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The Impact of CO2/HCO3 Availability on Anaplerotic Flux in PDHC

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The Impact of CO2/HCO3 Availability on Anaplerotic Flux in PDHC bioRxiv preprint doi: https://doi.org/10.1101/663856; this version posted June 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. - 1 The impact of CO2/HCO3 availability on anaplerotic flux in PDHC- 2 deficient Corynebacterium glutamicum strains 3 a a a a b# 4 Aileen Krüger , Johanna Wiechert , Cornelia Gätgens , Tino Polen , Regina Mahr 5 and Julia Frunzkea# 6 7 a Institut für Bio- und Geowissenschaften, IBG-1: Biotechnology, Forschungszentrum 8 Jülich, 52425 Jülich, Germany 9 b SenseUp GmbH, c/o Campus Forschungszentrum Jülich, 52425 Jülich, Germany 10 - 11 Running Head: Influence of CO2/HCO3 on anaplerotic flux 12 13 A.K. and J.W. contributed equally to this work. 14 15 #Address correspondence to Julia Frunzke, [email protected], and Regina 16 Mahr, [email protected]. 17 1 bioRxiv preprint doi: https://doi.org/10.1101/663856; this version posted June 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 18 Abstract 19 The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative 20 decarboxylation of pyruvate yielding acetyl-CoA and CO2. The PDHC-deficient 21 Corynebacterium glutamicum strain ΔaceE is therefore lacking an important 22 decarboxylation step in central metabolism. Additional inactivation of pyc, encoding 23 pyruvate carboxylase, resulted in a >15 hour lag phase in the presence of glucose, 24 while no growth defect was observed on gluconeogenetic substrates like acetate. 25 Growth was successfully restored by deletion of ptsG encoding the glucose-specific 26 permease of the PTS system, thereby linking the observed phenotype to the 27 increased sensitivity of strain ΔaceE Δpyc to glucose catabolism. In the following, 28 strain ΔaceE Δpyc was used to systematically study the impact of perturbations of - 29 the intracellular CO2/HCO3 pool on growth and anaplerotic flux. Remarkably, all - - 30 measures leading to enhanced CO2/HCO3 levels, such as external addition of HCO3 31 , increasing the pH, or rerouting metabolic flux via pentose phosphate pathway, at 32 least partially eliminated the lag phase of strain ΔaceE Δpyc on glucose medium. In - 33 accordance, inactivation of the urease enzyme, lowering the intracellular CO2/HCO3 34 pool, led to an even longer lag phase accompanied with the excretion of L-valine and 35 L-alanine. Transcriptome analysis as well as an adaptive laboratory evolution 36 experiment of strain ΔaceE Δpyc revealed the reduction of glucose uptake as a key 37 adaptive measure to enhance growth on glucose/acetate mixtures. Altogether, our - 38 results highlight the significant impact of the intracellular CO2/HCO3 pool on 39 metabolic flux distribution, which becomes especially evident in engineered strains 40 suffering from low endogenous CO2 production rates as exemplified by PDHC- 41 deficient strains. 42 2 bioRxiv preprint doi: https://doi.org/10.1101/663856; this version posted June 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 43 Importance: CO2 is a ubiquitous product of cellular metabolism and an essential 44 substrate for carboxylation reactions. The pyruvate dehydrogenase complex (PDHC) - 45 catalyzes a central metabolic reaction contributing to the intracellular CO2/HCO3 pool 46 in many organisms. In this study, we used a PDHC-deficient strain of 47 Corynebacterium glutamicum, which was additionally lacking pyruvate carboxylase 48 (ΔaceE Δpyc). This strain featured a >15 h lag phase during growth on glucose- 49 acetate mixtures. We used this strain to systematically assess the impact of - 50 alterations in the intracellular CO2/HCO3 pool on growth on glucose-containing - 51 medium. Remarkably, all measures enhancing the CO2/HCO3 levels successfully - 52 restored growth emphasizing the strong impact of the intracellular CO2/HCO3 pool on 53 metabolic flux especially in strains suffering from low endogenous CO2 production 54 rates. 55 56 3 bioRxiv preprint doi: https://doi.org/10.1101/663856; this version posted June 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 57 Introduction 58 CO2 is an inevitable product and at the same time an essential substrate of microbial - 2- 59 metabolism. In water, CO2 is in equilibrium with HCO3 and CO3 , which is influenced 60 by the pH of the medium (1). As a product of decarboxylating reactions and substrate - 61 for carboxylations, CO2 and bicarbonate (HCO3 ) are involved in various metabolic - 62 processes. Consequently, the intracellular CO2/HCO3 pool has a significant impact 63 on metabolic fluxes like e.g. anaplerosis, especially in the early phase of cultivations 64 when cell density is low (2). 65 During aerobic growth, the pyruvate dehydrogenase complex (PDHC), which is 66 conserved in various microbial species, catalyzes an important reaction contributing - 67 to the intracellular CO2/HCO3 pool (3, 4). The PDHC is a multienzyme complex 68 belonging to the family of 2-oxo acid dehydrogenase complexes also comprising the 69 2-ketoglutarate dehydrogenase complex (ODHC) and the branched-chain 2-ketoacid 70 dehydrogenase (3, 5). In particular, PDHC catalyzes the oxidative decarboxylation of 71 pyruvate to acetyl-CoA, thereby producing one molecule CO2 and one molecule 72 NADH per mol pyruvate. 73 The Gram-positive actinobacterium Corynebacterium glutamicum represents an 74 important platform strain used in industrial biotechnology for the production of amino 75 acids, proteins and various other value-added products (6-10). In this organism, the 76 PDHC complex represents a central target to engineer metabolic pathways of 77 pyruvate-derived products including L-valine, isobutanol, ketoisovalerate and L- 78 lysine. To this end, different studies focused on the reduction or complete 79 abolishment of PDHC activity to improve precursor availability (4, 11-13). Due to the 80 deficiency of PDHC activity, however, cells are not able to grow on glucose as single 81 carbon source which can be circumvented by the addition of acetyl-CoA refueling 82 substrates such as acetate. In presence of both carbon sources glucose and acetate, 4 bioRxiv preprint doi: https://doi.org/10.1101/663856; this version posted June 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 83 PDHC-deficient strains initially form biomass from acetate and subsequently, convert 84 glucose into products (e.g. L-valine or L-alanine) in the stationary phase (12). In 85 contrast to Escherichia coli or Bacillus subtilis, C. glutamicum does not normally 86 show the typical diauxic growth behaviour, but prefers co-metabolization of many 87 carbon sources (10, 14). Co-utilization of glucose and acetate has been studied in 88 detail, showing that consumption rates of both carbon sources decrease, but the total 89 carbon consumption is comparable to growth on either carbon source alone which is 90 regulated by SugR activity (15). Further examples for co-metabolization of glucose 91 with other sugars or organic acids include fructose (16), lactate (17) as well as 92 pyruvate (14) and gluconate (18). It was shown, that upon growth on glucose-acetate 93 mixtures, the glyoxylate shunt is required to fuel the oxaloacetate pool in the wild type 94 (15). Here it is important to note that the glyoxylate shunt is active in the presence of 95 acetate, but repressed by glucose (19, 20). 96 For growth on glycolytic carbon sources, organisms depend on TCA cycle 97 replenishing reactions constituting the anaplerotic node (or phosphoenolpyruvate- 98 pyruvate-oxaloacetate node), comprised of different carboxylating and 99 decarboxylating reactions (21). Consequently, flux via these reactions is influenced - 100 by the intracellular CO2/HCO3 pool. In contrast to most other organisms, C. 101 glutamicum possesses both anaplerotic carboxylases namely the 102 phosphoenolpyruvate carboxylase (PEPCx) (EC 4.1.1.31) encoded by the ppc gene 103 (22, 23) and the pyruvate carboxylase (PCx) (EC 6.4.1.1) encoded by pyc (21, 24). 104 These two C3-carboxylating enzymes catalyze bicarbonate-dependent reactions 105 yielding oxaloacetate from PEP or pyruvate, respectively. In C. glutamicum, it was 106 shown that these two enzymes could replace each other to a certain extent 107 depending on the intracellular concentrations of the respective effectors for each 108 enzyme. However, under standard conditions during growth on glucose, the biotin- 5 bioRxiv preprint doi: https://doi.org/10.1101/663856; this version posted June 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 109 containing PCx contributes to the main anaplerotic activity of 90% compared to 110 PEPCx (24). Remarkably, the Michaelis-Menten constants of both carboxylases are 111 about 30-fold higher in comparison to Escherichia coli PEP carboxylase (25). - 112 Apparently, a low CO2/HCO3 pool may thus limit anaplerotic flux, which is of special 113 relevance in early phases when biomass concentration is low, but high aeration may 114 strip dissolved CO2 from the medium. 115 Several studies revealed the inhibitory effect of low pCO2 on microbial growth (2, 26, 116 27). In the case of C. glutamicum, low pCO2 pressures led to a significant drop in 117 growth rate in turbidostatic continuous cultures (28) as well as batch cultures (29). It 118 was further demonstrated that the reduced flux through anaplerotic reactions under - 119 low CO2/HCO3 levels led to an increased production of the pyruvate-derived amino 120 acids L-alanine and L-valine (29).
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