Microbial cell factories,Biotechnology has found its entry into medicine (red) and agriculture (green), and now a new wave of industry (white), also called industrial biotechnology

1. Microbial cell factories
By
Assist. Prof
Dr. Berciyal Golda. P
Vicas

2. Biotechnology: old
2
its underlying processes have been used by mankind
for thousands of years. e.g. the production of wine and
cheese.

3. Biotechnology: new
Modern biotechnology uses enhanced micro-organism
like yeast, bacteria as ‘cell factories’, as well as
enzymes, to produce a variety of goods.
Downstream
Bioprocess
Products
3
Biomass
Microorganism

4. Three Waves of Modern Biotechnology
1980’s-
4
1990’s- 2000’s-
Red Biotechnology
Medicines
Green Biotechnology
Transgenic plants
White Biotechnology
Industrial products
Biotechnology has found its entry into medicine (red)
and agriculture (green), and now a new wave of
industry (white), also called industrial biotechnology.

5. Medical Biotechnol.
Agricultural Biotechnol.
Industrial Biotechnol.
People
Health
Foods
？
Technology
Demand
Red Biotechnology
Green Biotechnology
White Biotechnology
Why Industrial Biotechnology?
5

6. Refinery provides the essential
chemicals and energy for
modern society
Fossil: the basis of modern civilization
6

7. 7
The challenges for socio-economic
People
Environment
pollution
Resource
depletion
Climate
change
25
20
15
10
5
0
1990
China CO2 emission
2000 2010 2020 2030 2050 2075 2100年
Environmental costs (white pollution, greenhouse,
etc)

8. Modern
Industry
Chemicals, pesticide,
fine chemicals
Rubber, fuel, nylon
Plastics, chemicals
Antibiotics, Vitamins
amino acids, enzymes
Oil
Coal
Petrochemical
Coal industries
Green & sustainable
Biochemical
Engineering
The solutions: Biotechnol. process
-- from Petroleum to Biomass as materials
8
Renewable
Corn Cassava
biomass
industry exhaust

9. Definition of Industrial Biotechnology
application of modern biotechnology for the industrial
production of chemical substances and bio-energy,
using living cells and their enzymes, resulting in
inherently clean processes within minimum waste
generation and energy use.
9
Nutraceuticals
Fine chemicals
Bulk chemicals

10. Can biotechnology be competent?
Fuels
C1-C6
Platform
chemicals
Polymers
C2 ~P
ADP ATP
C2 aldehyde
NADH NAD+
C2 CoA
Pi+O2 CO2
NADH
NAD+
C1
2
NAD+
NADH CO
C3
C3
~P H2O
C3 aldehyde~P
NAD+ NADH ADP ATP
ADP
C3 keto
ATP
C3 ~P
+
ADP ATP NADH NAD
C3 diol
H2O NADH NAD+
Raw
biomass
C6 ~P
ATP ADP
C5 ~P
C7 ~P
C4 ~P
ADP
ATP
NADPH
NADP+
Phenyl keto acids
C3 Hydroxy
C3 Acryloyl CoA
NADH
acids
NAD+
C3 acrylic acid
CO2
H2
C4 CoA
C3 alcohol
NADH NAD+
C4 alcohol
Aromatic
compounds
Polyketone
PHA
C4
C6
CO2
NAD+
NADH
CO2
C4 CoA
CO2
C6 sugar transport
glucose
C5 sugar
(xylose and
arabinose
transform
hydrolysis
10
hydrolysis
transport
transform
C3 ketone
C2 acids
C2 alcohol
C5 keto acids
acids

11. Hydrocarbons
CnH2n+2
11
Bio-products fit with current industries
Petroleum
• Bio-ethanol
Fuel & Energy • Bio-diesel
• Biogas, H2
Chemical
industries
• Bulk chemicals
• Fine chemicals
• Bio-polymers
• Gasoline
• Kerosine
• Diesel
• Basic chemicals
• Fine chemicals
• Polymers
HO
Biomass
OH OH
H
OH OH O
Carbohydrates
C (H O)
n 2 n
• Oxygenate
• Chiral chemicals

12. 12
Sugar
• glucose
• fructose
• xylose
• arabinose
Polymers
SG
C2
C3
C5
C6
C4
starch
Hemicell
ulose
Cellulose
Protein
Carbonhydrates Thermochem
Biomass
H2
methane
Itaconic acid
Levulinic acid
Fumaric acid
Succinate
Aspartate
Malic acid
Citric acid
Gluconate
Sorbitol
Lactic, glycerol
acrylic acid
3-hydroxy-
propionic acid
Ethanol, ethylene
Microorganism
Modern
Chemical
Indust.
Lignin
Lipid
Bio-products fit with current industries
Platform chem.

13. 13
Chemical synthesis
Pharmaceuticals
Pesticides
Fine chemicals
Food additives
Feed additives
Cosmetic
Organic
substrate
I.B. reduces the processes and costs
Bioprocess
 Materials < 37%
 Energy < 30%
 CO2 em. <63%
1,3-
PDO
Traditional
Chem.
Biotechnology
Fermentation

14. 14
G3P Pyruvate
Glucose
TCA
DHAP 3-PGA Gly
Glycerol
pathway from
yeast
Native pathway in E. coli
1,3-PDO
3-PHA
1,3-PDO pathway
from Klebsiella
pneumoniae
• Modify >70 genes, 18 genes knockout or overexpressed
• Titer > 135 g/L, productivity >3.5 g/L/h
• First industrialization example of bio-based chemical
Case: 1,3-propanediol bio-production

15. 15
Efficient
Genetic
Manipulation
Protein
Engineering
& Screening
System
Metabolic
Engineering
Rapid
Microbial
Evolution
Advanced Biocatalysts
Nutraceuticals
Fine chemicals
Bulk chemicals
Fuel
Biotech makes industry green

16. Plant secondary metabolites
More than 2,000 kinds
of plant natural
products such as
isoprenoids, alkaloids
and flavonoids, which
are all plant secondary
metabolites, have been
used by human as
flavors, fragrances and
medicines.
(Chang et al., 2006)

17. (Marienhagen, et al., 2013)
Schematic overview of biosynthetic routes and
precursors of the plant natural products

18. Isoprenoids
Isoprenoids comprise the largest class of the natural product products,
encompassing more than 5,000 known compounds with an extremely
diverse array of chemical structures, such as taxol, artemisinin, and
carotenoids.

19. Alkaloids
Alkaloids are nitrogen-containing, low-molecular-weight
compounds and known for their medicinal use.

20. Glucosinolate
Glucosinolates constitute a natural class of organic
compounds that contain sulfur and nitrogen and are derived
from glucose and an amino acid.

21. Natural diversity of glucosinolates
They are synthesized from certain amino acids.
About 132 different glucosinolates are known to occur
naturally in plants.
Aliphatic glucosinolates derived from mainly methionine, but
also alanine, leucine, isoleucine, or valine.
Aromatic glucosinolates include indolic glucosinolates,
derived from tryptophan, phenylalanine and tyrosine.

22. Enzymatic activation
The plants contain the endogenous thioglucosidases, called
myrosinase, which, in the presence of water, cleaves off the
glucose group from a glucosinolate. The remaining molecule
then quickly converts to an isothiocyanate, a nitrile, or
a thiocyanate; these are the active substances that serve as
defense for the plant.
To prevent damage to the plant itself, the myrosinase and
glucosinolates are stored in separate compartments of the cell
and come together mainly under conditions of physical injury.

23. Sulphoraphane
(SFN)
Benzyl Isothiocyanate
(BITC)
Phenylethyl
Isothiocyanate
(PEITC)
Isothiocyanate was known for anti-carcinogenic activity,
antimicrobial activity, and anti-inflammatory activity .
Studies have suggested inverse relations between the intake of
cruciferous vegetables, such as broccoli, and cancer incidence.
The following compounds contribute to this function.
Isothiocyanates

24. Natural diversity of isothiocyanates
Its variation in the side group that is responsible for the
variation in the biological activities of these plant compounds.
Some glucosinolates:
• Sinigrin is the precursor to allyl isothiocyanate
• Glucotropaeolin is the precursor to benzyl isothiocyanate
• Gluconasturtiin is the precursor to phenethyl isothiocyanate
• Glucoraphanin is the precursor to sulforaphane

25.  Natural diversity makes the extraction of separate pure
compound high costly
 The enzymes of entire pathway in plants stored in separate
compartments of the cell, make the synthesis inefficient
 Extraction process is complicated with high pollution
Shortcomings of natural extraction

26. 26
中国科学院微生物研究所
The comparison between plants and
microorganisms as metabolic engineering platform
plant E. coli
Growing rate slow rapid
Costs high low
Yield low high
Purity low high
Extraction
process
difficult simple
on an industrial scale

27. 27
中国科学院微生物研究所
Microbial production of ITCs
-- Benzyl Isothiocyanate as a case --
E .col
i
G ene m i
ni
ng P rotei
n m odi
fi
cat
i
on
P athw ay desi
gn
F uncti
onaly
expressed enzym es
P henyl
al
ani
ne B enzyli
sot
hi
ocyanate

28. 28
中国科学院微生物研究所
P450 enzymes
Glucotropaeolin
The original biosynthesis pathway in plant
myrosinase

29. 29
中国科学院微生物研究所
The difficulties and solution strategies for P450
modules
2. Absence of electron transfer machinery
linker peptide: (G4S)n
Linker
peptide
ATR1： NADPH--cytochrome P450 reductase 1
ATR2：NADPH--cytochrome P450 reductase 2
1. Translational incompatibility of the membrane signal
Modification of N-terminal
membrane –binding domain
Fused with corresponding
reductase via linker peptide
This enzyme is required for electron
transfer from NADP to cytochrome P450
in microsomes.

30. 中国科学院微生物研究所
Functional expression of CYP79A2
The first eight
modified amino acid
from bovine-derived
CYP17α was
confirmed to be
advantageous for
anchorage to the
membrane for E.coli
73-711 amino acids were
the functional domain
without transmembrane
sequence
the 25 to 74 amino
acids from sorghum
derived CYP79A1
were proven to be
effective for higher
expression level
40-529 amino acids were the functional
domain
30

31. 中国科学院微生物研究所
Functional expression of CYP83B1
ATR1： NADPH--cytochrome P450 reductase 1
ATR2：NADPH--cytochrome P450 reductase 2
31

32. 中国科学院微生物研究所
The biosynthesis pathway of benzyl isothiocyanate
32

33. 中国科学院微生物研究所
Design the pathway and reduce the step
Glutathione
GSTF GGP1
Spontaneous reaction
Cysteine
Pathway in plants
33

34. 中国科学院微生物研究所
C-S lyase was not redundant
unlike the other enzymes in plant.
SUR1 is the only C-S lyase for this
pathway in plants.
We tried to optimize the induction
conditions but still failed to get the
functional enzyme for inclusion
body.
Selection and expression of C-S lyase
SUR1
In plants
34

35. 中国科学院微生物研究所
Selection and expression of C-S lyase
The biosynthesis of methionine in E. coli involves cystathionine β-
lyase which catalyzes a reaction similar to SUR1.
35

36. 中国科学院微生物研究所
8 1
0
m
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MetC
1
2 1
4 m
in
Pyruvic acid
MetC (β-lyase) was chosen for substituting SUR1
KDa
94
36
66
45
33
26
M 1 2

37. 中国科学院微生物研究所
Condon-optimized UGT74B1 from Broccoli rapa was successfully
expressed and purified which shows a high affinity for various
types of thiohydroximate. UGT74B1 Marker
UGT: UDP-glucose:thiohydroximate S-lucosyltransferase
37

38. 中国科学院微生物研究所
SOT16、SOT18 both show catalyze activity on benzyl-derived
glucosinolate. We failed to obtain the soluble SOT16 and select
Arabidopsis thaliana ecotype Col-0 derived SOT18 which was
confirmed to have higher Vmax compared to the other variants.
SOT18 Marker
SOT: desulfoglucosinolate:PAPS sulfotransferase
38

39. 中国科学院微生物研究所
Selection of suitable myrosinase
 Glycosylation modification was essential for plant-
derived myrosinases to form activity configuration.
 Myrosinase from Brevicoryne brassicae which need no
secondary modification was selected to express in
E.coli.
39

40. 中国科学院微生物研究所
The activity of
Myrosinase from
Brevicoryne
brassicae
was confirmed
40

41. 中国科学院微生物研究所
CYP79A2
O2
NADPH
CO2
H2O
NADP+
CYP83B1
O2 CO2
NADPH H2O
NADP +
SOT18
PAP PAPS
UGT74B1
UDP UDP-D-
glucose
BMYR
• Black indicate the
original element
• Red indicate the
modified in E.coli
41
PLP
design
The modified biosynthesis pathway of benzyl
isothiocyanate in E. coli
All enzymes have been functionally expressed in E.coli

42. 中国科学院微生物研究所
As a proof of
concept,
successful
biosynthesis
of BITC in vitro
by functionally
expressed
enzymes from
different
sources was
confirmed.
42

43. 43
中国科学院微生物研究所
Conclusion
ACS Synth. Biol. (2016)

44. 44
中国科学院微生物研究所
Next……
Functionalizing the synthesis in vivo
 Cofactor supplement: cysteine and PAPS
 Coenzyme balance and supplement: NADPH
 Remove susbtrate feedback inhibition and degadation: cysteine

45. 中国科学院微生物研究所
45
Pyruvate
+ NH3+
H2S
tnaA
ATP AMP
ATP regenaration
Regeneration
Remove feedback inhibition
cysQ
Cofactor supplement
Remove product
inhibition
Knock-out
degradation path.
SAT-m from
Arabidopsis

46. 中国科学院微生物研究所
NADPH balance (P450 enzymes needs NADPH)
46
Expression of zwf increases
PPP pathway for NADPH
supplement

47. 中国科学院微生物研究所
sulfide
O-Acetylserine
SA
T-m
serine
zwf
Glucose-6-P Glucose
47
Schematic map of BITC synthesis in vivo
PAPS regeneration

48. 中国科学院微生物研究所
Feixia Liu
48
Acknowledgement

49. Thanks for your
attention！
Bo Yu
E-mail: yub@im.ac.cn
49