The use of microorganisms

to add value.
“Biotechnology” Today
• Biological products produced by recombinant DNA techniques
• Specialty drug discovery and development
• Diagnostics
• IVD
• Molecular probes
• “Theranostics”

• Life sciences Instrumentation

• Technology-based “platform” companies
• Stem cell
• Genomics
• RNAi
• Drug Delivery

• Agribusiness
• Nutraceuticals

Essentially anything fits the mold these days…

Type of Cells used in Bio-Pharmaceutical
production for industry.

https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-014-0141-0
An industrial biotechnologists role?
• Knowing gene for the protein you want is great,
• but what cell line to use? What clone from that cell line is best?

• 60 or more nutritional components in culture media, how many

combinations?
• When to feed them? Inducers, promoters?

• What temperature? What oxygen level? CO2? pH any shifts?

• When to harvest?
• A strategy of multi-factorial design is the natural way to attack this
type of problem, but is difficult to execute in cell culture because
the parameters interact strongly-requiring a lot of experiments.
• This means models!
Objectives of protein expression in
industrial production
• Safe and controllable fermentation
• Cheap fermentation
• High level of recombinant production (g/L
not mg/L)
• Easy recovery of expressed proteins
• No degradation of expressed protein
A typical E. coli plasmid vector

• Blue/white screening
• SP6 or T7 RNA polymerase promoters
• Amp resistance selection
Microbial expression hosts – E. coli
ADVANTAGES
• Numerous specific vectors (l phage
and pUC plasmid derivatives)
• Very high transformation efficiencies
• Very well understood fermentations
• Simple cell recovery and lysis

DISADVANTAGES
• Not naturally competent
• Low expression yields
• No glycosylation of rProtein
• rProtein not exported
Microbial expression hosts – Saccharomyces cerevisiae

DISADVANTAGES
ADVANTAGES
• Low-medium
• Moderate
transformation
expression yields
efficiency
• Limited
• rProtein
range of
glycosylated
vector systems
• rGenes
• rProtein
must beexported
stably incorporated
• Very well understood fermentations
• Simple cell recovery and lysis
IB production of Insulin
Microbial expression hosts – Pichia pastoris and
Kluveromyces sp.
ADVANTAGES
• Moderate expression yields
• rProtein glycosylated
• rProtein exported
• Simple cell recovery
DISADVANTAGES
• Low-medium transformation
efficiencies
• Difficult fermentations
• Limited range of vector
systems
• rGenes must be stably
incorporated
• Unusual codon usages
Microbial expression hosts – Trichoderma reesii

ADVANTAGES DISADVANTAGES
• High expression yields • Low-medium transformation
• rProtein glycosylated efficiencies
• rProtein exported • Difficult fermentations
• Simple cell recovery • Limited range of vector
systems
• rGenes must be stably
incorporated
• Unusual codon usages
 Microbial expression hosts – Bacillus
ADVANTAGES
• Very high expression yields (>10
g/L)
• Simple fermentations
• rProtein exported
• Simple cell recovery
• Naturally competent
DISADVANTAGES
• Limited range of vector systems
• Optimised vector systems are
proprietary
• No glycosylation
• Variable transformation
efficiencies
Insect cell expression
(Specific host-vector system; Sf9 cultured insect cells and Baculovirus vectors)

• Disadvantages
Advantages
• Higher
Highly specific
eukaryote-like
transformation
glycosylation
system (Baculovirus)
• Fastidious
Scaleable to
culture
large volumes
• Very slow doubling time (20h)
Animal cell culture
• Advantages • Disadvantages
• Exact glycosylation • Some grow as adherent layer (anchorage
• Can be transformed to continuous cell dependent)
lines • Fastidious growth requirements
• Some (e.g., CHO cells) grow in suspension • Very slow doubling time (15-25h)
culture
• Very susceptible to contamination
• Some carcinoma-derived cells (e.g., HeLa
• Very sensitive to shear stress
cells) have rapid growth rates
• Extracellular expression
Plant cell culture
• Advantages
• Disadvantages
• Suspension cultures
• Restricted range of
• Low aeration required transformation systems
• rProtein directed to vacuole • Agrobacterium for dicots
• Biolistics for monocots
• Specific plant viruses
• Very slow doubling times (15h
– 48h)
• Very sensitive to shear stress
• Grow heterotrophically but
require light for optimum
growth
Other expression systems
• Tobacco plant expression
• Using recombinant tobacco mosaic virus
• Tissue specific promoters
• Scale-up of production as for normal agriculture

• Whole animal expression

• Transformation of unfertilized embryo
• Use of tissue-specific promotors (e.g., lactose-specific promoter for
expression of proteins in milk)
 Expression development
• Multicopy plasmids
• Multiple selective (resistance) markers (Ampicillin, Tetracycline, Kanamycin,
Thiostreptin)
• Different promoters (ITPG, Trp, temperature-inducible, aTet)
• Multiple promoters (shuttle vectors for different hosts)
• Multiple ori sequences
• N-terminal tags (e.g., polyHis) with cleavage site
Vectors designed for protein recovery:
eg: His Tags

• Vector contains polyGCA

• rProtein has polyHis Tag
• Recovery with Ni-NTA
affinity matrices
 After discovery comes development, lots
and lots of it!

Expression
Media Process
System Clone
Development
Flasks Optimization Scale Up
Developme Evaluation
* **
nt
• Identify target, • Screen and • Develop optimal • Optimize • Scale up process
isolate gene, and select the growth and conditions for for use in large
develop highest production biomanufacturing bioreactors for
expression producing and media for each process in a “scale- production of
system most stable cell line down” version therapeutic
clone
 Biotechnology & Antibiotic Production
• Microorganisms are commonly use for Antibiotic production
• This process can be enhanced by :

1
Increasing the output of microorganisms

2 Optimise production process

Microbial Biotechnology, 2nd edition, 2007

 Genetic Engineering - Strain Enhancement
Fleming 1929
3 mg/L

Eli Lilly Ltd.

Advancements
7000 mg/L
in molecular
biology

Medema et al. 2011

Microbial Biotechnology, 2nd edition, 2007
Yield & Production Rate (PR)

Source: Science Museum, London

“Out of the pan and into the deep tank

fermenter”: Scientists from Pfizer opted to
use deep-tank fermenters instead of shallow
based pans which increased yields by a
factor of five. (Walsh, 2008)

HELP! In 1941 the UK government

sought help from the US scientific
community for mass producing
antibiotics. (Walsh, 2008)
Yield & Production Rate (PR)
• The Importance of Deep Tank Fermentation: “It is the biggest single failing of the myth
about penicillin that it ignores the technological breakthrough of deep fermentation, a
breakthrough that was every bit as vital to the successful development of penicillin as any
of the more dramatic laboratory work.” Wilson, D. (1976) ‘Searching for penicillin’.

(Walsh, 2008)

Source: davidmoore.org.uk
Yield & Range
• Yields for the yield (g/L)
antibiotic penicillin
increased by 3
orders of magnitude
from 1945 to 1985
by virtue of
selective breeding
of Penicillium
strains (Schmidt-
Dengler, 2014).
• 20 families of
antibiotics, 200
antibiotics in
commercial use. (Schmidt-Dengler, 2014)
(Kozhuharova, 2007)
Yield & Production Rate (PR)
Penicillin Production: Post WWII vs Today
Post WWII Today

laboratory flasks reaching 100,000-

Vessels
(2 to 3 litre vessels) 150,000 litres.

Penicillium
Organism Penicillium notatum
chrysogenum
Yield 1 mg/L 50 g/L

Submerged culture
Culture Surface liquid culture
techniques
(Najafpour, 2007)
Conditions
controlled not very well tightly
 Cost

• Lowering the

Billions (Internal Units)

Costs: The cost of

Average Unit Value in $

antibiotics has
decreased
dramatically by
virtue of mass
production
methods
pioneered during
the war.
(Schmidt-Dengler, 2014)
What do you culture them on?
• How will synthetic biology be considered?

• Directed evolution commonly used.

Growth of Cell culture
https://www.alliedmarketresearch.com/RNA-based-therapeutics-market
Engineering alcohol tolerance in yeast, Volume: 346, Issue: 6205, Pages: 71-75, DOI: (10.1126/science.1257859)