Increased Butyrate Production in Clostridium saccharoperbutylacetonicum from Lignocellulose-Derived Sugars

Abstract

Butyrate is produced by chemical synthesis based on crude oil, produced by microbial fermentation, or extracted from animal fats (M. Dwidar, J.-Y. Park, R. J. Mitchell, and B.-I. Sang, The Scientific World Journal, 2012:471417, 2012, https://doi.org/10.1100/2012/471417). Butyrate production by anaerobic bacteria is highly favorable since waste or sustainable resources can be used as the substrates. For this purpose, the native hyper-butanol producer Clostridium saccharoperbutylacetonicum N1-4(HMT) was used as a chassis strain due to its broad substrate spectrum. BLASTp analysis of the predicted proteome of C. saccharoperbutylacetonicum N1-4(HMT) resulted in the identification of gene products potentially involved in acetone-butanol-ethanol (ABE) fermentation. Their participation in ABE fermentation was either confirmed or disproven by the parallel production of acids or solvents and the respective transcript levels obtained by transcriptome analysis of this strain. The genes encoding phosphotransacetylase (pta) and butyraldehyde dehydrogenase (bld) were deleted to reduce acetate and alcohol formation. The genes located in the butyryl-CoA synthesis (bcs) operon encoding crotonase, butyryl-CoA dehydrogenase with electron-transferring protein subunits α and β, and 3-hydroxybutyryl-CoA dehydrogenase were overexpressed to channel the flux further towards butyrate formation. Thereby, the native hyper-butanol producer C. saccharoperbutylacetonicum N1-4(HMT) was converted into the hyper-butyrate producer C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_BCS_P_bgaL]. The transcription pattern following deletion and overexpression was characterized by a second transcriptomic study, revealing partial compensation for the deletion. Furthermore, this strain was characterized in pH-controlled fermentations with either glucose or Excello, a substrate yielded from spruce biomass. Butyrate was the main product, with maximum butyrate concentrations of 11.7 g·L^-1 and 14.3 g·L^-1, respectively. Minimal amounts of by-products were detected. IMPORTANCE Platform chemicals such as butyrate are usually produced chemically from crude oil, resulting in the carry-over of harmful compounds. The selective production of butyrate using sustainable resources or waste without harmful by-products can be achieved by bacteria such as clostridia. The hyper-butanol producer Clostridium saccharoperbutylacetonicum N1-4(HMT) was converted into a hyper-butyrate producer. Butyrate production with very small amounts of by-products was established with glucose and the sustainable lignocellulosic sugar substrate Excello extracted from spruce biomass by the biorefinery Borregaard (Sarpsborg, Norway).

Keywords: Clostridium saccharoperbutylacetonicum; Excello; butyrate; fermentation; lignocellulosic sugars; metabolic engineering; solvents; transcriptome analysis.

FIG 1

Transcriptomic data from wild type C. saccharoperbutylacetonicum. Data are shown as log₂ fold changes compared to the early exponential growth phase and as normalized read counts determined by DeSeq2 (52). Filled mauve bars, after the butyrate peak; filled orange bars, after the acetate peak; filled yellow bars, stationary growth phase compared to the early exponential growth phase; open purple bars, early exponential growth phase; open mauve bars, after the butyrate peak; open orange bars, after the acetate peak; open yellow bars, stationary growth phase. (Data modified from reference .) Pta, phosphotransacetylase; Ptb, phosphotransbutyrylase (of interest is Cspa_c02520); Ack, acetate kinase; Buk, butyrate kinase (of interest is Cspa_c02530); Crt, crotonase; Hbd, 3-hydroxybutyryl-CoA dehydrogenase (of interest is Cspa_c04370); CtfA, CoA transferase subunit α; CtfB, CoA transferase subunit β; Adc, acetoacetate decarboxylase; Ald/Adh/AdhE, mono- and bifunctional aldehyde and alcohol dehydrogenases (of interest is butyraldehyde dehydrogenase encoded by Cspa_c56880); Bcd, butyryl-CoA dehydrogenase; Ldh, lactate dehydrogenase; ThlA, thiolase.

FIG 2

Growth experiment using different C. saccharoperbutylacetonicum deletion strains. Bacterial growth (OD₆₀₀), pH, and concentrations of the substrates and products throughout the experiment are shown. Open gray diamonds, wild type C. saccharoperbutylacetonicum; orange triangles, C. saccharoperbutylacetonicum Δpta; inverted red triangles, C. saccharoperbutylacetonicum Δbld; black-and-white circles, C. saccharoperbutylacetonicum ΔbldΔpta. Error bars represent standard deviations (n = 3). The colored asterisks mark significant differences between the strain represented by the curve and the strain represented by the color of the asterisk (P < 0.05 by one-way analysis of variance [ANOVA]). (Modified from reference .)

FIG 3

Growth experiment using C. saccharoperbutylacetonicum strains optimized for butyrate production. Bacterial growth (OD₆₀₀), pH, and concentrations of the substrates and products throughout the experiment are shown. Open gray diamonds, C. saccharoperbutylacetonicum wild type; black-and-white circles, C. saccharoperbutylacetonicum ΔbldΔpta; black squares, C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151] (vector control); upward- and downward-pointing lilac triangles, C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_PB_P_bgaL]; open and closed blue circles, C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_BCS_P_bgaL]; leftward- and rightward-pointing purple triangles, C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_BCS-PB_P_bgaL]; dashed lines with empty symbols, non-induced strains; straight lines with half-filled and filled symbols, induced strains; dashed-dotted red lines, time of induction with 20 mM lactose; upper lines in the lactate-lactose panel, lactose. Error bars represent standard deviations (n = 3). (Modified from reference .) The colored asterisks mark significant differences between the strain represented by the curve and the strain represented by the color of the asterisk, and empty asterisks represent the vector strain or noninduced strains (P < 0.05 by one-way ANOVA).

FIG 4

Transcriptomic data from the optimized butyrate producer C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_BCS_P_bgaL]. Data are shown as log₂ fold changes compared to C. saccharoperbutylacetonicum wild type and as normalized read counts determined using DeSeq2 (52). Black-and-white-striped bars, C. saccharoperbutylacetonicum ΔbldΔpta; closed light-blue bars, non-induced, C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_BCS_P_bgaL]; closed dark-blue bars, induced C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_BCS_P_bgaL], open gray bars, C. saccharoperbutylacetonicum wild type; open black bars, C. saccharoperbutylacetonicum ΔbldΔpta; open light-blue bars, non-induced, C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_BCS_P_bgaL] open dark-blue bars, C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_BCS_P_bgaL], induced. Error bars represent standard deviations (n = 3). Sampling during the exponential/early stationary growth phase are shown (OD₆₀₀ of C. saccharoperbutylacetonicum wild type, 3.25/6.93; OD₆₀₀ of C. saccharoperbutylacetonicum ΔbldΔpta, 3.82/5.35; OD₆₀₀ of non-induced C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_BCS_P_bgaL], 1.66/2.65; OD₆₀₀ of induced C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_BCS_P_bgaL], 1.58/2.99).

FIG 5

Controlled fermentation on glucose using C. saccharoperbutylacetonicum strains optimized for butyrate production. Bacterial growth (OD₆₀₀), pH, and concentrations of the substrates and products throughout the experiment are shown. Open gray diamonds, C. saccharoperbutylacetonicum wild type; black squares, C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151] (vector control); blue circles, C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_BCS_P_bgaL]; dashed-dotted blue line, time of induction with 20 mM lactose; upper line in the lactate-lactose panel, lactose. Error bars represent standard deviations (n = 3). The colored asterisks mark significant differences between the strain represented by the curve and the strain represented by the color of the asterisk (P < 0.05 by one-way ANOVA).

FIG 6

Controlled fermentation on Excello using C. saccharoperbutylacetonicum strains optimized for butyrate production. Bacterial growth (OD₆₀₀), pH, and concentrations of the main substrate (i.e., glucose) and products throughout the experiment are shown. Open gray diamonds, C. saccharoperbutylacetonicum wild type (glucose) (n = 1); gray-and-white diamonds, C. saccharoperbutylacetonicum wild type (Excello) (n = 1); black squares, C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151] (vector control) (n = 1); blue circles, C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_BCS_P_bgaL] (n = 2) (error bars represent standard deviations); dashed-dotted blue lines, time of induction with 20 mM lactose and inoculation of wild type C. saccharoperbutylacetonicum.