1. Engineering microbial cell factories for biosynthesis of isoprenoid molecules: beyond lycopene
Daniel Klein-Marcuschamer, Parayil Kumaran Ajikumar and Gregory Stephanopoulos

2. Table of Contents Introduction Lycopene, Isoprenoids, Carotenoids
Taxol
Lycopene, Isoprenoids, Carotenoids
Background, Pathway
Three Problems
Substrate availability
Intermediate build-up
Increase storage capacity
Conclusion
First I’ll give a brief introduction into metabolic engineering and synthetic biology. Then I’ll give some background on the three classes of compounds that feature heavily in the paper. Alex will then talk about three problems that researchers are beginning to address in the lycopene pathway.

3. The paper starts off with an interesting analogy
The paper starts off with an interesting analogy. It talks about all the molecules that we’ve discovered in nature for medicine, agriculture, polymer synthesis and compares them to boxes in a warehouse. We’ve discovered a lot of these boxes:

4. There’s aspirin from willow bark

5. Nicotine from nightshade

6. The antibiotic streptomycin

7. We talked about opium a couple weeks ago

8. Penicillin

9. And of course, there’s taxol, or paclitaxel

10. The important thing to remember is that we’ve only harnessed the potential from a fraction of these boxes, either because we have yet to find the chemical, or there are barriers to its production.

11. Taxol
Take taxol for example. Its structure is extremely complicated, it’s got aromatic rings, esters, ketones, hydroxyls, ethers, and even an amide.

12. Taxol
It’s synthesis is even more complicated. It starts with a framework, there are multiple modifications, and very precise reaction conditions. Basically what I’m trying to say is:

13. It’s Complicated Taxol
It’s complicated. Making molecules like this takes time, money, and a lot of brain power. But that’s not to say

14. It’s Complicated = $$$$ Taxol
It’s complicated. Making molecules like this takes time, money, and a lot of brain power. But that’s not to say it hasn’t been done. The market for these kind of molecules is huge, in the hundreds of billions of dollars. Which means that any process to shave off a few seconds or pennies can be a process worth pursuing.

15. Metabolic Engineering: ‘de novo engineered microbes’
One such process is metabolic engineering, where microbes are engineered to make useful compounds with useful yields. Essentially there are three steps.
First of all, the foreign pathway is introduced into the host of choice.

16. Metabolic Engineering: ‘de novo engineered microbes’
Then you eliminate the superfluous reactions, products, or anything that will impede the making of your product.

17. Metabolic Engineering: ‘de novo engineered microbes’
Finally, you re-engineer the regulatory network to express your product in useful quantities. This can be done in a couple ways. It can be random, usually through large high throughput screens. Or, if you have a better idea of how the pathway works, you can do it in a directed fashion.

18. Metabolic Engineering: ‘de novo engineered microbes’
Finally, you re-engineer the regulatory network to express your product in useful quantities. This can be done in a couple ways. It can be random, usually through large high throughput screens. Or, if you have a better idea of how the pathway works, you can do it in a directed fashion.

19. Metabolic Engineering: ‘de novo engineered microbes’
Finally, you re-engineer the regulatory network to express your product in useful quantities. This can be done in a couple ways. It can be random, usually through large high throughput screens. Or, if you have a better idea of how the pathway works, you can do it in a directed fashion.

20. Metabolic Engineering: ‘de novo engineered microbes’
Finally, you re-engineer the regulatory network to express your product in useful quantities. This can be done in a couple ways. It can be random, usually through large high throughput screens. Or, if you have a better idea of how the pathway works, you can do it in a directed fashion.

21. Metabolic Engineering: ‘de novo engineered microbes’
Finally, you re-engineer the regulatory network to express your product in useful quantities. This can be done in a couple ways. It can be random, usually through large high throughput screens. Or, if you have a better idea of how the pathway works, you can do it in a directed fashion.

22. Metabolic Engineering: ‘de novo engineered microbes’
But, it usually isn’t as simple as it sounds. Many compounds are produced through complex, multistep, or branching pathways, with different enzymes and requirement for each step.

23. Metabolic Engineering: ‘de novo engineered microbes’
These enzymes can be composed of multiple subunits, all of which can be required for efficient production.

24. Metabolic Engineering: ‘de novo engineered microbes’
Finally, these enzymes can require a variety of cofactors in order to function properly, such as ATP, NADH, or NAPDH

25. Isoprenoid Lycopene Carotenoid
The isoprenoid superfamily is an important candidate for this metabolic engineering, and they are of course, the focus of this paper. It gets a bit complicated to tell the difference between isoprenoids, carotenoids, and lycopene, so before I get started I wanted to explain the difference.

26. Isoprenoid Lycopene Carotenoid
The isoprenoid superfamily is an important candidate for this metabolic engineering, and they are of course, the focus of this paper. It gets a bit complicated to tell the difference between isoprenoids, carotenoids, and lycopene, so before I get started I wanted to explain the difference.

27. Isoprenoid Lycopene Carotenoid
The isoprenoid superfamily is an important candidate for this metabolic engineering, and they are of course, the focus of this paper. It gets a bit complicated to tell the difference between isoprenoids, carotenoids, and lycopene, so before I get started I wanted to explain the difference.

28. ? Isoprenoid Lycopene Carotenoid
The isoprenoid superfamily is an important candidate for this metabolic engineering, and they are of course, the focus of this paper. It gets a bit complicated to tell the difference between isoprenoids, carotenoids, and lycopene, so before I get started I wanted to explain the difference.

29. Isoprenoids
Isoprenoids is the name for the superfamily of lipids that are made of repeating 5 carbon isoprene units. These carbons are mixed and matched and modified to make a variety of different compounds.
Isoprenoids are found in all classes of living beings.

30. Isoprenoids x
Isoprenoids is the name for the superfamily of lipids that are made of repeating 5 carbon isoprene units. These carbons are mixed and matched and modified to make a variety of different compounds.
Isoprenoids are found in all classes of living beings.

31. Isoprenoids x
They are used as drugs, flavours, fragrances, pigments, antioxidants, and natural polymers. Even steroids and sterols are made from isoprenoid precusors.
Isoprenoids make up menthol, cannabis, and mustard seeds, and give smell to eucalyptus, cinnamon and ginger.

32. Isoprenoids x
They are used as drugs, flavours, fragrances, pigments, antioxidants, and natural polymers. Even steroids and sterols are made from isoprenoid precusors.
Isoprenoids make up menthol, cannabis, and mustard seeds, and give smell to eucalyptus, cinnamon and ginger.

33. Isoprenoids x
They are used as drugs, flavours, fragrances, pigments, antioxidants, and natural polymers. Even steroids and sterols are made from isoprenoid precusors.
Isoprenoids make up menthol, cannabis, and mustard seeds, and give smell to eucalyptus, cinnamon and ginger.

34. Isoprenoids x
They are used as drugs, flavours, fragrances, pigments, antioxidants, and natural polymers. Even steroids and sterols are made from isoprenoid precusors.
Isoprenoids make up menthol, cannabis, and mustard seeds, and give smell to eucalyptus, cinnamon and ginger.

35. Carotenoids 8
Carotenoids are a type of isoprenoid. They are referred to as Tetraterpenoid because they are specifically made up of 8 isoprene units (40C).
Like isoprenoids, they serve as pigments, food colorants, neutraceuticals, cosmetic products, and pharmaceuticals

36. Carotenoids 8 Tetraterpenoid
Carotenoids are a type of isoprenoid. They are referred to as Tetraterpenoid because they are specifically made up of 8 isoprene units (40C).
Like isoprenoids, they serve as pigments, food colorants, neutraceuticals, cosmetic products, and pharmaceuticals
Tetraterpenoid

37. Lycopene
Narrowing our focus even more, lycopene Is a type of carotenoid. It is a bright red pigment found in tomatoes, watermelons, papayas, and beans. Because of the nature of the isoprenoid pathway, it is an important intermediate in the production of other compounds of the same family.

38. MEP & MVA Pathways Methylerythritol Phosphate Mevalonic Acid
The isoprenoid pathway can be broken down into three parts. First, the methylerythritol phosphate and Mevalonic acid pathways produce the precusors to the make isoprenoids. These pathways are mutually exclusive, and occur in bacteria, algae, and higher plants. The products of these pathways are two isomers IPP and DMAPP. These two, nearly identical molecules go on to make lycopene, and other important isoprenoids.

39. MEP & MVA Pathways IPP & DMAPP Methylerythritol Phosphate
Mevalonic Acid
IPP & DMAPP
Isopentenyl Pyrophosphate
Dimethylallyl Diphosphate
The isoprenoid pathway can be broken down into three parts. First, the methylerythritol phosphate and Mevalonic acid pathways produce the precusors to the make isoprenoids. These pathways are mutually exclusive, and occur in bacteria, algae, and higher plants. The products of these pathways are two isomers IPP and DMAPP. These two, nearly identical molecules go on to make lycopene, and other important isoprenoids.

40. MEP & MVA Pathways IPP & DMAPP Isoprenoids Methylerythritol Phosphate
Mevalonic Acid
IPP & DMAPP
Isopentenyl Pyrophosphate
Dimethylallyl Diphosphate
The isoprenoid pathway can be broken down into three parts. First, the methylerythritol phosphate and Mevalonic acid pathways produce the precusors to the make isoprenoids. These pathways are mutually exclusive, and occur in bacteria, algae, and higher plants. The products of these pathways are two isomers IPP and DMAPP. These two, nearly identical molecules go on to make lycopene, and other important isoprenoids.
Isoprenoids

41. The big picture
Now if we take a step backwards, the isoprenoid family consists of a large number of useful products for medicine, food, and generalized industrial production. It has attracted a lot of attention from researchers who try to tweak the pathway to yield higher amounts of metabolites and a more efficient pathway in general. The tools that are developed using this pathway can be useful in a variety of other organisms too. Alex is going to talk about three problems researchers are trying to solve in a second, but first I wanted to explain why the isoprenoid pathway is such a good choice for this sort of experimentation.
Other than the useful products, two factors contribute to this pathway’s favorability in metabolic engineering. The carotenoid enzymes are often promiscuous, meaning that they are less substrate-specific than other enzymes.

42. And secondly, because carotenoids are pigments, its possible to use colorimetric screening.

43. Though the paper didn’t go into specifics, another paper by Sharpe and DiCosimo explains one way of setting up this reporter system and shows how it can be applied to synthetic biology in a more general sense. They introduced carotenoid genes randomly via transposons to identify which integration sites were the most effective – by examining the colour that was produced.

44. The big picture
Now Alex is going to explain various methods that researchers have used to increase the yield of isoprenoids.
Picture from:

45. The Three Problems: Ensuring Substrate Availability
Balancing Intermediate Pools
Improving Storage Capacity for End Products

46. Ensuring Substrate Availability
Focus on increasing the availability of substrates (IPP, DMAPP, FPP).

47. Ensuring Substrate Availability
Focus on increasing the availability of substrates (IPP, DMAPP, FPP).

48. A) Stoichiometric Flux Balance Analysis
A method of modelling a metabolic network to identify enzymes which heavily influence carbon flux.
(PT5-idi / PT5-ispDF / PT5-dxs)
Came across a triple knock-out mutant, seen above.
37% increase in lycopene production.

49. A) Stoichiometric Flux Balance Analysis
A method of modelling a metabolic network to identify enzymes which heavily influence carbon flux.
(PT5-idi- / PT5-ispDF- / PT5-dxs-)
37% increase in lycopene production.

50. B) Metabolic Control Structure
Diverts carbon flux to lycopene during the stationary phase of growth.
PT5-idi and PT5-pps under glnAp2.
glnAp2 is activated during the late exponential phase in response to increased carbon flux and induction of sigma factor RpoS, which activates as the population approaches stationary phase.

51. B) Metabolic Control Structure
Diverts carbon flux to lycopene during the stationary phase of growth.
PT5-idi and PT5-pps under glnAp2.
glnAp2 responds to the increased carbon flux associated with the late exponential phase.
RpoS induction also plays a role in carotenoid accumulation.

52. C) Rational Strain Design
Mevalonate pathway from S. cerevisiae cloned into E. coli.
Amorphadiene synthase.
Evasion of regulatory constraints and increased availability of metabolic precursors.
Clones were rescued by amorphadiene synthase, FPP  Amorphadiene, reducing carbon flux to IPP. Did the same with Streptomyces, didn’t share results.

53. C) Rational Strain Design
Mevalonate pathway from S. cerevisiae cloned into E. coli.
Amorphadiene synthase.

54. C) Rational Strain Design
Mevalonate pathway from S. cerevisiae cloned into E. coli.
Amorphadiene synthase.
FPP  Amorphadiene

55. C) Rational Strain Design
Mevalonate pathway from S. cerevisiae cloned into E. coli.
Amorphadiene synthase.
FPP  Amorphadiene

56. D) Modification of Native Proteins
Monoterpenes and diterpenes produced from substrates geranyl diphosphate (GPP) and geranylgeranyl diphosphate (GGPP).

57. D) Modification of Native Proteins
Monoterpenes and diterpenes produced from substrates geranyl diphosphate (GPP) and geranylgeranyl diphosphate (GGPP).
FPP synthase (ispA) point mutant.
This isn’t right!
Capable of using these two compounds as substrates and therefore produce monoterpenes and diterpenes.

59. E) Randomized Overexpression
Eight clones of random enzymes in the MEP/DOXP pathway.
T5-phage promoter.

60. E) Randomized Overexpression
Eight clones of random enzymes in the MEP/DOXP pathway.
T5-phage promoter  crtEBIY operon.
Screened for increased B-carotene accumulation.

61. http://www. discoveryandinnovation

62. F) Knockout Library Approach
Screened an E. coli genomic library in the presence of carotenogenic genes.
appY and crl.
Identified native genes which enhance lycopene accumulation

63. F) Knockout Library Approach
Screened an E. coli genomic library in the presence of carotenogenic genes.
appY and crl.
Who would’ve thought?
When overexpressed!

64. G) Investigation of Carotenogenic Genes
Tao et. al (2005) produced a transposon mutagenesis library in E. coli.
crtEXYIB operon on a ColE1 plasmid.

65. G) Investigation of Carotenogenic Genes
Tao et. al (2005) produced a transposon mutagenesis library in E. coli.
crtEXYIB operon on a ColE1 plasmid.
HIGHER COPY # = MORE CAROTENOIDS BUT NOT LINEAR.
Multi-copy vectors can lead to energy deficiencies and are not always the right choice.

66. Balancing Intermediate Pools

67. A) mRNA Stability
Smolke et. al (2001) altered mRNA processing signals on crtY /crtI.
300-fold variation in ϐ-carotene and lycopene production.
(ϐ-carotene from lycopene) and (lycopene synthesis).

68. Library of mRNA processing signals.
Auxotrophic reporter strain expressing GFP.
Decreased HMG-CoA synthase / HMG-CoA reductase mRNA levels = more mevalonate production!
Made a library of intergenic and mRNA processing regions of the enzymes required to produce mevalonate – reporter strain which needed mevalonate to live, expressing GFP. Decreased enzyme levels (2 examples) = more mevalonate – therefore overexpression not always required, contained expression.

69. Library of mRNA processing signals.
Auxotrophic reporter strain expressing GFP.
Decreased HMG-CoA synthase / HMG-CoA reductase mRNA levels = more mevalonate production!

70. B) Operon mRNA Levels and PermutationS
FPP  zeaxanthin, Bacillus subtilis.
Nishizaki et. al! 5 genes that convert FPP  Zeaxanthin.

71. B) Operon mRNA Levels and PermutationS
FPP  zeaxanthin, Bacillus subtilis.
One variant showed a 35% increase in zeaxanthin production.
Nishizaki et. al! Rearranging the operon has an effect on mRNA levels – in this case, the order of the genes in the operon which worked best is the order in which the enzymes work – not always the case.

72. C) Global mRNA Manipulation
Random mutagenesis of housekeeping E. coli sigma factor (σD).
Some strains demonstrated increased carotenoid yields by up to 50%.

73. C) Global mRNA Manipulation
Random mutagenesis of housekeeping E. coli sigma factor (σD).
Some strains demonstrated increased carotenoid yields by up to 50%.
Take home message: gene deletion and overexpression are not always ideal solutions for managing intermediate pools.

74. Improving Storage Capacity FOR End Products
Isoprenoid end products can potentially be toxic at high concentrations.

75. Improving Storage Capacity FOR End Products
Isoprenoid end products can potentially be toxic at high concentrations.
Detergents and the absence of light implicated in affecting lycopene yields.

76. Improving Storage Capacity FOR End Products
Isoprenoid end products can potentially be toxic at high concentrations.
Detergents and the absence of light implicated in affecting lycopene yields.

77. IN CONCLUSION
Increased energy costs + environmental concerns with traditional methods of chemical synthesis = microbes!

78. IN CONCLUSION
Increased energy costs + environmental concerns with traditional methods of chemical synthesis = microbes!
So how do we optimize this method of compound biosynthesis?

79. IN CONCLUSION
Increased energy costs + environmental concerns with traditional methods of chemical synthesis = microbes!