CHAPTER 3.

PRODUCTION OF ENZYMES:
FERMENTATION AND GENETIC ENGINEERING
Henry Eric Spinnler

To cite this version:

Henry Eric Spinnler. CHAPTER 3. PRODUCTION OF ENZYMES: FERMENTATION AND GE-
NETIC ENGINEERING. Selim Kermasha; Michael N.A. Eskin. Enzymes : Novel Biotechnological
Approaches for the Food Industry, 1, Elsevier, 2021, 978-0-12-800217-9. �10.1016/C2013-0-13507-6�.
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BIOTECHNOLOGICAL APPROACHES FOR THE USE OF ENZYMES IN FOOD INDUSTRY

Selim Kermasha & Michael N.A. Eskin

(Editors)
-----------------------------------------------

Henry-Eric Spinnler

AgroParis Tech, Paris, France

CHAPTER 3. PRODUCTION OF ENZYMES: FERMENTATION AND GENETIC ENGINEERING

Abstract
Producing a pure enzyme requires it to be overproduced. To obtain this overproduction, several
drawbacks must be overcome. The first drawback is the amplification of a strains capacity to use the
biological machinery, to produce the targeted enzyme as the major protein of the metabolism. For
ease of recovery, the secretion of the enzyme is researched. Consequently, organisms having specific
post-translational systems of protein secretion are preferred. These systems are particularly well
developed in fungi and as such, are preferred for industrial scale production. Moreover, fungi are
able to grow at low pH and easily cultivated in liquid medium and/or by solid state fermentation.
The recovery, the purification and the formulation of the protein preparation to maintain the
enzyme activity, for as long as possible, are also described in this chapter.

3.1. Introduction

Because enzymes are largely used as processing aids in food production, in washing
powders and in chemical manufacturing, their production by fermentation has been seen as
an opportunity to produce enzyme concentrates on a large scale. The progress in genetic
engineering and DNA edition has been used to build strains with a modified genome
particularly efficient for these production processes. One of the goals has been to maximise
the specific enzymatic activity of certain strains. This has been done by incorporating a gene
or increasing the number of copies of the gene coding the enzyme protein sequence, into
the genome. Another step has involved the overexpression of these genes in the
transcription phase to obtain increased enzymic activity. However, as these operations have
been intracellular, a third goal has been to improve the efficiency of the enzymatic recovery.
This has been done by selecting organisms that have the capacity to secrete the protein
outside the cell, into a culture broth. Figure 3.1 shows the key steps in a process using
specific organisms. Bacteria such as Bacillus sp. (Cai et al., 2019) and fungi like Trichoderma
reesei, Aspergillus orizae or Yarrowia lipolytica (Archer and Peberdy, 1997) are species
commonly used. By increasing the enzyme titre produced, it has reduced the risk of

1
unexpected secondary activities in the culture broth and has facilitated an efficient process
of enzyme extraction/ purification, which is a fourth step. The fifth step which is not the
easiest has been to harvest and stabilise the enzyme produced.

Due to their ability to secrete the protein outside the cell, manufacturing companies have
prefered using organisms mentioned above. In addition, companies have controlled and
optimized the culture media used for growing these organisms and, it has been done for
safety reasons (no toxins produced), optimising harvest conditions and for general ease of
use for industrial scale production. This chapter will attempt to cover the different aspects
of this process so as to facilitate the commercialisation of these enzymes.

Figure 3.1

3.2. Enzyme Screening

Several steps are necessary to achieve efficient enzyme production but the first is to
develop an efficient screening method to easily select strains of micro-organism producing
the researched enzyme.

Traditional systems using screening on agar plates were shown to be efficient. This has now
been surpassed when automatic microplate readers with a capacity to read over ninety-six
wells became available. With both systems the main challenge was to obtain a semi-
quantitative indication of the enzyme activity, obtained via a signal such as a colour reaction
for example. If necessary, a quantitative measurement of the activity could be obtained
subsequently, by more accurate and reliable systems. This could include labelled substrates
such as fluorogenic and chromogenic substrates that generate a reporter molecule by -
elimination, fluorescence resonance energy transfer (FRET) substrates and isotopic labels for
screening of enantioselectivity.

Depending on the enzyme researched, several strategies to detect activity have been used
(Goddard and Reymond, 2004). These include:

- using a substrate releasing a chromophore or a fluorophore when the activity is present,

as is the case for sugar hydrolase (with paranitrophenyl glycosides), proteases (with
azocasein), peptidases (using para nitro-anilide derivated peptides or amino-acids
releasing a coloured nitro-aniline when a peptidase was present).

- using a specific reactivity of the reaction product (e.g. CuCl2 because Cu++ reacts with
fatty acids, resulting in a bright blue colour, therefore enabling the detection of lipolitic
activities)

- using pH indicators when pH change is expected and

- using fluorochromes such as Sybr Green or coumarin derivatives (Goddard and Reymond,
2004)

2
3.3. Microbial Strain Selection

When an efficient screening method is usable it becomes easier to screen culture

collections. At first a diversity of wild type strains has to be screened to obtain high yielding
strains. However, the ability to modify genomes opened up possibilities in the second part
of the 20th century. Several companies used this technology to create strains dedicated to
the production of specific enzymes. In addition, fungi were preferred in the process due to
their ability to grow in low pH environments. Fungi were used to limit the growth of ‘short
generation time’ bacterial contaminants that usually grow at neutral pH.

3.4. Strain Improvement

To develop the first generation of hypersecreting strains three main steps are necessary.
One needs to i) prepare the receiving strain that is to be used industrially; ii) construct a
vector in E. coli (the donor strain) and iii) transfer the researched gene(s) from the donor
strain into the chromosomes of the receiving strain.

The strategy usually used is to insert the enzyme coding gene into cassettes built in the
genome of the production strain. In Trichoderma reesei or in Aspergillus niger, genetic
cassettes have been built by excision of known enzymes from the genome of the wild type
strain (e.g. Trichoderma cellulases or Aspergillus glucanases) (van Dijck et al., 2003).

3.4.1. Preparation of the Strain of Production (Cassette construction)

On the sites of known genes of the wild type fungal strain, a zone, sensitive to a different
restriction enzyme was inserted, while the gene coding for the known enzymes was
removed. Up to seven cassettes were built on the fungal genome which means, on each
cassette a sequence sensitive to a different restriction enzyme was positioned. This zone
was flanked by small nucleotidic sequences that helped the insertion of the gene of interest
at the right place, including a nucleotidic zone sensitive to another restriction enzyme other
than the one initially present in the cassette. The insertion was done from a vector and
provided a high number of copies of the targeted gene in the production strain (up to 120
copies).

Figure 3.2

3.4.2. Preparation of the Vector

The vector used to transfer the gene coding the enzyme is usually built in E. coli. If the
original gene is coming from a species very different from the species used for the
overproduction (e.g. initial gene coming from a prokaryote and produced by a yeast), a
change of the sequence in order to avoid problems with codon bias that could happen
between very different species should be applied using tables before to insert the gene of
interest in E. coli. The plasmid usually bears a marker gene and is in targeted zones that can
be opened with specific restriction enzymes, so that foreign genes could be inserted easily.

3
Markers need to be installed when constructing a vector so that the transformed clones
could be easily identified.

For a while, genes generating resistance to antibiotics were used as markers but this
approach should be avoided for enzymes used for food applications. If this approach is
unavoidable, these genes should be deleted in the final strain.

It should be stressed that this marker gene can enable the use of a specific substrate. For
example, the addition of the amdS gene in the plasmid coding the acetamidase, gives the
strain the capability to uptake acetamide as carbon source. In this case, the capability of the
transformed strains to use acetamide indicates the efficiency of the plasmid transfer (Selten
et al, 1995). Figure 3.3 shows the construction of such a plasmid.

Figure 3.3

3.4.3. Construction of the Overproducing Strain

This construction should meet several objectives to ultimately obtain a stable overproducing
strain. This means i) successfully inserting the ‘gene coding the enzyme’ on at least one of
the chromosomes ii) being able to have a hyperproduction of the enzyme, rendering this
protein the most significant one produced quantitatively, in the culture supernatant iii)
avoiding disrupting the general metabolism of the producing strain, iv) obtaining a protein
that will be processed by the post-traductional modification system, v) finally showing that
all foreign DNA, with the exception of the enzyme coding genes have been removed during
the strain construction process.

Figure 3.4

The inserted sequences are amplified by PCR and sequenced in order to check that no
sequence of E. coli remains in the production strain.

This approach was built for the construction of enzymes used as food processing aids and
was done so following very strict safety assessment guidelines. The idea was to have strains
produce enzymes which no longer needed to be tested on animals to demonstrate the
safety of the products. Irrespective and, at least in Europe, the present legislation still
obliges companies to test the enzyme preparations in animals to evaluate for different types
of toxicity (e.g. subchronical toxcity for 9 weeks). These tests must be completed prior to
permission being granted for use in food preparations.

3.4.4. The Revolution of CrispR- Cas9 Editing for the Construction of Fungal Strains that
Hyperproduce Enzyme

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated

(Cas) systems, are systems containing cas genes and CRISPR array(s) being naturally derived
from the adaptive immune system of prokaryotes. The cas genes are short sequences that

4
originated from foreign genetic material, called spacers, interspaced with identical
palindromic repeats (Jinek et al., 2012). The cas genes code proteins responsible for
acquisition of new foreign sequences into the CRISPR array(s) as well as for disruption of
exogenous DNA through the activity of Cas proteins bearing endonuclease activity, such as
the Cas9, Cas12 or Cas 13 proteins (Mota et al., 2020). Over the past decade, the ability to
reprogram Cas proteins by creating crRNAs (guide RNAs) designed to bind and/or cleave
specific DNA or RNA sequences has made them useful in a variety of applications, including
genome editing and modulation, specific control of gene expression and nucleic acid
detection (Mota et al., 2020).

Figure 3.5

n the CRISPR/Cas9 system, the Cas9 protein forms a complex with two RNA molecules:
CRISPR RNA (crRNA), encoded by the random spacers found in the CRISPR array(s), and
transactivating CRISPR RNA (tracrRNA). It has been shown, that these two RNAs form a dual-
tracrRNA:crRNA, which can also be designed as a single guide RNA (sgRNA) for genome
editing purposes (Jinek et al., 2012). The Cas9-sgRNA complex is guided to a target DNA via
homology of the crRNA to the protospacer region of the target sequence (Fig. 3.5).

The Cas9 nuclease subsequently binds the target DNA through interaction with the
protospacer adjacent motif (PAM) immediately downstream of the protospacer sequence. If
the crRNA part of the sgRNA sequence successfully pairs with the target DNA, the Cas9
nuclease will perform a double strand break (DSB) three nucleotides upstream of the PAM
motif, which can be repaired. The simple design and construction of a single guide RNA for
precise genome editing has rapidly expanded the toolbox of molecular methods, such as
Zinc Finger Nucleases (ZFN) and Transcription Activator-Like Effector Nucleases (TALENs)
(Kim and Kim, 2014).

CRISPR/Cas9 is a cost-efficient and simple platform to perform genetic manipulations with a

single enzymatic activity guided by a predesigned sgRNA molecule. Therefore, it has become
a common genome editing method in a variety of organisms and has been reported to work
in numerous filamentous fungi. In filamentous fungi, this system has been used to target
genes involved in enzyme production (Q. Liu et al., 2017) (Katayama et al., 2018) (Liu et al.,
2015) (R. Liu et al., 2017). The system can also be used to efficiently change functionnal
properties of the proteins encoded by the target gene by on site genomic mutation (e.g. in
Aspergillus niger) (Kun et al., 2020).

Once the overproducing strain is obtained a validation of the strain stability is necessary.
Strains with a high number of copies of a gene are often not very stable. It seems that there
is an economy of the cell that leads to the removal or inactivation of non-compulsory genes.
The high number of copies is not necessary and some are often deleted after a limited
number of generations. Irrespective, it is important to set up a proper strain stock of the
efficient strain, in order to keep the best activity obtained

5
3.5. Growth Requirements of Microorganisms

3.5.1. Media Development

The knowledge of the strain growth requirements and regulation systems are prerequisites
to controlling the over-expression of enzyme production. Sugar and nitrogen substrates are
quantitatively used during the process. By-products of the food industry such as molasses or
corn steep liquors can be efficient substrates and more affordable than purer substrates
such as pure sugars or protein hydrolysates (peptones or tryptones). Yeast extracts and their
content in vitamins (especially B vitamins) are also commonly used. However, the
complexity of the medium can be a drawback for the future purification of the enzymatic
activity at the end of the fermentation process.

Another substrate that should be provided in significant amounts is oxygen. The oxygen
transfer from gas to micro-organisms can be a limiting step especially when filamentous
fungi are used. This will be discussed later.

3.5.2. Nutritional Requirements

3.5.2.1. Effect of Carbon Sources

Apart from their influence on growth rates the availability of carbon sources has a
significant metabolic regulation effect. It starts by the expression of the substrate uptake,
but may also influence the pathway expression. Some of the enzyme activities are
associated with growth and, in this case, easy to use sugars such as sucrose, fructose or
maltose can be efficient for enzyme production. However, the availability of glucose, even if
very efficient on cell growth is also one of the most repressive substrates for a large number
of genes and as a consequence limits the production of many enzymes. So, the use of other
sugars such as lactose or maltose or the use of glycerol can be a better choice to develop a
full expression of the strain potentiality.

Arora et al, (2018) have shown the diversity of by-products that could be used for the
production of enzymes by solid phase fermentation (see 3.5). Substrates as diverse as apple
or tomato pomace, different types of bran, linseed oil cake, palm straw, citrus pulp or peel,
etc. can be used and offer the advantage of being cheap substrates as well as being
substrates easily used by fungi.

3.5.2.2. Effect of Nitrogen Sources

Nitrogen is often added in the mineral form with salts such as ammonium sulfate or
ammonium nitrate. Organic nitrogen can also be used with simple substrates such as urea
but peptone or tryptone of different proteins, even if they are significantly more expensive
are also very efficient. Other efficient complex sources of nitrogen are used for industrial
processes such as sugar slop or spent or corn steep liquor which are also reach in nitrogen.
However, a complete analysis of the flow sheet costs should be done to take the right

6
economical decision because the more complex the medium the more difficult and costly
could be the purification process.

Yeast extract is providing nitrogen, but it is also providing a large diversity of vitamins
especially B vitamins.

3.5.2.3. Effect of salts and oligoelements

Phosphate and sulphate sources are usually added to the culture medium as phosphorus
and sulphur are significant constituent atoms of living cells.

Metal ions and halogens can be efficient bio-chemical mediators and necessary oligo-
elements for efficient enzyme production (e.g. sodium, potassium, calcium, manganese,
magnesium, iron, copper, molybdenum, cobalt, iodine, chloride,...) however some
contaminant metals must be absent in enzyme preparations for food applications. It is the
case for lead, cadmium, arsenic, mercury and the concentration of these metals must be
controlled regularly for these applications.

3.6. Process Design and Optimization

Automated liquid fermentation equipment available on the market nowadays, enables

organisations to control temperature, pressure, pH and weight during industrial scale
production.

Sensors are also available for biomass measurement but are mainly adapted for monitoring
bacterial or yeast growth rather than filamentous fungi. Such sensors are not adapted to
measuring the biomass of fungi because the fungi usually form pellets when the liquid
medium that it is in is agitated, a step that is necessary for providing oxygen to the micro-
organisms. Special geometry of reactors called “air lift” are favouring oxygen transfer
without increasing the shearing stress associated to high oxygen transfer with propellers.

Figure 3.6

It can be interesting to produce enzymes by solid state fermentation (SSF). A recent review
(Arora et al., 2018) showed the diversity of enzymes and efficiency when using such a
method for producing enzymes. Different types of solid state bioreactors are used : tray
bioreactors, packed bed bioreactor, air pressure pulsation bioreactors and mixed SSF
bioreactors. A diverse range of enzymes has been produced with such reactors. The best
results were obtained for enzymes such as amylase, pullulanase and xylanase.

In this case the enzymes are more concentrated in the solution that can be obtained by
pressing the solid substrate (such as sugar beet pulps). However such equipment is less
common than the liquid fermentation ones. The control of sterility, relative humidity and
temperature can be an issue when scaled up for industrial production. However this
technique is used by several companies.

7
3.7. Downstream Processes

3.7.1. Concentration and Purification of Enzymes

At the end of fermentation, the purification of enzymes is facilitated by the titre and can be
hindered by the complexity of the medium. Most often enzymes in the culture supernatant
are concentrated by tangential ultrafiltration and then precipitated using salts such as
ammonium sulfate. It is followed by a desalination process, using membrane techniques.
As for the use in food, the regulations stipulate that there are maximum permissible limits
for heavy metals and there must be an absence of pathogenic micro-organisms (such as
Salmonella, Staphylococcus aureus, sulfito-reducting Clostridiae,…).

3.7.2. Product Formulation and Standardization

The activity of the concentrated form is measured in order to standardise the number of
enzyme units per pack. Some enzymes are sensitive to oxidation and they should be
protected by antioxidants such as sulphites or ascorbic acid. They are often mixed with
carriers agents such as dry starch or dextrines and granulated in order to facilitate their use.

3.8. Conclusion

The screening methods are still prerequisites for selecting overproducing strains but the
recent progress in genome knowledge and genome expression have made genetic
engineering and automated protein engineering easier. It has contributed to the
improvement of specific qualities and quantities of enzymes produced including producing
them at reasonable prices. The revolutionary CRISPR/Cas gene technology also helps these
operations, however in some cases the insertion sites are not as specific as expected.

One of the critical steps is to keep the built strain efficiency for a long duration. The number
of copies of the interesting gene can be deleted when the number of generations become
high. As far as we know the systems regulating the excision of genes with low interest for
the producing cells are not completely elucidated and seem to be difficult to inactivate.

The production of enzymes in liquid medium are now quite controlled. However, production
of enzymes by filamentous fungi, requires large amounts of oxygen. Oxygen transfer
presents technical problems such as shear stress damaging the mycelia and energy cost that
can be critical at large scale. New techniques of solid state fermentation of agro industry by-
products are now at an industrial scale. Even if they are more difficult to control than liquid
fermentation, obtaining high enzyme concentrations is possible. Moreover, solid state
fermentations save water and has shown to reduce sterilisation, downstream processes and
water treatment costs.

Downstream processes can be very specific depending on the type of enzyme and its
sensitivity to environmental factors. Levels of purity is sometime a significant factor for

8
some applications and secondary enzymatic activities present in the preparation can be a
problem.

The simultaneous development of these techniques has helped to develop a large number
of applications first for cleaning powders, then for food and feed applications, and more
recently for the lignocellulose breakdown and the production of specific chemicals.

9
 References

Archer, D.B. and Peberdy, J.F. (1997). The Molecular Biology of Secreted Enzyme Production
by Fungi. Crit. Rev. Biotechnol. 17, 273-306.

Arora, S., Rani, R. and Ghosh, S. (2018). Bioreactors in solid state fermentation technology:
Design, applications and engineering aspects. J. Biotechnol. 269, 16=34.

Baker, S.E. (2018). Protein hyperproduction in fungi by design. Appl. Microbiol. Biotechnol.
102, 8621-8628.

Cai, D., Rao, Y., Zhan, Y., Wang, Q. and Chen, S. (2019). Engineering Bacillus for efficient
production of heterologous protein: current progress, challenge and prospect. J. Appl.
Microbiol. 126, 1632-1642.

Fiedler, M.R.M., Barthel, L., Kubisch, C., Nai, C. and Meyer, V. (2018). Construction of an
improved Aspergillus niger platform for enhanced glucoamylase secretion. Microb Cell
Fact. 17, 95 (Doi: 10.1186/s12934-018-0941-8).

Goddard, J.-P. and Reymond, J.-L. (2004). Recent advances in enzyme assays. Trends
Biotechnol. 22, 363-370.

Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A. and Charpentier, E. (2012). A
programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Sci.
337, 816-821.

Katayama, T., Nakamura, H., Zhang, Y., Pascal, A., Fujii, W., and Maruyama, J., (2018). Forced
Recycling of an AMA1-Based Genome-Editing Plasmid Allows for Efficient Multiple Gene
Deletion/Integration in the Industrial Filamentous Fungus Aspergillus oryzae. Appl.
Environ. Microbiol. 85, e01896-18 (Doi: 10.1128/AEM.01896-18).

Kim, H. and Kim, J.-S. (2014). A guide to genome engineering with programmable nucleases.
Nat. Rev. Genet. 15, 321-334.

Kun, R.S., Gomes, A.C.S., Hilden, K.S., Cerezo, S.S., Makela, M.R. and de Vries, R.P. (2019).
Developments and opportunities in fungal strain engineering for the production of novel
enzymes and enzyme cocktails for plant biomass degradation. Biotechnol. Adv. 37 (Dot:
10.1016/j.biotechadv.2019.02.017).

Kun, R.S., Meng, J., Salazar-Cerezo, S., Makela, M.R. de Vries, R.P., and Garrigues, S. (2020).
CRISPR/Cas9 facilitates rapid generation of constitutive forms of transcription factors in
Aspergillus niger through specific on-site genomic mutations resulting in increased
saccharification of plant biomass. Enzyme Microb. Technol. 136 (Doi:
10.1016/j.enzmictec.2020.109508).

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Liu, Q., Gao, R., Li, J., Lin, L., Zhao, J., Sun, W. and Tian, C. (2017). Development of a genome-
editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its
application to hyper-cellulase production strain engineering. Biotechnol. Biofuels. 10 (Doi:
10.1186/s13068-016-0693-9).

Liu, R., Chen, L., Jiang, Y., Zhou, Z. and Zou, G. (2015). Efficient genome editing in
filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system. Cell Discov. 1 (Doi:
10.1038/celldisc.2015.7

Liu, R., Chen, L., Jiang, Y., Zou, G. and Zhou, Z. (2017). A novel transcription factor specifically
regulates GH11 xylanase genes in Trichoderma reesei. Biotechnol. Biofuels. 10. (Doi:
10.1186/s13068-017-0878-x).

Mota, D.S., Marques, J.M., Guimarães, J.M., Mariúba and L.A.M. (2020). Research Article
CRISPR/Cas Class 2 systems and their applications in biotechnological processes. Genet.
Mol. Res. 19 (Doi: 10.4238/gmr18478).

Selten, G.C.M., van Gorcom, R.F.M. and Swinkels, B.W. (1995). Selection marker gene free
recombinant strains : a method for obtaining them and the use of these strains. Eur.
Patent Appl. EP 00635574 A1.

van Dijck, P.W.M., Selten, G.C.M. and Hempenius, R.A. (2003). On the safety of a new
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11
 Carbon catabolite Genes
repression

Fungal morphology
Transcripts

Nutrient uptake regulation

Protein
G-protein-coupled receptors,
synthesis
« transceptors »
Enzyme
Translocation and recovery,
secretion Enzyme concentration,
ER stress and Unfolded
Protein Response (UPR) secretion purification
and
formulation

Figure 3.1. Key steps to overproduce a ready to use specific enzyme (Baker, 2018; Fiedler et
al., 2018).

12
 copies).

Fig. 3.2. Visualization of all seven marked glaA loci of A. niger strains, in which the glaA
genes were removed. All gene sequences of the seven glucoamylase loci were
removed by using prop rietary technology. Subsequently they were partly filled
using the same methodology with sequences from the 30 regions of the original
locus. Due to the fact that these sequences varied in length and were decorated
with a specific restriction site the seven marked loci can be detected individually
by PCR using a set of specific primers and separation on a gel electrophoretic
system (van Dijck et al, 2003).

13
 .

Fig 3.3. Example of the marker-gene free insertion of an expression unit. The expression unit,
a linear piece of DNA, is integrated into one of the seven marked loci by homology
of the 30 regions of the loci. By varying the conditions of transformation of the
expression units multiple copies of the gene of interest arranged in tandem can be
integrated in a marked locus. Current practise is that both the gene of interest and the
amdS selection marker are on different expression units. So the transformation is
carried out with a mixture of expression units carrying the gene of interest and
expression units carrying the amdS marker. By selection on agar plates containing
acetamide as sole carbon source the transformants are selected. By counter-selection
on agar plates containing fluoro-acetate variants can be selected from these
transformants which have lost the amdS marker but which have retained (multiple
copies of) the gene of interest (van Dijck et al, 2003).

14
 .

Figure 3.4. Selection of natural variants. The natural process of gene conversion is used to
select for variants that have acquired a set of multiple gene copies of the gene of
interest in any the remaining empty marked loci. The filling-up process in the
several selection rounds can be followed by analysis in a gel electrophoretic
system (van Dijck et al, 2003).

15
 (Mota et al., 2020).

Figure 3.5. Utilization of the CRISPR/Cas9 system in filamentous fungi for increased enzyme
production. The artificial sgRNA is composed of a guide sequence (in green) and
a scaffold sequence (in yellow), which are the corresponding parts of the
naturally occurring crRNA and tracrRNA, respectively. The sgRNA forms a
complex with the Cas9 nuclease (in blue), which will be directed to a targeted
locus of a gene of interest. In case there is sequence homology between the guide
sequence and the target sequence upstream of a PAM sequence (in red), a double
strand break (DSB) will be performed. The DSB can be repaired in a targeted
manner using repair templates, which can result in precise gene editing (Kun et
al., 2019).

16
Fermen- Pre-Culture
Seed
tation Fermentation Fermentation Killing Off
Fermentation

Down- Polish and

Polish and
stream Ultra-filtration Germ Broth Filtration
germ filtration
Filtration

Granulation
Formu- Granulated
and
lation product
standardisation

Figure 3.6. Example of a process sheet used for food enzyme production.

17