The key to acetate: metabolic fluxes of acetic acid bacteria under cocoa pulp fermentation-simulating conditions

Abstract

Acetic acid bacteria (AAB) play an important role during cocoa fermentation, as their main product, acetate, is a major driver for the development of the desired cocoa flavors. Here, we investigated the specialized metabolism of these bacteria under cocoa pulp fermentation-simulating conditions. A carefully designed combination of parallel 13C isotope labeling experiments allowed the elucidation of intracellular fluxes in the complex environment of cocoa pulp, when lactate and ethanol were included as primary substrates among undefined ingredients. We demonstrate that AAB exhibit a functionally separated metabolism during coconsumption of two-carbon and three-carbon substrates. Acetate is almost exclusively derived from ethanol, while lactate serves for the formation of acetoin and biomass building blocks. Although this is suboptimal for cellular energetics, this allows maximized growth and conversion rates. The functional separation results from a lack of phosphoenolpyruvate carboxykinase and malic enzymes, typically present in bacteria to interconnect metabolism. In fact, gluconeogenesis is driven by pyruvate phosphate dikinase. Consequently, a balanced ratio of lactate and ethanol is important for the optimum performance of AAB. As lactate and ethanol are individually supplied by lactic acid bacteria and yeasts during the initial phase of cocoa fermentation, respectively, this underlines the importance of a well-balanced microbial consortium for a successful fermentation process. Indeed, AAB performed the best and produced the largest amounts of acetate in mixed culture experiments when lactic acid bacteria and yeasts were both present.

FIG 1

Metabolic network of the central carbon metabolism of Acetobacter pasteurianus. The enzymatic reactions and their corresponding genes and the definitions of the abbreviations for the enzymes are given in Table S1 in the supplemental material. G6P, glucose-6-phosphate; 6PGl, 6-phosphogluconate; P5P, pentose-5-phosphate; F6P, fructose-6-phosphate; E4P, erythrose-4-phosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate; S7P, sucrose-7-phosphate; 3PG, 3-phosphoglycerate; AcLac, acetolactate; EtOH, ethanol; Acd, acetoin dehydrogenase; AcCoA, acetyl coenzyme A; OGDC, alpha-ketoglutarate dehydrogenase; AarC, succinyl-CoA:acetate CoA transferase; Ac, acetate; Cit, citrate; AKG, α-ketoglutarate; SucCoa, succinyl-CoA; Suc, succinyl; Mal, malate; OAA, oxaloacetate.

FIG 2

Labeling strategy used to elucidate metabolic fluxes of Acetobacter pasteurianus and Acetobacter ghanensis DSM 18895 in cocoa pulp simulation medium using specifically enriched isotopic tracer substances and mass spectral labeling analysis of proteinogenic amino acids and extracellular products. (A) Theoretical ¹³C-labeled mass isotopomer distribution of alanine obtained by de novo synthesis from the labeled substrates ([U-¹³C]lactate and [U-¹³C]ethanol) or amino acid uptake (¹²C). (B and C) Contribution of lactate and ethanol to formation of amino acid precursors. (D) Elucidation of specific metabolic routes of [3-¹³C]lactate utilization. Black circles, ¹³C-labeled carbon atoms; white circles, unlabeled carbon (¹²C); PP, pentose phosphate; Rel. intensity, relative intensity.

FIG 3

GC/MS analysis of the ¹³C labeling pattern of acetoin in culture supernatant. (A) Total ion current of the sample after derivatization with PFBHA and BSTFA, with acetoin eluting after 9.42 min; (B) the corresponding mass spectrum of PFBHA-BSTFA-acetoin; (C) chemical structure of PFBHA-BSTFA-acetoin and the corresponding fragments M-15 and M-43.

FIG 4

Profile of the first growth phase of A. pasteurianus NCC 316 grown in cocoa pulp simulation medium. The data are means from biological duplicates. Black circles, lactate; white circles, acetate; white diamonds, acetoin; black squares, ethanol; white squares, CDW; white stars, pyruvate; black line, dissolved oxygen.

FIG 5

(A) Relative contribution of lactate and ethanol to biosynthesis of amino acids. Squares, fractions originating from lactate and ethanol that were found in the proteinogenic amino acids of A. pasteurianus NCC 316 (left) and A. ghanensis DSM 18895 (right) during the first growth phase in cocoa pulp simulation medium. The full data are given in Table S5 in the supplemental material. (B) Relative contribution (percent) of lactate and ethanol to formation of acetate and acetoin in A. pasteurianus NCC 316 (upper values) and A. ghanensis DSM 18895 (lower values). Violet circles, nodes between the modules of lactate and ethanol metabolism.

FIG 6

Biosynthesis of phosphoenolpyruvate from lactate or ethanol via phosphoenolpyruvate carboxykinase (A) and pyruvate phosphate dikinase (B) by A. pasteurianus NCC 316. (A) Carbon transition in the reaction of phosphoenolpyruvate carboxykinase and experimental labeling profiles of oxaloacetate and phosphoenolpyruvate resulting from growth on a mixture of [¹²C₃]lactate and [¹³C₂]ethanol. Experimental data are derived from the fragments of aspartate and tyrosine at m/z 302 (Asp 302, Tyr 302) containing carbon atoms C-1 and C-2, as described in the supplemental material. (B) Carbon transition in the reaction of pyruvate phosphate dikinase and experimental labeling profiles of pyruvate and phosphoenolpyruvate resulting from growth on a mixture of [¹³C₃]lactate and [¹²C₂]ethanol. Experimental data are derived from the fragments of valine and tyrosine at m/z 302 (Val 302, Tyr 302) containing carbon atoms C-1 and C-2, as described in the supplemental material.

FIG 7

Phosphoenolpyruvate-pyruvate-oxaloacetate node in Corynebacterium glutamicum and Bacillus subtilis (A), Escherichia coli (B), and A. pasteurianus (C). Abbreviations for the enzymes are given in Table S1 the supplemental material. MDH, malate dehydrogenase.

FIG 8

Metabolic fluxes of A. pasteurianus NCC 316 during the first growth phase in cocoa pulp simulation medium. The data are given as relative fluxes, normalized to the cumulative uptake flux of lactate and ethanol (see Tables S10 and S11 in the supplemental material). The relative flux intensity is illustrated by the arrow thickness, and the individual contribution of lactate and ethanol to the fluxes is indicated by the arrow color. Solid arrows correspond to central metabolic fluxes, and dashed arrows correspond to the drain of precursors into biomass. The net direction of a reversible reaction is indicated by a small black arrow. The fluxes were derived via metabolite and isotopomer balancing recruiting extracellular fluxes (see Table S11 in the supplemental material) and the ¹³C labeling pattern of proteinogenic amino acids and acetate. Simulated and experimental mass isotopomer distributions are presented in Fig. S3 in the supplemental material. In addition to the net fluxes (in large type), 90% confidence intervals from Monte Carlo analysis are shown (in smaller type). [x], extracellular substrates and products.