Chemostat Cultures Research Articles

Multi‐omic profiling ­of EPO‐producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production

ABSTRACTChinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi‐omics approach was applied to study the production of erythropoietin (EPO) in a panel of CHO‐K1 cells under growth‐limited and unlimited conditions in batch and chemostat cultures. Physiological characterization of the EPO‐producing cells included global transcriptome analysis, targeted metabolome analysis, including intracellular pools of glycolytic intermediates, NAD(P)H/NAD(P)+, adenine nucleotide phosphates (ANP), and extracellular concentrations of sugars, organic acids, and amino acids. Potential impact of EPO expression on the protein secretory pathway was assessed at multiple stages using quantitative PCR (qPCR), reverse transcription PCR (qRT‐PCR), Western blots (WB), and global gene expression analysis to assess EPO gene copy numbers, EPO gene expression, intracellular EPO retention, and differentially expressed genes functionally related to secretory protein processing, respectively. We found no evidence supporting the existence of production bottlenecks in energy metabolism (i.e., glycolytic metabolites, NAD(P)H/NAD(P)+ and ANPs) in batch culture or in the secretory protein production pathway (i.e., gene dosage, transcription and post‐translational processing of EPO) in chemostat culture at specific productivities up to 5 pg/cell/day. Time‐course analysis of high‐ and low‐producing clones in chemostat culture revealed rapid adaptation of transcription levels of amino acid catabolic genes in favor of EPO production within nine generations. Interestingly, the adaptation was followed by an increase in specific EPO productivity. Biotechnol. Bioeng. 2015;112: 2373–2387. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Nitrite-Oxidizing Bacterium Nitrobacter winogradskyi Produces N-Acyl-Homoserine Lactone Autoinducers.

Nitrobacter winogradskyi is a chemolithotrophic bacterium that plays a role in the nitrogen cycle by oxidizing nitrite to nitrate. Here, we demonstrate a functional N-acyl-homoserine lactone (acyl-HSL) synthase in this bacterium. The N. winogradskyi genome contains genes encoding a putative acyl-HSL autoinducer synthase (nwi0626, nwiI) and a putative acyl-HSL autoinducer receptor (nwi0627, nwiR) with amino acid sequences 38 to 78% identical to those in Rhodopseudomonas palustris and other Rhizobiales. Expression of nwiI and nwiR correlated with acyl-HSL production during culture. N. winogradskyi produces two distinct acyl-HSLs, N-decanoyl-l-homoserine lactone (C10-HSL) and a monounsaturated acyl-HSL (C10:1-HSL), in a cell-density- and growth phase-dependent manner, during batch and chemostat culture. The acyl-HSLs were detected by bioassay and identified by ultraperformance liquid chromatography with information-dependent acquisition mass spectrometry (UPLC-IDA-MS). The C=C bond in C10:1-HSL was confirmed by conversion into bromohydrin and detection by UPLC-IDA-MS.

Time-series analysis of the transcriptome and proteome of Escherichia coli upon glucose repression

Time-series transcript- and protein-profiles were measured upon initiation of carbon catabolite repression in Escherichia coli, in order to investigate the extent of post-transcriptional control in this prototypical response. A glucose-limited chemostat culture was used as the CCR-free reference condition. Stopping the pump and simultaneously adding a pulse of glucose, that saturated the cells for at least 1h, was used to initiate the glucose response. Samples were collected and subjected to quantitative time-series analysis of both the transcriptome (using microarray analysis) and the proteome (through a combination of 15N-metabolic labeling and mass spectrometry). Changes in the transcriptome and corresponding proteome were analyzed using statistical procedures designed specifically for time-series data. By comparison of the two sets of data, a total of 96 genes were identified that are post-transcriptionally regulated. This gene list provides candidates for future in-depth investigation of the molecular mechanisms involved in post-transcriptional regulation during carbon catabolite repression in E. coli, like the involvement of small RNAs.

Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae

In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni2+-dependent enzyme and Saccharomyces cerevisiae does not express native Ni2+-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2Δ S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01µmolmin−1mg protein−1 and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni2+ concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni2+-dependent enzymes can be functionally expressed in S. cerevisiae.

Aerobic Hydrogen Production via Nitrogenase in Azotobacter vinelandii CA6.

The diazotroph Azotobacter vinelandii possesses three distinct nitrogenase isoenzymes, all of which produce molecular hydrogen as a by-product. In batch cultures, A. vinelandii strain CA6, a mutant of strain CA, displays multiple phenotypes distinct from its parent: tolerance to tungstate, impaired growth and molybdate transport, and increased hydrogen evolution. Determining and comparing the genomic sequences of strains CA and CA6 revealed a large deletion in CA6's genome, encompassing genes related to molybdate and iron transport and hydrogen reoxidation. A series of iron uptake analyses and chemostat culture experiments confirmed iron transport impairment and showed that the addition of fixed nitrogen (ammonia) resulted in cessation of hydrogen production. Additional chemostat experiments compared the hydrogen-producing parameters of different strains: in iron-sufficient, tungstate-free conditions, strain CA6's yields were identical to those of a strain lacking only a single hydrogenase gene. However, in the presence of tungstate, CA6 produced several times more hydrogen. A. vinelandii may hold promise for developing a novel strategy for production of hydrogen as an energy compound.

Heavy water and (15) N labelling with NanoSIMS analysis reveals growth rate-dependent metabolic heterogeneity in chemostats.

To measure single-cell microbial activity and substrate utilization patterns in environmental systems, we employ a new technique using stable isotope labelling of microbial populations with heavy water (a passive tracer) and (15) N ammonium in combination with multi-isotope imaging mass spectrometry. We demonstrate simultaneous NanoSIMS analysis of hydrogen, carbon and nitrogen at high spatial and mass resolution, and report calibration data linking single-cell isotopic compositions to the corresponding bulk isotopic equivalents for Pseudomonas aeruginosa and Staphylococcus aureus. Our results show that heavy water is capable of quantifying in situ single-cell microbial activities ranging from generational time scales of minutes to years, with only light isotopic incorporation (∼0.1 atom % (2) H). Applying this approach to study the rates of fatty acid biosynthesis by single cells of S. aureus growing at different rates in chemostat culture (∼6 h, 1 day and 2 week generation times), we observe the greatest anabolic activity diversity in the slowest growing populations. By using heavy water to constrain cellular growth activity, we can further infer the relative contributions of ammonium versus amino acid assimilation to the cellular nitrogen pool. The approach described here can be applied to disentangle individual cell activities even in nutritionally complex environments.

Nitrite Control over Dissimilatory Nitrate/Nitrite Reduction Pathways in Shewanella loihica Strain PV-4.

Shewanella loihica strain PV-4 harbors both a functional denitrification (NO3 (-)→N2) and a respiratory ammonification (NO3 (-)→NH4 (+)) pathway. Batch and chemostat experiments revealed that NO2 (-) affects pathway selection and the formation of reduced products. Strain PV-4 cells grown with NO2 (-) as the sole electron acceptor produced exclusively NH4 (+). With NO3 (-) as the electron acceptor, denitrification predominated and N2O accounted for ∼90% of reduced products in the presence of acetylene. Chemostat experiments demonstrated that the NO2 (-):NO3 (-) ratio affected the distribution of reduced products, and respiratory ammonification dominated at high NO2 (-):NO3 (-) ratios, whereas low NO2 (-):NO3 (-) ratios favored denitrification. The NO2 (-):NO3 (-) ratios affected nirK transcript abundance, a measure of denitrification activity, in the chemostat experiments, and cells grown at a NO2 (-):NO3 (-) ratio of 3 had ∼37-fold fewer nirK transcripts per cell than cells grown with NO3 (-) as the sole electron acceptor. In contrast, the transcription of nrfA, implicated in NO2 (-)-to-NH4 (+) reduction, remained statistically unchanged under continuous cultivation conditions at NO2 (-):NO3 (-) ratios below 3. At NO2 (-):NO3 (-) ratios above 3, both nirK and nrfA transcript numbers decreased and the chemostat culture washed out, presumably due to NO2 (-) toxicity. These findings implicate NO2 (-) as a relevant modulator of NO3 (-) fate in S. loihica strain PV-4, and, by extension, suggest that NO2 (-) is a relevant determinant for N retention (i.e., ammonification) versus N loss and greenhouse gas emission (i.e., denitrification).

Adaptive responses of Bacillus cereus ATCC14579 cells upon exposure to acid conditions involve ATPase activity to maintain their internal pH.

This study examined the involvement of ATPase activity in the acid tolerance response (ATR) of Bacillus cereus ATCC14579 strain. In the current work, B. cereus cells were grown in anaerobic chemostat culture at external pH (pHe) 7.0 or 5.5 and at a growth rate of 0.2 h−1. Population reduction and internal pH (pHi) after acid shock at pH 4.0 was examined either with or without ATPase inhibitor N,N’-dicyclohexylcarbodiimide (DCCD) and ionophores valinomycin and nigericin. Population reduction after acid shock at pH 4.0 was strongly limited in cells grown at pH 5.5 (acid-adapted cells) compared with cells grown at pH 7.0 (unadapted cells), indicating that B. cereus cells grown at low pHe were able to induce a significant ATR and Exercise-induced increase in ATPase activity. However, DCCD and ionophores had a negative effect on the ability of B. cereus cells to survive and maintain their pHi during acid shock. When acid shock was achieved after DCCD treatment, pHi was markedly dropped in unadapted and acid-adapted cells. The ATPase activity was also significantly inhibited by DCCD and ionophores in acid-adapted cells. Furthermore, transcriptional analysis revealed that atpB (ATP beta chain) transcripts was increased in acid-adapted cells compared to unadapted cells before and after acid shock. Our data demonstrate that B. cereus is able to induce an ATR during growth at low pH. These adaptations depend on the ATPase activity induction and pHi homeostasis. Our data demonstrate that the ATPase enzyme can be implicated in the cytoplasmic pH regulation and in acid tolerance of B. cereus acid-adapted cells.

Development of tools for quantitative intracellular metabolomics of Aspergillus niger chemostat cultures.

In view of the high citric acid production capacity of Aspergillus niger, it should be well suited as a cell factory for the production of other relevant acids as succinic, fumaric, itaconic and malic. Quantitative metabolomics is an important omics tool in a synthetic biology approach to develop A. niger for the production of these acids. Such studies require well defined and tightly controlled cultivation conditions and proper rapid sampling, sample processing and analysis methods. In this study we present the development of a chemostat for homogeneous steady state cultivation of A. niger, equipped with a new dedicated rapid sampling device. A quenching method for quantitative metabolomics in A. niger based on cold methanol was evaluated using balances and optimized with the aim of avoiding metabolite leakage during sample processing. The optimization was based on measurements of the intermediates of the glycolysis, TCA and PPP pathways and amino acids, using a balance approach. Leakage was found to be absent at −20 °C for a 40 % (v/v) methanol concentration in water. Under these conditions the average metabolite recovery was close to 100 %. When comparing A. niger and Penicillium chrysogenum metabolomes, under the same cultivation conditions, similar metabolite fingerprints were found in both fungi, except for the intracellular citrate level which is higher for A. niger.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-015-0781-z) contains supplementary material, which is available to authorized users.

Cloning and expression of gamma carbonic anhydrase from Serratia sp. ISTD04 for sequestration of carbon dioxide and formation of calcite

Bacterial strains isolated from marble mines rock and enriched in the chemostat culture with different concentrations of sodium bicarbonate. The enriched consortium had six bacterial isolates. One of bacterium isolate showed carbonic anhydrase (CA) activity by catalyzing the reversible hydration reaction of carbon dioxide to bicarbonate. The bacterium was identified as Serratia sp. by 16S rRNA sequence analysis. The carbonic anhydrase gene from Serratia sp. was found to be homologous with gamma carbonic anhydrase. The carbonic anhydrase gene was cloned in PET21b(+) and expressed it in recombinant Escherichia coli BL21 (DE3) with His-tag at the C-terminus. The recombinant protein was purified efficiently by using one-step nickel affinity chromatography. Expected size of carbonic anhydrase was approximately 29kDa in SDS–PAGE gel. Recombinant carbonic anhydrase enzyme was used for biomineralization-based conversion of atmospheric CO2 into valuable calcite minerals. The calcification was confirmed by using XRD, FTIR, EDX and SEM analysis.

Microbial conversion of synthetic and food waste-derived volatile fatty acids to lipids

Lipid accumulation in the oleaginous yeast Cryptococcus albidus was evaluated using mixtures of volatile fatty acids (VFA) as substrates. In general, batch growth under nitrogen limitation led to higher lipid accumulation using synthetic VFA. During batch growth, an initial COD:N ratio of 25:1mg COD:mg N led to maximum intracellular lipid accumulation (28.3±0.7% g/g dry cell weight), which is the maximum reported for C. albidus using VFA as the carbon source, without compromising growth kinetics. At this feed COD:N ratio, chemostat cultures fed with synthetic VFA yielded statistically similar intracellular lipid content as batch cultures (29.9±1.9%, g/g). However, batch cultures fed with VFA produced from the fermentation of food waste, yielded a lower lipid content (14.9±0.1%, g/g). The lipid composition obtained with synthetic and food-waste-derived VFA was similar to commercial biodiesel feedstock. We therefore demonstrate the feasibility of linking biochemical waste treatment and biofuel production using VFA as key intermediates.

Characterizing the metabolic trade-off in Nitrosomonas europaea in response to changes in inorganic carbon supply.

The link between the nitrogen and one-carbon cycles forms the metabolic basis for energy and biomass synthesis in autotrophic nitrifying organisms, which in turn are crucial players in engineered nitrogen removal processes. To understand how autotrophic nitrifying organisms respond to inorganic carbon (IC) conditions that could be encountered in engineered partially nitrifying systems, we investigated the response of one of the most extensively studied model ammonia oxidizing bacteria, Nitrosomonas europaea (ATCC19718), to three IC availability conditions: excess gaseous and excess ionic IC supply (40× stoichiometric requirement), excess gaseous IC supply (4× stoichiometric requirement in gaseous form only), and limiting IC supply (0.25× stoichiometric requirement). We found that, when switching from excess gaseous and excess ionic IC supply to excess gaseous IC supply, N. europaea chemostat cultures demonstrated an acclimation period that was characterized by transient decreases in the ammonia removal efficiency and transient peaks in the specific oxygen uptake rate. Limiting IC supply led to permanent reactor failures (characterized by biomass washout and failure of ammonia removal) that were preceded by similar decreases in the ammonia removal efficiency and peaks in the specific oxygen uptake rate. Notably, both excess gaseous IC supply and limiting IC supply elicited a previously undocumented increase in nitric and nitrous oxide emissions. Further, gene expression patterns suggested that excess gaseous IC supply and limiting IC supply led to consistent up-regulation of ammonia respiration genes and carbon assimilation genes. Under these conditions, interrogation of the N. europaea proteome revealed increased levels of carbon fixation and transport proteins and decreased levels of ammonia oxidation proteins (active in energy synthesis pathways). Together, the results indicated that N. europaea mobilized enhanced IC scavenging pathways for biosynthesis and turned down respiratory pathways for energy synthesis, when challenged with excess gaseous IC supply and limiting IC supply.

13C-based metabolic flux analysis of Saccharomyces cerevisiae with a reduced Crabtree effect

Saccharomyces cerevisiae shows a Crabtree effect that produces ethanol in a high glucose concentration even under fully aerobic condition. For efficient production of cake yeast or compressed yeast for baking, ethanol by-production is not desired since glucose limited chemostat or fed-batch cultivations are performed to suppress the Crabtree effect. In this study, the (13)C-based metabolic flux analysis ((13)C-MFA) was performed for the S288C derived S. cerevisiae strain to characterize a metabolic state under the reduced Crabtree effect. S. cerevisiae cells were cultured at a low dilution rate (0.1 h(-1)) under the glucose-limited chemostat condition. The estimated metabolic flux distribution showed that the acetyl-CoA in mitochondria was mainly produced from pyruvate by pyruvate dehydrogenase (PDH) reaction and that the level of the metabolic flux through the pentose phosphate pathway was much higher than that of the Embden-Meyerhof-Parnas pathway, which contributes to high biomass yield at low dilution rate by supplying NADPH required for cell growth.

The multifactorial interplay of diet, the microbiome and appetite control: current knowledge and future challenges.

The recent availability of high-throughput nucleic acid sequencing technologies has rapidly advanced approaches to analysing the role of the gut microbiome in governance of human health, including gut health, and also metabolic, cardiovascular and mental health, inter alia. Recent scientific studies suggest that energy intake (EI) perturbations at the population level cannot account for the current obesity epidemic, and significant work is investigating the potential role of the microbiome, and in particular its metabolic products, notably SCFA, predominantly acetate, propionate and butyrate, the last of which is an energy source for the epithelium of the large intestine. The energy yield from dietary residues may be a significant factor influencing energy balance. This review posits that the contribution towards EI is governed by EI diet composition (not just fibre), the composition of the microbiome and by the levels of physical activity. Furthermore, we hypothesise that these factors do not exist in a steady state, but rather are dynamic, with both short- and medium-term effects on appetite regulation. We suggest that the existing modelling strategies for bacterial dynamics, specifically for growth in chemostat culture, are of utility in understanding the dynamic interplay of diet, activity and microbiomic organisation. Such approaches may be informative in optimising the application of dietary and microbial therapy to promote health.

Metabolic profiling and stress response of anaerobic and respiratory cultures of Lactobacillus plantarum C17 grown in a chemically defined medium

We investigated the effect of anaerobic (AN) and respiratory conditions (RS; oxygen, hemin and menaquinone) on the growth, enzymatic activities (POX, NOX, NPR), metabolic profile (1H-NMR spectroscopy), and response to oxidative (catalase, tolerance of H2O2) and heat stresses of Lactobacillus plantarum C17 (wild-type) and its natural oxidative stress-tolerant mutant C17-m58, using chemostat cultivation in a modified chemically defined medium (mCDM). Respiratory growth impaired biomass production and tolerance of heat stress in mCDM compared with cultivation in the complex Weissella medium broth (WMB). POX activity, oxygen consumption, and H2O2 tolerance were higher in respiratory cells and in C17-m58. Significant amounts of formate and acetate were found in AN cultures of wild-type and mutant strains, confirming the activation of an anaerobic pyruvate formate lyase–acetate kinase (PFL-ACK) pathway in L. plantarum. The presence of succinate supports the presence of an incomplete TCA cycle in this species. High concentrations of pyruvate were measured in RS supernatants, indicating reduced functionality of POX in vivo (35 °C) and the need to clarify the regulation and control of oxygen metabolism. Adipate was found in respiratory samples, likely because of lipid peroxidation, and its production by respiring lactic acid bacteria may be investigated and exploited for biotechnological uses.

Physiology of the fuel ethanol strain Saccharomyces cerevisiae PE-2 at low pH indicates a context-dependent performance relevant for industrial applications.

Selected Saccharomyces cerevisiae strains are used in Brazil to produce the hitherto most energetically efficient first-generation fuel ethanol. Although genome and some transcriptome data are available for some of these strains, quantitative physiological data are lacking. This study investigates the physiology of S.cerevisiae strain PE-2, widely used in the Brazilian fuel ethanol industry, in comparison with CEN.PK113-7D, a reference laboratory strain, focusing on tolerance to low pH and acetic acid stress. Both strains were grown in anaerobic bioreactors, operated as batch, chemostat or dynamic continuous cultures. Despite their different backgrounds, biomass and product formation by the two strains were similar under a range of conditions (pH 5 or pH <3, with or without 105mM acetic acid added). PE-2 displayed a remarkably higher fitness than CEN.PK113-7D during batch cultivation on complex Yeast extract - Peptone - Dextrose medium at low pH (2.7). Kinetics of viability loss of non-growing cells, incubated at pH 1.5, indicated a superior survival of glucose-depleted PE-2 cells, when compared with either CEN.PK113-7D or a commercial bakers' strain. These results indicate that the sulfuric acid washing step, used in the fuel ethanol industry to decrease bacterial contamination due to non-aseptic operation, might have exerted an important selective pressure on the microbial populations present in such environments.

Chemostat cultivation and transcriptional analyses of Clostridium acetobutylicum mutants with defects in the acid and acetone biosynthetic pathways.

Clostridium acetobutylicum is a model organism for the biotechnologically important acetone-butanol-ethanol (ABE) fermentation. With the objective to rationally develop strains with improved butanol production, detailed insights into the physiological and genetic mechanisms of solvent production are required. Therefore, pH-controlled phosphate-limited chemostat cultivation and DNA microarray technology were employed for an in-depth analysis of knockout mutants with defects in the central fermentative metabolism. The set of studied mutants included strains with inactivated phosphotransacetylase (pta), phosphotransbutyrylase (ptb), and acetoacetate decarboxylase (adc) encoding genes, as well as an adc/pta double knockout mutant. A comprehensive physiological characterization of the mutants was performed by continuous cultivation, allowing for a well-defined separation of acidogenic and solventogenic growth, combined with the advantage of the high reproducibility of steady-state conditions. The ptb-negative strain C. acetobutylicum ptb::int(87) exhibited the most striking metabolite profile: Sizable amounts of butanol (29 ± 1.3 mM) were already produced during acidogenic growth. The product patterns of the mutants as well as accompanying transcriptomic data are presented and discussed.

Evaluation of bactericidal activity of Hannon honey on slowly growing bacteria in the chemostat.

There is renewed interest in the therapeutic use of honey, including use in the treatment of infected wounds and burn patients. In this study, we have assessed the antibacterial activity of Libyan floral Hannon honey on Escherichia coli and Staphylococcus aureus, both known to infect wounds. The effects of four concentrations (5%–30%) of honey were compared with that of four antibiotics (ampicillin, tetracycline, polymyxin, and ciprofloxacin) on the growth of these bacteria at early log, mid log, and late log phases. It has been shown that E. coli and S. aureus are to some degree susceptible during mid log phase compared with late log phase, demonstrated by their complete resistance to antibiotics. Chemostat culture was used to investigate the effect of honey on E. coli grown at a steady state with specific growth rates between 0.1 to 0.5 hour−1. The rate of killing was distinctively clear during the two stages of growth monitored: there was a relatively moderate reduction at the slow growth phase (0.1 to 0.3 hour−1), while a dramatic reduction was obtained at the fast growth phase (0.3 to 0.5 hour−1), reaching a complete reduction at 0.5 hour−1. These results complement data using the cup-cut technique. The antibacterial effect of honey was concentration and time dependent, the bactericidal effect was indeed observed at low concentrations, it demonstrates that the honey has more impact on slow growing bacteria than antibiotics have. We suggest that more reduction could be achieved at higher concentrations of honey. These results may have important clinical implications, such as for the management of wound and burn patients.

Increasing ATP conservation in maltose consuming yeast, a challenge for industrial organic acid production in non-aerated reactors

Anaerobic fermentation processes are economically attractive for industry, as costs for aeration and stirring are greatly reduced. An example of such a process is (bio)ethanol production by Saccharomyces cerevisiae, in which a low but positive ATP gain leads to elevated product yields. However, for other fermentative pathways, such as in an engineered homolactic strain of S. cerevisiae, the net ATP gain for the fermentation of glucose to lactate is null due to the requirement of ATP for product export. Therefore, increasing the conservation of ATP is of major importance for such 'zero-ATP pathways'. One opportunity to increase ATP conservation arises when disaccharides are used as a substrate. In many industrial microorganisms, disaccharides are cleaved by hydrolysis, which results in the dissipation of energy that is available in this cleaving reaction. However, phosphorolytic cleavage could be used to replace the hydrolysis of disaccharides, thereby increasing the ATP yield. In this study, growth of S. cerevisiae on maltose was used as a model. All known native maltose metabolism genes were removed and replaced by a maltose phosphorylase (Lactobacillus sanfranciscensis) and a single overexpressed copy of the native MAL11 maltose transporter. Because maltose phosphorylase cleaves maltose into glucose and β-glucose-1-phosphate, additionally a β-phosphoglucomutase (Lactococcus lactis) was co-expressed in this strain. Anaerobic maltose-limited chemostat cultures showed that replacement of maltose hydrolysis by phosphorolysis increased the biomass yield of the mutant strain by 26% over the wild type strain, demonstrating the potential of phosphorolysis to improve ATP conservation of disaccharide metabolism in industrial microorganisms [1].

Evaluation of gene expression and alginate production in response to oxygen transfer in continuous culture of Azotobacter vinelandii.

Alginates are polysaccharides used as food additives and encapsulation agents in biotechnology, and their functional properties depend on its molecular weight. In this study, different steady-states in continuous cultures of A. vinelandii were established to determine the effect of the dilution rate (D) and the agitation rate on alginate production and expression of genes involved in alginate polymerization and depolymerization. Both, the agitation and dilution rates, determined the partitioning of the carbon utilization from sucrose into alginate and CO2 under oxygen-limiting conditions. A low D (0.07 h−1) and 500 rpm resulted in the highest carbon utilization into alginate (25%). Quantitative real-time polymerase chain reaction was used to determine the transcription level of six genes involved in alginate polymerization and depolymerization. In chemostat cultures at 0.07 h−1, the gene expression was affected by changes in the agitation rate. By increasing the agitation rate from 400 to 600 rpm, the algE7 gene expression decreased tenfold, whereas alyA1, algL and alyA2 gene expression increased between 1.5 and 2.8 times under similar conditions evaluated. Chemostat at 0.07 h−1 showed a highest alginate molecular weight (580 kDa) at 500 rpm whereas similar molecular weights (480 kDa) were obtained at 400 and 600 rpm. The highest molecular weight was not explained by changes in the expression of alg8 and alg44 (genes involved in alginate polymerization). Nonetheless, a different expression pattern observed for lyases could explain the highest alginate molecular weight obtained. Overall, the results suggest that the control of alginate molecular weight in A. vinelandii cells growing in continuous mode is determined by a balance between the gene expression of intracellular and extracellular lyases in response to oxygen availability. These findings better our understanding of the biosynthesis of bacterial alginate and help us progress toward obtain tailor-made alginates.