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  • 1
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    Production of biobutanol from cellulose hydrolysate by the Escherichia coli co-culture system (2016)
    Oxford University Press
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    Publication Date: 2016-02-12
    Description: The commercialization of the n -butanol bioprocess is largely dependent on the price of feedstocks. Renewable cellulose appears to be an appealing feedstock. The microbial production of n -butanol still remains challenging because of the limited availability of intracellular NADH. To address this issue, an Escherichia coli strain carrying the clostridial CoA-dependent pathway was supplied with heterologous formate dehydrogenase. With the cellulose hydrolysate of rice straw, this single strain produced cellulosic biobutanol with a production yield at 35% of the theoretical and a productivity of 0.093 g L –1  h –1 . In an alternative method, the production involved a co-culture system consisting of two separate strains provided with the full CoA-dependent pathway. This system achieved a production yield and productivity reaching 62.8% of the theoretical and 0.163 g L –1  h –1 , respectively. The result indicates that the E. coli co-culture system is technically promising for the production of cellulosic biobutanol.
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 2
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    Pseudomonas putida KT2440 markerless gene deletion using a combination of {lambda} Red recombineering and Cre/loxP site-specific recombination (2016)
    Oxford University Press
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    Publication Date: 2016-02-07
    Description: Pseudomonas putida KT2440 is a saprophytic, environmental microorganism that plays important roles in the biodegradation of environmental toxic compounds and production of polymers, chemicals and secondary metabolites. Gene deletion of KT2440 usually involves cloning of the flanking homologous fragments of the gene of interest into a suicide vector followed by transferring into KT2440 via triparental conjugation. Selection and counterselection steps are then employed to generate gene deletion mutant. However, these methods are tedious and are not suitable for the manipulation of multiple genes simultaneously. Herein, a two-step, markerless gene deletion method is presented. First, homologous armsflanked loxP-neo-loxP was knocked-in to replace the gene of interest, then the kanamycin resistance marker is removed by Cre recombinase catalyzed site-specific recombination. Both two-plasmid and one-plasmid gene systems were established. MekR/P mekA regulated gene expression system was found to be suitable for tight Cre expression in one-plasmid deletion system. The straightforward, time saving and highly efficient markerless gene deletion strategy has the potential to facilitate the genetics and functional genomics study of P. putida KT2440.
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 3
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    Synergistic effect of calcium and zinc on glucose/xylose utilization and butanol tolerance of Clostridium acetobutylicum (2016)
    Oxford University Press
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    Publication Date: 2016-02-25
    Description: Biobutanol outperforms bioethanol as an advanced biofuel, but is not economically competitive in terms of its titer, yield and productivity associated with feedstocks and energy cost. In this work, the synergistic effect of calcium and zinc was investigated in the acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum using glucose, xylose and glucose/xylose mixtures as carbon source(s). Significant improvements associated with enhanced glucose/xylose utilization, cell growth, acids re-assimilation and butanol biosynthesis were achieved. Especially, the maximum butanol and ABE production of 16.1 and 25.9 g L –1 were achieved from 69.3 g L –1 glucose with butanol/ABE productivities of 0.40 and 0.65 g L –1 h –1 compared to those of 11.7 and 19.4 g/L with 0.18 and 0.30 g L –1 h –1 obtained in the control respectively without any supplement. More importantly, zinc was significantly involved in the butanol tolerance based on the improved xylose utilization under various butanol-shock conditions (2, 4, 6, 8 and 10 g L –1 butanol). Under the same conditions, calcium and zinc co-supplementation led to the best xylose utilization and butanol production. These results suggested that calcium and zinc could play synergistic roles improving ABE fermentation by C. acetobutylicum .
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 4
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    A Pseudomonas putida double mutant deficient in butanol assimilation: a promising step for engineering a biological biofuel production platform (2016)
    Oxford University Press
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    Publication Date: 2016-02-20
    Description: Biological production in heterologous hosts is of interest for the production of the C4 alcohol (butanol) and other chemicals. However, some hurdles need to be overcome in order to achieve an economically viable process; these include avoiding the consumption of butanol and maintaining tolerance to this solvent during production. Pseudomonas putida is a potential host for solvent production; in order to further adapt P. putida to this role, we generated mini-Tn 5 mutant libraries in strain BIRD-1 that do not consume butanol. We analyzed the insertion site of the mini-Tn 5 in a mutant that was deficient in assimilation of butanol using arbitrary PCR followed by Sanger sequencing and found that the transposon was inserted in the malate synthase B gene. Here, we show that in a second round of mutagenesis a double mutant unable to take up butanol had an insertion in a gene coding for a multisensor hybrid histidine kinase. The genetic context of the histidine kinase sensor revealed the presence of a set of genes potentially involved in butanol assimilation; qRT-PCR analysis showed induction of this set of genes in the wild type and the malate synthase mutant but not in the double mutant.
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 5
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    Frontiers in microbial 1-butanol and isobutanol production (2016)
    Oxford University Press
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    Publication Date: 2016-02-20
    Description: The heavy dependence on petroleum-derived fuel has raised concerns about energy sustainability and climate change, which have prompted researchers to explore fuel production from renewable sources. 1-Butanol and isobutanol are promising biofuels that have favorable properties and can also serve as solvents or chemical feedstocks. Microbial production of these alcohols provides great opportunities to access a wide spectrum of renewable resources. In recent years, research has improved the native 1-butanol production and has engineered isobutanol production in various organisms to explore metabolic diversity and a broad range of substrates. This review focuses on progress in metabolic engineering for the production of these two compounds using various resources.
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 6
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    Industrial production of acetone and butanol by fermentation--100 years later (2016)
    Oxford University Press
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    Publication Date: 2016-06-08
    Description: Microbial production of acetone and butanol was one of the first large-scale industrial fermentation processes of global importance. During the first part of the 20th century, it was indeed the second largest fermentation process, superseded in importance only by the ethanol fermentation. After a rapid decline after the 1950s, acetone-butanol-ethanol (ABE) fermentation has recently gained renewed interest in the context of biorefinery approaches for the production of fuels and chemicals from renewable resources. The availability of new methods and knowledge opens many new doors for industrial microbiology, and a comprehensive view on this process is worthwhile due to the new interest. This thematic issue of FEMS Microbiology Letters, dedicated to the 100th anniversary of the first industrial exploitation of Chaim Weizmann's ABE fermentation process, covers the main aspects of old and new developments, thereby outlining a model development in biotechnology. All major aspects of industrial microbiology are exemplified by this single process. This includes new technologies, such as the latest developments in metabolic engineering, the exploitation of biodiversity and discoveries of new regulatory systems such as for microbial stress tolerance, as well as technological aspects, such as bio- and down-stream processing.
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 7
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    Construction and characterization of three protein-targeting expression system in Lactobacillus casei (2016)
    Oxford University Press
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    Publication Date: 2016-03-13
    Description: We previously reported that the β-1,4-Mannanase ( manB ) gene from Bacillus pumilus functions as a good reporter gene in Lactobacillus casei . Two vectors were constructed. One carries the signal peptide of secretion protein Usp45 (SP Usp45 ) from Lactococcus lactis (pELSH), and the other carries the full-length S-layer protein, SlpA, from L. acidophilus (pELWH). In this work, another vector, pELSPH, was constructed to include the signal peptide of protein SlpA (SP SlpA ), and the capacity of all three vectors to drive expression of the manB gene in L. casei was evaluated. The results showed that SP Usp45 is functionally recognized and processed by the L. casei secretion machinery. The SP Usp45 -mediated secretion efficiency was ~87%, and SP SlpA drove the export of secreted ManB with ~80% efficiency. SP SlpA secretion was highly efficient, and expressed SlpA was anchored to the cell wall by an unknown secretion mechanism. Full-length SlpA drove the cell wall-anchored expression of an SlpA-ManB fusion protein but at a much lower level than that of protein SlpA.
    Keywords: Biotechnology & Synthetic Biology
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    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 8
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    Hetero-oligomeric MspA pores in Mycobacterium smegmatis (2016)
    Oxford University Press
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    Publication Date: 2016-03-13
    Description: The porin MspA of Mycobacterium smegmatis is a biological nanopore used for DNA sequencing. The octameric MspA pore can be isolated from M. smegmatis in milligram quantities, is extremely stable against denaturation and rapidly inserts into lipid membranes. Here, we show that MspA pores composed of different Msp subunits are formed in M. smegmatis and that hetero-oligomers of different Msp monomers increase the heterogeneity of MspA pores designed for DNA sequencing. To improve the quality of preparations of mutant MspA proteins, all four msp genes were deleted from the M. smegmatis genome after insertion of an inducible porin gene from M. tuberculosis. In the msp quadruple mutant M. smegmatis ML712 no Msp porins were detected and mutant MspA proteins were produced at wild-type levels. Lipid bilayer experiments demonstrated that MspA pores isolated from ML712 formed functional channels and had a narrower conductance distribution than pores purified from M. smegmatis with background msp expression. Thus, the M. smegmatis msp quadruple mutant improves the homogeneity of MspA pores designed for DNA sequencing and might also facilitate the identification and functional characterization of other mycobacterial pore proteins.
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 9
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    Lichen-forming fungus Caloplaca flavoruscens inhibits transcription factors and chromatin remodeling system in fungi (2016)
    Oxford University Press
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    Publication Date: 2016-05-20
    Description: Lichen-forming fungi and extracts derived from them have been used as alternative medicine sources for millennia and recently there has been a renewed interest in their known bioactive properties for anticancer agents, cosmetics and antibiotics. Although lichen-forming fungus-derived compounds are biologically and commercially valuable, few studies have been performed to determine their modes of action. This study used chemical-genetic and chemogenomic high-throughput analyses to gain insight into the modes of action of Caloplaca flavoruscens extracts. High-throughput screening of 575 lichen extracts was performed and 39 extracts were identified which inhibited yeast growth. A C. flavoruscens extract was selected as a promising antifungal and was subjected to genome-wide haploinsufficiency profiling and homozygous profiling assays. These screens revealed that yeast deletion strains lacking Rsc8, Pro1 and Toa2 were sensitive to three concentrations (IC 25.5 , IC 25 and IC 50 , respectively) of C. flavoruscens extract. Gene-enrichment analysis of the data showed that C. flavoruscens extracts appear to perturb transcription and chromatin remodeling.
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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  • 10
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    Cytotoxic damage of soybean agglutinin on intestinal epithelial cells of broiler chicks: in vitro protection by Bifidobacterium infantis CRL1395 (2016)
    Oxford University Press
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    Publication Date: 2016-05-20
    Description: Plant lectins, which are proteins/glycoproteins present in a wide range of vegetables, fruits, cereals and beans, are resistant to digestive enzymes and food cooking temperatures. They bind reversibly to specific glycosidic residues expressed on the membrane of intestinal epithelial cells (IEC) and cause anti-nutritional effects in humans and animals. Soybean lectin (SBA) has been detected in poultry diets, and its ability to bind to the intestinal epithelium has been reported. The development of new methods for removing SBA from feeds or to prevent interaction with the intestinal mucosa is of interest. In this study, the in vitro cytotoxicity of SBA on IEC of chicks was demonstrated for the first time. The LD 50 , assessed after 2 h exposure of IEC to SBA, was 6.13 μg mL –1 . The ability of Bifidobacterium infantis CRL1395 to bind SBA on the bacterial envelope was confirmed, and prevention of IEC cytotoxicity by lectin removal was demonstrated. Safety of B. infantis CRL1395, resistance to gastrointestinal stress and adhesion were also determined. It was concluded that the early administration of B. infantis CRL1395 to chicks would effectively reduce the toxicity of SBA. Besides, it would favour the colonization of the gut with a beneficial microbiota.
    Keywords: Biotechnology & Synthetic Biology
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
    Published by Oxford University Press
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