Enzyme technology: from discovery of enzymes to development of

production strains

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What are enzymes?

Enzymes are:

– biological catalysts in the form of proteins, which drive chemical reactions in living cells

– fully biodegradable, replacing chemicals in industrial production

– reducing resource usage and increasing efficiency

Enzymes as Industrial Products

• Exoenzymes

– Enzymes that are produced in such large amounts that

they are excreted into the medium instead of being held

within the cell; they are extracellular

– Can digest insoluble polymers such as cellulose, protein,

and starch

• Enzymes are useful as industrial catalysts

– High substrate specificity

Enzymes as Industrial Products

• Enzymes are produced from fungi and bacteria

– Bacterial proteases are used in laundry detergents (can also

contain amylases, lipases, and reductases)

• Isolated from alkaliphilic bacteria

– Amylases and glucoamylases are applied in 1. generation

bioethanol (starch based ethanol)

• Isolated from filamentous fungi

– Cellulases are applied in 2. generation bioethanol

• Isolated from filamentous fungi

Industrial Enzymes:

An enzyme that can be produced in yields sufficient to use in large scale industrial processes in such a way as to be relatively pure and economical to produce.

>98% of such enzyme products are secreted enzymes

Microbial Enzymes and Their Applications

Enzymes as Industrial Products

• Extremozymes

– Enzymes that function at some environmental extreme

(i.e., pH or temperature)

– Produced by extremophiles

e.g. Acid-Tolerant Enzymes

Figure 25.15b

Examples of Extremozymes: Thermostable Enzymes

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Strategies

1. Screening for enzyme activities

a) classical

b) metagenomic

2. Other screening (e.g. signal trapping)

3. Targeted gene finding

a) in genomic data

b) in cDNA libraries

4. Synthetic biology

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Enzyme discovery

The first step in enzyme discovery is to determine the operating conditions of the industrial enzyme.

This includes:

Is the enzyme intended to improve the performance of an existing product?

Is the enzyme intended for a new application?

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Enzyme discovery

Important parameters that should be addressed for the enzyme:

Importancy of the stability of the enzyme.

Identification of potential inhibitors that can inhibit the enzyme activity.

The activity and specificity of the enzyme.

Temperature profile in the operating conditions.

pH optimum in the operating conditions.

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Classical screening for enzymes

Examples:

Enzymes for detergent use must show optimal performance at high pH and temperatur of 20-50oC.

Some feed enzymes must show extreme stability at high temp. because they are formulated together with the feed. At the same time they have to resist low pH because of the animal stomach.

For biomass conversion, enzymes are interesting that work at high temperature because they in general show higher stability and higher efficiency. This will reduce the cost of enzyme production.

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Classical Screening

Classical microbial screening is based on culturing a broad diversity of microorganisms – based on a variety in

taxonomy

phylogeny

physiology

biochemistry

ecology

The trick is to make a screening strategy that reflects the desired properties of the enzymes or organisms

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Exotic microorganisms from exotic envirnonments?

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Microbial Biodiversity

• only the tip of the iceberg is known so far

• 1 g soil contains > 4000 different genomes

• so far only 1-5 % of all microbes have been cultured

• 3,5 billion years of evolution

• all ecological habitats are populated with microbes

• adaptations to all environmental challenges

• enormous potential of yet undiscovered phyiological & biochemical traits

• Bacteria

• Fungi

Described Species

– 72.000

– 4.000

Estimated Species

– 1.500.000

– 2-3.000.000

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Environmental sampling

Collection of environmental samples: • Enrichment in the lab • Enrichment in situ • Isolation of microbes • Pure cultivation in the Laboratory

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Culture collections

The DSMZ microbial collections contain over 13 000 cultures representing some 6 900 species and 1 400 genera (Archaea, Bacteria, plasmids, phages, yeasts, fungi). Unique subcollections are held in the prokaryotes groups of acidophiles, alkaliphiles, halophiles, methanogens, myxobacteria, phototrophs, thermophiles, and sulfate reducers. Selected strains for antibiotic testing, quality control, bioassay, bioremediation, and strains with certificate are included.

The ATCC Bacteriology Collection is the most diversified assemblage of prokaryotes in the world, containing nearly 18,000 strains in more than 750 genera. Every important prokaryotic physiological group is represented. The Collection holds more than 3,600 type cultures of validly described species, forming the basis for systematic bacteriology, and nearly 500 bacteriophages.

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Classical screening

Secreted enzymes from microorganisms (fungi, bacteria and archaea) have the highest probability to live up to the often extreme conditions.

In contrast to intracellular enzymes, the secreted enzymes have evolved to work in (often extreme) environments by natural selection over thousands of years.

For a ”typical” microbial genome ,1-3% of the genes encode secreted

proteins

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Screening for enzyme activity

There are several parameters that can be tested for:

Different substrates

Natural substrates

Synthetic substrates

Incubation conditions

Different Temperatures

Different pH

Different methods to test enzyme activity

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Screening for enzyme activity

Growth conditions inducing enzyme production Growth on selective media

Agar plates 96wells 384wells 1536wells

Detection of enzyme activity

High through put

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Screening for enzyme activity

A common way is development of a simple assay: e.g. We are interested in identifying microorganisms that are able to efficiently

degrade the polysaccharides in Sugarcane Bagasse to monosaccharides. Therefore we are interested in identifying e.g. fungi that express high

amounts of cellulases, and xylanases when they grow on Sugarcane Bagasse.

We therefore constructed a medium containing Sugarcane Bagasse as the

only carbon source, and identified a specific set of parameters (fixed temperature, pH, cultivation time) for the screening process.

Under these conditions we screened hundreds of fungi in order to identify

the best enzyme producers.

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Identification of good enzyme producers

During the first screening program, several good enzyme producers were identified.

Next step is to identify more specific parameters for the enzymes of interest. This could be conditions under which the enzymes are intended to function: High temperature (e.g. 50-55oC) Low pH (< 4.5) High pressure (2 bar) Environment with many proteases The second screening setup could therefore include these parameters in order to

identify the best or the 5-10 best enzyme producers.

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Development of better enzyme producers

During the second screening setup a few microorganism are identified, that are able to grow under the conditions selected.

Some are growing well, but do not produce enzymes in high yields.

Some do not grow well, but produce good yields of enzyme compared to the growth rate.

Next step could be to mutate the selected strains to grow better and produce enzymes in higher amounts.

Different possibilities exists in order to make mutations in the genome of a microorganism.

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Genome walking

Isolation of fungi from different habitats

Isolation of genes encoding selected enzymes

Cloning and expression of selected genes in a relevant host

Enzyme activity studies

Evaluation of biomass conversion efficacy of enzymes

Screening of fungi for enzyme activity

Enzyme discovery for biomass conversion

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Production

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Improving strategies

Three different strategies for improving the properties of individual cellulase components:

1) Mutagenesis of cells or spores by UV or chemicals.

2) Mutagenesis of specific genes using ”Rational design” in which detailed knowledge to the protein structure and structure-function relationship is required (lecture in Fungal Biotechnology).

3) Mutagenesis of specific genes using ”Directed evolution” (irrational design) in which no knowledge to the protein structure and structure-function relationship is required (lecture in Fungal Biotechnology).

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Elevate the yield by classical mutagenesis

Selected transformants are mutated by:

• UV light.

• Gamma light.

• Chemical mutagenesis, e.g. nitrosoguanidine.

Mutants is tested for:

• Higher yield.

• Loss of specific enzyme activity.

• Loss of specific metabolites.

• Changed morphology.

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1 pH 12

100oC

5oC

Higher temp. tolerance Higher stability Different pH optimum

Low temp. tolerance Low stability

Evolution by mutagenesis

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Screening for good growth and high yield producing mutants

UV

Mutagenese of spores or cells

growth i microtiter plates

isolation of good mutants

test in shake kolbs test i lab.tanks

assay

test i pilot / production

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Why use recombinant technology to enzyme production

Many enzymes cannot be produced in sufficient amounts by classical methods

Many donor organisms are not ideal for fermentation in steel fermentors

Using a few selected host organisms it is possible to develop a high expertise in handling these in such a way that they will be ideal for production

The host strain can be modified in such a way that only the desired product is produced.

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Classical Screening

Advantages:

The biodiversity is enormeous in nature - new organisms and enzymes can be found.

Culture collections contain many organisms – better enzymes can be found.

Gene banks contain many genes – information in order to develop variants – e.g. better enzymes.

Disadvantages:

Time consuming process – easy to

overlook superior organisms ”needle in a haystack”.

It is expensive to keep a culture collection.

Large collection of genes – often lack of heterologous systems for gene expression.

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Fungal genome sequencing projects

www.cazy.org

Mycoparasites Cellulolytic fungi Plant pathogens/ molds Mycorrhiza White rot fungi Brown rot fungi Yeasts Truffle (eatable)

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Fungal genome sequencing projects BG CBHs EGs

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The uncultivated world

Molecular, culture-independent methods has revealed a microbial diversity in soil and sea water that was unknown 2 decades ago.

New divisions of bacteria and Archaea has been discovered and it is thought that only less than 1 % of prokaryotes in soil are cultivable.

Also new fungi has been discovered.

Molecular biology methods provides powerful new approaches to study the microbial diversity in the biosphere.

Metagenomics

Metagenomics is the genomic analysis of the collective genomes of an assemblage of organisms (enviromental genomics, community genomics).

Metagenomic libraries contains DNA extracted directly from the environment providing sequences, giving phylogenetic and functional information.

Phylogenetic markers (e.g. 16S rRNA for bacteria, 18S or ITS rRNA for fungi) provide indications of the origin of the DNA.

Microbial Species Diversity

Figure 23.1

High Species Richness and Low to Moderate Abundance

Low Species Richness and High Abundance

Classical versus metagenomic screening

Lorenz et al., 2002.

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Cloning of genes (lecture in Fungal Biotechnology )

Grow culture

Isolate DNA Restriction enzyme digest Ligate into

plasmid vector

Transformation of host bacterium

Transformation of fungal host

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Proteins

Protein P 1

Protein P 2

Signal Sequence Secreted

Not secreted

Expression vectors

• A selection marker in order to select Aspergillus transformants is necessary

• amdS: acetamid to acetic acid and ammonium • Function as selection marker if no other ammonium is present.

•A strong promoter is necessary. •Amylase hybrid promoters are very strong artificial

promoters.

• A terminator is necessary in order to terminate gene transcription

• Tamg is a strong terminator

Expression vector: • Well characterised • Minimal in size • Allow efficient expression

plasmid 7643 bp

E. coli selection

Gene of interest

amdS

Promoter

ColE1 origin

Tamg

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Optimisation of protein expression

Protein P 1

Protein P 2

Protein P 3

host 1 host 2 host 3 host 4

Different promoters / Different signal sequences / host combinations Evaluation of yield

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Screening for growth and high yield producing transformants

growth i microtiter plates

isolation of suitable transformants

test in shaking flasks

test i lab tanks

assay

test in pilot / production

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SDS-PAGE of transformants grown in shake flasks

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Strain characterization in lab fermentors

• Tank size 2 – 10 L

• Computer control and data

• Model experiments in well defined medium

• Downscaling of production fermentation methods

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Fermentation proces

• Use of GM microorganisms

• GM microorganisms are used as cell factories for production of enzymes

• GMOs (production strains) are only helpers in the process

• The product is separated

from the GM microorganism

in order to make a clean product

without GMO.

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Diagram for enzyme production

Harvest and test extracellular enzymes

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1.5 ml

10,000 rpm; 5 min

1 ml

15 µl

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AZurine-CrossLinked Polysaccharides - how does it work?

Remazolbrilliant Blue R

AZCL substrates for enzyme assays

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AZCL-Cellulose – an example

1. Aspergillus – CF - agar

2. Fusarium – CF - agar

3. Penicillium – CF - agar

4. Trichoderma – CF - agar

5. Aspergillus – SB - agar

6. Fusarium – SB - agar

7. Penicillium – SB - agar

8. Trichoderma – SB - agar

9. Aspergillus – SBP - agar

10. Fusarium – SBP - agar

11. Penicillium – SBP - agar

12. Trichoderma – SBP - agar

13. Aspergillus – CF - liq

14. Fusarium – CF - liq

15. Penicillium – CF - liq

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Comparison of different enzyme profiles

arabinan

cellulose xylan

galactan

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T. asperellum Enzyme production on different substrates

Results from transcriptome analysis

Comparison between the transcriptome based secretome and wheat bran composition

Conclusions from the paper

• Wheat bran was the best medium for enzyme production

• In total 175 glycosyl hydrolases from 48 GH families were produced