Development of genetic transformation and heterologous expression system in carboxydotrophic thermophilic acetogen Moorella thermoacetica

Abstract

To develop a microbial production platform based on hydrogen and carbon dioxide, a genetic transformation system for the thermophilic acetogen Moorella thermoacetica ATCC39073 was developed. The uracil auxotrophic strain dpyrF was constructed by disrupting pyrF for orotate monophosphate decarboxylase. The transformation plasmids were methylated by restriction methylases of M. thermoacetica to avoid the decomposition of introduced plasmids by restriction-modification system. Reintroduction of native pyrF into the mutant by homologous recombination ensured recovery from uracil auxotrophy. To test heterologous gene expression in dpyrF, the lactate dehydrogenase (LDH) gene (T-ldh) from Thermoanaerobacter pseudethanolicus ATCC33223 was electroporated into dpyrF with a promoter of the glyceraldehyde-3-phosphate dehydrogenase (G3PD) gene of M. thermoacetica ATCC39073. The resulting transformant (C31) successfully transcribed T-ldh and exhibited higher LDH activity than ATCC39073 and dpyrF, yielding 6.8 mM of lactate from fructose, whereas ATCC39073 did not produce lactate.

Homolactic Acid Fermentation by the Genetically Engineered Thermophilic Homoacetogen Moorella thermoacetica ATCC 39073

“…In the ΔpduL1::ldh mutant, the acetate yield was almost the same as that of the wild-type strain. The lactate yield, which was only 0.036 mol/mol, was comparable to that of the mutant M. thermoacetica C31, in which only T-ldh was introduced into the ΔpyrF parental mutant strain (14). In the ΔpduL2::ldh mutant, the lactate yield increased significantly to 0.73 mol/mol, and the acetate yield decreased to 0.77 mol/mol.…”

“…In previous studies, we constructed a mutant that produced lactate from fructose by introducing an exogenous lactate dehydrogenase gene into M. thermoacetica. However, the lactate yield was significantly lower than the acetate yield (14). This result demonstrated that the acetate-producing pathway of M. thermoacetica should be disrupted for the efficient production of target metabolites.…”

“…We have thus focused on thermophilic acetogens as host microorganisms for the production of biofuels (11)(12)(13), and we developed a system of genetic transformation for M. thermoacetica ATCC 39073 (14,15) and related strains (16) to manipulate the metabolic pathways of thermophilic acetogens genetically. In previous studies, we constructed a mutant that produced lactate from fructose by introducing an exogenous lactate dehydrogenase gene into M. thermoacetica.…”

Trash to treasure: production of biofuels and commodity chemicals via syngas fermenting microorganisms

“…However, there has been remarkable progress in the development of genetic systems for numerous industrially important anaerobes, especially clostridia, in recent years. Protocols for gene deletion via double crossover homologous recombination were recently developed for the syngas fermenting C. ljungdahlii [39 ] and M. thermoacetica [40][41][42]. Uracil-auxotrophic mutants of M. thermoacetica [41 ] and C. thermocellum [43] were constructed allowing for both positive-and counter-selection of desired recombinants.…”

“…Protocols for gene deletion via double crossover homologous recombination were recently developed for the syngas fermenting C. ljungdahlii [39 ] and M. thermoacetica [40][41][42]. Uracil-auxotrophic mutants of M. thermoacetica [41 ] and C. thermocellum [43] were constructed allowing for both positive-and counter-selection of desired recombinants. Moreover, methods employing bacterial group II intron for gene deletion [44,45,46 ] and a Bacillus subtilis resolvase [47-49] were developed as universal genetic tools for Clostridium species.…”