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1.  Generation of food-grade recombinant Lactobacillus casei delivering Myxococcus xanthus prolyl endopeptidase 
Applied microbiology and biotechnology  2014;98(15):6689-6700.
Prolyl endopeptidases (PEP), a family of serine proteases with the ability to hydrolyze the peptide bond on the carboxyl side of an internal proline residue, are able to degrade immunotoxic peptides responsible for celiac disease (CD), such as a 33-residue gluten peptide (33-mer). Oral administration of PEP has been suggested as a potential therapeutic approach for CD, although delivery of the enzyme to the small intestine requires intrinsic gastric stability or advanced formulation technologies. We have engineered two food-grade Lactobacillus casei strains to deliver PEP in an in vitro model of small intestine environment. One strain secretes PEP into the extracellular medium, whereas the other retains PEP in the intracellular environment. The strain that secretes PEP into the extracellular medium is the most effective to degrade the 33-mer and is resistant to simulated gastrointestinal stress. Our results suggest that in a future, after more studies and clinical trials, an engineered food-grade Lactobacillus strain may be useful as a vector for in situ production of PEP in the upper small intestine of CD patients.
doi:10.1007/s00253-014-5730-7
PMCID: PMC4393947  PMID: 24752841
Celiac disease; gluten; prolyl endopeptidase; Myxococcus xanthus; heterologous expression; Lactobacillus casei
2.  Transformable facultative thermophile Geobacillus stearothermophilus NUB3621 as a host strain for metabolic engineering 
Applied microbiology and biotechnology  2014;98(15):6715-6723.
Metabolic engineers develop inexpensive enantioselective syntheses of high-value compounds, but their designs are sometimes confounded by the misfolding of heterologously expressed proteins. Geobacillus stearothermophilus NUB3621 is a readily transformable facultative thermophile. It could be used to express and properly fold proteins derived from its many mesophilic or thermophilic Bacillaceae relatives or to direct the evolution of thermophilic variants of mesophilic proteins. Moreover, its capacity for high-temperature growth should accelerate chemical transformation rates in accordance with the Arrhenius equation and reduce the risks of microbial contamination. Its tendency to sporulate in response to nutrient depletion lowers the costs of storage and transportation. Here, we present a draft genome sequence of G. stearothermophilus NUB3621 and describe inducible and constitutive expression plasmids that function in this organism. These tools will help us and others to exploit the natural advantages of this system for metabolic engineering applications.
doi:10.1007/s00253-014-5746-z
PMCID: PMC4251812  PMID: 24788326
Geobacillus stearothermophilus; Facultative thermophile; Genome; Expression system
3.  The substrate tolerance of alcohol oxidases 
Applied Microbiology and Biotechnology  2015;99(16):6617-6642.
Alcohols are a rich source of compounds from renewable sources, but they have to be activated in order to allow the modification of their carbon backbone. The latter can be achieved via oxidation to the corresponding aldehydes or ketones. As an alternative to (thermodynamically disfavoured) nicotinamide-dependent alcohol dehydrogenases, alcohol oxidases make use of molecular oxygen but their application is under-represented in synthetic biotransformations. In this review, the mechanism of copper-containing and flavoprotein alcohol oxidases is discussed in view of their ability to accept electronically activated or non-activated alcohols and their propensity towards over-oxidation of aldehydes yielding carboxylic acids. In order to facilitate the selection of the optimal enzyme for a given biocatalytic application, the substrate tolerance of alcohol oxidases is compiled and discussed: Substrates are classified into groups (non-activated prim- and sec-alcohols; activated allylic, cinnamic and benzylic alcohols; hydroxy acids; sugar alcohols; nucleotide alcohols; sterols) together with suitable alcohol oxidases, their microbial source, relative activities and (stereo)selectivities.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-015-6699-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-015-6699-6
PMCID: PMC4513209  PMID: 26153139
Oxidation; Biocatalysis; Alcohol oxidase; Substrate tolerance; Flavoprotein; Cu-containing oxidase
4.  Insect Antimicrobial Peptides and Their Applications 
Applied microbiology and biotechnology  2014;98(13):5807-5822.
Insects are one of the major sources of antimicrobial peptides/proteins (AMPs). Since observation of antimicrobial activity in the hemolymph of pupae from the giant silk moths Samia Cynthia and Hyalophora cecropia in 1974 and purification of first insect AMP (cecropin) from H. cecropia pupae in 1980, over 150 insect AMPs have been purified or identified. Most insect AMPs are small and cationic, and they show activities against bacteria and/or fungi, as well as some parasites and viruses. Insect AMPs can be classified into four families based on their structures or unique sequences: the α-helical peptides (cecropin and moricin), cysteine-rich peptides (insect defensin and drosomycin), proline-rich peptides (apidaecin, drosocin and lebocin), and glycine-rich peptides/proteins (attacin and gloverin). Among insect AMPs, defensins, cecropins, proline-rich peptides and attacins are common, while gloverins and moricins have been identified only in Lepidoptera. Most active AMPs are small peptides of 20–50 residues, which are generated from larger inactive precursor proteins or pro-proteins, but gloverins (~14 kDa) and attacins (~20 kDa) are large antimicrobial proteins. In this mini-review, we will discuss current knowledge and recent progress in several classes of insect AMPs, including insect defensins, cecropins, attacins, lebocins and other proline-rich peptides, gloverins, and moricins, with a focus on structural-functional relationships and their potential applications.
doi:10.1007/s00253-014-5792-6
PMCID: PMC4083081  PMID: 24811407
alpha-helical peptide; cysteine-rich peptide; glycine-rich peptide; proline-rich peptide; lipopolysaccharide; conformational changes
5.  Pseudomonas putida—a versatile host for the production of natural products 
Applied Microbiology and Biotechnology  2015;99(15):6197-6214.
The biosynthesis of natural products by heterologous expression of biosynthetic pathways in amenable production strains enables biotechnological access to a variety of valuable compounds by conversion of renewable resources. Pseudomonas putida has emerged as a microbial laboratory work horse, with elaborated techniques for cultivation and genetic manipulation available. Beyond that, this bacterium offers several particular advantages with regard to natural product biosynthesis, notably a versatile intrinsic metabolism with diverse enzymatic capacities as well as an outstanding tolerance to xenobiotics. Therefore, it has been applied for recombinant biosynthesis of several valuable natural products. This review provides an overview of applications of P. putida as a host organism for the recombinant biosynthesis of such natural products, including rhamnolipids, terpenoids, polyketides and non-ribosomal peptides, and other amino acid-derived compounds. The focus is on de novo natural product synthesis from intrinsic building blocks by means of heterologous gene expression and strain engineering. Finally, the future potential of the bacterium as a chassis organism for synthetic microbiology is pointed out.
doi:10.1007/s00253-015-6745-4
PMCID: PMC4495716  PMID: 26099332
Pseudomonas putida; Heterologous pathway expression; Recombinant biosynthesis; Natural products
6.  Microbially influenced corrosion communities associated with fuel-grade ethanol environments 
Applied Microbiology and Biotechnology  2015;99(16):6945-6957.
Microbially influenced corrosion (MIC) is a costly problem that impacts hydrocarbon production and processing equipment, water distribution systems, ships, railcars, and other types of metallic infrastructure. In particular, MIC is known to cause considerable damage to hydrocarbon fuel infrastructure including production, transportation, and storage systems, often times with catastrophic environmental contamination results. As the production and use of alternative fuels such as fuel-grade ethanol (FGE) increase, it is important to consider MIC of engineered materials exposed to these “newer fuels” as they enter existing infrastructure. Reports of suspected MIC in systems handling FGE and water prompted an investigation of the microbial diversity associated with these environments. Small subunit ribosomal RNA gene pyrosequencing surveys indicate that acetic-acid-producing bacteria (Acetobacter spp. and Gluconacetobacter spp.) are prevalent in environments exposed to FGE and water. Other microbes previously implicated in corrosion, such as sulfate-reducing bacteria and methanogens, were also identified. In addition, acetic-acid-producing microbes and sulfate-reducing microbes were cultivated from sampled environments containing FGE and water. Results indicate that complex microbial communities form in these FGE environments and could cause significant MIC-related damage that may be difficult to control. How to better manage these microbial communities will be a defining aspect of improving mitigation of global infrastructure corrosion.
doi:10.1007/s00253-015-6729-4
PMCID: PMC4513208  PMID: 26092755
Microbial diversity; Fuel-grade ethanol; Microbiologically influenced corrosion; Pyrosequencing
7.  Accumulation and metabolism of selenium by yeast cells 
Applied Microbiology and Biotechnology  2015;99(13):5373-5382.
This paper examines the process of selenium bioaccumulation and selenium metabolism in yeast cells. Yeast cells can bind elements in ionic from the environment and permanently integrate them into their cellular structure. Up to now, Saccharomyces cerevisiae, Candida utilis, and Yarrowia lipolytica yeasts have been used primarily in biotechnological studies to evaluate binding of minerals. Yeast cells are able to bind selenium in the form of both organic and inorganic compounds. The process of bioaccumulation of selenium by microorganisms occurs through two mechanisms: extracellular binding by ligands of membrane assembly and intracellular accumulation associated with the transport of ions across the cytoplasmic membrane into the cell interior. During intracellular metabolism of selenium, oxidation, reduction, methylation, and selenoprotein synthesis processes are involved, as exemplified by detoxification processes that allow yeasts to survive under culture conditions involving the elevated selenium concentrations which were observed. Selenium yeasts represent probably the best absorbed form of this element. In turn, in terms of wide application, the inclusion of yeast with accumulated selenium may aid in lessening selenium deficiency in a diet.
doi:10.1007/s00253-015-6650-x
PMCID: PMC4464373  PMID: 26003453
Selenium; Accumulation; Yeast; Cells
8.  O-antigen Polymerase Adopts a Distributive Mechanism for Lipopolysaccharide Biosynthesis 
Bacterial Lipopolysaccharide (LPS) is an essential cell envelope component for Gram-negative bacteria. As the most variable region of LPS, O-antigens serve as important virulence determinants for many bacteria and represent a promising carbohydrate source for glycoconjugate vaccines. In the Wzy-dependent O-antigen biosynthetic pathway, the integral membrane protein Wzy was shown to be the sole enzyme responsible for polymerization of O-repeat unit. Its catalytic mechanism, however, remains elusive. Herein, Wzy was successfully overexpressed in E. coli with an N-terminal His10-tag. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed that the Wzy protein exists in its native confirmation as a dimer. Subsequently, we chemoenzymatically synthesized the substrates of Wzy, the lipid-PP linked repeat units. Together with an optimized O-antigen visualization method, we monitored the production of reaction intermediates at varying times. We present here our result as the first biochemical evidence that wzy functions in a distributive manner.
doi:10.1007/s00253-014-5552-7
PMCID: PMC3989392  PMID: 24557568
Wzy; integral membrane protein; overexpression; distributive mechanism
9.  Enhanced C30 carotenoid production in Bacillus subtilis by systematic overexpression of MEP pathway genes 
Applied Microbiology and Biotechnology  2015;99(14):5907-5915.
Creating novel biosynthetic pathways and modulating the synthesis of important compounds are one of the hallmarks of synthetic biology. Understanding the key parameters controlling the flux of chemicals throughout a metabolic pathway is one of the challenges ahead. Isoprenoids are the most functionally and structurally diverse group of natural products from which numerous medicines and relevant fine chemicals are derived. The well-characterized and broadly used production organism Bacillus subtilis forms an ideal background for creating and studying novel synthetic routes. In comparison to other bacteria, B. subtilis emits the volatile compound isoprene, the smallest representative of isoprenoids, in high concentrations and thus represents an interesting starting point for an isoprenoid cell factory. In this study, the effect of systematic overexpression of the genes involved in the methylerythritol phosphate (MEP) pathway on isoprenoid production in B. subtilis was investigated. B. subtilis strains harboring a plasmid containing C30 carotenoid synthetic genes, crtM and crtN, were combined with pHCMC04G plasmids carrying various synthetic operons of the MEP pathway genes. The levels of produced carotenoids, diaponeurosporene and diapolycopene, were used as indication of the role of the various enzymes on the flux of the MEP pathway. It was shown that the production of carotenoids can be increased significantly by overexpressing the MEP pathway enzymes. More broadly, the strains developed in this study can be used as a starting point for various isoprenoid cell factories.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-015-6531-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-015-6531-3
PMCID: PMC4480331  PMID: 25851715
Terpenoids; MEP; Bacillus subtilis; Isoprene; Carotenoid
10.  Benchmarking of commercially available CHO cell culture media for antibody production 
Applied Microbiology and Biotechnology  2015;99(11):4645-4657.
In this study, eight commercially available, chemically defined Chinese hamster ovary (CHO) cell culture media from different vendors were evaluated in batch culture using an IgG-producing CHO DG44 cell line as a model. Medium adaptation revealed that the occurrence of even small aggregates might be a good indicator of cell growth performance in subsequent high cell density cultures. Batch experiments confirmed that the culture medium has a significant impact on bioprocess performance, but high amino acid concentrations alone were not sufficient to ensure superior cell growth and high antibody production. However, some key amino acids that were limiting in most media could be identified. Unbalanced glucose and amino acids led to high cell-specific lactate and ammonium production rates. In some media, persistently high glucose concentrations probably induced the suppression of respiration and oxidative phosphorylation, known as Crabtree effect, which resulted in high cell-specific glycolysis rates along with a continuous and high lactate production. In additional experiments, two of the eight basal media were supplemented with feeds from two different manufacturers in six combinations, in order to understand the combined impact of media and feeds on cell metabolism in a CHO fed-batch process. Cell growth, nutrient consumption and metabolite production rates, antibody production, and IgG quality were evaluated in detail. Concentrated feed supplements boosted cell concentrations almost threefold and antibody titers up to sevenfold. Depending on the fed-batch strategy, fourfold higher peak cell concentrations and eightfold increased IgG titers (up to 5.8 g/L) were achieved. The glycolytic flux was remarkably similar among the fed-batches; however, substantially different specific lactate production rates were observed in the different media and feed combinations. Further analysis revealed that in addition to the feed additives, the basal medium can make a considerable contribution to the ammonium metabolism of the cells. The glycosylation of the recombinant antibody was influenced by the selection of basal medium and feeds. Differences of up to 50 % in the monogalacto-fucosylated (G1F) and high mannose fraction of the IgG were observed.
doi:10.1007/s00253-015-6514-4
PMCID: PMC4435641  PMID: 25846330
Chinese hamster ovary (CHO); Cell culture medium; Antibody production; Batch; Fed-batch; Medium benchmarking
11.  Comprehensive mapping infection-enhancing epitopes of dengue pr protein using polyclonal antibody against prM 
Applied Microbiology and Biotechnology  2015;99(14):5917-5927.
Dengue vaccine development is considered a global public health priority, but the antibody-dependent enhancement (ADE) issues have critically restricted vaccine development. Recent findings have demonstrated that pre-membrane (prM) protein was involved in dengue virus (DENV) infection enhancement. Although the importance of prM antibodies have been well characterized, only a few epitopes in DENV prM protein have ever been identified. In this study, we screened five potential linear epitopes located at positions pr1 (1-16aa), pr3 (13-28aa), pr4 (19-34aa), pr9 (49-64aa), and pr10 (55-70aa) in pr protein using peptide scanning and comprehensive bioinformatics analysis. Then, we found that only pr4 (19-34aa) could elicit high-titer antibodies in Balb/c mice, and this epitope could react with sera from DENV2-infected patients, suggesting that specific antibodies against epitope peptide pr4 were elicited in both DENV-infected mice and human. In addition, our data demonstrated that anti-pr4 sera showed limited neutralizing activity but significant ADE activity toward standard DENV serotypes and imDENV. Hence, it seems responsible to hypothesize that anti-pr4 serum was infection-enhancing antibody and pr4 was infection-enhancing epitope. In conclusion, we characterized a novel infection-enhancing epitope on dengue pr protein, a finding that may provide new insight into the pathogenesis of DENV infection and contribute to dengue vaccine design.
doi:10.1007/s00253-015-6538-9
PMCID: PMC4480844  PMID: 25822571
Dengue virus; pr protein; Epitope; Synthetic peptides; Antibody-dependent enhancement
12.  Antimicrobial photodynamic therapy with fulleropyrrolidine: photoinactivation mechanism of Staphylococcus aureus, in vitro and in vivo studies 
A family of N-methylpyrrolidinium fullerene iodide salts has been intensively studied to determine their applicability in antimicrobial photodynamic therapy (APDT). This study examined in vitro the efficacy of a C60 fullerene functionalized with one methylpyrrolidinium group to kill upon irradiation with white light gram-negative and gram-positive bacteria, as well as fungal cells, and the corresponding mechanism of the fullerene bactericidal action. The in vitro studies revealed that the high antistaphylococcal efficacy of functionalized fullerene could be linked to their ability to photogenerate singlet oxygen and superoxide anion. Following Staphylococcus aureus photoinactivation, no modifications of its genomic DNA were detected. In contrast, photodamage of the cell envelope seemed to be a dominant mechanism of bactericidal action. In in vivo studies, a 2 log10 reduction in the average bioluminescent radiance between treated and non-treated mice was reached. One day post APDT treatment, moist and abundant growth of bacteria could be observed on wounds of non-fulleropyrrolidine and dark control mice. APDT-treated wounds stayed visibly clear up to the third day. Moreover, cytotoxicity test on human dermal keratinocytes revealed great safety of using the sensitizer toward eukaryotic cells. These data indicate potential application of functionalized fullerene as antistaphylococcal sensitizer for superficial infections.
doi:10.1007/s00253-015-6539-8
PMCID: PMC4428807  PMID: 25820601
Cytotoxicity; Fulleropyrrolidine; Mouse model; Photoinactivation; Staphylococcus aureus; Wound infection
13.  In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas aeruginosa strains from cystic fibrosis patients 
Applied Microbiology and Biotechnology  2015;99(14):6021-6033.
The goal of the study was to determine the relationship between in vitro/in vivo efficacy of environmental Pseudomonas phages and certain phenotypical properties of Pseudomonas aeruginosa (PA) strains. We studied the diversity between particular isolates and determined phage sensitivity in vitro and in vivo in the Galleria mellonella insect model. Twenty-eight lytic bacteriophages specific for PA were tested against 121 CF PA isolates including 29 mucoid PA strains. Most strains from cystic fibrosis (CF) patients were lysed by at least three phages (93.6 %), but completely insensitive strains were also present (6.4 %). Two phages PA5oct and KT28 exhibited high rates of lytic potency on 55–68 % of PA strains (72–86 % of mucoid isolates). We further explored phage activity against six PA strains (CF and non-CF) in vitro, comparing clonal differences in phage susceptibility with bacterial properties such as the ability to form biofilms, mucosity, twitching motility, and biochemical profiles. We observed the relationship between variation in phage susceptibility and Fourier transform infrared spectroscopy (FTIR) analysis in the spectra window of carbohydrates. The protective efficacy of two selected phages against PA PAO1 and 0038 infection was confirmed in vivo in G. mellonella larvae. Generally, the wax moth model results confirmed the data from in vitro assays, but in massive infection of CF isolates, the application of lytic phages probably led to the release of toxic compound causing an increase in larvae mortality. We assumed that apart of in vitro phage activity testing, a simple and convenient wax moth larvae model should be applied for the evaluation of in vivo effectiveness of particular phage preparations.
doi:10.1007/s00253-015-6492-6
PMCID: PMC4480334  PMID: 25758956
Myoviridae bacteriophages; Phage treatment; Pseudomonas aeruginosa; Cystic fibrosis; Galleria mellonella model
14.  Lactic acid bacteria—20 years exploring their potential as live vectors for mucosal vaccination 
Lactic acid bacteria (LAB) are a diverse group of Gram-positive, nonsporulating, low G + C content bacteria. Many of them have been given generally regarded as safe status. Over the past two decades, intensive genetic and molecular research carried out on LAB, mainly Lactococcus lactis and some species of the Lactobacillus genus, has revealed new, potential biomedical LAB applications, including the use of LAB as adjuvants, immunostimulators, or therapeutic drug delivery systems, or as factories to produce therapeutic molecules. LAB enable immunization via the mucosal route, which increases effectiveness against pathogens that use the mucosa as the major route of entry into the human body. In this review, we concentrate on the encouraging application of Lactococcus and Lactobacillus genera for the development of live mucosal vaccines. First, we present the progress that has recently been made in the field of developing tools for LAB genetic manipulations, which has resulted in the successful expression of many bacterial, parasitic, and viral antigens in LAB strains. Next, we discuss the factors influencing the efficacy of the constructed vaccine prototypes that have been tested in various animal models. Apart from the research focused on an application of live LABs as carriers of foreign antigens, a lot of work has been recently done on the potential usage of nonliving, nonrecombinant L. lactis designated as Gram-positive enhancer matrix (GEM), as a delivery system for mucosal vaccination. The advantages and disadvantages of both strategies are also presented.
doi:10.1007/s00253-015-6498-0
PMCID: PMC4365182  PMID: 25750046
Lactic acid bacteria; Antigens; DNA vaccine; GEM particles; Immunoprophylaxis
15.  Synthesis and assembly of fungal melanin 
Melanin is a unique pigment with myriad functions that is found in all biological kingdoms. It is multifunctional, providing defense against environmental stresses such as ultraviolet (UV) light, oxidizing agents and ionizing radiation. Melanin contributes to the ability of fungi to survive in harsh environments. In addition, it plays a role in fungal pathogenesis. Melanin is an amorphous polymer that is produced by one of two synthetic pathways. Fungi may synthesize melanin from endogenous substrate via a 1,8-dihydroxynaphthalene (DHN) intermediate. Alternatively, some fungi produce melanin from l-3,4-dihydroxyphenylalanine (l-dopa). The detailed chemical structure of melanin is not known. However, microscopic studies show that it has an overall granular structure. In fungi, melanin granules are localized to the cell wall where they are likely cross-linked to polysaccharides. Recent studies suggest the fungal melanin may be synthesized in internal vesicles akin to mammalian melanosomes and transported to the cell wall. Potential applications of melanin take advantage of melanin's radioprotective properties and propensity to bind to a variety of substances.
doi:10.1007/s00253-011-3777-2
PMCID: PMC4318813  PMID: 22173481
Fungi; Melanin; Cell wall; Vesicle; Chitin; Radioprotection
16.  [No title available] 
PMCID: PMC3968793  PMID: 24402415
17.  [No title available] 
PMCID: PMC4096848  PMID: 24292079
18.  Effect and behaviour of different substrates in relation to the formation of aerobic granular sludge 
Applied Microbiology and Biotechnology  2015;99(12):5257-5268.
When aerobic granular sludge is applied for industrial wastewater treatment, different soluble substrates can be present. For stable granular sludge formation on volatile fatty acids (e.g. acetate), production of storage polymers under anaerobic feeding conditions has been shown to be important. This prevents direct aerobic growth on readily available chemical oxygen demand (COD), which is thought to result in unstable granule formation. Here, we investigate the impact of acetate, methanol, butanol, propanol, propionaldehyde, and valeraldehyde on granular sludge formation at 35 °C. Methanogenic archaea, growing on methanol, were present in the aerobic granular sludge system. Methanol was completely converted to methane and carbon dioxide by the methanogenic archaeum Methanomethylovorans uponensis during the 1-h anaerobic feeding period, despite the relative high dissolved oxygen concentration (3.5 mg O2 L−1) during the subsequent 2-h aeration period. Propionaldehyde and valeraldehyde were fully disproportionated anaerobically into their corresponding carboxylic acids and alcohols. The organic acids produced were converted to storage polymers, while the alcohols (produced and from influent) were absorbed onto the granular sludge matrix and converted aerobically. Our observations show that easy biodegradable substrates not converted anaerobically into storage polymers could lead to unstable granular sludge formation. However, when the easy biodegradable COD is absorbed in the granules and/or when the substrate is converted by relatively slow growing bacteria in the aerobic period, stable granulation can occur.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-014-6358-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-014-6358-3
PMCID: PMC4445487  PMID: 25616527
Aerobic granular sludge; Methanol; Alcohol; Aldehyde; Methanogens; Granule formation; Industrial wastewater; Disproportionation; Feeding strategies
19.  The impact of oxygen on the final alcohol content of wine fermented by a mixed starter culture 
We have developed a wine fermentation procedure that takes advantage of the metabolic features of a previously characterized Metschnikowia pulcherrima strain in order to reduce ethanol production. It involves the use of M. pulcherrima/Saccharomyces cerevisiae mixed cultures, controlled oxygenation conditions during the first 48 h of fermentation, and anaerobic conditions thereafter. The influence of different oxygenation regimes and initial inoculum composition on yeast physiology and final ethanol content was studied. The impact of oxygenation on yeast physiology goes beyond the first aerated step and influences yields and survival rates during the anaerobic stage. The activity of M. pulcherrima in mixed oxygenated cultures resulted in a clear reduction in ethanol yield, as compared to S. cerevisiae. Despite relatively low initial cell numbers, S. cerevisiae always predominated in mixed cultures by the end of the fermentation process. Strain replacement was faster under low oxygenation levels. M. pulcherrima confers an additional advantage in terms of dissolved oxygen, which drops to zero after a few hours of culture, even under highly aerated conditions, and this holds true for mixed cultures. Alcohol reduction values about 3.7 % (v/v) were obtained for mixed cultures under high aeration, but they were associated to unacceptable volatile acidity levels. In contrast, under optimized conditions, only 0.35 g/L acetic acid was produced, for an alcohol reduction of 2.2 % (v/v), and almost null dissolved oxygen during the process.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-014-6321-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-014-6321-3
PMCID: PMC4428804  PMID: 25582558
Low alcohol; Non-Saccharomyces yeast; Respiratory quotient; Dissolved oxygen; Volatile acidity
20.  An updated view on horseradish peroxidases: recombinant production and biotechnological applications 
Horseradish peroxidase has been the subject of scientific research for centuries. It has been used exhaustively as reporter enzyme in diagnostics and histochemistry and still plays a major role in these applications. Numerous studies have been conducted on the role of horseradish peroxidase in the plant and its catalytic mechanism. However, little progress has been made in its recombinant production. Until now, commercial preparations of horseradish peroxidase are still isolated from plant roots. These preparations are commonly mixtures of various isoenzymes of which only a small fraction has been described so far. The composition of isoenzymes in these mixed isolates is subjected to uncontrollable environmental conditions. Nowadays, horseradish peroxidase regains interest due to its broad applicability in the fields of medicine, life sciences, and biotechnology in cancer therapy, biosensor systems, bioremediation, and biocatalysis. These medically and commercially relevant applications, the recent discovery of new natural isoenzymes with different biochemical properties, as well as the challenges in recombinant production render this enzyme particularly interesting for future biotechnological solutions. Therefore, we reviewed previous studies as well as current developments with biotechnological emphasis on new applications and the major remaining biotechnological challenge—the efficient recombinant production of horseradish peroxidase enzymes.
doi:10.1007/s00253-014-6346-7
PMCID: PMC4322221  PMID: 25575885
Horseradish peroxidase; Plant peroxidase; Recombinant protein production; Diagnostics; Biosensor; Indole-3-acetic acid; Cancer treatment
21.  Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome 
Applied Microbiology and Biotechnology  2015;99(13):5475-5485.
Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78 % following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-014-6355-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-014-6355-6
PMCID: PMC4464377  PMID: 25575887
Rumen; Lipolysis; Fatty acid; Lipase; Esterase; Bacteria; Functional metagenomic
22.  Sustainable source of omega-3 eicosapentaenoic acid from metabolically engineered Yarrowia lipolytica: from fundamental research to commercial production 
The omega-3 fatty acids, cis-5, 8, 11, 14, and 17-eicosapentaenoic acid (C20:5; EPA) and cis-4, 7, 10, 13, 16, and 19-docosahexaenoic acid (C22:6; DHA), have wide-ranging benefits in improving heart health, immune function, mental health, and infant cognitive development. Currently, the major source for EPA and DHA is from fish oil, and a minor source of DHA is from microalgae. With the increased demand for EPA and DHA, DuPont has developed a clean and sustainable source of the omega-3 fatty acid EPA through fermentation using metabolically engineered strains of Yarrowia lipolytica. In this mini-review, we will focus on DuPont’s technology for EPA production. Specifically, EPA biosynthetic and supporting pathways have been introduced into the oleaginous yeast to synthesize and accumulate EPA under fermentation conditions. This Yarrowia platform can also produce tailored omega-3 (EPA, DHA) and/or omega-6 (ARA, GLA) fatty acid mixtures in the cellular lipid profiles. Fundamental research such as metabolic engineering for strain construction, high-throughput screening for strain selection, fermentation process development, and process scale-up were all needed to achieve the high levels of EPA titer, rate, and yield required for commercial application. Here, we summarize how we have combined the fundamental bioscience and the industrial engineering skills to achieve large-scale production of Yarrowia biomass containing high amounts of EPA, which led to two commercial products, New Harvest™ EPA oil and Verlasso® salmon.
doi:10.1007/s00253-014-6318-y
PMCID: PMC4322222  PMID: 25567511
Yarrowia lipolytica; Omega-3 fatty acid; Metabolic engineering; Fermentation; Commercialization
23.  Staphylococcus epidermidis in the human skin microbiome mediates fermentation to inhibit the growth of Propionibacterium acnes: Implications of probiotics in acne vulgaris 
Increasing evidence demonstrates that commensal microorganisms in the human skin microbiome help fight pathogens and maintain homeostasis of the microbiome. However, it is unclear how these microorganisms maintain biological balance when one of them overgrows. The overgrowth of Propionibacterium acnes (P. acnes), a commensal skin bacterium, has been associated with the progression of acne vulgaris. Our results demonstrate that skin microorganisms can mediate fermentation of glycerol, which is naturally produced in skin, to enhance their inhibitory effects on P. acnes growth. The skin microorganisms, most of which have been identified as Staphylococcus epidermidis (S. epidermidis), in the microbiome of human fingerprints can ferment glycerol and create inhibition zones to repel a colony of overgrown P. acnes. Succinic acid, one of four short-chain fatty acids (SCFAs) detected in fermented media by nuclear magnetic resonance (NMR) analysis, effectively inhibits the growth of P. acnes in vitro and in vivo. Both intralesional injection and topical application of succinic acid to P. acnes-induced lesions markedly suppress the P. acnes-induced inflammation in mice. We demonstrate for the first time that bacterial members in the skin microbiome can undergo fermentation to rein in the overgrowth of P. acnes. The concept of bacterial interference between P. acnes and S. epidermidis via fermentation can be applied to develop probiotics against acne vulgaris and other skin diseases. In addition, it will open up an entirely new area of study for the biological function of the skin microbiome in promoting human health.
doi:10.1007/s00253-013-5394-8
PMCID: PMC3888247  PMID: 24265031
Acne; Fermentation; P. acnes; Probiotic; S. epidermidis; Skin Microbiome
24.  Cloning, expression, and purification of insect (Sitophilus oryzae) alpha-amylase, able to digest granular starch, in Yarrowia lipolytica host 
Raw-starch-digesting enzymes (RSDE) are of major importance for industrial applications, as their usage greatly simplifies the starch processing pipeline. To date, only microbial RSDE have gained considerable attention, since only microbial production of enzymes meets industrial demands. In this study, α-amylase from rice weevil (Sitophilus oryzae), the major rice pest, was cloned and expressed in Yarrowia lipolytica Po1g strain. The enzyme was secreted into the culture medium, and the peak activity (81 AU/L) was reached after only 29 h of culturing in 5-L bioreactors. Through simple purification procedure of ammonium sulfate precipitation and affinity chromatography, it was possible to purify the enzyme to apparent homogeneity (25-fold purification factor, at 5 % yield). The optimal conditions for the α-amylase activity were pH 5.0 and a temperature of 40 °C. The α-amylase studied here did not show any obligate requirement for Ca2+ ions. The recombinant α-amylase appeared to efficiently digest granular starch from pea, amaranth, waxy corn, and waxy rice.
doi:10.1007/s00253-014-6314-2
PMCID: PMC4342842  PMID: 25547839
Amylase; Granular starch; Rice weevil; Yarrowia lipolytica; Recombinant protein expression
25.  Asymmetric reduction of ketones and β-keto esters by (S)-1-phenylethanol dehydrogenase from denitrifying bacterium Aromatoleum aromaticum 
Applied Microbiology and Biotechnology  2014;99(12):5055-5069.
Enzyme-catalyzed enantioselective reductions of ketones and keto esters have become popular for the production of homochiral building blocks which are valuable synthons for the preparation of biologically active compounds at industrial scale. Among many kinds of biocatalysts, dehydrogenases/reductases from various microorganisms have been used to prepare optically pure enantiomers from carbonyl compounds. (S)-1-phenylethanol dehydrogenase (PEDH) was found in the denitrifying bacterium Aromatoleum aromaticum (strain EbN1) and belongs to the short-chain dehydrogenase/reductase family. It catalyzes the stereospecific oxidation of (S)-1-phenylethanol to acetophenone during anaerobic ethylbenzene mineralization, but also the reverse reaction, i.e., NADH-dependent enantioselective reduction of acetophenone to (S)-1-phenylethanol. In this work, we present the application of PEDH for asymmetric reduction of 42 prochiral ketones and 11 β-keto esters to enantiopure secondary alcohols. The high enantioselectivity of the reaction is explained by docking experiments and analysis of the interaction and binding energies of the theoretical enzyme-substrate complexes leading to the respective (S)- or (R)-alcohols. The conversions were carried out in a batch reactor using Escherichia coli cells with heterologously produced PEDH as whole-cell catalysts and isopropanol as reaction solvent and cosubstrate for NADH recovery. Ketones were converted to the respective secondary alcohols with excellent enantiomeric excesses and high productivities. Moreover, the progress of product formation was studied for nine para-substituted acetophenone derivatives and described by neural network models, which allow to predict reactor behavior and provides insight on enzyme reactivity. Finally, equilibrium constants for conversion of these substrates were derived from the progress curves of the reactions. The obtained values matched very well with theoretical predictions.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-014-6309-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s00253-014-6309-z
PMCID: PMC4445480  PMID: 25549618
Alcohol dehydrogenase; Optically pure alcohols; Hydrogen-transfer biocatalysis; Cofactor regeneration; Stereoselective bioreduction

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