Design and construction of synthetic organelles
Minimal cells
Compartmentalization in biocatalysis:
• Strategy for enabling competing pathways
• Selective regulation of enzymes by localization
• Substrate channeling of intermediates between
enzymatic steps
• Sequestering volatile and toxic compounds
• Formation of specific microenvironments
Design and construction of synthetic organelles
An important area of synthetic biology is production of important chemicals, such as pharmaceuticals, materials and biofuels from
cheap and sustainable biomass. This requires high productivity and yields of engineered pathways. One promising strategy is to
repurpose organelles or protein complexes as cellular factories for improving the performance of engineered pathways.
Substrate chanelling in tryptophan synthase
Lipid-based organelles
Other means of cellular compartmentalization
Proteinaceous bacterial microcompartments
• carboxysomes
Organelle: physically delimited compartment within a cell
• propanediol-utilizing microcompartment
• encapsulins
Biomolecular condensates
In a mixture of two types of molecules, LLPS leads to the formation of two
phases akin to droplets of oil appearing from a mixture of oil and water.
Proteins can undergo a similar phase separation. LLPS underpins the
biogenesis of a wide array of membraneless organelles within cells.
Core design principles for synthetic organelles
Targeting – selective targeting of desired biochemical activities
(enzymes) into the compartment. Often based on protein-protein
interactions through the use of signaling sequences.
Chemical environment – result of th einterplay between permeability
an dcombined enzymatic aktivity. It will determine concentration of
substrates and products, as wel as general properties such as pH.
Permeability – selectivity of surrounding membrane or protein shell
that directly affects what can diffuse accross or be transported in and
out of the compartment.
Biogenesis – proces of organelle self-assembly. It will determine
organelle size, shape and copy number.
Carboxysomes in cyanobacteria
The cyanobacterial carbon concentrating mechanims is a single-cell, bipartite system that first generates a high intracellular
bicarbonate (HCO3−) pool through action of membrane-bound inorganic carbon (Ci) transporters and CO2-converting complexes.
This HCO3- pool is then utilized by subcellular micro-compartments called carboxysomes, which encapsulate the cell’s complement
of Rubisco. The carboxysome’s outer protein shell enables diffusional influx of HCO3− and RuBP, where the former is converted to
CO2 by a localized carbonic anhydrase (CA).
shell-bound Rubisco
carboxysomal carbonic anhydrase
interlinks Rubisco and the shell
Carboxysome of Cyanobium
• exclusion of O2 as the Rubisco competing substrate from the lumen
• CO2 chanelling from carbonic anhydrase to Rubisco via tight clustering
• raising local pH around Rubisco to increase its catalytic activity
Repurposing propanediol-utilizing microcompartment for ethanol production
The Pdu microcompartment shell is built from hexameric tiles composed of PduA, B, J, K, U and T (purple) that form the facets of the structure whereas pentameric tiles
(PduN, cyan) form the vertices. 1,2-Propanediol enters the shell through pores in the shell proteins and is metabolized to propionyl-CoA (red box), which leaves the
compartment and is further converted to propionate. Enzymes that are encapsulated within the metabolosome contain short signaling peptides. Changing the specificity of
the Pdu microcompartment is achieved by stripping out the Pdu pathway and replacing it with the required pathway e.g. ethanol production (green box). Fusion of signaling
peptides to the new pathway enzymes – pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) facilitates internalization of the heterologous proteins an ethanol
production. 1,2-PD = 1,2-propanediol, PA = propionaldehyde, P-OH = 1-propanol, PCoA = propionyl-CoA, POI = protein of interest, SP = signalling peptide.
Lawrence et al., ACS Synth Biol 2014
Engineering prokaryotic encapsulins as synthetic organelles in yeast
Encapsulins – procaryotic proteins capabale of assembling into a 20-30 nm icosahedral nanocompartments. They
represent minimal versions of microcompartments – they consists of a single shelĺ protein and can be targeted with
different cargos. However, their small size limits their tageting to one or two enzymes, so they can be used for
engineering only very short pathways.
Encapsulin compartments represent a modular platform,
orthogonal to existing organelles, for programming synthetic
compartmentalization in eukaryotes.
Lau et al., Nat Comm 2018
Phase separation sequesters the machinery for repurposing stop codons
Inside the designer orthogonal organelle, nonnatural amino acids can be added during protein synthesis at the location of
the Amber stop codon. Outside the organelle, the stop codon still halts protein synthesis.
Reinkemeier et al., Science 2019
The assemblers: proteins capable of phase separation fused to the assmbled components - kinesin motors (localization to microtubules),
suppressor tRNA synthase, and MCP protiens (bind to ms2 loops on targeted mRNA)
Polymersomes are synthetic analogues of liposomes and are constituted of amphiphilic block copolymer membrane. Whilst
most properties are similar for both carriers, polymersomes exhibit a high versatility and an enhanced stability.
Towards artificial organelles: liposomes and polymersomes
Synthetic endosymbiosis: inspired by kleptoplasty
Kleptoplasty: a symbiotic phenomenon whereby chloroplasts
from algae, are sequestered by host organisms.
Microinjection of cyanobacteria into zebrafish embryo
• no adverse immune response
• cyanobacteria expressing listeriolysin and
invasin to escape lysosome digestion were able
to proliferate in macrophages for several days
Agapakis et al., PLoS One 2011
Minimal cell
• A cell whose genome only encodes the minimal set of genes necessary for the cell to survive
• The genes in minimal cell are, by definition, essential
• In reductionism approach is a minimal cell key to learn the first principles of cellular biology by mapping
function of all genese and componets – then it may be possible to achieve a complete understanding pf
what it take to be alive.
• A minimal cell has all of the machinery for independent cellular life – there is no redundancy.
• With this knowledge, it may be possible to model the minimal cell´s behaviuor on computer. And from there
one may be able to build cells that are more complex.
Mycoplasma as a model for minimal cell
• Mycoplasmas are a group of bacteria characterized by the lack of cell wall, obligate parasitic lifestyle, metabolic
simplicity, and small genomes.
• Mycoplasma did not evolved as the simplest form of cellular life. They descent from a conventional bacteria (like B.
subtilis or S. aureus) through massive gene loss due to adopted parasitic lifestyles in highly nutrient rich and stable
environment.
• Mycoplasma genitalium has the smallest genome with 580,076 bps encoding 507 genes
• Because nutriens are imported rather than synthesized, all that Mycoplasma do is synthesize DNA, RNA and protein.
Determining a minimal set of genes: comparative genomics
1996: Gram-positive and -negative bacteria - 256 orthologous genes specify core functions
2003: all sequenced organisms - 65 orthologous genes
2004: 147 prokaryotic genomes available – less than 50 commob orthologous genes (mostly translation)
2012: 20 strains of Mycoplasma family – core of only 196 orthologs
Nonorthologous gene displacement – orthologs evolved too far to be recognizable as such, or an essential function was originally
provided by two redundant genes that separated in the course of evolution.
Determining a minimal set of genes: genetic approach
Essential genes (E) – the cell in which essential gene is inactivated cannot be propagated
Nonessential genes (NE) – can be inactivated without affecting the viability or growth rate (in a specific environment)
Quasi-essential genes (QE) – theri dirsuption impairs growth. They are important for robust growth, but not strictly essential.
Transposon insertion mutagenesis in Mycoplasma
M. genitalium 580 kb 507 101 406
M. pneumoniae 816 kb 739 259 480
M. pulmonis 963 kb 589 321 468
Genome
size
Total
genes
NE
genes
Total - NE
genes
M. mycoides JCVI-Syn1.0 1080 kb 901 432 469
Design and synthesis of a minimal bacterial genome
Hutchison et al., Science 2016
M. mycoides JCVI-Syn1.0
(1078 kb, 863 protein and 38 RNA-coding genes)
M. mycoides JCVI-Syn3.0
(531 kb, 438 protein and 35 RNA-coding genes)
3x reiterative cycles
doubling time 60 min doubling time 180 min
Insertional mutagenesis indetified additional 53 NE genes – extrapolation to NE equals to 0 predicts 413 essential genes.
Genes retained in the Syn3.0 genome
Comparison of protein coding genes with other bacteria
Functional classes of protein coding genes
Of the 91 genes of unclear function, 30 are essential, 32 are quasi-essential, and 29 are non-essential. Those 30 essential
genes could represent new biological mechanisms not yet defined and should motivate the search to discover their function.
Metabolic reconstruction of the minimal cell
Breuer et al., eLife 2019
• 338 reactions
• 304 metabolites
• 155 gene products
Recommended reading:
Hutchison C. A. et al. (2019) Design and synthesis of a minimal bacterial genome. Nature 351:1414
Lau Y.H. et al. (2018) Prokaryotic nanocompartments form synthetic organelles in a eukaryote. Nat Comm 9:1311