United States of America, As Represented by the Secretary of Agriculture

The United States of America As Represented by the Secretary of

Timothy D Leathers

Gregory L Cote

Neil P Price

Thomas M Hohn

James D Jones

Melinda S Nunnally

Christopher D Skory

Kenneth M Bischoff

G Thomas Hayman

Pennapa Manitchotpisit

The present disclosure provides methods for producing bioproducts from novel genetically altered strains of. Methods and materials for the construction of these strains, examination of the bioproducts and analysis and isolation of the bioproducts from genetically altered strains is provided. Genetically alteredstrains in which one or more genes encoding biosynthetic enzymes are knocked out is detailed and the benefits of using such strains described.

Methods and strains for producing bioproducts in

Compounds, called liamocins from, having the general structure in Formula 1 are disclosed. where Ris either COCHor H; and Ris between two to ten O-linked 3,5-dihydroxydecanoate; and Rcan be a polyol (e.g., L- or D-glycerol, L- or D-threitol, L- or D-erythritol, L- or D-arabitol, L- or D-xylitol, L- or D-lyxitol, L- or D-ribitol, L- or D-allitol, L- or D-altritol, L- or D-mannitol, L- or D-iditol, L- or D-gulitol, L- or D-glucitol (also called sorbitol), L- or D-galactitol (also called dulcitol), and L- or D-talitol), 2-amino-D-mannitol, 2N-acetylamino-D-mannitol, L-rhamnitol, or D-fucitol; except when Ris D-mannitol, Ris not 2 nor 3 O-linked 3,5-dihydroxydecanoate chains. These liamocins described above in addition to D-mannitol liamocin A1, D-mannitol liamocin A2, D-mannitol liamocin B1, and D-mannitol liamocin B2, alone or in combination with each other, can be used to kill certain bacteria and to treat certain bacterial infections.

Oils having antibacterial activity

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Novel compounds, called liamocins from, having the general structure in Formula 1 are disclosed. where Ris either COCHor H; and Ris between two to ten O-linked 3,5-dihydroxydecanoate; and Rcan be a polyol (e.g., L- or D-glycerol, L- or D-threitol, L- or D-erythritol, L- or D-arabitol, L- or D-xylitol, L- or D-lyxitol, L- or D-ribitol, L- or D-allitol, L- or D-altritol, L- or D-mannitol, L- or D-iditol, L- or D-gulitol, L- or D-glucitol (also called sorbitol), L- or D-galactitol (also called dulcitol), and L- or D-talitol), 2-amino-D-mannitol, 2N-acetylamino-D-mannitol, L-rhamnitol, or D-fucitol; except when Ris D-mannitol, Ris not 2 nor 3 O-linked 3,5-dihydroxydecanoate chains. These liamocins described above in addition to D-mannitol liamocin A1, D-mannitol liamocin A2, D-mannitol liamocin B1, and D-mannitol liamocin B2, alone or in combination with each other, can be used to kill certain bacteria and to treat certain bacterial infections.

Four newspp. isolates (NRRL 21966, NRRL 21967, NRRL 21968, and NRRL 21969) are capable of essentially quantitative conversion of native alternan to a polymeric modified form having a lower apparent molecular weight than native alternan. A fifth isolate (NRRL 30489) obtained from a survey of deposited organisms and classified as ahas the same ability. The modified alternan has rheological properties similar to ultrasonicated alternan and is produced without the expense of ultrasonication. It would have utility as a substitute for gum arabic for uses such as bulking agents and extenders in foods and cosmetics.

Modified alternan

Four newspp. isolates (NRRL 21966, NRRL 21967, NRRL 21968, and NRRL 21969) are capable of essentially quantitative conversion of native alternan to a polymeric modified form having a lower apparent molecular weight than native alternan. A fifth isolate (NRRL 30489) obtained from a survey of deposited organisms and classified as asubgenushas the same ability. The modified alternan has rheological properties similar to ultrasonicated alternan and is produced without the expense of ultrasonication. It would have utility as a substitute for gum arabic for uses such as bulking agents and extenders in foods and cosmetics.

The instant invention is drawn towards transformed strains of effective for the production of lycopene. The transformed strains comprise an expression cassette having three genes encoding, respectively, geranylgeranyl-pyrophosphate synthase, phytoene synthase and phytoene desaturase (i.e. Tri5crtE, Tri5crtB and Tri5crtl). The transformed strains of of the instant invention produce lycopene at levels of up to 0.5 milligrams per gram culture dry weight.

Fusarium sporotrichioides strains for production of lycopene

Four new Penicillium spp. isolates (NRRL 21966, NRRL 21967, NRRL 21968, and NRRL 21969) are capable of essentially quantitative conversion of native alternan to a polymeric modified form having a lower apparent molecular weight than native alternan. A fifth isolate (NRRL 30489) obtained from a survey of deposited organisms and classified as a Penicillium subgenus Biverticillium has the same ability. The modified alternan has rheological properties similar to ultrasonicated alternan and is produced without the expense of ultrasonication. It would have utility as a substitute for gum arabic for uses such as bulking agents and extenders in foods and cosmetics.

Penicillium isolates for modifying alternan

The instant invention is drawn towards transformed strains of effective for the production of B-carotene. The transformed strains comprise an expression cassette having four genes encoding, respectively, geranylgeranyl-pyrophosphate synthase, phytoene synthase, phytoene desaturase and lycopene cyclase (i.e. Tri5crtE, Tri5crtB, TriScrtI and CrtY). The transformed strains of of the instant invention produce B-carotene at levels of up to 3-4 milligrams per gram of fungus (dry weight).

Fusarium sporotrichioides strains for production of B-carotene

A method which combines the use of polymerase chain reaction (PCR) or oligonucleotide linkers and restriction enzymes which cleave degenerate or variable recognition site sequences to allow the cloning of multiple DNA sequences into a vector is disclosed. In this invention, a plurality of unrelated DNA sequences may be directionally cloned within a single vector by adding onto the ends of the sequences, restriction sites with specific sequences which are cleaved by corresponding restriction endonucleases which recognize degenerate or variable recognition sites and which generate cohesive ends upon cleavage. The compatibility (or ability to anneal) of the cohesive ends on different DNA sequences is controlled by the choice of the nucleotide sequence within the recognition sequences of the restriction endonucleases, allowing the DNA sequences to be inserted or joined in any desired orientation. These restriction sites may be selectively incorporated onto either or both end of any DNA sequence of interest using oligonucleotidelinkers or using PCR by adding the restriction sites onto the termini of the 5&prime; and/or 3&prime; primers.

System for the sequential, directional cloning of multiple DNA sequences

A rapid screening procedure to select microorganism strains that produce a high proportion of alternan to dextan and a high proportion of alternansucrase to dextransucrase is described. Strains selected by the method are useful to produce a high proportion of alternansucrase to dextransucrase and to produce a high proportion of alternan to dextran, either fermentatively by cultivation of the strains on sucrose, or enzymatically by using alternansucrase produced by the strains of the invention. The selected strains can also be used to obtain microorganism strains having further improvements.

Rapid screening method to select microorganism strains that produce a

A rapid screening procedure to select microorganism strains that produce a high proportion of alternan to dextran and a high proportion of alternansucrase to dextransucrase is described. Strains selected by the method are useful to produce a high proportion of alternansucrase to dextransucrase and to produce a high proportion of alternan to dextran, either fermentatively by cultivation of the strains on sucrose, or enzymatically by using alternansucrase produced by the strains of the invention. The selected strains can also be used to obtain microorganism strains having further improvements.

Growing community of inventors

Dunlap, IL, United States of America

Timothy D Leathers