CHOICE OF MICROBE

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NUTRITIONAL BOOSTERS:

NUTRITIONAL BOOSTERS

OVERVIEW:

OVERVIEW ANIMAL FEEDS LOW IN LYSINE AND METHIONINE ADDITION OF AMINO ACIDS FROM MICROBIAL SOURCES IS INEXPENSIVE. CERTAIN AMINO ACIDS SUCH AS TRYPTOPHAN AND SEROTONIN HAVE PHARMACOLOGICAL EFFECTS. MORE THAN 70,000 TONS EACH OF LYSINE AND METHIONINE. PRODUCED EVERY YEAR.MAJOR PRODUCERS-JAPAN,EUROPE,CHINA,US.

1.FROM MEAT OR OTHER PROTEIN RICH FOODS…….. SEPARARTION OF SPECIFIC AMINO ACIDS IS DIFFICULT AND EXPENSIVE!!! 2. PRODUCTION OF AMINO ACIDS SYNTHETICALLY…… DIFFICULT TO SYNTHESIZE SPECIFIC STERIOISOMERS OF AN AMINO ACID!!! 3.USE OF MICROBES AND MICROBIAL ENZYMES…….. RIGID CONTROL OVER THE OPTICAL CHARACTERSTICS AS WELL AS INEXPENSIVE METHOD!!!:

1.FROM MEAT OR OTHER PROTEIN RICH FOODS…….. SEPARARTION OF SPECIFIC AMINO ACIDS IS DIFFICULT AND EXPENSIVE!!! 2. PRODUCTION OF AMINO ACIDS SYNTHETICALLY…… DIFFICULT TO SYNTHESIZE SPECIFIC STERIOISOMERS OF AN AMINO ACID!!! 3.USE OF MICROBES AND MICROBIAL ENZYMES…….. RIGID CONTROL OVER THE OPTICAL CHARACTERSTICS AS WELL AS INEXPENSIVE METHOD!!! HOW TO OBTAIN AMINO ACIDS???

CHOICE OF MICROBE:

CHOICE OF MICROBE MICROBES CAN BE USED TO PRODUCE LARGE AMOUNTS OF AN END PRODUCT OF FERMENTATION. (e.g. ETHANOL ). REQUIREMENTS SUPPLY OF SUFFICIENT FERMENTABLE SUBSTRATES. CORRECT ENVIRONMENT.

1. MICROBES DO NOT NORMALLY SECRETE AMINO ACIDS BECAUSE THEY ARE REQUIRED WITHIN THE CELL. 2. ALSO,SECRETION OF AMINO ACIDS IS ENERGETICALLY UNWISE FOR THE MICROBES BECAUSE AMINO ACIDS SYNTHESIS TYPICALLY REQUIRES THE COORDINATED ACTIVITY OF SEVERAL ENZYMES. 3.SINCE, SYNTHESIS OF AMINO ACIDS SERVES NO PURPOSE FOR THE MICROBE SO ISOLATION OF A BACTERIUM THAT NATURALLY OVERPRODUCES AN AMINO ACID IS EXTERMELY RARE. :

1. MICROBES DO NOT NORMALLY SECRETE AMINO ACIDS BECAUSE THEY ARE REQUIRED WITHIN THE CELL. 2. ALSO,SECRETION OF AMINO ACIDS IS ENERGETICALLY UNWISE FOR THE MICROBES BECAUSE AMINO ACIDS SYNTHESIS TYPICALLY REQUIRES THE COORDINATED ACTIVITY OF SEVERAL ENZYMES. 3.SINCE, SYNTHESIS OF AMINO ACIDS SERVES NO PURPOSE FOR THE MICROBE SO ISOLATION OF A BACTERIUM THAT NATURALLY OVERPRODUCES AN AMINO ACID IS EXTERMELY RARE. BUT ….

SO….??????? TO ACHIEVE OVERPRODUCTION OF AMINO ACIDS……. WE NEED TO ARTIFICALLY MANIPULATE AMINO ACID METABOLISM…!!! :

SO….??????? TO ACHIEVE OVERPRODUCTION OF AMINO ACIDS……. WE NEED TO ARTIFICALLY MANIPULATE AMINO ACID METABOLISM…!!! CHOICE OF MICROORGANISM IS CRITICAL E . coli –AN IDEAL MICROBE.

THIS, IT IS NOT DIFFFICULT TO SEPARATE PRODUCT FROM CELLS,IF DESIRED.:

THIS, IT IS NOT DIFFFICULT TO SEPARATE PRODUCT FROM CELLS,IF DESIRED.

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AMINO ACID PRODUCTION IN E.COLI IS CONTROLLED THROUGH…….

REGULATION OF ENZYME ACTIVITY THROUGH FEEDBACK INHIBITION:

REGULATION OF ENZYME ACTIVITY THROUGH FEEDBACK INHIBITION PRODUCTION OF GLUTAMATE AND PROLINE BY Serratia marcescens AND THE ASSOCIATED ENERGY COSTS.

REGULATION OF TRANSCRIPTION:

REGULATION OF TRANSCRIPTION REGULATION OF AMINO ACID , PROLINE SYNTHESIS BY CO-REPRESSION.

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ATTENUATION A COMPLEX MODE OF REGULATION THAT ACTS DURING REGULATION

E.coli:

E.coli OVERPRODUCTION OF AN AMINO ACID CAN OCCUR ONLY IF ALL OF THESE LEVELS OF REGULATION ARE OVERCOME. POSSIBLE AND SUCCESSFULLY ACCOMPLISHED. BETTER RESULTS OBTAINED USING OTHER BACTERIA.

Serratia marcescens:

Serratia marcescens MEMBER OF ENTEROBACTERIACEAE FAMILY MUCH EASIER TO CONVERT TO AN AMINO ACID OVERPRODUCER FEEDBACK INHIBITION IS THE MOST IMPORTANT MECHANISM OF REGULATION OF AMINO ACID SYNTHESIS.

PROLINE: MUTAGENESIS LEADS TO OVERPRODUCTION:

PROLINE: MUTAGENESIS LEADS TO OVERPRODUCTION PRODUCED THROUGH A RELATIVELY SIMPLE PATHWAY THAT USES GLUTAMATE AS THE INITIAL SUBSTRATE. IN DEVELOPING A STRAIN OF S.MARCESCENS THAT OVERPRODUCES PROLINE…. OBSTACLE TO OVERCOME FEEDBACK INHIBITION. HOW??? MUTANTS WERE PLATED ONTO MEDIA CONTAING PROLINE ANALOGS….WILD TYPE CELLS---DIED AS THE ANALOGS SHUT DOWN PROLINE BIOSYNTHESIS THROUGH FEEDBACK INHIBITION. MUTANTS SURVIVED----OVERPRODUCED PROLINE.

STEPS LEADING TO OVERPRODUCTION OF PROLINE:

STEPS LEADING TO OVERPRODUCTION OF PROLINE 1.a) THE Put GENE THAT CODES FOR THE ENZYME,PROLINE OXIDASE THATDEGRADES PROLINE WAS DELETED FROM THE CHROMOSOME OF S.MARCESCENS. b ) THIS ENZYME ALLOWS TO SCAVENGE PROLINE FROM ITS ENVIRONMENT AND USE IT AS A CARBON AND NITROGEN SOURCE. c ) THIS IS ENZYME IS UNDESIREABLE AND UNNECCESSARY IN A PROLINE OVERPRODUCER.

Slide 20:

2.a ) GROWTH OF S. MARCESCENS IN A HIGH SALT MEDIUM RESULTS IN FURTHER INCREASE IN RATE OF PROLINE PRODUCTION. b ) IT USES PROLINE AS AN OSMOPROTECTANT WHEN GROWING IN OSMOTICALLY STRESSED ENVIRONMENT. c ) BACTERIA PRODUCED 60-75 g/l OF PROLINE AND 20 % OF THE CARBON FLOW IS DIRECTED TO PROLINE PRODUCTION. osmoprotectants accumulate within cells to high concentration without adversely affecting cellular metabolism. it decreases water activity and prevents water dehydration.

3. RECOMBINANT TECHNIQUES WERE APPLIED IN WHICH GENES CODING FOR PROLINE SYNTHESIS WERE CLONED AND INSERTED INTO PLASMIDS. THEY WERE MAINTAINED IN HIGH COPY NUMBER THAT RESULTED IN A FURTHER 50-60% INCREASE IN THE RATE OF PROLINE PRODUCTION.:

3. RECOMBINANT TECHNIQUES WERE APPLIED IN WHICH GENES CODING FOR PROLINE SYNTHESIS WERE CLONED AND INSERTED INTO PLASMIDS. THEY WERE MAINTAINED IN HIGH COPY NUMBER THAT RESULTED IN A FURTHER 50-60% INCREASE IN THE RATE OF PROLINE PRODUCTION.

Slide 22:

LYSINE, AN ESSENTIAL AMINO ACID USED TO SUPPLEMENT CEREALS AND BREADS, WAS ORIGINALLY PRODUCED IN A TWO-STEP MICROBIAL PROCESS. THIS HAS BEEN REPLACED BY A SINGLE-STEP FERMENTATION IN WHICH THE BACTERIUM Corynebacterium glutamicum , BLOCKED IN THE SYNTHESIS OF HOMOSERINE, ACCUMULATES LYSINE. OVER 44 G/LITER CAN BE PRODUCED IN A 3 DAY FERMENTATION. LYSINE

METHIONINE:

METHIONINE A RECOMBINANT MICROORGANISM COMPRISING GENETIC ALTERATIONS IN EACH OF AT LEAST FIVE GENES CHOSEN FROM HOM/ BR , METX, METY, METB, METH, METE, METF AND ZWF, WHEREIN THE GENETIC ALTERATIONS LEAD TO OVEREXPRESSION OF THE AT LEAST FIVE GENES, THEREBY RESULTING IN AN INCREASED METHIONINE PRODUCTION BY THE MICROORGANISM RELATIVE TO THE METHIONINE PRODUCED IN ABSENCE OF THE GENETIC ALTERATIONS IN THE AT LEAST FIVE GENES. MICRORGANISM IS CORYNEBACTERIUM GLUTAMICUM METHIONINE, ALPHA-L-AMINO-GAMMA-METHYLTHIO-N-BUTYRIC ACID, IS NUTRITIONALLY ESSENTIAL FOR MAMMALS AND FOWLS. IT CANNOT BE SYNTHESIZED INTERNALLY, BUT MAY BE ADDED TO FOOD AND FEED MATERIALS TO IMPROVE THE PROTEIN QUALITY (PHAM ET AL., 1992). METHIONINE, IN MEDICINE AND PHARMACEUTICALS, IS USED AS AN ANTIDOTE IN PARACETAMOL TO PREVENT LIVER DAMAGE AND ALSO IN TREATMENT OF PARKINSON’S DISEASE

Nutrition Boosters Foods and preparation methods that increase nutrient density and digestibility:

Nutrition Boosters Foods and preparation methods that increase nutrient density and digestibility Grandma knew best

hELPFUL sUPPLEMENTS:

Digestive Enzymes Probiotics Cod liver oil/fish oil/EFAs Magnesium Calcium Zinc B6 Vitamins A, C, and E CoQ10 Glutathione/NAC Methylation: B12, folinic or 5MTHF, TMG/DMG Transfer factor Amino acids hELPFUL sUPPLEMENTS

GOOD WAYS TO BOOST NUTRIENT LEVELS:

GOOD WAYS TO BOOST NUTRIENT LEVELS Cook and puree orange vegetables (or any). Freeze in ice cube trays and add to smoothies Cook and puree any vegetables and add to meatballs, meat patties, meatloaf, or pasta sauce Cook allowable grains or gluten-free pasta in homemade broth Nettles can be consumed as a tea, or added to a homemade broth Seaweed - Add kombu or other sea vegetable to cooking grains, soups, tomato sauce, even boiling pasta

JUICING:

JUICING Higher concentration of nutrients Chlorophyll and phytonutrients Get nutrients without needing to eat/chew vegetables Children that like liquids, juices and smoothies Add vegetable juice to smoothies. Add a bit of fruit to vegetable juice for flavor or added sweetness Add supplements to vegetable juice (instead of fruit juices) Preparation tip Start with Add as you evolve taste Go cautiously (high sugar) Cucumber Celery Fennel Ginger Lemon Green apples Parsley, cilantro Kale or other greens Cabbage (ulcers) Cranberries Carrot Beet Fruit: Apple, pear

FERMENTED FOODS – RICH IN PROBIOTICS:

©Julie Matthews, CNC 2007 FERMENTED FOODS – RICH IN PROBIOTICS Dairy-free: Raw sauerkraut Beverages (contain yeast that kills candida): Kombucha Coconut juice kefir “Sodas” (hibiscus/rosehip tea with kefir starter) Nut milk yogurt Dairy: Milk-based yogurt/kefir

COMPANIES AND THEIR PRODUCTS:

COMPANIES AND THEIR PRODUCTS NUTRABIO RIBOSE- -BIOENERGY D-RIBOSE.INCREASES MUSCLE AND HEART ENERGY.IMPROVES ATHLETIC PERFORMANCE. SPEEDS MUSCLE RECOVERY. INCREASES ENERGY RESERVES. NUTRABIO L-ARGININE ETHYL ESTER DIHCL- PROMOTES INCREASED NITRIC OXIDE (NO) PRODUCTION FOR ENHANCED BLOOD FLOW AND BETTER DELIVERY OF OXYGEN AND NUTRIENTS TO WORKING MUSCLE NUTRABIO BCCA 5500 - IMPROVES PROTEIN SYNTHESIS IMPROVES NITROGEN RETENTION SPEEDS MUSCLE RECOVERY BOOSTS MUSCLE GROWTH

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Hubei Xinyuan Biotechnology Co., Ltd. Hanlong Amino-Acids Hubei Baofeng Bio-chemical Co., Ltd HENOGEN Chengdu Baishixing Science and Technology Industry Co., Ltd. Langen Suzhou Co., Ltd

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THANK YOU

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