1

TBT4170v2015

Kapittel 4White Biotechnology:

Cells as Synthetic Factories

Part 2

2

4.9 Chemical Synthesis Versus Microbial Production

In Japan glutamate was first chemically synthesized from acrylonitrile usingcobolt carbonyl catalysts in what is know as Strecker synthesis

Lysine was produced chemically from aminocaprolactam

Challenge: the chemical process results in equal amounts of D and L forms (racemic mixtures) - only L-glutamate provides the desired taste

----------------------

Acetone-Butanol-Ethanol (ABE) fermentation using Clostridium acetobutylicumintroduced during 1st. World War, especially acetone was limiting for productionof cordite (a mix of nitroglycerin and cellulose) (Check out Box 6.6 p. 188 and Ch. 6.9).- large industry until 1940-50s when prices for petrochemicals fell below those ofstarch and molasses, but this might change in the future?

5

4.10 L-Ascorbic Acid (Vitamin C)Chemico-Biotechnological method

6

Recent developments of pure biotechnological methods – Genetic/ MetabolicEngineeringErwinia sp

convert glucoseto 2,5 diketo-D-gluconic acid in a three enzymeprocess

Corynebacteriumsp has a 2,5 diketo-D-gluconicacid reductasethat convert2,5DKG to 2-keto-L-gluconic acid, that caneasily be turnedinto a vitamin C ring molecule

-2,5DKG reductaseheterologousexpressed in Erwinia The recombinant Erwinia cells produce about 120 grams (?) of 2-KLG

within 120 hours with a glucose conversion rate of 60%

7

Reading material:Box 4.5 Biotech History: Vitamin C and Reichstein’s Fly

Daily recommendation: 150 mg Vit C

1936: Roche sold appr 370 kg vitC for 1140 CHF/ kg1938: 550 CHF/ kg, 1940: 390 CHF, 1950: 102 CHF, early 60’ies: 80 CHFCurrently: 20 CHF/ kg (i.e. 50 times cheaper than 70 years ago)Today:$ 600 million marked

Most other vitamins are produced by purely chemical processes, e.g the carrotpigment -carotene and astaxanthin (salmon feed additive), but biotechnologicalmethods are available, challenge: to be economically competitive to the chemicalprocess

Industrial strains of Pseudomonas denitrificans and Propionibacterium shermaniiproduce cobalamin, a precursor for cyanocobalamin – vitB12, at a rate > 50 000 times of wild type strains (see later 4.13 how this is obtained) – culture is heatedin presence of cyanide, resulting in cyanocobalamin

The fungus Asbya gossypii, a filamentous ascomycete, produces riboflavin (Vit B2). Industrial strains produces > 20 000 times more than wild typeRecombinant Bacillus subtilis is an alternative (see next four slides – not pensum)

4.11 Aspartame – Sweet Success of a Dipeptide Ester• Is a methyl ester of the two amino acids aspartate and phenylalanine, both can

be produced by bacteria or enzymatically, and then bonded chemically• 200x the sweeting power of cane sugar, 4 kcal/g which is less than one

hundred of sugar• Challenge: starts decompose after 6-9 months, and it is more expensive than

saccarin and enzyme-produced fructose• Can aspartame be produced heterologously by recombinant bacteria at a

lower price?• Worldwide production was 14 000 tons, worth $ 859 million in 2004

4.12 Immobilized Cells Producing Amino Acids and Organic Acids

• Deep frozen Escherichia coli cells enclosed in a gel synthesized 600 tonsaspartate from fumaric acid

• 50% decrase in activity after 120 days, compared to ten days for free cells• Production with immobilized cells cost 60% of free cell production• 2 tons in 1000 l reactor/ day• Partially membrane disruption, substrates can be transported into cells,

enzymes remain stable• Alcohol production with yeast

13

4.13 Mutations as A Way of Targeting Microbial ProgrammingUndesirable traits of the wild type must be eliminated, desirable propertiesenhanced, and perhaps even new properties added- See slide 19-21 Part 1

Natural mutation frequency low, mutagens (e.g. nitrosoamines) added to cell population,-> targeted selection strategies to isolate mutants over producingrequested biochemical (usually several rounds of mutagenesis

13

Box 4.7

14

Reading material:Box 4.8 Biotech History: Alexander Fleming, Penicillin and theBeginnings of the Antibiotics Industry

4.14 Penicillium notatum – Alexander Fleming’s Miraculous Fungus

- discovered by accident?

Louis Pasteur: Dans les champs de l'observation le hasard ne favorise que les espritspréparés. In the fields of observation chance favors only the prepared mind.

Lecture, University of Lille (7 December 1854)Alternate translations of this or similar statements include:Chance favors the prepared mind.Fortune favors the prepared mind.In the field of observation, chance favors the prepared mind.Where observation is concerned, chance favors only the prepared mind.

15

-lactams

Over 100 -lactams, mostly penicillins and cephalosporins, have been approved for human use and they account for over half of the antibiotics produced worldwide.

They target the synthesis of the bacterial cell wall peptidoglycan (inhibit the crosslinking transpeptidation reaction).

Penicillin was discovered by Fleming in 1928 following his famous observation of an inhibitory zone surrounding a fungal contaminant, Penicillium notatum, on a plate of Staphylococcus aureus.

Penicillin exhibits the properties of a typical secondary metabolite, being formed at or near the end of exponential growth phase

P. notatum produces small amounts of penicillin, 1 mg/ l in solid-state fermentations. A 20-25 fold increase in yield was achieved when corn steep liquor was incorporated into the fermentation medium. Even greater yields were obtained with P. chrysogenum.

16

Further increases in yield were achieved when production went over to submerged fermentation. Since 1940s, penicillin yield and fermentation has been vastly improved by extensive (random) mutation and selection of producer strains. Penicillin fermentations now produce yields in excess of 50 g/l , a 50 000-fold increase from the original isolate.

Not pensumPenicillin production is usually via a fed-batch process carried out aseptically in stirred tank fermentors of 40 to 200 m3, although airlift systems are sometimes used. The oxygen level is very important and must be maintained at a rather constant level which is difficult since the viscosity increases as the fermentation progresses.

Carbon sources: glucose, lactose, sucrose, ethanol and vegetable oils. In the past, a mixture of glucose and lactose was used, the former producing good growth but poor penicillin yields while the latter had the opposite effect.

Corn steep liquor is used as nitrogen source, additional nutrients and side-chain precursors.

Inoculum development is usually initiated by adding lyophilized spores to a small fermentorat a concentration of 5x103 spores/ml. Fungal mycelium can then be grown up through one or two further stages until there is sufficient to inoculate the production fermentor.

19

Reading material:Box 4.9 How Penicillin Works – Enzyme Inhibitors as MolecularSpoilsports

Penicillin prevents the formation of peptid crosslinks in the bacterial cell wall, by posingas a perfect imitation pf peptide bridge

Consequence: cell walls becomes weakerand cannot withstand the osmotic pressure anymore

20

Reading material:Box 4.11 BiotechnologicallyProducedAntibiotics – Sites and Modes ofAction

21

4.15 Screening – Biotechnologists In Search of Molds

- for novel bioactivities- for high performance strains

Some Actinomycetes bacteria–not only beautiful, they kill other bacteria

Filamentous morphology Collection of carotenoid producing bacteria

22

4.16 What’s On the Microbial Menu?

Extreme metabolic diversity in the Microbial World

Monomers like sugars, amino acids and fatty/ organic acids are usually easilyconsumed.

Polymers like starch, cellulose, proteins are more challenging since they must be broken down to mono-/di-mers before taken up by the microbes.- Extracellular ensymes (eg. Amylases, proteases) must be synthesized and

excreted (external digestion process), only a limited fraction of microbescarries the property to do this

Reading material:Box 4.10 Preserving Techniques – Heat, Frost, and the Exclusion ofAir

23

4.17 A Modern Biofactory

Box 4.12 Bioreactors –Creative Space for Microbes

24

25

4.18 Heat, Cold, and Dry Conditions Keep Microbes at Bay

Heat – to sterilize reactors and other equipement, to prevent contamination, alsofor preservation of food (canning, pasteurization)

Use overpressure to prevent entry of contaminating organisms

Cold – also used to stop microbial growth, but does not kill them, used to store microorganisms (in 10% glycerol, -80°C)

Water – also needed for microorganisms

pH – both for prevention of microbial growthbut also used for selective growthconditions

Not pensum

26

4.19 Downstream ProcessingNot pensum:

Unit operations:Adsorption, centrifugation, chromatography, crystallisation, dialysis, distillation, drying,evaporation, filtration, flocculation, flotation,homogenisation, humidification, microfiltration,milling, precipitation, sedimentation,solvent extraction, and ultrafiltration

The purpose of downstream processing is to concentrate and purify product for sale

Sequence of steps:• Cell removal: - filtration, centrifugation• Primary isolation:- remove components with properties significantly different

from those of product, adsorption, liquid extraction, precipitation• Purification:- highly selective unit operation, chromatography, ultrafiltration,

fractional precipitation• Final isolation:- crystallization, centrifugation, filtration, drying

27

4.20 Streptomycin and Cephalosporins – The Next Generationof Antibiotics

After discovery and introduction ofpenicillin for treatment of Gram positivebacteria - triggered and intense searchfor other natural antibiotics

Two motives- Penicillin – not active against Gram negative bacteria- it turned out that some Gram positive are or can become resistant to penicillin

Selman Abraham Waksman, Rutgers University«Waksman took a novel and systematic approach, and unlike Fleming whose discovery of penicillin was more or less a chance find, he and his students grew a series of isolated soil microbes on agar plates growing under a variety of culture conditions. They screened them by looking for growth inhibition zones surrounding single microbial colonies.- Isolated a new antibiotic (streptomycin) produced by actinomycetes of the Streptomyces genus

28

Giuseppe Brotzu, Institute of Hygiene, Cagliari, Italy- discovered the mold Cephalosporium acremonium (renamed to Acremonium

chrysogenum) produced several antibiotics inhibiting a whole host of bacteria- one of them, Cephalosporin C,

proved particularly effective againstpenicillin-resistant Gram positivepathogens

Key numbers:Up to 8000 antibiotics have beenisolated from microorganisms,4000 from higher organisms

Over 60 000 tons of penicillinsand cephalosporins are producedper year worldwide

Box 4.13 Primary and Secondary Metabolites

29

4.21 The Race Against Microbial Resistance

Resistance mechanism to penicillin:- synthesis of -lactamase that break up the -

lactam ring in penicillin or cephalosporin intoinactive acids

One successful solution: - production of semisynthetic antibiotics

But, it is a continuous battle:- antibiotics is an evolutionary challenge for the

microbes, - with mutation, selection, survival ofthe fittest, and of the best-adapted

Bacteria exchange plasmids that carry genes for resistance

4.22 Cyclosporin – A Microbial Product Used in Transplants

From 1957, employes at the Swizz pharmaceutial company Sandoz, were asked to bring back a plastic bag with soil samples from all over the world

1970: the fungus Tolypocladium inflatum was isolated from soil sampled from Wisconsin, USA and Hardangervidda, Norway- the isolated substance (24-556) was, disappointingly, only slightly active against

other fungi- but it was only slightly toxic for lab animals, probably the reason why it underwent

further testing- 1972: the compound suppressed the human

immune system

Cyclosporin is synthesized by a nonribosomal peptidesynthetase – eleven aminoacids

Cyclosporin A: binds to specific receptors in T-lymphocytes, leading to blockade of interleukin-2 (required for activation ofthe immune system) production

31

Reading materialBox 4.14 Biotech History: Mexico, the Father of the Pill and the Race for Cortisone

4.23 Steroid Hormones – Cortisone and the Contraceptive Pill

Merck & Co developed a 37 steps protocol for production of cortisone from bovine bile acid, $260/g

Through microbial hydroxylation by the fungus Rhizopus arrhizus it was possible to cut down to 11 steps, $ 9,70/g- More environmental/ sustainable process: ambient temperature and pressure,

less usage of non-hydrous solvents, finally $ 1,30/g

Furthermore, mexican supplier of diosgenin (starter substance, isolated from wildyam) increased the price ten-fold- Soon (two years) the pharmaceutical industry had developed alternative

bioproduction processes (microbial degradation of byproducts from soy beanproduction, the steroids sitosterol and stigmasterol)

«In the coming decades, we will see more combined biological and chemicalprocesses» Sustainable Chemistry