Bioprocess Technology Lecture # 1, 2, 3, 4, 5, 6 & 7 (MKM 10.09.2013)

Bioprocess Technology
(Industrial biotechnology)
Biological Science
Non-Biological Science
&
Engineering
Biotechnology
&
Biochemical engineering
Product
Biotransformation /
Fermentation
by
Bio-agent
(enzymes/organisms)
Biomaterial /
biomass
Renewable / non-renewable
Bioreactor/ fermentation
Food processing
Immobilized enzymes
Detoxification of wastes
Bye product utilization
Biosensor
Several others
Upstream processing:
Selection and preparation of the suitable biosystem
[enzyme/organism(microbe)]
Selection and preparation of the substrate (for enzyme) / raw
material (for proper growth of the organism/microbe) for
product formation

Fermentation and Biotransformation:
Immobilization of the catalytic enzyme or growth of the
candidate microbe in a large bioreactor (usually > 100 lit) and
the target product formation by the single enzyme /pathway
enzymes
Design of the bioreactor, monitoring and controlling the
fermentation /biotransformation is very critical for yield of the
target product

Downstream processing:
Purification of the target metabolite or molecule from either
the immobilized material/ the cell mass/ the culture medium
Bioprocess Technology/ Industrial biotechnology
Microbial Cell Factories (MCFs)
for chemicals & fuels

Microorganisms have the diverse metabolic pathways and pathway
enzymes to produce value-added chemicals, fuels and other bulk
products from simple, readily available and inexpensive starting
material.

Currently, these fuels and chemical products are derived from the
non-renewable resources, like petroleum or other fossil reserves.

However, the lignocellulosic biomass-derived sugars are renewable
resources.

Microbes have the capacity to utilize these biomass-derived sugars
to convert them into varieties of chemicals, drugs and fuels.
What is MCF?
Glucose
Transformation of renewable biomass-derived sugars
to chemicals by MCF
Ref: Science 2010, 330: 1355-1358
Critical considerations for development of a successful MCFs
(i) Cost and availability of starting materials (e.g., carbon substrates);

(ii) Metabolic route and corresponding genes encoding the enzymes in the
pathway to produce the desired product;
[Lack of well characterized pathway & enzymes, poor activity of the selected pathway
enzymes, metabolic burden, unfavorable cofactor balance. Thus designing and
engineering the pathway and the pathway enzymes followed by experimental
validation]

(i) Most appropriate microbial host;

(ii) Robust and responsive genetic control system for the desired pathways
and chosen host;

(iii) Methods for debugging and debottlenecking the constructed/designed
pathway; and

(iv) Bioprocess optimization: ways to maximize yields, titers, and
productivities
Lecture # 1, delivered up to this slide on 23.07.2013
1) Improvement of the upstream processes
i) Development and Improvement of the microbial strain
including the pathway and the enzymes involved for the
target metabolite/product
ii), iii), iv) etc. for other considerations in upstream
processes

2) Improvement in fermentation/ biotransformation
processes; designing, monitoring and controlling the
bioreactor.

3) Improvement of the downstream processes
Optimization of the bioprocess technology
Strain development and improvement
Biotransformation in large-scale by natural microbes is less than optimum
and not economic.
Thus, strain improvement is essential and vital

1) Screening & selection from naturally occurring diversity and/
artificially induced genetic mutation:

2) Genetic or metabolic engineering: up-regulation of the desired
pathway or down-regulation of the competitive pathway to increase
the metabolic flux towards a desirable/targeted metabolite or
product by gene manipulation.

3) Rational design & engineering of the metabolic pathway: Recent
development in the re-design and model-based engineering of the
naturally exiting pathway and the pathway enzymes in one specific
host, and combination of enzymes and pathways from different hosts.
Optimization of the bioprocess technology:
Naturally occurring vs. engineered MCF
Improved or engineered microbial strains coupled with the
optimization of the upstream and downstream processes have
enabled successful production of the desired metabolites (natural or
novel)
Optimization of the bioprocess technology:
Naturally occurring vs. engineered MCF
Thus, MCFs:
now, become a complementary and
in future, may be rival to the synthetic organic chemistry
Design & engineering of the pathway for microbial cell
factories (MCFs): Conceptual strategies
a) Selection of specific production pathway
(Analogy: one can reach the destination by different routes)

b) Selection of suitable enzyme(s) for the pathway
(Analogy: each route has set of stations with bypasses)

c) Pathway optimization to enhance the product yield/titer
(Analogy: One combination of route, station and bypass for cost-
effective way/better comfort of journey/specific purpose)

a) Selection of specific production pathway:

Identification and selection of the target
metabolite, the suitable pathway and the
desired substrate/feedstock/biomass from several
possibilities at each level.

Bioinformatics approach- Several computational
tools are available and

Experimental validation in small-scale is crucial
b) Selection of suitable
enzyme(s) for the pathway using
various enzyme information
databases.

Selected Enzymes:
1) clearly known activity for the
substrate to specific
metabolite conversion
2) promiscuous activity for
structurally and chemically
similar substrates (or similar
catalytic reactions with
different substrate)

Bioinformatics approach &
Experimental verification
c) Pathway optimization to enhance the
product yield/titer:

1) Genetic/Metabolic engineering by up-
regulation or down-regulation of the
desired enzyme(s) by over-expression
or gene-knockout, respectively.
2) Protein engineering to enhance the
activity (catalytic turn over) and
specificity (substrate binding) of the
pathway enzyme.
3) Cofactor balancing by effective
recycling of suitable cofactor involved
in the target metabolite production.

Bioinformatics approach &
Experimental verification
Ref: Current Opinion of Structural Biology 2011, 21: 1-7
factories (MCFs): Operational strategies
a) Recruitment of partial pathways from independent sources

b) Using engineered or promiscuous enzymes,

c) de novo design or retro-biosynthetic approach
Ref: Current Opinion of Biotechnology 2008, 19:468-474
a) Recruitment of partial pathways from
independent sources and co-localization
in a single host to produce a known target
product
b) Using engineered or
promiscuous enzymes,
new pathways can be
constructed for the
production of novel,
non-natural products
M
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c) de novo design or retro-biosynthetic
approach considers the biotransformation of
the functional group rather than entire
structure, exploiting the tremendous natural
diversity of enzyme-catalyzed reactions
occurring across many living systems.
This can result into enhanced yield/titer of
already known product/metabolite
This can also lead to the production of novel
metabolites for which natural pathways have
not been elucidated.
This strategy just started to develop, but the
situation is analogous to the retro-synthesis
scheme widely practiced by organic chemists.
1) Combinations of existing pathways from different
organisms/strains into a single host

2) Engineering of existing pathway with naturally occurring
promiscuous enzymes or engineered enzymes or in combination

3) de novo pathway design using synthetic & systems biology

factories (MCFs): Objectives/outcomes/examples
1) Combinations of existing pathways from different
organisms/strains having individual native enzyme activities into a
single host:

Outcome: new/hybrid pathway
to produce the same natural
product/metabolite
Example: 4-step pathway to produce isopropanol from acetyl-CoA,
was re-constructed in E. coli using enzyme encoding genes from E.
coli, Clostridia acetobutylicum and C. beijerinckii (Ref: Appl Environ
Micrbiol 2007, 73:7814-7818).
2) Engineering of existing pathway with naturally occurring
promiscuous enzymes or engineered enzymes or in combination
Outcome: new pathway and novel product/metabolite
Example: Synthesis of novel 1,2,4-butanetriol from xylose in E. coli
using combination of promiscuous and engineered enzymes from
Pseudomonous fragi, E. coli and P. putida (Ref: J Am Chem Soc 2003,
125:12998-12999).
3) de novo pathway design
Outcome: new pathway and novel product/metabolite or/and
Increasing yield of naturally-existing (already known) product/metabolite
Previously described two operational strategies are based upon the naturally
occurring known or promiscuous enzymes of the pathways.

Most recent approaches, such as BNICE (Biochemical Network Integrated
Computational Explorer*) and others go beyond the current knowledge
boundaries to explore the entire space of possible metabolic pathways
generated based upon in silico design using the enzyme reaction rules and
starting or target metabolites.
* Trends in Biotechnology (2010) 28: 501508
Examples: Production of artemisinic acid (precursor of anti-malerial drug)
and taxadiene (precursor of anticancer drug) in S. cerevisiae; Production of
polylactic acid (PLA, biodegradable thermoplastic) and taxadiene in E. coli.
factories (MCFs): de novo pathway design
Trends in Biotechnology (2010) 28: 501508
DREAMS of metabolism = Discovery, Retrosynthesis, Evolution, Aalysis of the pathways,
Mining of omics , Selection of targets for enzyme engg.
Overview of various high-throughput databases available for
metabolic pathway engineering
FEBS Letters (2010) 584: 2556-2564
FEBS Letters (2010) 584: 2556-2564
FEBS Letters (2010) 584: 2556-2564
FEBS Letters (2010) 584: 2556-2564
FEBS Letters (2010) 584: 2556-2564
FEBS Letters (2010) 584: 2556-2564
FEBS Letters (2010) 584: 2556-2564
FEBS Letters (2010) 584: 2556-2564
Integration of system biology, synthetic biology and
evolutionary engineering into metabolic engineering
Trends in Biotechnology August 2011, Vol. 29, No. 8 (pp 370-378)
Systems metabolic engineering resulted:

Successful development of several designer pathways for
fine/value-added chemicals and biofuels production in MCFs

Development of a few synthetic microbes with minimum
genome for basic and applied research
Systems biology is an integrative theoretical and experimental
discipline that studies biological system at a holistic level .

Experimental systems biology utilize high through-put and genome-
wide tools at whole-cell or sub-cellular levels, and include methods to
profile genome, transcriptome, proteome, metabolome, and fluxome,
which are the hierarchical components for the flow of information for
life from genotype to phenotype.

Theoretical systems biology allows mathematical description of the
biological network that can be computationally simulated to predict
systematically the phenotypes of an organism under various conditions
of interest for several possible applications, including systems
metabolic engineering.
Synthetic biology aims at creating novel functional parts, modules,
circuits, and/or organisms using synthetic DNAs (bio-bricks) and
mathematical/ logical methodologies.

It has been shown to be practical and useful in various biotechnological
applications, e.g. production of various chemicals and materials that are
heterologous to the original host strain.

More sophisticated and optimal design of such smart biological
systems will be a continued challenge of synthetic biology for metabolic
engineering.
Evolutionary engineering is defined as a technique to select or
screen cells that have desired phenotypes from variant cell libraries
that are created either adaptively or randomly.

To optimize metabolic pathways by evolutionary engineering,
mutagenesis followed by selective breeding, i.e. selection based on the
desired phenotype are required.

Some objectives of evolution can include
better cell growth,
utilization of desired carbon sources,
higher yield of product,
good tolerance to the product or inhibitory intermediate metabolite,
elimination of byproduct formation.
Complementarily and synergistically overcomes the weaknesses of each technology.

Allows the creation of novel enzymes and metabolic pathways and gene regulatory circuits.

Fine-tuning and optimization of the metabolic fluxes that lead to increased yield and
concentration of a desired product.

Increased tolerance of cells to the product, harmful medium components or inhibitory
intermediate metabolite.

Formation of fewer or no byproducts.

Enhanced utilization of desired carbon substrates (biomass).

Development of cost-effective fermentation and downstream processes.

The starting point of systems metabolic engineering can be decided based on several criteria
such as the availability of native producer, biosynthetic pathways, enzymes, and others.

Several cycles of systems metabolic engineering can be performed until the efficiencies of
production of the desired chemicals and materials become satisfactory.
Advantages of strain development by systems metabolic engineering
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What is the need for universal platform microbe for MCF ?
No straightforward way to extract the natural products (desired metabolite) from the native
producer at economic level

Most of the native producers are not cultivable in laboratory/ fermentor [Only 1% of bacteria
and 5% of fungi are cultivable in lab conditions]

Even if some are cultivable in laboratory, extensive optimization procedures are required to
standardize their growth conditions

Many organisms grow slowly and low-yielder of the desired product/metabolite

Lack of genetic and physiological information in these native producers and suitable
engineering tools to genetically manipulate these organisms to improve the product yield and
productivity
Some platform microbes are:
Escherichia coli
Saccharomyces cerevisiae
Bacillus subtilis
Pseudomonas putida
Streptomyces spp.
Certain problems of over-production of secondary metabolites in plants, but advantageous in
microbes
Example of systems metabolic engineering approach: Saccharomyces
cerevisiae platform for production of value-added compounds
Ref: J Ind Microbiol Biotechnol 2011
Native pathway in yeast
Anti-malerial drug
Anti-cancer drug
IPP = isopentenyl pyrophosphate;
DMAPP = dimethylallyl pyrophosphate;
GPP = geranyl pyrophospahe;
GGPP = geranylgeranyl pyrophosphate
FPP = farnesyl pyrophosphate;
ADS = amorphadiene synthase
P450 = cytochrome P450 monooxygenase;
CPR = cytochrome P450 reductase
Titer: From 115 mg/L to 1g/L
Titer: From 204 g/L to 8.7 mg/L
Engineered yeast to
produce
increased level of FPP and
reduced level of sterols
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E.g., terpenoids
Two genes added from Taxus chinensis
Example of systems metabolic engineering approach: Saccharomyces
cerevisiae platform for production of value-added compounds
Ref: J Ind Microbiol Biotechnol 2011
Native pathway in yeast
Anti-malerial drug
Anti-cancer drug
IPP = isopentenyl pyrophosphate;
DMAPP = dimethylallyl pyrophosphate;
GPP = geranyl pyrophospahe;
GGPP = geranylgeranyl pyrophosphate
FPP = farnesyl pyrophosphate;
ADS = amorphadiene synthase
P450 = cytochrome P450 monooxygenase;
CPR = cytochrome P450 reductase
Titer: From 115 mg/L to 1g/L
Titer: From 204 g/L to 8.7 mg/L
Engineered yeast to
produce
increased level of FPP and
reduced level of sterols
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E.g., terpenoids
Two genes added from Taxus chinensis
Example of systems metabolic engineering approach: Escherichia coli
platform for production of value-added compounds
Production of taxadiene 5 -ol, the precursor of taxol
Multivariate modular pathway
engineering, metabolomics and
synthetic biology approach.

Relative expression levels of two
modules are optimized to balance the
overall flux distribution for enhanced
production of the desired metabolite
along with the synthetic taxadiene 5-
hydroxylase with evolved N-terminal
transmembrane domain.

Metabolomics identified that indole is
the inhibitor of E. coli cell growth and
taxadiene synthesis.
Titer: About 1g/L of taxadiene
Taxa-4,11-diene GGPP
GLC = glucose
Example of systems metabolic engineering approach: Escherichia coli
platform for production of value-added compounds
Production of polylactic acid (PLA)
The artificially evolved heterologous
enzymes- propionate Coenzyme A (CoA)
transferase can produce lactyl-CoA from
lactate; and polyhydroxyalkanoate (PHA)
synthase produces polylactic acid (PLA)
from lactyl-CoA.

Besides PLA homopolymer, the engineered
PHA synthase can also make copolymer
poly(3-hydroxybutyrate-co-lactic acid) or
P(3HB-co-LA).

To increase the flux towards the precursors
of these biopolymers, the central carbon
metabolic pathways have been engineered
based upon genome-scale simulation by
eliminating acetate kinase (ackA), PEP
carboxylase (ppc) and alcohol
dehydrogenase (adhE) along with the
overexpression of lactate dehydrogenase
(ldhA) and acetyl-CoA synthetase (acs).
Yield: PLA 11 wt% ; P(3HB-co-LA) 56 wt% of dry cell wt
Trends in Biotechnology August 2011,
Vol. 29, No. 8 (pp 370-378)
Successful development and operation of MCFs depend upon the
devices or machines made up of constituent parts- genes,
enzymes, reactions and metabolites.

Pathway enzymes are the important machine parts of the MCFs.

Suitable genetic transformation technique is required to introduce
the foreign or modified genes encoding the enzymes of the
metabolic pathway.

Control over their expression is important to maximize yields and
titers.

All these genes need not to be highly expressed, but must be
produced in catalytic amounts sufficient to adequately transform the
metabolic intermediates into the desired products at a sufficient rate.
The key issue: Genetic regulation of the pathway enzymes
High level expression of one gene dose not necessarily increase the
pathway flux.

However, coordinated and balanced expressions of all the genes
involved in the particular metabolic pathway are crucial for
minimizing the accumulation of the toxic metabolite and maximizing
the product formation.

Whether transgene(s) integrated in the genome or not
Copy number of the plasmid bearing the genes of interest
Promoter strength & types (constitutive or inducible)
Presence of terminator
Proper uses of activator and repressor
Specificity/efficiency of ribosome binding site (RBS)
Codon usage
mRNA stability
The key issue: Genetic regulation of the pathway enzymes (Contd..)
Over-expression or underexpression of the genes may be problem:
Expression of the desired genes at too high a level will rob the cell of
metabolites that might otherwise be used to produce the desired
molecule of interest, particularly important for production of low-
margin chemicals, while under-expressed genes will create pathway
bottlenecks.

When intermediate metabolite is toxic/inhibitory:
Furthermore, because intermediates of a foreign metabolic pathway can
be toxic to the heterologous host, which results in decreased production
of the desired final compound, it is essential that the relative levels of
the enzymes be coordinated.
The key issue: Genetic regulation of the pathway enzymes (Contd..)
Use of regulatory RNA (encoded by gene R) or protein
(encoded by gene P) to modulate the metabolic pathway
that has a toxic/inhibitory metabolite
Ref: Science 2010, 330: 1355-1358
Ref: Science 2010, 330: 1355-1358
Binding of the regulatory RNA and protein to the toxic metabolite
down-regulate the toxic metabolite synthesis and up-regulate the
product formation
Ref: Science 2010, 330: 1355-1358
MCFs for production of building block
chemicals & biopolymers
MCFs for production of building block chemicals & biopolymers
Microorganisms are endowed with capabilities to synthesize a wide range of building
block chemicals, i.e. monomers and polymers/co-polymers from varieties of carbon
sources.
These monomers and biopolymers/bio-co-polymers serve diverse biological
functions in natural hosts.

They have varying chemical and material properties suitable for numerous industrial
(food/feed & non-food/feed) and medical applications.

Due to increasing concerns on the environmental pollution (because of chemical
industries), adverse climate change (affecting plants as bioreactors) and depletion of
fossil-fuel (and stored chemicals), MCFs are considered as the suitable platform for
production of these chemicals from renewable biomass.
Enhanced production and isolation of the particular monomeric chemicals that may
be used for synthesis of biopolymer outside the host cells or
direct synthesis of tailor-made biopolymers with highly applicable material
properties has been possible by superior strain or MCFs coupled with modern
fermentation technology and advanced downstream processes.

Recent advances in systems metabolic engineering helped us to develop superior
strains or MCFs.
(A) Production of building block chemicals: A few examples
1) Succinic acid producers:
Natural strains are mostly rumen bacteria.
Natural isolates of Actinobacillus succinogenes, Anaerobiospirillum
succiniciproducens, Mannheimia succiniciproducens, Corynebacterium
glutamicum and their engineered derivatives can produce 52106 g/L
of succinic acid.

Metabolically engineered, acid-tolerant (cells grow at low pH, which is
required for industrial production and purification), osmo-tolerant yeast
Yarrowia lipolytica can produce a titer of 45.5 g/L succinic acid

Systems metabolic engineered E. coli can produce 87 g/L succinic acid
Succinic acid is commonly used as surfactant, precursor of other
chemicals (e.g. 1,4-butanediol = BDO) and poly(butylene succinate) =
PBS polymer
(A) Production of building block chemicals: A few successful examples
2) Lactic acid producers:
Natural isolates of Lactobacillus plantarum, Lactococcus lactis,
Lactobacillus delbrueckii, and Lactobacillus casei and their engineered
derivatives can produce 30135 g/L of lactic acid.
Metabolically engineered E. coli can produce 138 g/L lactic acid
3) Itaconic acid producers:
Natural isolates of Aspergillus terreus after strain improvement by
random mutagenesis produce 82 g/L of itaconic acid.
Systems metabolic engineered E. coli can produce 4 g/L itaconic acid
Lactic acid is the precursor of polylactic acid (PLA) homopolymer and
poly(3-hydroxybutyrate-co-lactic acid) or P(3HB-co-LA) copolymer
Itaconic acid is the precursor of polyitaconate homopolymer and
poly(acrylate-co-itaconate) copolymer
4) Glucaric acid producers:
Naturally present in fruits, vegetables and mammals, but not produced
by natural microbes.
Systems metabolic engineered E. coli with a synthetic polypeptide
scaffolds composed of protein-protein interaction domains can produce
2.37 g/L glucaric acid
Glucaric acid is the precursor of poly(hexamethylene glucaramide) and
poly(glucaramide), a type of water-resistant hydroxylated nylon
PtsG= PEP-dependent glucose phosphotransferase; Ino1= myo-inositol-1-phosphate
synthase; SuhB= inositol monophosphatase; MIOX= myo-inositol oxygenase; Udh=
uronate dehydrogenase
5) Adipic acid producers:
It is the most important dicarboxylic acid for industrial uses. Naturally
rarely present (in some plants and historically first isolated from
oxidation of fats?), but not produced by natural microbes.
Systems metabolic engineered E. coli can produce 36.8 g/L muconic
acid, which after chemical hydrogenation produces adipic acid.
Adipic acid is the precursor of nylon-4,6 and nylon-6,6 polymer and
polyurethane
DHS= 3-dehydroshikimic acid; PCA= protocatechuic acid; CTL= catechol; MCA= cis,cis-
muconic acid; AroZ= DHS dehydratase; AroY= PCA decarboxylase; CatA= CTL 1,2-
dioxygenase
6) Isoprene producers:
It is a 5-carbon diene known as 2-methyl-1,3-butadiene. Naturally present in
all classes of living organisms as different modified forms or polymers.
Systems metabolic engineered E. coli can produce 60 g/L isoprene.
Isoprene is the precursor of the largest class of naturally occurring isoprenoids or terpenoids
molecules having medicinal and industrial values. Its polymer, i.e. poly(isoprene) mainly
extracted from rubber tree.
MvaE= AACoA thiolase/HMGCoA reductase; MvaS= Mev synthase; HMG-CoA= 3-hydroxy-3-methyl-glutaryl-
CoA; MVK= Mev kinase; PMK= phosphomevalonate kinase; MVD= diphosphomevalonate decarboxylase; Idi=
isopentenyl diphosphate isomerase; IspS= isoprene synthase
(B) Production of biopolymers: A few successful examples
Some microbes naturally produce biopolymers or can be engineered to produce the
biopolymers directly by fermentation/transformation without the chemical catalytic
process.
One step microbial biopolymer formation has several advantages including the
control on composition of polymer, particularly the heteropolymer by balancing the
ratio of various monomers, and circumvent the costly and environmentally harmful
chemical catalytic process
1. Polysaccharides: Intracellular- Glycogen
Extracellular- Alginate, Xanthan , Dextran, Curdlan,
Gellan, Cellulose, Hyaluronic acid
2. Polyamides: Intracellular- Cyanophycin
Extracellular- Poly- -glutamate, -poly-L-lysine
3. Polyesters: Intracellular- Polyhydroxyalkanoate (PHA), Polylactic acid
(PLA, not natural in microbes)
4. Polyanhydrides: Intracellular- Polyphosphate
Four major classes of naturally occurring microbial biopolymers & a few examples:
Bacterial biopolymer synthesis pathways from intermediates of
central metabolism
Nature Review Microbiology (2010) 8: 578- 592
Polymer Primary structure Main component Natural producer Industrial
applications
Glycogen -(1,6)-branched -(1,4 )- linked
homopolymer
Glucose Bacteria and
archaea
----------
Alginate -(1,4)-linked non-repeating
heteropolymer
Mannuronic acid
and guluronic acid
Pseudomonas spp.
and Azotobacter spp.
Biomaterial (for example, as a
tissue scaffold or for drug delivery)
Xanthan -(1,4)-linked repeating
heteropolymer consisting of
pentasaccharide units
Glucose, mannose
and glucuronate
Xanthomonas spp. Food additive (for example, as a
thickener or an emulsifier)
Dextran -(1,2)/-(1,3)/ -(1,4)-branched
-(1,6)-linked homopolymer
Glucose Leuconostoc spp. and
Streptococcus spp.
Blood plasma extender and
chromatography media
Curdlan -(1,3)-linked
homopolymer
Glucose Agrobacterium spp.,
Rhizobium spp. etc
Food additive (for example, as a
thickener or a gelling agent)
Gellan -(1,3)-linked repeating
tetrasaccharide units
Glucose, rhamnose
and glucuronate
Sphingomonas spp. Culture media additive, food
additive (for example, as a gelling
agent) or for encapsulation
Cellulose -(1,4)-linked homopolymer Glucose Alpha-, Beta- and
Gamma- proteobacteria,
Gram-positive bacteria
Food, diaphragms of acoustic
transducers and wound dressing
Hyaluronic
acid
-(1,4)-linked repeating
disaccharide units
Glucuronate and N-
acetyl glucosamine
Streptococcus spp. and
Pasteurella multocida
Cosmetics, viscosupplementation,
tissue repair and drug delivery
Cyanophycin
granule
Repeating heteropolymer
consisting of dipeptide units
Aspartate and
arginine
Cyanobacteria,
Acinetobacter spp. , etc.
Dispersant and water softener
(after removal of arginyl residues)
Poly-
-glutamate
Homopolymer d-glutamate and/or
L-glutamate
Few Gram +, Gram -
bacteria, & few archaea
Replacement of polyacrylate,
thickener, humectant, drug
delivery and cosmetics
Examples of bacterial polymers, features and their applications
Polymer Primary structure Main component Natural producer Industrial
applications
Glycogen -(1,6)-branched -(1,4 )- linked
homopolymer
Glucose Bacteria and
archaea
----------
Alginate -(1,4)-linked non-repeating
heteropolymer
Mannuronic acid
and guluronic acid
Pseudomonas spp.
and Azotobacter spp.
Biomaterial (for example, as a
tissue scaffold or for drug delivery)
Xanthan -(1,4)-linked repeating
pentasaccharide units
Glucose, mannose
and glucuronate
Xanthomonas spp. Food additive (for example, as a
thickener or an emulsifier)
Dextran -(1,2)/-(1,3)/ -(1,4)-branched
-(1,6)-linked homopolymer
Glucose Leuconostoc spp. and
Streptococcus spp.
Blood plasma extender and
chromatography media
Curdlan -(1,3)-linked
homopolymer
Glucose Agrobacterium spp.,
Rhizobium spp. etc
Food additive (for example, as a
thickener or a gelling agent)
Gellan -(1,3)-linked repeating
tetrasaccharide units
Glucose, rhamnose
and glucuronate
Sphingomonas spp. Culture media additive, food
additive (for example, as a gelling
agent) or for encapsulation
Cellulose -(1,4)-linked homopolymer Glucose Alpha-, Beta- and
Gamma- proteobacteria,
Gram-positive bacteria
Food, diaphragms of acoustic
transducers and wound dressing
Hyaluronic
acid
-(1,4)-linked repeating
disaccharide units
Glucuronate and N-
acetyl glucosamine
Streptococcus spp. and
Pasteurella multocida
Cosmetics, viscosupplementation,
tissue repair and drug delivery
Cyanophycin
granule
Repeating heteropolymer
consisting of dipeptide units
Aspartate and
arginine
Cyanobacteria,
Acinetobacter spp. , etc.
Dispersant and water softener
(after removal of arginyl residues)
Poly-
-glutamate
Homopolymer D-glutamate and/or
L-glutamate
Few Gram +, Gram -
bacteria, & few archaea
Replacement of polyacrylate,
thickener, humectant, drug
delivery and cosmetics
Examples of bacterial polymers, features and their applications
Glycerol, whey, corn steep liquor etc. are industrial waste by-products

How the whey, a waste by-product of cheese- and yogurt-making
industries is converted into economically valuable biopolymer?
Example of waste by-product utilization to make biopolymer
Whey contains ~94% water, ~ 5% lactose, rest small amount of
proteins, minerals etc.
Enormous quantities are generated by diary industry
Disposal is a problem and cause ground-water contamination &
environmental pollution
Releasing into rivers and lakes cause available oxygen depletion,
killing aquatic organisms
When used in processed food product, may be a problem to lactose
intolerant people
Recovering the solid component from whey is cost-expensive
Thus, effective way of whey utilization was sought for
Xanthomonas campestris is a natural producer of xanthan gum. The wild type
X. campestris utilizes glucose, sucrose and starch, but not lactose as carbon.
The genes encoding for lactose catabolism enzymes from E. coli were cloned
onto a broad-host range plasmid and under the transcriptional control of X.
campestris bacteriophage promoter.
lacZ encodes -galactosidase enzyme that cleaves the disaccharide lactose
into glucose and galactose
lacY encodes -galactoside permease, a membrane-bound transport protein
that pumps lactose into the cell
This recombinant plasmid was then introduced into X. campestris by
triparental mating.
How E. coli lactose catabolism was introduced into other bacteria
capable of synthesizing biopolymer?
Mol Biotech book Glick et al.
How E. coli lactose catabolism & biopolymer synthesizing genes of
other bacteria were put together in E. coli?
Several bacteria like Alcaligenes eutrophus, Ralstonia eutropha, Pesudomonas sp.,
Azotobacter sp. naturally synthesize polyhydroxybutyrate (PBA) , a type of
polyhydroxylalkanote (PHA), but not E. coli.
The PBA synthesizing genes from Azotobacter sp. were cloned onto the plasmid
under the transcriptional control of lac promoter.
This recombinant plasmid was then introduced into an E. coli strain that has the
genes for lactose uptake and assimilation, but not the lactose repressor gene. Thus,
this transformed E. coli cells express the lactose catabolism and PBA biosynthesizing
constitutively.
phaA for 3-ketothiolase, phaB for acetoacetyl-CoA reductase, phaC for
polyhydroxylalkanoate synthase
The engineered E. coli can grow on whey or corn steep liquor (a by-product of corn
processing, and source of nitrogen, amino acids, vitamins and other nutrients) to
produce PBA upto 73% of dry cell weight in fed-batch culture.
Mol Biotech book Glick et al.
On 03.09.2013, Prof. D. Das took my class. Hence, I took two classes
(lecture # 7 & 8) on 10.09.2013

Bioprocess Technology Lecture # 1, 2, 3, 4, 5, 6 & 7 (MKM 10.09.2013)

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Bioprocess Technology Lecture # 1, 2, 3, 4, 5, 6 & 7 (MKM 10.09.2013)

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