Genetic engineering of a thermophilic acetogen, Moorella thermoacetica Y72, to enable acetoin production

Abstract

Acetogens are among the key microorganisms involved in the bioproduction of commodity chemicals from diverse carbon resources, such as biomass and waste gas. Thermophilic acetogens are particularly attractive because fermentation at higher temperatures offers multiple advantages. However, the main target product is acetic acid. Therefore, it is necessary to reshape metabolism using genetic engineering to produce the desired chemicals with varied carbon lengths. Although such metabolic engineering has been hampered by the difficulty involved in genetic modification, a model thermophilic acetogen, M. thermoacetica ATCC 39073, is the case with a few successful cases of C2 and C3 compound production, other than acetate. This brief report attempts to expand the product spectrum to include C4 compounds by using strain Y72 of Moorella thermoacetica. Strain Y72 is a strain related to the type strain ATCC 39073 and has been reported to have a less stringent restriction-modification system, which could alleviate the cumbersome transformation process. A simplified procedure successfully introduced a key enzyme for acetoin (a C4 chemical) production, and the resulting strains produced acetoin from sugars and gaseous substrates. The culture profile revealed varied acetoin yields depending on the type of substrate and culture conditions, implying the need for further engineering in the future. Thus, the use of a user-friendly chassis could benefit the genetic engineering of M. thermoacetica.

Keywords: C4 chemicals; acetoin; biomass; gaseous substrates; genetic engineering; thermophilic acetogen.

FIGURE 1

Design of metabolic pathways for acetoin production in Moorella thermoacetica. Pathways for acetate and acetoin production from sugars (A) and gaseous substrates (B). The substrates are colored orange, and the key enzymatic reaction of bsALDC for acetoin production is shown in red.

FIGURE 2

DNA construction for introducing bsALDC into the chromosome of Moorella thermoacetica Y72 by homologous recombination. (A) shows the schematic representation of the constructed plasmids and the homologous recombination events in the pyrF or pduL2 region. (B) shows the agarose gel electrophoresis following PCR amplification of the pyrF or pduL2 region. The size shifts of the amplified DNA confirmed the successful introduction of the bsALDC constructs in the pyrF or pduL2 region. 1.9 kb of the pyrF region was shifted to 2.7, and 0.9 kb of the pduL2 region was shifted to 2.3 kb. M, DNA size marker; lane 1, the Y72-Km strain; lane 2, the Y72-pyrF::bsALDC strain; lane 3, the Y72-pduL2::bsALDC strain.

FIGURE 3

Acetoin production from sugars. The cell growth (OD), substrate consumption, acetate and acetoin productions were monitored over time in the fructose-supplemented culture. The Y72-pyrF::bsALDC (A) and the Y72-pduL2::bsALDC (B) strains are shown, respectively. (C) The acetoin and acetate production yields by the Y72-pduL2::bsALDC strain from fructose were compared between 55°C and 60°C. (D) The acetoin and acetate production yields by the Y72-pduL2::bsALDC strain were compared between hexose (fructose) and pentose (xylose) sugars. Data are presented as the mean with the SDs of three biological replicates. Some error bars are smaller than the symbols of data plots.

FIGURE 4

Acetoin production from gaseous substrates. The cell growth (OD) and the production of acetate, acetoin, and formate were monitored over time. Syngas (A) or CO₂+H₂ gas (B) was supplemented in the head space of culture vials. Data are presented as the mean with SDs of three (A) and two (B) biological replicates, respectively. Some error bars are smaller than the symbols of data plots. Fructose concentration was shown in (B) to indicate its absence and the residual amount from the seed culture.