de-methyl of M3 and then hydrolyzed into M6.

Keywords: ZJ0273; Biodegradation pathway; Degradation intermediate; Amycolatopsis ; Carbon 14-labeled

RETRACTED ARTICLE: Simultaneous expression of antibody light and heavy chains in Pichia pastoris: improving retransformation outcome by linearizing vector at a different site by Yueming Qian; Xuankuo Xu; Zizhuo Xing; Li You; Vineeta Dhar; Nan-Xin Qian; Chao Huang; Bruce Eagan; Shih-Hsie Pan; Zheng Jian Li (pp. 1381-1381).

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Applied Microbiology and Biotechnology (v.96, #5)

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Applied Microbiology and Biotechnology (v.96, #5)