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

8/11/2019 Bioprocess Technology Lecture # 1, 2, 3, 4, 5, 6 & 7 (MKM 10.09.2013)

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Bioprocess Technology

(Industrial biotechnology)


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Biological ScienceNon-Biological Science

&

Engineering

Biotechnology

&

Biochemical engineering


Product

Biotransformation /

Fermentation

by

Bio-agent

(enzymes/organisms)

Biomaterial /

biomass

Renewable / non-renewable


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Bioreactor/ fermentation

Food processing

Immobilized enzymes

Detoxification of wastes

Bye product utilization

Biosensor

Several others


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Upstream processing:

Selection and preparation of the suitable biosystem

[enzyme/organism(microbe)]

Selection and preparation of the substrate (for enzyme) / rawmaterial (for proper growth of the organism/microbe) for

product formation

Fermentation and Biotransformation:

Immobilization of the catalytic enzyme or growth of thecandidate 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 thetarget 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


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Microbial Cell Factories (MCFs)

for chemicals & fuels


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Microorganisms have the diverse metabolic pathways and pathwayenzymes 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?


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Glucose

Transformation of renewable biomass-derived sugars

to chemicals by MCF

Ref: Science 2010, 330: 1355-1358


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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


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Lecture # 1, delivered up to this slide on 23.07.2013


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1) Improvement of the upstream processes

i) Development and Improvement of the microbial strainincluding the pathwayand 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


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Strain development and improvement


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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


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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


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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)


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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 validationin small-scale is crucial


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factories (MCFs): Conceptual strategiesb) Selection of suitable

enzyme(s) for the pathway using

various enzyme information

databases.

Selected Enzymes:

1) clearly known activityfor thesubstrate to specific

metabolite conversion

2) promiscuous activity for

structurally and chemically

similar substrates (or similarcatalytic reactions with

different substrate)

Bioinformatics approach &

Experimental verification


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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 effectiverecycling of suitable cofactor involved

in the target metabolite production.

Bioinformatics approach &

Experimental verification


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Ref: Current Opinion of Structural Biology 2011, 21: 1-7


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i & i i f h h f i bi l ll


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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

D i & i i f h h f i bi l ll


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Ref: Current Opinion of Biotechnology 2008, 19:468-474

a) Recruitment of partial pathways fromindependent sources and co-localization

in a single host to produce a known target

product

D i & i i f h h f i bi l ll


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b) Using engineered orpromiscuous enzymes,

new pathways can be

constructed for the

production of novel,non-natural products

M1

M2

D i & i i f th th f i bi l ll


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c) de novo design or retro-biosynthetic

approach considers the biotransformation ofthe 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.



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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 novopathway design using synthetic & systems biology


factories (MCFs): Objectives/outcomes/examples



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1) Combinations of existing pathways from different

organisms/strains having individual native enzyme activities into asingle host:



Outcome: new/hybrid pathway

to produce the same naturalproduct/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).

Desi n & en ineerin of the path a for mi robial ell


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2) Engineering of existing pathway with naturally occurring

promiscuous enzymes or engineered enzymes or in combination



Outcome:new pathwayand 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. coliand P. putida(Ref: J Am Chem Soc 2003,

125:12998-12999).



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3) de novo pathway design



Outcome:new pathwayand 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 silicodesign 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.


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factories (MCFs): de novopathway design

Trends in Biotechnology (2010) 28: 501508



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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


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metabolic pathway engineering

FEBS Letters (2010) 584: 2556-2564



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Overview of various high throughput databases available for


FEBS Letters (2010) 584: 2556-2564



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FEBS Letters (2010) 584: 2556-2564



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FEBS Letters (2010) 584: 2556-2564



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FEBS Letters (2010) 584: 2556-2564



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FEBS Letters (2010) 584: 2556-2564



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FEBS Letters (2010) 584: 2556-2564



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FEBS Letters (2010) 584: 2556-2564

Integration of system biology, synthetic biology and


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Integration of system biology, synthetic biology and

evolutionary engineering into metabolic engineering

Trends in Biotechnology August 2011, Vol. 29, No. 8 (pp 370-378)


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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 minimumgenome for basic and applied research

Systems biology is an integrative theoretical and experimental


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Systems biology is an integrative theoretical and experimentaldiscipline 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 phenotypesof an organism under various conditions

of interest for several possible applications, including systemsmetabolic engineering.


Synthetic biology aims at creating novel functional parts modules


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Synthetic biologyaims 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 biologicalsystemswill be a continued challenge of synthetic biology for metabolic

engineering.


Evolutionary engineering is defined as a technique to select or


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Evolutionary engineering is defined as a technique to select orscreen 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 objectivesof evolution can includebetter cell growth,

utilization of desired carbon sources,

higher yield of product,

good tolerance to the product or inhibitory intermediate metabolite,elimination of byproduct formation.


Advantages of strain development by systems metabolic engineering


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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 andconcentration 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.



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Trendsin

BiotechnologyAugust2011,Vol.29,No.8

(pp

37

0-378)


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This is Prof Bahadurs class, he was absent & told me to take this class.

What is the need for universal platform microbe for MCF ?


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pNo straightforward wayto extract the natural products (desired metabolite) from the native

producerat 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-yielderof the desired product/metabolite

Lack of genetic and physiological information in these native producers and suitable

engineering tools to genetically manipulatethese organisms to improve the product yield and

productivity

Some platform microbes are:

Escherichia coliSaccharomyces 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


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cerevisiaeplatform 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

Twomoregenesaddedf

romA

rtemisiaannua

E.g., terpenoids

Two genes added from Taxus chinensis

Example of systems metabolic engineering approach: Saccharomyces


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cerevisiaeplatform 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

TwomoregenesaddedfromA

rtemisiaannua

E.g., terpenoids

Two genes added from Taxus chinensis

Example of systems metabolic engineering approach: Escherichia coli


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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 metabolitealong with the synthetic taxadiene 5-

hydroxylase with evolved N-terminal

transmembrane domain.

Metabolomics identified that indole isthe inhibitorof E. colicell growth and

taxadiene synthesis.

Titer: About 1g/L of taxadiene

Taxa-4,11-dieneGGPP

GLC = glucose


Example of systems metabolic engineering approach: Escherichia coli


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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) orP(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 wtTrends in Biotechnology August 2011,Vol. 29, No. 8 (pp 370-378)

The key issue: Genetic regulation of the pathway enzymes


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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 themetabolic 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 (Contd..)


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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 plasmidbearing the genes of interest

Promoterstrength & types (constitutive or inducible)

Presence of terminator Proper uses of activatorand repressor

Specificity/efficiency of ribosome binding site (RBS)

Codonusage

mRNAstability


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The key issue: Genetic regulation of the pathway enzymes (Contd..)


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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 desiredmolecule 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.

Use of regulatory RNA (encoded by gene R) or protein


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(encoded by gene P) to modulate the metabolic pathway

that has a toxic/inhibitory metabolite

Ref: Science 2010, 330: 1355-1358



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g y ( y g ) p



Ref: Science 2010, 330: 1355-1358



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g y ( y g ) p



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


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MCFs for production of building block

chemicals & biopolymers

MCFs for production of building block chemicals & biopolymersMicroorganisms are endowed with capabilities to synthesize a wide range of building


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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 propertiessuitable 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 offossil-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 technologyand advanced downstream processes.

Recent advances in systems metabolic engineering helped us to develop superior

strains or MCFs.


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MCFs for production of building block chemicals & biopolymers


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(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

glutamicumand their engineered derivativescan 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 engineeredE. colican produce 87 g/Lsuccinic acid

Succinic acid is commonly used as surfactant, precursor of other

chemicals (e.g. 1,4-butanediol = BDO) and poly(butylene succinate) =

PBS polymer



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(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 caseiand their engineered

derivativescan produce 30135 g/Lof lactic acid.

Metabolically engineeredE. colican produce 138 g/Llactic acid

3) Itaconic acid producers:

Natural isolates of Aspergillus terreus after strain improvement by

random mutagenesis produce 82 g/Lof itaconic acid.Systems metabolic engineeredE. colican produce 4g/Litaconic acid

Lactic acid is the precursorof polylactic acid (PLA) homopolymer andpoly(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



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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/Lglucaric acid

Glucaric acid is the precursorof 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


( ) d f b ld bl k h l f f l l


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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


( ) d i f b ildi bl k h i l f f l l


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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 engineeredE. colican produce 60 g/Lisoprene.

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


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MCFs for production of building block chemicals & biopolymers(B) Production of biopolymers: A few successful examples


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(B) Production of biopolymers: A few successful examples

Some microbes naturallyproduce biopolymers or can be engineeredto produce the

biopolymers directlyby 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 ofcentral metabolism


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central metabolism

Nature Review Microbiology (2010) 8: 578- 592

l l d d l

Examples of bacterial polymers, features and their applications


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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.

andAzotobacter 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

Xanthomonasspp. Food additive (for example, as a

thickener or an emulsifier)

Dextran -(1,2)/-(1,3)/ -(1,4)-branched

-(1,6)-linked homopolymer

Glucose Leuconostocspp. 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

Hyaluronicacid

-(1,4)-linked repeatingheteropolymer consisting of

disaccharide units

Glucuronate and N-acetyl glucosamine

Streptococcusspp. andPasteurella multocida

Cosmetics, viscosupplementation,tissue repair and drug delivery

Cyanophycin

granule

Repeating heteropolymer

consisting of dipeptide units

Aspartate and

arginine

Cyanobacteria,

Acinetobacterspp. , 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


P l P i M i N l d I d i l

Examples of bacterial polymers, features and their applications


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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.

andAzotobacter spp.

Biomaterial (for example, as a

tissue scaffold or for drug delivery)

Xanthan -(1,4)-linked repeating


pentasaccharide units

Glucose, mannose

and glucuronate

Xanthomonasspp. Food additive (for example, as a

thickener or an emulsifier)

Dextran -(1,2)/-(1,3)/ -(1,4)-branched

-(1,6)-linked homopolymer

Glucose Leuconostocspp. 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

Hyaluronicacid

-(1,4)-linked repeatingheteropolymer consisting of

disaccharide units

Glucuronate and N-acetyl glucosamine

Streptococcusspp. andPasteurella multocida

Cosmetics, viscosupplementation,tissue repair and drug delivery

Cyanophycin

granule

Repeating heteropolymer

consisting of dipeptide units

Aspartate and

arginine

Cyanobacteria,

Acinetobacterspp. , 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


Gl l h t li t i d t i l t b d t

Example of waste by-product utilization to make biopolymer


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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?

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 lactoseintolerant people

Recovering the solid component from whey is cost-expensive

Thus, effective way of whey utilization was sought for

How E. coli lactose catabolism was introduced into other bacteriacapable of synthesizing biopolymer?


80/82

Xanthomonas campestrisis a natural producer of xanthan gum. The wild type

X. campestrisutilizes 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.

campestrisbacteriophage 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.

capable of synthesizing biopolymer?

Mol Biotech book Glick et al.

How E. coli lactose catabolism & biopolymer synthesizing genes ofother bacteria were put together in E. coli?


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other bacteria were put together in E. coli?

Several bacteria like Alcaligenes eutrophus, Ralstonia eutropha, Pesudomonassp.,

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 lacpromoter.

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 biosynthesizingconstitutively.

phaA for 3-ketothiolase, phaB for acetoacetyl-CoA reductase, phaC for

polyhydroxylalkanoate synthase

The engineered E. colican 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 weightin fed-batch culture.

Mol Biotech book Glick et al.


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On 03.09.2013, Prof. D. Das took my class. Hence, I took two classes

(lecture # 7 & 8) on 10.09.2013

Documents

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