Keywords

11.1 Introduction

The strictly aerobic α-proteobacterium Gluconobacter oxydans is used for a variety of industrial applications for reasons of its unusual metabolic capabilities, in particular, the incomplete oxidation of organic substrates. The main industrial applications of G. oxydans are the production of vitamin C, dihydroxyacetone, 6-amino-l-sorbose (a key intermediate for the synthesis of the anti-diabetic drug miglitol), shikimate, and 3-dehydroshikimate (Adachi et al. 2003b; Deppenmeier et al. 2002; Gupta et al. 2001; Macauley et al. 2001; Mamlouk and Gullo 2013; Nishikura-Imamura et al. 2014; Pappenberger and Hohmann 2014; Raspor and Goranovič 2008; Saichana et al. 2015). A set of membrane-integral dehydrogenases enables G. oxydans to partially oxidize diverse sugars, sugar alcohols, and other reduced compounds in one or more steps in the periplasm. The resulting products accumulate in the culture medium, often causing its acidification. The catalytic centers of these dehydrogenases are located in the periplasm and deliver electrons or electrons and protons to the respiratory chain (Matsushita et al. 1994, 2004). The rate of these oxidations is usually quite high, resulting in a high demand of G. oxydans for oxygen. Only a small fraction of the sugars or sugar alcohols is transported into the cell and catabolized in the cytoplasm via the pentose phosphate pathway (PPP) and the Entner–Doudoroff pathway (EDP). Because of the absence of a gene encoding 6-phosphofructokinase , the Embden–Meyerhof–Parnas pathway (EMP) is nonfunctional (Prust et al. 2005). Similarly, the tricarboxylic acid (TCA) cycle is incomplete because the genes for succinate dehydrogenase and also succinyl-CoA synthetase are lacking. Furthermore, the genome contains neither the key genes of the glyoxylate cycle nor a gene for a gluconeogenetic phosphoenolpyruvate (PEP)-forming enzyme (Deppenmeier and Ehrenreich 2009; Prust et al. 2005).

This chapter summarizes recent studies on central carbon metabolism and respiration in G. oxydans 621H (ATCC 621H is identical to DSM2343), including transcriptome analyses with DNA microarrays and 13C-based carbon flux analyses.

11.2 Cytoplasmic Sugar Metabolism

11.2.1 Pentose Phosphate Pathway and the Entner–Doudoroff Pathway

The importance of the PPP and the EDP for cytoplasmic sugar catabolism in G. oxydans has long been a matter of interest (Asai 1968; Hauge et al. 1955; Olijve and Kok 1979a, b; Rauch et al. 2010; Shinjoh et al. 1990; Tonouchi et al. 2003). Studies of the dissimilation pathway of l-[U-14C]sorbose by the 2-ketogulonate-producing strain UV10, derived from Gluconobacter melanogenus IFO 3293 (now G. oxydans NBRC 3293), showed that 40 % of the metabolized substrate was converted to l4CO2, which was mainly generated via the PPP (Shinjoh et al. 1990). In vitro characterization of enzymes involved in the central metabolism of G. oxydans revealed dual cofactor specificities of the key PPP enzymes glucose 6-phosphate dehydrogenase (GOX0145) and 6-phosphogluconate dehydrogenase (GOX1705). Under physiological conditions, these enzymes were found to be NADP+- and NAD+ dependent, respectively (Adachi et al. 1982; Rauch et al. 2010; Tonouchi et al. 2003). Lack of cofactor specificity in the oxidative PPP seemingly is not an uncommon characteristic, as it was also observed in 13C-based metabolic flux analyses of six other bacterial species (Fuhrer and Sauer 2009). Transaldolase (Tal) and glucose 6-phosphate isomerase (Pgi) form a bifunctional enzyme in G. oxydans (Sugiyama et al. 2003). Overexpression of the pgi/tal gene brought about increased growth rates and final cell densities in strains G. oxydans IFO 3293 and G. oxydans N44-1 (a derivative of IFO 3293) with sorbitol as the carbon source (Bremus et al. 2008a, b). Furthermore, it was found that the addition of purified Tal-Pgi or ribulokinase increased xylitol production from d-arabitol by a cell-free system of G. oxydans, presumably by improving NADH supply via an enhanced activity of the oxidative PPP (Sugiyama et al. 2003).

Recently, an analysis of the cytoplasmic catabolism of fructose formed by oxidation of mannitol, one of the preferred carbon sources of G. oxydans, was carried out with a Δgnd mutant lacking 6-phosphogluconate dehydrogenase and thus the oxidative PPP and a Δedd-eda mutant lacking 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase , and thus the EDP (Richhardt et al. 2012). Marker-free gene deletion was accomplished by a method based on G. oxydans Δupp, which lacks the upp gene for uracil phosphoribosyltransferase (Peters et al. 2013). A scheme of the central carbon metabolism of G. oxydans, including the reactions involved in mannitol catabolism, is shown in Fig. 11.1. In the first exponential growth phase, mannitol is oxidized to fructose, which accumulates in the medium. In the second growth phase, part of the fructose is taken up by the cells and another part is oxidized to 5-ketofructose (Fig. 11.2). Growth experiments using media with mannitol as an energy and carbon source and yeast extract as supplement revealed that neither of the two pathways, PPP or EDP , is essential for survival of G. oxydans. However, the growth characteristics of the two mutants under controlled conditions showed that the PPP is the main route for cytoplasmic fructose catabolism, whereas the EDP is dispensable and even unfavorable (Fig. 11.2). The Δedd-eda mutant formed 24 % more cell mass than the reference strain. The longer period of almost unimpaired exponential growth of the EDP mutant is probably the result of the lack of an adjustment phase during transition from growth phase I to II. This transition is connected with de novo protein synthesis, as was shown in an earlier study (Olijve and Kok 1979a). In contrast, deletion of gnd (6-phosphogluconate dehydrogenase) severely inhibited growth and caused a strong selection pressure for secondary mutations inactivating glucose 6-phosphate dehydrogenase , thus also preventing fructose catabolism via the EDP . These Δgnd zwf* mutants were almost totally disabled in fructose catabolism but still produced about 14 % of the carbon dioxide of the reference strain, possibly by catabolizing substrates from the yeast extract. The selection pressure of the Δgnd mutant of G. oxydans for secondary zwf mutations inactivating glucose 6-phosphate dehydrogenase could be caused by increased concentrations of 6-phosphogluconate or KDPG, which were shown to have an inhibitory effect on growth of Escherichia coli (Fuhrman et al. 1998) and Pseudomonas cepacia (Allenza and Lessie 1982). Overexpression of gnd in the reference strain improved biomass formation in a manner similar to deletion of edd-eda, further confirming the importance of the PPP for cytoplasmic fructose catabolism (Richhardt et al. 2012).

Fig. 11.1
figure 1

Scheme of the central carbon metabolism of Gluconobacter oxydans with the substrate mannitol. Glucose-6-P glucose 6-phosphate, fructose-1,6-P2 fructose 1,6-bisphosphate, DHAP dihydroxyacetone phosphate, UQ ubiquinone, PEP phosphoenolpyruvate

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Fig. 11.2
figure 2

(a–c) Growth of G. oxydans deletion strains (open symbols) and the parent strain G. oxydans Δupp (filled symbols) on mannitol: Δgnd (a); Δgnd zwf* (b); Δedd-eda (c). (d–f) Substrate consumption and product formation of G. oxydans deletion strains (dashed lines) and the reference strain Δupp (closed lines): Δgnd (d); Δgnd zwf* (e); Δedd-eda (f). Cells were cultivated in mannitol medium at 15 % dissolved oxygen at pH 6. Mean values and standard deviations of three independent cultures are shown. Mannitol (red); fructose (green); 5-ketofructose (blue); acetate (yellow). (Modified from Richhardt et al. 2012)

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Glucose catabolism by G. oxydans proceeds in two phases, comprising rapid periplasmic oxidation of glucose to gluconate together with cytoplasmic 5-ketogluconate production (phase I) followed by periplasmic oxidation of gluconate to 2-ketogluconate and consumption of 5-ketogluconate (phase II) (Fig. 11.3a). Only a small amount of glucose and part of the gluconate are taken up into the cells. To determine the roles of the PPP and the EDP for intracellular glucose and gluconate catabolism, the growth parameters of the mutants Δgnd, Δgnd zwf*, and Δedd-eda were determined in a bioreactor at pH 6 and 15 % dissolved oxygen (Richhardt et al. 2013a). In the presence of yeast extract, neither of the two pathways was essential for growth with glucose. However, the PPP mutants showed a reduced growth rate in phase I and completely lacked growth phase II. In contrast, the EDP mutant showed the same growth behavior as the reference strain. These results again demonstrate that the PPP is of major importance for cytoplasmic glucose and gluconate catabolism, whereas the EDP is dispensable. Up to now, the mechanism by which glucose is transported into the cell has not been known for G. oxydans. The presence of an incomplete PEP:carbohydrate phosphotransferase system (PTS) lacking the EIIC and EIIB components suggests a regulatory function rather than a transporter function. It was recently shown that the predicted HPr kinase (GOX0816) of G. oxydans phosphorylates HPr at Ser54 (Zhang et al. 2014), supporting a regulatory function of the PTS components.

Fig. 11.3
figure 3

13C-Metabolic flux analysis of G. oxydans during growth on glucose. a Growth (optical density at 600 nm) and glucose consumption (upper graph), product formation and consumption (lower graph). b Periplasmic and cytoplasmic carbon fluxes of nonphosphorylated intermediates. c Cytoplasmic carbon fluxes in central metabolism during growth phases I and II. Flux values given in b and c are related to 100 % glucose uptake (growth phase I) or 100 % gluconate uptake (growth phase II)

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11.2.2 13C-Metabolic Flux Analyses

The distribution and regulation of periplasmic and cytoplasmic carbon fluxes in G. oxydans 621H cultivated on glucose was studied by 13C-based metabolic flux analysis (Hanke et al. 2013). Cells were cultivated with specifically 13C-labeled glucose (Fig. 11.3a), and intracellular metabolites were analyzed for their labeling pattern by liquid chromatography–mass spectrometry (LC-MS). In growth phase I, 90 % of the glucose was oxidized periplasmatically to gluconate and partially further to 2-ketogluconate (Fig. 11.3b). Of the glucose taken up by the cells, 9 % was phosphorylated to glucose 6-phosphate, whereas 91 % was oxidized by cytoplasmic glucose dehydrogenase to gluconate (Fig. 11.3b, c). Of the gluconate formed in this way or taken up into the cells by a gluconate permease (GOX2188), 70 % was oxidized to 5-ketogluconate and 30 % was phosphorylated to 6-phosphogluconate. In growth phase II, 87 % of gluconate was oxidized to 2-ketogluconate in the periplasm and 13 % was taken up by the cells and almost completely converted to 6-phosphogluconate (Fig. 11.3b, c). 13C-Metabolic flux analysis (MFA) showed that 6-phosphogluconate is catabolized primarily via the oxidative PPP in both phase I and II (62 % and 93 %, respectively), and demonstrated a cyclic carbon flux through the oxidative PPP , as shown by the positive flux from fructose 6-phosphate to glucose 6-phosphate (indicated in red in Fig. 11.3c).

A cyclic operation of the PPP , as shown for G. oxydans, was also observed for Gluconacetobacter (Ga.) oboediens (recently renamed as Komagataeibacter oboediens) and Gluconacetobacter xylinus (recently renamed as Komagataeibacter xylinus) (Sarkar et al. 2010; Zhong et al. 2013). Carbon flux analyses with Ga. oboediens cultivated on glucose (15 g l−1) and acetate (5.24 g l−1) by 13C-labeling experiments showed that in spite of the presence of a pfk gene encoding phosphofructokinase, the activity of this enzyme was very low, leading to a negative carbon flux through the phosphoglucose isomerase (Pgi)-catalyzed reaction (i.e., a positive flux from fructose 6-phosphate to glucose 6-phosphate) and cyclization of the oxidative PPP (Sarkar et al. 2010). The same was observed in another study with Ga. xylinus, where negative fluxes through Pgi were determined in cells grown on the carbon sources glucose, fructose, and glycerol (Zhong et al. 2013).

Cocoa bean fermentation is a mixed-culture process, consisting initially of fermentations by yeast and lactic acid bacteria followed by oxidation of the fermentation products ethanol and lactic acid into acetic acid and acetoin by several Acetobacter strains, of which A. pasteurianus is the prominent one (Moens et al. 2014). A 13C-based carbon flux analysis of Acetobacter during cocoa pulp fermentation-simulating conditions revealed a functionally separated metabolism during co-consumption of ethanol and lactate. Acetate was almost exclusively derived from ethanol, whereas lactate served for formation of acetoin and biomass building blocks. This switch was attributed to the lack of phosphoenolpyruvate carboxykinase and malic enzyme activities, which prevents conversion of oxaloacetate and malate formed by acetate metabolism in the TCA cycle to PEP and pyruvate and subsequently to acetoin (Adler et al. 2014). Lactate, on the other hand, can be converted to pyruvate, which is then used for acetoin formation or, after conversion to PEP by pyruvate phosphate dikinase, for gluconeogenesis. The inability of conversion of TCA cycle intermediates to PEP resembles the situation in G. oxydans, where in addition no enzyme for conversion of pyruvate to PEP is present.

11.2.3 Transcriptome Analyses

For a better understanding of the two growth phases observed for G. oxydans during cultivation on glucose, a transcriptome comparison was performed using RNA isolated from cells harvested in phase I and phase II (Hanke et al. 2013). The DNA microarray analyses revealed 454 genes showing differential expression: 227 genes had an mRNA ratio (phase II/phase I) ≥2.0, and 227 genes had an mRNA ratio ≤0.5 (Hanke et al. 2013). Several genes encoding proteins that feed electrons into the respiratory chain showed increased mRNA levels in phase II, such as those for a PQQ-containing myo-inositol dehydrogenase (GOX1857), for the membrane-bound gluconate 2-dehydrogenase (GOX1230 and GOX1231), and for the type II NADH dehydrogenase (GOX1675). One of the two terminal oxidases of the respiratory chain of G. oxydans, the cytochrome bd ubiquinol oxidase, renamed “cyanide-insensitive oxidase CIO ” (GOX0278 and GOX0279) (Miura et al. 2013), was also upregulated in growth phase II. The genes encoding the membrane-integral pyridine nucleotide transhydrogenase (pntA1A2B, GOX0310-0312) belonged to the most strongly upregulated genes in phase II. The genes downstream of pntB, GOX0313 and GOX0314, encode putative alcohol dehydrogenases and showed comparable mRNA ratios as the pntA1A2B genes. The G. oxydans genome contains three gene clusters coding for subunits of F1Fo-ATP synthases. The clusters GOX1110 to GOX1113 and GOX1310 to GOX1314 encode the subunits of the Fo part and the F1 part of an ATP synthase, which is an orthologue of the ATP synthases of Acetobacter pasteurianus IFO 3283-01, Gluconacetobacter diazotrophicus PAL 5, and other α-Proteobacteria. Both these clusters showed decreased expression in phase II. The genes of the third cluster, GOX2167 to GOX 2175, might code for a Na+-translocating F1Fo-ATP synthase (Dibrova et al. 2010), and showed an increased expression in phase II. Furthermore, the DNA microarray analysis revealed an increased expression of PPP genes in growth phase II, which correlated with an increased PPP flux in phase II. Moreover, genes possibly related to a general stress response displayed increased expression in growth phase II (Hanke et al. 2013).

Transcriptome studies aimed at understanding the influence of different carbon sources on global gene expression were also performed for A. aceti NBRC 14818 cultivated on either ethanol, or acetate, or glucose, or a mixture of ethanol and glucose (Sakurai et al. 2011). In contrast to G. oxydans, this species possesses all genes for the TCA cycle as well as the glyoxylate cycle and thus is able to completely oxidize ethanol and acetate and perform anaplerosis. Neither the gene for phosphofructokinase nor that for 2-keto-3-deoxy-6-phosphogluconate aldolase could be identified, suggesting that sugar catabolism presumably proceeds exclusively via the PPP . As genes for acetate kinase and phosphotransacetylase are absent, acetate activation occurs either via acetyl-CoA synthetase, for which two genes are present, or via succinyl-CoA:acetate CoA transferase. Growth on ethanol is diauxic, whereby in the first phase ethanol is oxidized to acetate and in the second phase acetate is oxidized to CO2. Glucose (40 g/l) was almost completely oxidized to the end product gluconate, and biomass formation was much lower than with 10 g/l ethanol, suggesting that the strain is unable to efficiently utilize glucose and gluconate as carbon and energy sources. The transcriptome studies revealed a variety of carbon source-specific responses. The TCA cycle genes displayed higher mRNA levels during growth on acetate or glucose than on ethanol or ethanol/glucose mixture. The glyoxylate cycle genes showed high expression during growth on ethanol, acetate, and the glucose/ethanol mixture, but were not expressed on glucose alone, in accord with the known function of this pathway. Ethanol triggered increased expression of several stress-responsive genes.

In a further study, the time-dependent transcriptome changes during cultivation of A. aceti NBRC 14818 on ethanol were analyzed (Sakurai et al. 2012). In line with the previous data, the TCA cycle genes showed low mRNA levels during oxidation of ethanol to acetate and were significantly upregulated in the transition and acetate oxidation phases. This result suggested that the switch from acetate accumulation to acetate oxidation might be controlled by changes in the metabolic flux through the TCA cycle .

11.3 Respiratory Energy Metabolism

Because its many membrane-bound dehydrogenases incompletely oxidize sugars, sugar alcohols, and other compounds stereo- and regioselectively in the periplasm, Gluconobacter oxydans has been used for decades in industrial biotechnology (Adachi et al. 2003a; De Ley et al. 1984; Matsushita et al. 1994, 2002, 2004; Saichana et al. 2015; Yakushi and Matsushita 2010). The membrane-bound dehydrogenases transfer the reducing equivalents to ubiquinone. Two quinol oxidases, cytochrome bo 3 and cytochrome bd, then catalyze transfer of the electrons from ubiquinol to molecular oxygen (Fig. 11.4). Based on the cyanide insensitivity of G. oxydans cytochrome bd, Matsushita and coworkers designated this enzyme as cyanide-insensitive oxidase CIO and the corresponding genes cioA and cioB instead of cydA and cydB (Mogi et al. 2009). In a recent study, they purified and characterized the terminal oxidases of G. oxydans and for comparison cytochrome bd of Escherichia coli (Miura et al. 2013) (cf. Chaps. 7 and 13, in this volume). Based on the reported data, a different designation for members of the CIO clade, cioAB, is used in the following text (Fig. 11.4).

Fig. 11.4
figure 4

Overview of the components of the respiratory chain of G. oxydans 621H. DH dehydrogenase, FAD flavin adenine dinucleotide, PQQ pyrroloquinoline quinone, MoCo molybdopterin cofactor, CIO cyanide-insensitive oxidase. *In the sequenced strain 621H, the sldD gene encoding the flavoprotein subunit of the sorbitol dehydrogenase contains a frameshift mutation in codon 70, resulting in fragmentation (GOX2095, GOX2096) and the absence of 30 amino acids (positions 70–100 in the native SldD protein) (Prust et al. 2005). The functionality of the resulting complex is therefore questionable. (Modified from Richhardt et al. 2013b)

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11.3.1 Roles of the Two Terminal Oxidases Cytochrome bo 3 and Cyanide-Insensitive Oxidase, CIO

To elucidate the role of the two terminal oxidases of G. oxydans, the in-frame deletion mutants ΔcioAB (previously termed ΔcydAB) and ΔcyoBACD were constructed and characterized with respect to growth, respiratory activity, and H+/O ratio (Richhardt et al. 2013b). Deletion of the cioAB genes had no obvious influence on growth, whereas the lack of the cyoBACD genes severely reduced the growth rate and the cell yield . Using a respiration activity monitoring system and adjusting different levels of oxygen availability, hints for a low oxygen affinity of CIO were obtained, which were supported by measurements of oxygen consumption in a respirometer. Our evidence that CIO of G. oxydans has a significantly lower oxygen affinity than cytochrome bo 3 oxidase is in agreement with recent biochemical data, wherein the K m values for oxygen of CIO and cytochrome bo 3 determined with oxymyoglobin were reported to be 21 μM and 3 μM, respectively (Miura et al. 2013).

The H+/O ratio of the ΔcyoBACD mutant with mannitol as substrate was 0.56 ± 0.11, and more than 50 % lower than that of the reference strain (1.26 ± 0.06) and the ΔcioAB mutant (1.31 ± 0.16), indicating that cytochrome bo 3 oxidase is the main component for proton extrusion via the respiratory chain (Richhardt et al. 2013b). In previous studies, H+/O ratio s of 1.7–2.2 were reported for G. oxydans cells supplied with glycerol, glucose, lactate, or ethanol at pH 6 (Matsushita et al. 1989). The differences could be caused by the use of other substrates or differences in the experimental conditions. For cytochrome bo 3 oxidase , an H+/e stoichiometry of 2 and therefore an H+/O ratio of 4 can be assumed (Puustinen et al. 1989), and for cytochrome bd oxidase an H+/e stoichiometry of 1 and an H+/O ratio of 2 (Jasaitis et al. 2000; Puustinen et al. 1991). As the CIO was not relevant for growth of G. oxydans and proton translocation under the conditions used by Richhardt et al. 2013b, a H+/O ratio approaching 4 might be expected for G. oxydans. However, both our results as well as those presented by Matsushita et al. 1989 are much lower than a value of 4. There are a number of very speculative hypotheses to explain the low H+/O ratio s determined for G. oxydans. (1) The cytoplasmic membrane of G. oxydans might be more leaky for protons than that of E. coli, causing a non-energy-conserving backflow of protons into the cells (Brown 1992). (2) The respiratory chain could involve a reverse electron transfer coupled to an influx of protons (van der Oost et al. 1995). (3) Cytochrome bo 3 oxidase might not function as a primary proton pump, but as a Na+ pump, as has been described for cytochrome bo 3 oxidase of Vitreoscilla sp. (Kim et al. 2005; Park et al. 1996). In this context it is interesting to note that G. oxydans possesses two different F1Fo-type ATP synthases (Dibrova et al. 2010), one of which might use Na+ as the coupling ion (Hanke et al. 2012; Prust et al. 2005). The comparative DNA microarray analysis of the cyoBACD deletion mutant versus the reference strain showed an upregulation of genes (GOX2167-2175) coding for the putative Na+-dependent F1Fo-ATPase (Richhardt et al. 2013b).

Plasmid-based overexpression of cyoBACD led to increased growth rates and growth yields in both the wild type and the ΔcyoBACD mutant, suggesting that cytochrome bo 3 might be a rate-limiting factor of the respiratory chain (Richhardt et al. 2013b). Although additional studies are required to clarify the positive effect of cytochrome bo 3 overproduction, the result shows that respiratory chain components are a promising starting point for further optimization of G. oxydans for its use in biotechnological applications. Recently, a comparison of the genome sequences of G. oxydans 621H and G. oxydans DSM 3504 revealed, among others, the presence of an additional type II NADH dehydrogenase gene (ndh2) in strain DSM3504, which reaches an optical density almost three times higher than that of G. oxydans 621H. Interestingly, plasmid-based expression of ndh2 from strain DSM3504 in strain 621H led to a significantly increased growth rate (Kostner et al. 2014), explained by a competition between membrane-bound dehydrogenases and NADH dehydrogenase for transferring electrons to ubiquinone, which could be altered in favor of the latter enzyme by increased ndh expression.

11.3.2 Influence of Oxygen on Global Gene Expression

G. oxydans is strictly aerobic; nevertheless, in its natural habitat, oxygen deprivation caused by the rapid oxygen consumption by G. oxydans itself is likely to occur very often. In a recent study, the genome-wide transcriptional responses of G. oxydans 621H to oxygen limitation were analyzed (Hanke et al. 2012). For that purpose, cells were cultivated in a bioreactor system for 6 h at a constant dissolved oxygen concentration (DOC) of 15 %, before gassing was switched to a mixture of 2 % O2 and 98 % N2, leading to a DOC of 0 %. Samples for RNA isolation were taken before and 4 h after the switch and used for comparative transcriptome analysis with DNA microarrays. Oxygen deprivation caused expression changes of almost 500 genes, 215 with a ≥2.0-fold increased and 271 with a ≥2.0-fold decreased mRNA ratio (oxygen limitation/oxygen excess). Accordingly, oxygen limitation triggered a strong response influencing transcription of about 20 % of all chromosomal genes, including many involved in respiration and oxidative phosphorylation.

Although several genes coding for respiratory dehydrogenases showed reduced mRNA levels, expression of the terminal oxidase genes was increased. An opposite regulation of the dehydrogenases, several of which are PQQ dependent, and the terminal oxidases represents an adequate response to oxygen limitation, as it probably allows the cells to reduce the electron flux into the respiratory chain on the one hand and to increase the capability to capture the limiting oxygen on the other. The reduced expression of genes involved in PQQ biosynthesis and the increased expression of genes required for heme biosynthesis fits the expression patterns of the dehydrogenases and the terminal oxidases.

The genes for the membrane-integral pyridine nucleotide transhydrogenase PntA1A2B were among the most strongly upregulated genes under oxygen limitation (10- to 15 fold). The enzyme either consumes the electrochemical proton gradient Δp for NADP+ reduction or generates Δp at the expense of NADPH oxidation (Jackson et al. 2002). As observed in another study (see earlier), the genes downstream of pntA1A2B (GOX0313, GOX0314) encoding putative zinc-containing alcohol dehydrogenases showed mRNA ratios similar to the pntA1A2B genes, suggesting that these five genes might form an operon and a functional connection between the encoded enzymes. Increased levels of these enzymes might allow the cells a rapid exchange between NAD+/NADH and NADP+/NADPH and reoxidation of NAD(P)H via reduction of aldehydes/ketones to the corresponding alcohols, which could be favorable under oxygen limitation. In fact, the enzyme encoded by GOX0313 was recently shown to function as medium-chain alcohol dehydrogenase oxidizing various primary alcohols, but having a preference for substrate reduction, reducing many aldehydes and α-diketones (Schweiger et al. 2013).

As mentioned previously, the G. oxydans genome contains genes for two F1Fo-ATP synthases, one of which might code for a Na+-translocating F1Fo-ATP synthase. The genes for the presumably H+-translocating F1Fo-ATP synthase showed a decreased expression under oxygen limitation, whereas those for the putative Na+-translocating enzyme had increased mRNA levels. Remarkably, an inverse regulation of the two F1Fo-ATP synthases was also observed in the transcriptome comparison of growth phase I and II during cultivation of G. oxydans on glucose (see earlier). Many genes encoding proteins involved in transcription and translation showed lower expression under oxygen limitation, including subunits of the RNA polymerase, elongation factors, and 45 ribosomal proteins. This response probably presents an adaptation to the reduced linear growth observed after shifting the cells from oxygen excess to oxygen limitation. Overall, the transcriptome comparison revealed a complex response of G. oxydans to oxygen starvation and raised many interesting questions that deserve more detailed studies, such as the function of the two inversely regulated F1Fo-ATP synthases.

11.3.3 The Enigmatic Function of the Cytochrome bc 1 Complex

G. oxydans possesses the genes qcrABC (GOX0565-0567) for a cytochrome bc 1 complex , cycA (GOX0258) for a soluble cytochrome c 552, GOX1863 for a protein with similarity to subunit I of cytochrome c oxidase , and ctaB (GOX1864) for heme o-synthetase (Matsutani et al. 2014; Prust et al. 2005; Sakurai et al. 2011). As the putative subunit I lacks histidine residues serving as CuB and heme a 3 ligands, it is unlikely to be functional. Moreover, genes for subunits II and III of cytochrome c oxidase are absent in the genome. To understand the function of a cytochrome bc 1 complex in the presumptive absence of a functional cytochrome c oxidase , a ΔqcrABC mutant of G. oxydans was constructed and analyzed (Hanke et al. 2012). Although the mutant showed the same growth behavior as the wild type during cultivation on mannitol at pH 6, a growth defect was observed at pH 4 under oxygen saturation, where the mutant showed a 18 % reduced growth rate and a 13 % lower final optical density. Comparison of the transcriptomes of the ΔqcrABC mutant versus the wild type at pH 4 revealed 51 differentially expressed genes. Interestingly, almost all the 45 genes with increased expression in the ΔqcrABC mutant at pH 4 (including the cytochrome bo 3 oxidase genes) were also upregulated in the wild type grown at pH 6 under oxygen limitation (Fig. 11.5). The results obtained for the ΔqcrABC mutant clearly suggest that the cytochrome bc 1 complex possesses a physiological function in G. oxydans, at least at acidic pH, but further studies are required to elucidate this function.

Fig. 11.5
figure 5

(a) Growth and substrate consumption of G. oxydans 621H wild type and the cytochrome bc 1 mutant ΔqcrABC at pH 4 on 80 g l−1 mannitol under oxygen saturation. (b) Selected mRNA ratios from two different DNA microarray experiments: I, strain ΔqcrABC versus wild type, both cultivated at pH 4 under oxygen saturation; II, wild type cultivated under oxygen limitation versus wild type cultivated under oxygen saturation, both at pH 6. Genes shown are an excerpt of 51 genes having identical regulation patterns. (a was modified from Hanke et al. 2012)

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Two sets of genes for the cytochrome bc 1 complex , one of which lacks the gene for the Rieske iron sulfur protein, were identified in Acetobacter aceti NBRC 14818 and A. pasteurianus NBRC 3283 (Azuma et al. 2009; Sakurai et al. 2011). In both species also a gene encoding a putative subunit I of cytochrome c oxidase (coxA) was identified, but genes for subunits II and III were absent in the genome (Sakurai et al. 2011). Further organisms containing the genes for a cytochrome bc 1 complex but presumably lacking a functional cytochrome c oxidase are Acetobacter pasteurianus 386B (Illeghems et al. 2013) and Zymomonas mobilis (Balodite et al. 2014; Charoensuk et al. 2011; Sootsuwan et al. 2008), all of which belong to the α-Proteobacteria. Accordingly, this situation is not specific for G. oxydans, but is widespread in related bacteria.

One idea regarding the function of the cytochrome bc 1 complex is to provide reduced cytochrome c for reduction of hydrogen peroxide to water by an enzyme called cytochrome c peroxidase. G. oxydans (GOX0998) and many other acetic acid bacteria with known genome sequences possess the corresponding ccpR gene. However, both in G. oxydans and in A. aceti NBRC 14818, the expression level of ccpR was low under the conditions tested (Hanke et al. 2012; Sakurai et al. 2011). In other bacteria, these enzymes are usually present in addition to other terminal electron acceptors for reduced cytochrome c (Atack and Kelly 2007) and there is currently no experimental evidence for such a function of the cytochrome bc 1 complex in the aforementioned species.

11.4 Concluding Remarks

In the past years, a more detailed understanding of the metabolic features of G. oxydans and several other acetic acid bacteria was obtained based on the availability of genome sequences, the application of omics technologies such as transcriptomics and fluxomics, and by the detailed analysis of defined mutants. The genome sequences revealed significant heterogeneity in the equipment of individual species with membrane-bound dehydrogenases, sugar catabolic pathways, the TCA cycle , and the glyoxylate cycle . These differences are reflected in the metabolic properties of the species. Gluconobacter oxydans is one of the acetic acid bacteria prominently applied in industrial production processes, which are often whole-cell biotransformations requiring prior biomass production. Because the cell yield of G. oxydans (0.09 gcdw/gglucose during bioreactor cultivation is very low (Krajewski et al. 2010; Richhardt et al. 2013a), biomass production is cost intensive and presents a disadvantage for industrial application. The results obtained in the studies summarized here disclose starting points for improving the biomass yield and broadening the application range of G. oxydans by metabolic engineering of cytoplasmic sugar catabolism and respiratory energy metabolism.