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Insights Into CO2 Fixation Pathway of Clostridium Autoethanogenum by Targeted Mutagenesis

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Insights Into CO2 Fixation Pathway of Clostridium Autoethanogenum by Targeted Mutagenesis

Fungmin Liew et al. mBio.

Abstract

The future sustainable production of chemicals and fuels from nonpetrochemical resources and reduction of greenhouse gas emissions are two of the greatest societal challenges. Gas fermentation, which utilizes the ability of acetogenic bacteria such as Clostridium autoethanogenum to grow and convert CO2 and CO into low-carbon fuels and chemicals, could potentially provide solutions to both. Acetogens fix these single-carbon gases via the Wood-Ljungdahl pathway. Two enzyme activities are predicted to be essential to the pathway: carbon monoxide dehydrogenase (CODH), which catalyzes the reversible oxidation of CO to CO2, and acetyl coenzyme A (acetyl-CoA) synthase (ACS), which combines with CODH to form a CODH/ACS complex for acetyl-CoA fixation. Despite their pivotal role in carbon fixation, their functions have not been confirmed in vivo By genetically manipulating all three CODH isogenes (acsA, cooS1, and cooS2) of C. autoethanogenum, we highlighted the functional redundancies of CODH by demonstrating that cooS1 and cooS2 are dispensable for autotrophy. Unexpectedly, the cooS1 inactivation strain showed a significantly reduced lag phase and a higher growth rate than the wild type on H2 and CO2 During heterotrophic growth on fructose, the acsA inactivation strain exhibited 61% reduced biomass and the abolishment of acetate production (a hallmark of acetogens), in favor of ethanol, lactate, and 2,3-butanediol production. A translational readthrough event was discovered in the uniquely truncated (compared to those of other acetogens) C. autoethanogenum acsA gene. Insights gained from studying the function of CODH enhance the overall understanding of autotrophy and can be used for optimization of biotechnological production of ethanol and other commodities via gas fermentation.

Importance: Gas fermentation is an emerging technology that converts the greenhouse gases CO2 and CO in industrial waste gases and gasified biomass into fuels and chemical commodities. Acetogenic bacteria such as Clostridium autoethanogenum are central to this bioprocess, but the molecular and genetic characterization of this microorganism is currently lacking. By targeting all three of the isogenes encoding carbon monoxide dehydrogenase (CODH) in C. autoethanogenum, we identified the most important CODH isogene for carbon fixation and demonstrated that genetic inactivation of CODH could improve autotrophic growth. This study shows that disabling of the Wood-Ljungdahl pathway via the inactivation of acsA (encodes CODH) significantly impairs heterotrophic growth and alters the product profile by abolishing acetate production. Moreover, we discovered a previously undescribed mechanism for controlling the production of this enzyme. This study provides valuable insights into the acetogenic pathway and can be used for the development of more efficient and productive strains for gas fermentation.

Figures

FIG 1
FIG 1
The acsA KO strain is unable to grow autotrophically on 200 kPa CO (A) or 130 kPa H2 plus 70 kPa CO2 (B). Symbols: black circles, WT (n = 4 for CO; n = 3 for H2-CO2); red squares, acsA KO strain (n = 3). Error bars show the standard error of the mean.
FIG 2
FIG 2
Growth, headspace pressure, and metabolite profiles of the C. autoethanogenum WT, acsA KO, and complementation strains on 10 g/liter fructose. Panels: A, growth profile; B, headspace pressure profile; C, acetate profile; D, ethanol profile; E, 2,3-butanediol profile; F, lactate profile. Black, WT; red, acsA KO strain; blue, complementation strain. n = 3. Error bars show the standard error of the mean.
FIG 3
FIG 3
Examination of AcsA translation pattern with FLAG-tagged protein in C. autoethanogenum and E. coli. (A) Western blot analysis of C. autoethanogenum transconjugant crude lysates. Lanes: M, Bio-Rad kaleidoscope Precision Plus protein ladder; 1, 13 µg of soluble lysate of pMTL8315-PacsA plasmid control; 2, 13 µg of soluble lysate of pMTL83151-PacsA-acsA(TGA)-FLAG; 3, 13 µg of soluble lysate of pMTL83151-PacsA-acsA(TCA)-FLAG; 4, 13 µg of soluble lysate of pMTL83151-PacsA-acsA(TAA)-FLAG; 5, 6.7 µg of insoluble lysate of pMTL83151-PacsA plasmid control; 6, 6.7 µg of insoluble lysate of pMTL83151-PacsA-FLAG-acsA(TGA). The red arrow indicates the position of a mature 69-kDa protein; the blue arrow indicates the position of the larger 44-kDa truncated protein. The ca. 70-kDa band present in all crude lysates from C. autoethanogenum is a consequence of nonspecific binding of the anti-FLAG antibody to a native C. autoethanogenum protein, most likely DnaK (encoded by CAETHG_2891), which shares 7/8 amino acid identity with FLAG and is of the appropriate predicted size. (B) Schematic showing the expected protein sizes of C. autoethanogenum AcsA in the event of translational readthrough or truncation. (C) Western blot analysis of E. coli transformant crude lysates. Lanes: M, Bio-Rad kaleidoscope Precision Plus protein ladder; 7, 15 µg of soluble lysate of pMTL83151-PacsA plasmid control; 8, 15 µg of soluble lysate of pMTL83151-PacsA-acsA(TGA)-FLAG; 9, 15 µg of soluble lysate of pMTL83151-PacsA-acsA(TCA)-FLAG; 10, 15 µg of soluble lysate of pMTL83151-PacsA-acsA(TAA)-FLAG; 11, 39 µg of soluble lysate of pMTL83151-PacsA-FLAG-acsA(TGA); 12, 38 µg of insoluble lysate of pMTL83151-PacsA-FLAG-acsA(TGA).
FIG 4
FIG 4
Changes in biomass, headspace, and metabolite levels between the start and finish of a mixotrophic-growth experiment with the acsA KO and WT strains on 10 g/liter fructose and 200 kPa CO. Panels: A, change in headspace pressure; B, change in headspace CO or CO2; C, change in growth based on OD600; D, change in metabolites. Columns: red, acsA KO strain; black, WT. n = 3. Error bars show the standard error of the mean.
FIG 5
FIG 5
Inactivation of CODH/ACS in C. autoethanogenum generates excess reducing equivalents that are consumed in biochemical reactions that lead to ethanol, 2,3-butanediol, and lactate formation.
FIG 6
FIG 6
Growth and metabolite profiles of the C. autoethanogenum WT and cooS1 and cooS2 KO strains on CO or H2-CO2. Panels: A, growth profile on CO; B, metabolite profile on CO; C, growth profile on H2-CO2; D, metabolite profile on H2-CO2. Columns: black, WT (n = 4 for CO, n = 3 for H2-CO2); green, cooS1 KO strain (n = 3); purple, cooS2 KO strain (n = 3). Error bars show the standard error of the mean.

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