Applying Genetic

Engineering to Brewing
Yeasts
Reason for genetic engineering

 Economic importance of alcoholic beverages

 Research on the genetic makeup of brewing yeasts and how the genotype
influences the characteristics of br̥ewing yeast.
 Yeast plasmids –naturally occurring and modified
 Insertion of selectable marker genes such as genes that code for resistance
to copper ions of the antibiotic gentamycin.
 Strong constitute promoters
 Presence of Leader sequence that facilitate excretion of enzyme from
yeast cells
 Reasons contd…

 Yeast plasmid are multi-copy –leading to high expression levels of

transgene.
 Unstable in host cells and transgene can be deleted
 Integrative vectors-process of homologous recombination.
Relevant targets for brewer’s yeast

 Giving yeasts the ability to hydrolyse dextrins

 Giving yeast the ability to degrade glucans
 Improving the fermentation rates of yeast
 Improving flocculation properties of brewing yeast
 Improving properties of brewing yeasts to allow reduction in beer
maturation periods
 Improving the production of flavor compounds by brewing yeast.
 Overcome – catabolite repression
 High gravity brewing
Giving yeasts the ability to hydrolyse dextrins
 Larger oligosaccahrides cannot be utilized.
 Light or lite beer
 Glucoamylase enzyme – commercially attached. – to bring the hydrolysis of
dextrins.
 Four strategies.
 1st strategy
 STA2 gene- glucoamylase from Saccaromyces cerviase var. diastaticus.
 CUP1 gene-coding for resistance to copper ions. – selection marker.
 Multi copy bacterial/yeast chimeric plasmid.
 Result- synthesis of extracellular glucoamylase.
 Limitations- unstable, growth and transformation rates reduced upon transformation,
glucoamylase lacked the debranching enzyme, limiting dextrin hydrolysis
 2nd strategy

 All yeast mutti copy plasmid,

 STA2
 Upstream-Constitutive promoter from the yeast PGK gene
 Downstream – PGK gene
 CUP1
 Result
 Produced ethanol at a similar rate to the original yeast and produced
a final concentration of ethanol in wort 1% greater.
 3rd strategy
 Gene coding for glucoamylase with ,1-6 hydrolytic activity.
 Suitable gene from the filamentous fungus –Aspergillus niger
 Fused to the coding sequence of a signal peptide of a known gene
 Flanked by constitutive promoter
 Terminator sequences of a gene coding for a known enzyme
 Also included are sequences of the HO gene of yeast chromosome –IV
 Result – stably integrated by homologous recombination into the HO gene.
 Significance –
 HO gene is redundant in brewers yeast- unlikely to effect the brewing
characteristics.

 Aspergillus food grade.

 4th strategy
 Multiple copies to be present and integrated into the chromosome-glucoamylase
gene from Aspergillus awamori
 Constitutive promoter and terminator sequence.
 Flanked by sequences from the yeast gene that codes for the rRNA.
 100-150 copies , arranged on Chromosome XII , as a series of tandem repeat.
 Result – Transformation was successful.
 Limitation- pilot scale trials that transformed yeast are not capable of utilizing dextrins.
 Glucoamylases from Aspergillus spp- heat stable.
 Unacceptable changes in flavor during long storage periods.
 Giving yeast the ability to utilize starches

 Complete degradation - -amylase and glucoamylase

 Engineer yeasts- hybrid enzyme.
 - -amylase- bacillus subtilis
 Glucoamylase -- Aspergillus awamori
 Downstream from a constitutive promoter and a yeast leader sequence.
 Result -Recombinant yeast was effective in hydrolyzing soluble starch and raw
potato starch- not effective in hydrolysis of maize starch.
 Limitation – defective glucoamylase debranching activity in the fusion protein.
 Giving yeast the ability to degrade glucans

 - glucans from barley- insoluble, cause hazes , sediments and gels in

beer.
 -glucanases – heat labile and are inactivated by the roasting
temperature .
 -glucanases-bacterium Bacillus cereus
Filamentous fungus Trichoderma reesei
Barley
 Enzyme from barley showed highest activity at the pH of beer.
 Gene from barley has been fused to a yeast signal sequence and
placed in an integrative vector downstream from a constitutive yeast
promoter.
 Result: reduced -glucan , improved filterability and reduced haze
formation, while flavour and foam stability of the beer was not affected.
 Improving the fermentation rates of
yeast
 Save time
 Lessen the chance of spoilage causing microorganism
 Major factor that limits rate of fermentation of maltose in genes coding for
Maltose permease (MALT) and -glucosidae (MALS) are repressed by glucose.
 Concentrated worts –high concentration of glucose , transcription of MALT and
MALS are repressed, and fermentation rates slowed.
 MALT and MALS have been cloned and inserted into multi copy plasmids under
the control of constitutively expressed.
 Result: MALT alone increased the rate of fermentation of maltose
 MALS gene alone did not increase the rate of fermentation of maltose.
 Improving flocculation properties of
brewing yeasts.
 Spontaneous flocculation of yeast at the end of the fermentation phase-
clarified beer.
 Flocculation does have a genetic basis.
 FLO1gene codes for a hydrophobic cell wall protein
 Used to transform non flocculent yeast with FLO1 gene in a multi copy and
integrative vectors under the control of constitutive promoters.
 Result: increased flocculation
 Limitation: flocculate during the fermentation period.
 Reducing cell concentration
 Reducing fermentation rates
 Improving properties of brewing yeasts to allow
reduction in beer maturation periods

Why the maturation period is required?

Off flavor compounds produced by

yeast can be converted by
biochemical and chemical
conversion into more palatable
compounds.
Flavours compounds are improved.
Target genes- ILV2
ILV5
ILV3
Combination of ILV3 and ILV5.
Methodology

 ILV2- acetolactate synthase activity is inhibited by herbicide sulphometuron

methyl . By isolating brewing yeasts resistant to the herbicide , organism
with altered (mutant) gene-ILV2. and were growing slowly on valine free
medium.
 Limitations – unacceptable slow rates of fermentation.
 Antisense ILV2
 Reduced levels of acetolactate syntahase mRNA with reduced levels of
enzyme activity.
 Limitation: unacceptable slow rates of fermentation.
 Transformed with ILV5-acetolactate reductoisomerase using multicopy and integrative
vectors.
 Outcome: increased levels of reductoisomerase activity and reduced levels of dicetyl
formation
 Transformed with ILV3 – dihydroxyacid dehydrayase
 Outcome: did not show reduced diacetyl levels , despite increased level of dehydratase.
 Transformation of yeast using a plasmid containing ILV5 and ILV3- did not result in lowered
diacetyl levels.
 Acetolactate decarboxylase (ALDC)- converts acetolactate to acetoin(no off flavor
development).
 Eneterobacter aerogenes
 Klebsiella treeigena
 Lactococcus lactis
 Acetobacter aceti var. xylinum.
 Improving the production of flavour compounds by
brewing yeasts

Sulphur dioxide contributes to

flavour stability-by sequestering
carbonyl compound.
MET 3 and MET 14
Antisense to MET 10 (methionine
causes repression of
transcription to MET 3 and MET
14 genes)
 Biotechnological Improvements –
catabolite repression
 Catabolite
repression –
prolong
fermentation
 Wort- large
amounts of
glucose,
maltose and
maltotriose
Use of mutagenesis strategy to isolate yeast
strain with impaired catabolite repression
Glucose Control

 When maltose or sucrose is present in the medium together with glucose,

intermittent lag phases exist between the depletion of glucose and the
initial consumption of sucrose or maltose, a phenomenon designated
glucose control.
 The molecular mechanism of Mig1(zinc finger protein )-mediated glucose
repression is a complicated regulatory cascade that eventually involves a
protein complex containing Ssn6, Tup1, and Mig1, where Mig1 directs the
complex to a specific consensus motif on the promoters of the target
genes .