Synthetic Biology Parts, Devices and Applications

14.3 ­roteinnBased Organelles 289

extremely high [11]. Interestingly, there is little information on the effect such
harsh conditions have on protein activity within the BMCs. Many metabolic
pathway intermediates, such as the aldehydes present in the PDU, EUT, and
many candidate biofuel pathways, will inevitably cause protein misfolding and
inactivate individual enzymes in the complex. A key open biological question is
therefore how the proteostasis of BMCs and their enzymatic content is regu­
lated. A single BMC is on the order of 0.2% of total cellular protein (estimated
from CB mass of ~250 MDa [75] and E. coli protein content from BioNumber
104879 [76]) and represents a tremendous investment for the cell. It remains to
be seen whether BMCs are surveilled via the cell’s proteostatic chaperones and,
if so, whether this entire “costly” complex is turned over at the level of single
inactive subunits or all at once.

14.3.1.4 Biogenesis
The critical information for biogenesis is an understanding of the genes and
expression levels that are necessary and sufficient for BMC self‐assembly and
function. Early genetic, cloning, and sequencing efforts revealed that BMCs
genes are often co‐localized together in operons but that the degree of co‐
localization varies with each BMC. For example, α‐CB genes cluster together as
a single regulon in the genome of H. neapolitanus, while the β‐CB regulon found
in many cyanobacterial strains is composed of five operons spread across the
genome (Figure 14.4). For this reason, successful efforts at reconstituting and
transgenically expressing fully functional BMCs in a heterologous host have
focused on those where genes are co‐localized in a single genomic island. For
example, screening of a C. freundii genomic DNA library identified a cosmid
capable of endowing E. coli with the ability to metabolize 1,2‐propanediol [71].
Sequencing of this cosmid and further molecular biology to narrow down the
candidate genes identified a minimal subset important for the heterologous
production of PDUs [50]. This has proven an important tool for studying the role
of each gene in defining PDU structure and for identifying signal sequences.
A  similar approach was successful for the α‐CB and EUT. Expression of the

Carbonic

anhydrase

Carbonic

anhydrase

ccaA

S. elongatus PCC7942 β-CB regulon

rbc

LS

RuBisCO

ccm

K2 LM NO

Shell and associated

ccm

K3K4

Shell

ccm

P

Shell

rbc

L S csoS2

RuBisCO

csoS3

csoS4 csoS1

A B C A B

Shell-associated

Shell

H. neapolitanus α-CB operon

D

Figure 14.4 Genetic organization of α‐ and β‐carboxysome regulons from two different
bacteria. Color scheme indicates relatedness.