Metabolic engineering of Clostridium autoethanogenum for selective alcohol production

Abstract

Gas fermentation using acetogenic bacteria such as Clostridium autoethanogenum offers an attractive route for production of fuel ethanol from industrial waste gases. Acetate reduction to acetaldehyde and further to ethanol via an aldehyde: ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase has been postulated alongside the classic pathway of ethanol formation via a bi-functional aldehyde/alcohol dehydrogenase (AdhE). Here we demonstrate that AOR is critical to ethanol formation in acetogens and inactivation of AdhE led to consistently enhanced autotrophic ethanol production (up to 180%). Using ClosTron and allelic exchange mutagenesis, which was demonstrated for the first time in an acetogen, we generated single mutants as well as double mutants for both aor and adhE isoforms to confirm the role of each gene. The aor1+2 double knockout strain lost the ability to convert exogenous acetate, propionate and butyrate into the corresponding alcohols, further highlighting the role of these enzymes in catalyzing the thermodynamically unfavourable reduction of carboxylic acids into alcohols.

Keywords: Aldehyde:ferredoxin oxidoreductase (AOR); Bi-functional aldehyde/alcohol dehydrogenase (AdhE); Clostridium autoethanogenum; Gas fermentation; Metabolic engineering.

Fig. 1

Autotrophic product formation in C. autoethanogenum. The ATP-efficient, indirect ethanol route employing phosphotransacetylase (Pta), acetate kinase (Ack) and aldehyde:ferredoxin oxidoreductase (AOR) are depicted in green. The direct ethanol biosynthesis route utilizing bi-functional aldehyde/alcohol dehydrogenase (AdhE), CoA-dependent acetaldehyde dehydrogenase (Ald) and alcohol dehydrogenase (Adh) is shown in red. AlsS = acetolactate synthase; 2,3-BDH =2,3-butanediol dehydrogenase; BudA = acetolactate decarboxylase; CODH = carbon monoxide dehydrogenase; CoFeSP = corrinoid iron sulphur protein; Fd_ox = oxidized ferredoxin; Fd_red = reduced ferredoxin; Hyt = NADP-dependent electron bifurcating hydrogenase; LdhA = lactate dehydrogenase; Nfn = transhydrogenase; Pfor = pyruvate:ferredoxin oxidoreductase; Rnf = H⁺-translocating ferredoxin: NAD⁺-oxidoreductase; WLP = Wood-Ljungdahl Pathway. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Screening and validation of the aor double KO strain with restored pyrE. (A) PCR screening of ∆aor2 and aor1 KO strain; (B) PCR screening of uracil autotrophic aor double KO strain for restored pyrE allele; (C) Southern Blot analysis of aor1 KO strain. M = NEB 2-log DNA ladder; 1 – 6= aor2-seq-F and aor2-seq-R primer pair; 7 – 12= aor1–559 s-F and aor1–559 s-R primer pair; 13 – 18= ACE-pyrE-F and ACE-pyrE-R primer pair; 1, 7 and 13= Non-template controls; 6, 12, 18 and 23= C. autoethanogenum WT genomic DNA control; 2 – 5, 8 – 11, 14 – 17= clones of aor double KO strain with restored pyrE; 19 – 22= HindIII digested genomic DNA of aor1 KO strain. Arrows and the accompanying numbers denote the fragment sizes of DNA ladder in kilobases.

Fig. 3

Growth, headspace pressure change and metabolite profiles of C. autoethanogenum WT (black circles), aor1 KO (red triangles), aor2 KO (green squares), and aor1+2 KO strains (blue diamonds) on 200 kPa CO. (A) Growth profile; (B) Change in headspace pressure from start to end of cultivation; (C) Acetate profile; (D) Ethanol profile; (E) 2,3-Butanediol profile; and (F) Lactate profile; For each strain n =4, except for aor2 KO n =3; Error bars = s.e.m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Growth, headspace pressure and metabolite profiles of C. autoethanogenum WT, and aor1+2 KO strain on H₂+CO₂. (A) Growth profile; (B) Change in headspace pressure; (C) Acetate profile; (D) Ethanol profile; Black circles = WT (n =4); Blue squares = aor1+2 KO strain (n =4); Error bars = s.e.m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Growth and metabolite profiles of C. autoethanogenum WT and adhE KO strains on fructose. (A) Growth profile; (B) Acetate profile; (C) Ethanol profile; and (D) 2,3-Butanediol profile. Black circles = WT (n =4); Red triangles = adhE1a KO strain (n =3); Green inverted triangles = adhE1b KO strain (n =3); Blue squares = adhE2 KO strain (n =3); Error bars = s.e.m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Growth and metabolite profiles of C. autoethanogenum WT and adhE KO strains on CO. (A) Growth profile; (B) Acetate profile; (C) Ethanol profile; and (D) 2,3-Butanediol profile. Black circles = WT (n =4); Red triangles = adhE1a KO strain (n =3); Green inverted triangles = adhE1b KO strain (n =2); Blue squares = adhE2 KO strain (n =3); Error bars = s.e.m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)