Danmarks Tekniske Universitet

Other

Technical University of Denmark

Babcock & Wilcox Vølund A/s

Jensen Peter Ruhdal

Peter Ruhdal Jensen

Kim Dam-Johansen

Jacky Leendert Snoep

Hans Victor Westerhoff

Niels Bech

Ambareesh Govind Phadnavis

Flemming Frandsen

Jensen B Koebmann

Karin Hammer

Jianming Liu

Christian Solem

Lars Blank

Ole Madsen

Søren Helmark

The present invention provides a genetically modified lactic acid bacterium capable of producing diacetyl under aerobic conditions. Additionally the invention provides a method for producing diacetyl using the genetically modified lactic acid bacterium under aerobic conditions in the presence of a source of iron-containing porphyrin and a metal ion selected from Fe, Feand Cu. The lactic acid bacterium is genetically modified by deletion of those genes in its genome that encode polypeptides having lactate dehydrogenase (E.C 1.1.1.27/E.C.1.1.1.28); &#x3b1;-acetolactate decarboxylase (E.C 4.1.1.5); water-forming NADH oxidase (E.C. 1.6.3.4); phosphotransacetylase (E.C.2.3.1.8) activity; and optionally devoid of or deleted for genes encoding polypeptides having diacetyl reductase ((R)-acetoin forming; EC:1.1.1.303); D-acetoin reductase; butanediol dehydrogenase ((R,R)-butane-2,3-diol forming; E.C. 1.1.1.4/1.1.1.-) and alcohol dehydrogenase (E.C. 1.2.1.10) activity. The invention provides for use of the genetically modified lactic acid bacterium for the production of diacetyl and a food product.

High-level production of diacetyl in a metabolically engineered lactic acid bacterium

The invention is directed to a genetically modified microorganism for the extracellular production of free fatty acids and esters thereof, wherein said microorganism is characterized by a modified lipid biosynthesis metabolic pathway: for example reduced fatty acyl-coA synthetase activity that enables the microorganism to overproduce and secrete of esters of fatty acids (Biodiesel) into the surrounding medium, using one or more of: glucose, starch, lignocellulose and a glycerol-based substrate, as a carbon source. The invention further provides a method for the extracellular production of free fatty acids and esters thereof, comprising the use of said genetically modified organism, and a growth medium adapted for said method.

Production of biodiesel by yeast from lignocellulose and glycerol

This invention relates to a boiler () drying, igniting and combusting refuse and producing steam () by heat exchange with flue gases (), said boiler defining a main flow direction () of gases, said boiler () comprising a separator element () and an end superheater (), the separator element () being adapted for separating said flue gases () into streams of a less-corrosive gas flow () and a corrosive gas flow (), said separator element () being located substantially in and along said main flow direction (), said end superheater () being located in proximity to said separator element () and in the flow () of said less-corrosive gas. Said separator element () comprises a plate () or a wall (), which in a number of said separator elements () forms a channel as another separator element. This provides for an increased lifetime of said superheater and makes the boiler provide a high and efficient electrical power output.

Boiler producing steam from flue gases under optimized conditions

A fast pyrolysis apparatus () for producing pyrolysis liquid, such as oil or tar, char and pyrolysis gas from biomass, such as straw, comprises a centrifuge chamber () and a rotor () arranged to impart rotation on the biomass in the centrifuge chamber to force the biomass outwardly under the action of centrifugal forces. A furnace () arranged coaxially around the centrifuge chamber () maintains the temperature at an outer reactive wall of the centrifuge chamber at an elevated temperature to effect the pyrolysis process at or near the reactive wall. The apparatus comprises a condenser () arranged coaxially with and surrounded by the centrifuge chamber (). The apparatus may be accommodated by a mobile unit for simultaneously collecting biomass from a field and processing the biomass in the apparatus.

Pyrolysis methods and apparatus

The production of biomass or a desired product from a cell can be improved by inducing conversion of ATP to ADP without primary effects on other cellular metabolites or functions which is achieved by expressing an uncoupled ATPase activity in the cell and incubating the cell with a suitable substrate to produce the biomass or product. This is conveniently done by expressing in the cell the soluble part (F) of the membrane bound (FFtype) HATPase or a portion of Fexhibiting ATPase activity. The organism from which the FATPase or portions thereof is derived, or in which the FATPase or portions thereof is expressed, may be selected from prokaryotes and eukaryotes. In particular the DNA encoding For a portion thereof may be derived from bacteria and eukaryotic microorganisms such as yeasts, other fungi and cell lines of higher organisms and be selected from the group consisting of the gene encoding the Fsubunit &#x3b2; or a portion thereof and various combinations of the gene or portion with the genes encoding the other Fsubunits or portions thereof. The method can be used i.a. for optimizing the formation of biomass or a desired product by a cell by expressing different levels of uncoupled ATPase activity in the cell, incubating the cell on a suitable substrate, measuring the conversion rate of substrate into biomass or the desired product at each level of ATPase expression, and choosing level of ATPase expression at which the conversion rate is optimized.

Method of improving the production of biomass or a desired product from a cell

An artificial promoter library (or a set of promoter sequences) for a selected organism or group of organisms is constructed as a mixture of double stranded DNA fragments, the sense strands of which comprise at least two consensus sequences of efficient promoters from said organism or group of organisms, or parts thereof comprising at least half of each, and surrounding intermediate nucleotide sequences (spacers) of variable length in which at least 7 nucleotides are selected randomly among the nucleobases A, T, C and G. The sense strands of the double stranded DNA fragments may also include a regulatory DNA sequence imparting a specific regulatory feature, such as activation by a change in the growth conditions, to the promoters of the library. Further, they may have a sequence comprising one or more recognition sites for restriction endonucleases added to one or both of their ends. The selected organism or group of organisms may be selected from prokaryotes and from eukaryotes; and in prokaryotes the consensus sequences to be retained most often will comprise the &#x2212;35 signal (&#x2212;35 to &#x2212;30): TTGACA and the &#x2212;10 signal (&#x2212;12 to &#x2212;7): TATAAT or parts of both comprising at least 3 conserved nucleotides of each, while in eukaryotes said consensus sequences should comprise a TATA box and at least one upstream activation sequence (UAS). Such artificial promoter libraries can be used, e.g., for optimizing the expression of specific genes in various selected organisms.

Artificial promoter libraries for selected organisms and promoters derived from such libraries

A method of enhancing biomass yield of a lactic acid bacterial species cell culture, comprising cultivating the cells in a process comprising the steps of providing conditions that results in a reduced glycolytic flux and providing conditions that enable the cells to have, under aerobic conditions, a respiratory metabolism. The increased yield of biomass may be the result of an increased yield of ATP which can be obtained by activating the native ATP synthase activity of the H -ATPase complex by lowering the ATP/ADP ratio, e.g. by carbon source limitation, and/or by increasing the proton gradient (membrane potential) of the cells, e.g. by enhancing or establishing an electron transport chain which can be achieved by enhancing expression of dehydrogenases or electron transport chain components, by adding to the medium a quinone or porphyrin compound or by enhancing the expression of the H -ATPase activity.

Method of improving biomass yield of lactic acid bacterial cultures

The present invention provides methods for modifying cells. In particular, methods are provided which may be used to express an uncoupled ATPase activity in cells. By implementing these methods, the conversion of ATP to ADP can be increased in a cell without primary effects, e.g., on the other cellular metabolites or functions. The methods may therefore be used to increased ADP dependent metabolism, e.g., of a substrate. Accordingly, the methods of the invention are useful for improving the production of a biomass or other product of the ADP dependent metabolism of a substrate.

Growing community of inventors

Gentofte, Denmark

Peter Ruhdal Jensen