Biological conversion of CO2 into the platform chemicals lactate and 3-hydroxypropionate using recombinant strains of Acetobacterium woodii

Autor: Beck, Matthias Hermann
Přispěvatelé: Dürre, Peter, Eikmanns, Bernhard
Jazyk: angličtina
Rok vydání: 2020
Předmět:
DOI: 10.18725/oparu-28743
Popis: The overall goal of this work was the production of 3 hydroxypropionate (3-HP) from CO2 + H2 using recombinant strains of Acetobacterium woodii. To accomplish the main goal via establishment of a lactate-converting 3-HP production pathway, the implementation of inducible promoter systems and the production of lactate were considered as corresponding milestones of this work. The �����glucuronidase-encoding gusA gene was successfully established as a reporter system and used to demonstrate the activity of the (inducible) promoters Pfac (IPTG), PbgaL (lactose), PackA���theo (theophylline), Ptet (anhydrotetracycline) and Ppta-ack. Tight regulation and inducibility was warranted using the tetracycline-inducible promoter system Ptet as well as the designed PackA-theo promoter system. The tetracycline-inducible promoter system Ptet was successfully used for the creation of recombinant lactate and 3-HP production strains of A. woodii. In addition, the native lactate metabolism had to be circumvented since lactate production from CO2 + H2 was only viable when A. woodii ��pyrE ��lctBCD was used as a chassis instead of the wild type strain.
Hence, A. woodii ��pyrE ��lctBCD was metabolically engineered to produce lactate via transformation using lactate-synthesis plasmids harboring a gene encoding a d-lactate dehydrogenase (ldhD) alone or in combination with the pyruvate:ferredoxin oxidoreductase encoding gene (nifJ). Under autotrophic conditions, highest lactate concentrations were recorded when using A. woodii ��pyrE ��lctBCD [pJIR750_Ptet_ldhD_LM], A. woodii ��pyrE ��lctBCD [pJIR750_Ptet_nifJ_ldhD], and A. woodii ��lctBCD ��pyrE::pyrE-Ptet-ldhD, producing 10.6, 10.1, and 10.2 mM lactate, respectively. Under heterotrophic conditions, highest yields of 1.22, 1.10, and 0.90 mole lactate per mole fructose were recorded using A. woodii ��pyrE ��lctBCD [pJIR750_Ptet_nifJ_ldhD], A. woodii ��pyrE ��lctBCD [pJIR750_Ptet_nifJCl_ldhD], and A. woodii ��lctBCD ��pyrE::pyrE-Ptet-ldhD, respectively. In addition, the production with A. woodii ��lctBCD ��pyrE::pyrE���Ptet-ldhD is the first demonstration of a recombinant production strain of A. woodii whose production is due to heterologous gene expression on the chromosomal level. Some recombinant A. woodii ��pyrE ��lctBCD strains showed depletion of lactate when high amounts of lactate were produced under heterotrophic conditions. This phenomenon was suspected to be explained by the thermodynamics of the lactate dehydrogenase reaction and the intracellular pyruvate/lactate ratio. Genes encoding key enzymes of the acrylate pathway and the synthetic 3-HP production pathway were identified via whole genome sequencing of the strains Clostridium homopropionicum LuHBu1 (DSM 5847) and Clostridium neopropionicum X4 (DSM 3847).
A. woodii ��pyrE ��lctBCD was metabolically engineered to produce 3-HP from lactate via transformation using 3-HP synthesis plasmids harboring genes encoding the lactoyl-CoA dehydratase (lcdCAB), propionyl-CoA transferase (pct) and the codon-optimized version of Caur_0101 encoding the enoyl-CoA hydratase (ehy_opt). Neither the production of enoyl-CoA hydratase (Ehy) nor lactate conversion into 3���HP were noticed using the non-optimized Ehy-encoding gene ehy (Caur_0101). In contrast, A. woodii ��pyrE ��lctBCD [pMTL83151_Ptet_LPE_opt] produced highest 3-HP concentrations of 1.8 and 0.9 mM growing under heterotrophic and autotrophic conditions, respectively. As one possibility to enable 3-HP production without supplementation of lactate, the two-plasmid system conceptualized in this work for A. woodii and realized by Flaiz (2018) was for the first time successfully established for pathway engineering of A. woodii. This was shown by creation of A. woodii ��pyrE ��lctBCD strains harboring pMTL83151_Ptet_LPE_opt and either pMTL82251_Ptet_ldhD_LM, pMTL82251_Ptet_nifJ_ldhD, or pMTL82251_Ptet_nifJCl_ldhD. Thus, A. woodii ��pyrE ��lctBCD was metabolically engineered to produce 3-HP without supplementation of lactate via transformation using either one or two 3-HP synthesis plasmids harboring the genes (nifJ), ldhD, lcdCAB, pct and either ehy_opt or phaJ (also Ehy-encoding). Some of the resulting strains growing under autotrophic conditions produced traces (< 0.5 mM) of 3���HP below the quantification limit. Under heterotrophic conditions, the highest and quantifiable concentration of 3-HP produced without supplementation of lactate was 0.8 mM. This was achieved using A. woodii ��pyrE ��lctBCD [pMTL83151_Ptet_3���HP_opt]. By production of lactate and conversion of lactate into 3-HP, each under autotrophic conditions, as well as by production of traces of 3-HP under autotrophic conditions without supplementation of lactate, it was successfully demonstrated that the connection of the Wood-Ljungdahl pathway to the synthetic lactate-converting 3-HP production pathway is feasible.
Databáze: OpenAIRE
Externí odkaz: