Synthesis Gas (Syngas)-Derived Medium-Chain-Length Polyhydroxyalkanoate Synthesis in Engineered Rhodospirillum rubrum
Authors: Heinrich Daniel , Raberg Matthias , Fricke Philipp , Kenny Shane T. , Morales-Gamez Laura , Babu Ramesh P. , O'Connor Kevin E. , Steinbüchel Alexander 
Published: August 2016

The purple nonsulfur alphaproteobacterium
Rhodospirillum rubrum
S1 was genetically engineered to synthesize a heteropolymer of mainly 3-hydroxydecanoic acid and 3-hydroxyoctanoic acid [P(3HD-
co
-3HO)] from CO- and CO
2
-containing artificial synthesis gas (syngas). For this, genes from
Pseudomonas putida
KT2440 coding for a 3-hydroxyacyl acyl carrier protein (ACP) thioesterase (
phaG
), a medium-chain-length (MCL) fatty acid coenzyme A (CoA) ligase (
PP_0763
), and an MCL polyhydroxyalkanoate (PHA) synthase (
phaC1
) were cloned and expressed under the control of the CO-inducible promoter P

cooF

from
R. rubrum
S1 in a PHA-negative mutant of
R. rubrum
. P(3HD-
co
-3HO) was accumulated to up to 7.1% (wt/wt) of the cell dry weight by a recombinant mutant strain utilizing exclusively the provided gaseous feedstock syngas. In addition to an increased synthesis of these medium-chain-length PHAs (PHA
MCL
), enhanced gene expression through the P

cooF

promoter also led to an increased molar fraction of 3HO in the synthesized copolymer compared with the P

lac

promoter, which regulated expression on the original vector. The recombinant strains were able to partially degrade the polymer, and the deletion of
phaZ2
, which codes for a PHA depolymerase most likely involved in intracellular PHA degradation, did not reduce mobilization of the accumulated polymer significantly. However, an amino acid exchange in the active site of PhaZ2 led to a slight increase in PHA
MCL
accumulation. The accumulated polymer was isolated; it exhibited a molecular mass of 124.3 kDa and a melting point of 49.6°C. With the metabolically engineered strains presented in this proof-of-principle study, we demonstrated the synthesis of elastomeric second-generation biopolymers from renewable feedstocks not competing with human nutrition.

IMPORTANCE
Polyhydroxyalkanoates (PHAs) are natural biodegradable polymers (biopolymers) showing properties similar to those of commonly produced petroleum-based nondegradable polymers. The utilization of cheap substrates for the microbial production of PHAs is crucial to lower production costs. Feedstock not competing with human nutrition is highly favorable. Syngas, a mixture of carbon monoxide, carbon dioxide, and hydrogen, can be obtained by pyrolysis of organic waste and can be utilized for PHA synthesis by several kinds of bacteria. Up to now, the biosynthesis of PHAs from syngas has been limited to short-chain-length PHAs, which results in a stiff and brittle material. In this study, the syngas-utilizing bacterium
Rhodospirillum rubrum
was genetically modified to synthesize a polymer which consisted of medium-chain-length constituents, resulting in a rubber-like material. This study reports the establishment of a microbial synthesis of these so-called medium-chain-length PHAs from syngas and therefore potentially extends the applications of syngas-derived PHAs.