ChE 275 Winter 2014 Lecture 1 (January 6)

Class Introduction & Impact of Biology and Biotechnology in Chemical Engineering

Focus of ChE 275

Basic understanding of biochemistry, cell biology and molecular biology. The flow of genetic information. Cell structure and function. Cell regulation and control strategies. Applications of biology in (chemical) engineering.

Key concepts
Specific noncovalent interactions and recognition events. Diverse roles of hydrophobic and hydrophilic interactions.

Selective transport of molecules.

Storage, use, and inter-conversion of chemical energy. Transduction, amplification, and modulation of external signals.

Key concepts
Multiple levels (and time scales) by which cells regulate protein activity. Biopolymers: functions, synthesis, and regulated degradation.

Reversibility, regulation and linkage of biological reactions.

Regulation and quality control of DNA, RNA and protein synthesis and processing. Recombinant DNA technology.

Assignments for ChE 275

Complete reading assignment (~ 20 pages) and reading test (by 7:00 am) on Blackboard before each MW class; OK to discuss with classmates, but complete RT independently.
First reading test on Mon Jan 13.

Lectures/class discussions will focus on key concepts and questions/issues raised on-line and in class. Closed-book quiz each Monday. Copies of previous quizzes will be provided. Final exam at 3:00 5:00 pm on March 19.

Office Hours
Shea (Silverman 3617) - before quiz each week:
Monday 11:00 AM 12:00 PM After class

TAs By appointment
Jennifer Schoborg (jschoborg@northwestern.edu) Sarah Wood (sarahwood2011@u.northwestern.edu)

Biotechnology and Bioengineering at NU

Molecular

Cellular
Amaral Broadbelt Jewett Kung Leonard Miller Shea Tyo

Amaral Grzybowski Leonard Miller Shea Tyo

Tissue-level Global; Systemslevel

Amaral Broadbelt Jewett

Leonard Miller Shea

Biotechnology and bioprocess engineering

Biotechnology is broadly defined as the use of biological systems or organisms for a socially desirable goal (healthcare, environment, etc.). Bioprocess engineering is the application of chemical (and other) engineering principles to design and optimize biological processes and biological catalysts to bring about desired chemical transformations. A key aspect of bioprocess engineering is the need to develop large-scale processes that are economical to operate and comply with FDA regulations.

Why study biology as a chemical engineer?

1. An individual cell can be treated as a micro-factory
Inputs, outputs, oxygen/mass transport, kinetics (enzyme), energy balances (ATP, NADH, etc.), and even control systems Capable of being optimized individually (metabolic engineering)

2. Much biological research requires systems level understanding

Biologic pharmaceuticals, disease treatment, microbial generation of commodity chemicals, bioremediation All levels of systems: individual cells, tissue, whole organisms, communities of organisms Engineers are well trained for such tasks

3.

Biological commercial processes require massive scale-up

Molecular/ bench-scale research Pilot scale Industrial scale

The story of penicillin

Discovery of penicillin biosynthesis by Penicillium notatum. (1928)
Not useful for medicine

The Problem A simple scratch could result in a lethal infection In WWII, cut, burn, bullet wounds frequently were infected in battlefield conditions Development of penicillin synthesis by Howard Florey and Ernst Chain (1939)
Grow the mold faster (reactants, optimal T) Extract the penicillin (separations) 1st Clinical Test in vivo

Pressing need for penicillin during WWII involvement of industry such as Merck, Pfizer, Squibb and USDA-NRRL.

Cartoon source: http://nobelprize.org/medicine/educational/penicillin/

The story of penicillin

Post WWII Low yield of penicillin from Penicillium notatum (ca. 0.001 g/l) and hence very inefficient process. Fermentation route vs. chemical route Chemical route initially preferred. Isolation of penicillin hyper-producing strain, Penicillium chrysogenum at NRRL. Penicillin fermentation in surface cultures. Penicillin fermentation in roller bottles. Development of submerged fermentation process by Pfizer.

Penicillin productivity
Mutation and breeding Molecular biology and breeding

New species

Bioprocess engineering improvements have contributed greatly to the increased productivity.

Products made by biotechnology

Vitamin B2 Polymers/plastics Ethanol Fuel Pharmaceuticals Vaccines

http://www.bio.org/ind/pubs/cleaner2004/CleanerReport.pdf

Vitamin B2 synthesis

http://www.bio.org/ind/pubs/cleaner2004/CleanerReport.pdf

http://www.bio.org/ind/pubs/cleaner2004/CleanerReport.pdf

http://www.bio.org/ind/pubs/cleaner2004/CleanerReport.pdf

http://www.bio.org/ind/pubs/cleaner2004/CleanerReport.pdf

DuPonts Sorona is one member of a family of polymers based on Bio-PDO corn-derived chemical/1,3-propanediol.

http://www.dupont.com/sorona/technologyplatform.html

A joint venture between DuPont and Tate & Lyle PLC has been formed to produce 1,3-propanediol (PDO), the key building block for DuPont Sorona polymer, using a proprietary fermentation and purification process based on corn sugar. This bio-based method uses less energy, reduces emissions and employs renewable resources instead of traditional petrochemical processes.

http://www2.dupont.com/Sorona/en_US/

DuPont is pursuing other bio-based compounds

Convert glucose to the bi-functional aromatic compound p-hydroxycinnamic acid (pHCA), which can serve as a building block for various industrial chemicals. Two-step process in engineered E. coli via tyrosine intermediate.

Omega-3 fatty acids (EPA and DHA; present in fish oil) in engineered Yarrowia lipolytica yeast grown to accumulate triglycerides. Production of high-value carotenoids from methane or methanol in engineered Methylomonas sp. 16a.
Abstracts submitted to Metabolic Engineering Conference

http://www.bio.org/ind/pubs/cleaner2004/CleanerReport.pdf

U.S. National Vision goals for biomass technologies

Appl. Microbiol. Biotechnol., 64:137, 2004

http://www.bio.org/ind/pubs/cleaner2004/CleanerReport.pdf

Life Cycle

Nature Biotechnology, 22: 671, 2004

Integration

Appl. Microbiol. Biotechnol., 64:137, 2004

http://www.bio.org/ind/pubs/cleaner2004/CleanerReport.pdf

Examples of therapeutic bioproducts

Taxol Penicillins

Erythropoietin

Insulin dimer and hexamer

Therapeutic Glycoproteins
Initially recovered from blood and other tissues (clotting factors and growth hormone). Production at low concentrations in primary cell culture (urokinase). Recombinant DNA technology developed in bacteria by Cohen and Boyer in 1973. Genetic engineering of animal cells to produce recombinant proteins became routine in the 1980's. tPA approved in 1987. EPO sales of $1.4 billion in 1994 (still in roller bottles). Many small reactors, but reactors in the 10,000+ L scale are used for high-volume products.

Monoclonal Antibodies
Polyclonal antibodies initially recovered from blood and produced in animals (-globulin and antisera) Hybridomas for production of monoclonal antibodies were developed by Khler and Milstein in 1975. Production expanded from mouse ascites to stirred cultures and hollow fiber reactors. Many of the recent advances in animal cell culture were developed using hybridomas. OKT3 (reversal of transplant rejection) approved in 1986. Used in many diagnostic assays. Production scale now exceeds 10,000 L for airlift and stirred vessels. New advances include fed-batch and highdensity perfusion systems. New therapeutic applications require doses of 215 mg/kg body weight and are providing much of the growth.

U.S. Biotech: 1994-2005

Sales up 4X R&D spending up 3X Employees up 2X

Source: Ernst & Young, LLP

2005-2006 Top Ten Biotech Drugs: US Sales >$30 Billion

Product (indications) Enbrel (arthritis, psoriasis, ankylosing spondylitis) Aranesp (anemia) Rituxan/MabThera (nonHodgkin lymphoma) Remicade (Crohn disease, arthritis) Procrit/Eprex (anemia) Herceptin (breast cancer) Epogen (anemia) Neulasta (neutropenia) Human insulinsc (diabetes) Avastin (colon cancer) Company Amgen, Wyeth, Takeda 2005 sales ($ billions) 2006 sales ($ billions) Change

$3.7

$4.4

20%

Amgen Biogen Idec, Genentech, Roche Johnson & Johnson, ScheringPlough Johnson & Johnson Genentech, Roche Amgen, Kirin Amgen NovoNordisk Genentech, Roche

$3.3

$4.1

26%

$3.3
$3.0 $3.3 $1.7 $2.8 $2.3 $2.5 $1.3

$3.9
$3.6 $3.2 $3.1 $2.9 $2.7 $2.5 $2.4

16%
20% -4% 82% 0% 18% 1% 77%

Vaccine Production
1950's: Polio vaccine produced in African Green Monkey kidney cells in roller bottles and flasks. 1960's: Foot-and-mouth disease vaccine produced in BHK cells adapted to suspension culture (1,000 4,000 L scale). 1970's 1980's: Microcarriers allowed for culture of adherent cells in stirred vessels (500 L scale). 1990's 2000's:
extensive use of larger stirred vessels automated roller bottle systems cell factories and cell cubes

2000's: New growth emphasis from potential for flu pandemic and bioterrorism; focus on cell lines.

Nature Biotechnology, 24: 284 (suppl.), 2006

Cell & Tissue Culture Applications

Cultured cells as final products:
"bone-marrow" transplantation immunotherapy

mesenchymal cell therapies

blood cells for transfusions neural stem and progenitor cells gene therapies

Cell & Tissue Culture Applications

Cells for encapsulation (pancreas).
Cultured tissues for transplantation (skin, chondrocytes, nerve regeneration).

Functional extracorporeal or implanted bioartificial organs (liver, kidney).

Model systems for toxicity and efficacy testing (liver, cornea).

Combination Devices- KeraCures KeraPac

Non-woven fabric combined with porous microcarrier beads and human keratinocytes (skin cells)
Placed directly on the wound, removed several days later

Active wound care

Covers & protects Promotes moist environment Active agents stimulate healing

http://www.keracure.com/kerapac.asp

Regenerative Medicine: Artificial Bladder

Tengion technology
Neo-bladder Patients cells (autologous)
minimizes immune rejection risk

Three phase II clinical trials initiated

http://www.tengion.com/technology/platform.cfm

Not only can biotech create It can also destroy.

http://www.bio.org/ind/pubs/cleaner2004/CleanerReport.pdf

Chlorinated hydrocarbons are excellent solvents, used in many industries.

Chlorinated hydrocarbons are very toxic and accumulate in ground water..

Several species of Dehaloccoides that are effective in breaking down chlorinated chemicals have had their genomes sequenced. This may allow for further optimization of chlorinated compound breakdown by Dehaloccoides, and may allow for use of selected genes in other bacteria.

Nature Biotechnology, 23: 1235, 2005

Explosives contaminates can also permeate ground water.

Nature Biotechnology, 24: 162, 2006

Incorporation of an NADPH-dependent nitroreductase from Enterobacter cloacae and the complex XplA enzyme (ferrodoxin and P450 domains) from Rhodocossus rhodochrous allows plants to degrade (and get energy from) TNT and RDX explosives. Endogenous plant enzymes catalyze the remaining reactions.

Why study biology as a chemical engineer?

1. An individual cell can be treated as a micro-factory
Inputs, outputs, oxygen/mass transport, kinetics (enzyme), energy balances (ATP, NADH, etc.), and even control systems Capable of being optimized individually (metabolic engineering)

2. Much biological research requires systems level understanding

Bi ol ogi c pharm ac euti c al s , di streatm eas e ent, m i c robi al generati on of c om m odi ty c hem i c al s , bi orem edi ati on Al l l ev el s of s y s tem s : i ndi v i dual c el l s , ti s s ue, whol e organi s m s , c om m uni ti es of organi s m s Engi neers are wel l trai ned for s uc h tas k s

3.

Biological commercial processes require massive scale-up

M o l e c u l a r/ b e n c h -s c a l e re s e a rc h Pi l o t s c a l e In d u s tri a l s c a l e

Highly Automated Production Facilities

Nature Biotechnology, 23: 1054, 2005

Impact of biology on ChE Depts.

More than half of incoming graduate students are interested in bio-related research. Many undergraduates are also interested in biology; parallel growth in BME Depts. Many of our students take jobs in bio-related positions or companies. Most ChE departments now require a class in biology for all students (13/16 in Big 10+). Many ChE departments have changed their name; some have developed new degrees or majors and most have bio-related options.

Employment Opportunities
R&D Development Commercialization

Discovery Product Research

Process Development Product Development Quality Assurance/Control Medical Writers Project Managers Intellectual Property

Product Managers Technical Sales Technical/Customer Support Scientific Liaisons Medical/Regulatory Affairs Quality Assurance/ Control Manufacturing & technical support

Process Research

Intellectual Property Clinical Monitors/Trainers

ChE 275 Winter 2014

Lecture 1a (January 6)
Cooper and Hausman Chapter 1
This chapter introduces a lot of material in overview format. We will go into much more detail on most of this during the quarter. Dont worry about all of the little details. .

Announcements
I will post powerpoint slides on Blackboard after lecture. TAs will be responsible for any dispute/regrades on weekly quizzes. See syllabus for which TA to contact on a given week. Lowest quiz will be dropped.

Outline
Evolution
Organic molecules (amino acids, metabolites) Macro-molecules (DNA, RNA, proteins) Membranes (phospho-lipid bilayer) Energy source (metabolism)

Fig. 1.1 Spontaneous formation of organic molecules

What was the bulk of the atmosphere composed during pre-biotic times?

1950s

Fig. 1.1 Spontaneous formation of organic molecules

Macromolecules
Nucleic Acids
RNA DNA

Which molecules can replicate themselves?

Proteins
Fig. 1.2 Self-replication of RNA
(specific noncovalent interactions)

Which came 1st, DNA or RNA?

Fig. 1.3 Enclosure of self-replicating RNA in a phospholipid membrane

(also has catalytic activity) Early membranes formed by simple amphiphiles were likely more leaky.

Fig. 1.4 Generation of metabolic energy

What was the source of energy to form the 1st organic molecules?

ATP <> ADP + Pi G0 = 7.3 kcal/mol

Fig. 1.4 Generation of metabolic energy

In what order did these mechanisms evolve?

Universal requirements for cells

What are the minimal attributes required for viable cells to exist?

Universal requirements for cells

What are the minimal attributes required for viable cells to exist?
A. Separation of cell contents from the environment.
B. Ability to transport nutrients into the cell. C. Ability to replicate with reasonable fidelity. D. Ability to generate energy to carry out cell functions. E. Functional molecules to carry out cell functions.

Universal requirements for cells

What are the minimal attributes required for viable cells to exist?
A. Separation of cell contents from the environment.
B. Ability to transport nutrients into the cell. C. Ability to replicate with reasonable fidelity. D. Ability to generate energy to carry out cell functions. E. Functional molecules to carry out cell functions.

Components DNA RNA Protein Lipid bi-layers ATP (chemical potential)

Conceptual model of protocell

(Mansy et al., Nature, 454:122, 2008)

Once we have the 1st cell, where do we go from here

Prokaryotes
Eubacteria Archeabacteria

Eukaryotes
Single-cell Multicelled
Plant Animal

Fig. 1.7 Evolution of cells

Species and Phylogenetic Trees

What are possible implications of this?

Bacterial Diversity
Bacteria, especially archaebacteria, have adapted to a wide range of environments; different bacteria can survive at: very high temperature very low temperature very acidic conditions very basic conditions

very high salt content

A black smoker at a hydrothermal vent deep under the ocean is home to a microbe from which a thermostable alpha amylase was recovered. The enzyme is now being used commercially for extracting ethanol from corn. (Source: Diversa) (Nature Biotechnol., 23:1199, 2005)

Fig. 1.5 Electron micrograph of E. coli

Diverse shapes

Ribosomes are present throughout the cell. Divide to form two new cells; may remain attached.

Bacillus cereus (SEM)

Streptococcus sanguis (SEM)

------- Present

At least three classes of dynamic polymers make up the bacterial cytoskeleton. Scientists continue to discover new things about bacteria.

Fig. 1.6 Structure of animal cells

Nuclear pore

(have their own DNA (partial) & ribosomes)

(both free and associated with the rough ER)

Eukaryotic cell nucleus and organelles

What are possible advantages for having organelles?

Eukaryotic cell nucleus and organelles

What are possible advantages for having organelles?
A. Creation of distinct environments (pH, redox, etc.).

B. Decreased diffusion distances; cells can be larger.

C. Localization of reactants for particular reactions. D. Protect DNA from damage.

What is the evidence that mitochondria and chloroplasts arose from prokaryotic endosymbionts?

Eukaryotic cell nucleus and organelles

What are possible advantages for having organelles?
A. Creation of distinct environments (pH, redox, etc.).

B. Decreased diffusion distances; cells can be larger.

C. Localization of reactants for particular reactions. D. Protect DNA from damage.

What is the evidence that mitochondria and chloroplasts arose from prokaryotic endosymbionts?
A. Their DNA and ribosomes resemble those of bacteria.
B. They are similar in size and replicate by simple division. C. Current endosymbionts show loss of DNA (similar size).

Endosymbionts have small genomes

Science, 314: 259, 2006 Science, 314: 267, 2006

Endosymbionts that lost substantial DNA may have remained as organelles.

Model organisms
What Why How

Experimental Biology an active field

Growing in the lab

Animal Plant Virus

Microscopy (only fluorescence / 2-photon)

81

Some model organisms

What is a genome sequence? Why useful for a model organism?

82

Which of the following are reasons that biologist define and study model organisms?
A. Model organisms are easy to grow and analyze in the lab. B. All model organisms require the same nutrients, so it is simple to compare. C. Model organisms have similar DNA to other organisms, so what we learn in a model organism applies to other organisms. D. Model organisms are more complex than others, so if we understand the model organism, the non-model organisms should be easy. E. We have lots of techniques to modify the DNA of model organisms that can be difficult in other organisms.

83

Bacterial model E. coli

Bacteria grow rapidly; dilute suspensions of single cells form colonies in solid media.

Fig. 1.13 Bacterial colonies

Colonies formed by mutants able to grow under adverse conditions can be readily isolated.

How? Grow at high T? Grow much faster?

Tougher to select for cells that dont84 grow.

Finding a mutant that is resistant to salt

1. Grow cells in high salt levels
1. What size colony would you expect for
1. Normal cell? 2. Salt-resistant mutant?

2. Analyze mutant
How would you find mutants with low salt resistance?
85

Fig. 1.14 EM of Saccharomyces cerevisiae

Nuclear membrane is not evident during cell division; rough ER is evident. Good model for studying cell division and organelle function, as well as transport into and out of the nucleus.

86

Fig. 1.15 Caenorhabditis elegans

C elegans has 959 somatic cells. What could we study in this organism?
87

Fig. 1.16 Drosophila melanogaster

Many genes related to development, including of limbs, were first identified in Drosophila. Gene names are often based on changes in fly appearance (e.g., wingless, frizzled).

88

Fig. 1.19 Zebrafish

Fins were the precursors of limbs.

Xenopus laevis eggs. How to decide which model system to use?

89

Fig. 1.20 Mouse as a model for human development

90 Model systems are often used in sequence during drug development

Model organisms
If you can choose only one model organism per study, which organism would be best suited for which study and why? Consider simplicity (why?) and appropriateness. Organisms:

E. coli (bacterium) S. cerevisiae (yeast) C. elegans (nematode or worm) D. melanogaster (fruit fly) M. musculus (mouse)
Studies:
a) effect of a specific gene on digestive system development b) transport of proteins through the nuclear membrane

c) how mutation of a specific gene affects limb development

d) role of protein misfolding in a neurodegenerative disease e) protein synthesis on ribosomes
91

Growing cells in culture

Unicellular organisms Easy
Why?

Multicellular organisms Hard

Animal Plant

92

Fig. 1.41 Culture of animal cells

Normal cells stop growing when they reach confluence. Transformed (tumor) cells and embryonic stem cells can grow indefinitely in culture. Lifetime in culture of normal cells is limited due to genetic damage and loss of telomeres.

93

Animal Cell Nutritional Requirements

In contrast to animal cells, yeast or bacterial cell cultures can be grown on fairly simple media without the need to add amino acids or various vitamins and hormones. What is/are the primary reason(s) for this?

94

Animal Cell Nutritional Requirements

In contrast to animal cells, yeast or bacterial cell cultures can be grown on fairly simple media without the need to add amino acids or various vitamins and hormones. What is/are the primary reason(s) for this? Animal cells come from organisms having multiple cell types with specialized functions, including metabolism. Animal cells typically live in association with other cells and receive stimulatory factors from other cells to regulate their growth. Animal cells lack the enzymes to synthesize some amino acids and vitamins (these are called essential).
95

Fig. 1.12 Light micrographs of selected animal cells

Fibroblasts in connective tissue

Red and white blood cells

96

Fig. 1.12 Light micrographs of selected animal cells

Isolated and cultured tissues can also serve as experimental models.

Why can cells look so different when they contain the same DNA?
97