Non-repressing Carbon Source Research Articles

Optimized Expression and Isolation of Recombinant Active Secreted Proteases Using Pichia pastoris.

Recombinant proteins of high quality are crucial starting materials for all downstream applications, but the inherent complexities of proteins and their expression and purification create significant challenges. The Pichia pastoris yeast is a highly useful eukaryotic protein expression system. Pichia's low cost, genetic tractability, rapid gene expression, and scalability make it an ideal expression system for foreign proteins. Here, we developed a protocol that has optimized the expression and isolation of a non-mammalian secreted metalloprotease, where we can routinely generate recombinant proteins that are pure and proteolytically active. We maximized growth and protein production by altering the feeding regime, through implementation of a non-fermentable and non-repressing carbon source during the methanol-induction phase. This approach increased biomass production and yielded milligrams of recombinant protein. Downstream applications involving active, recombinant fungal proteases, such as conjugation to nanoparticles and structure-related studies, are greatly facilitated with this improved, standardized approach. Graphical abstract.

Integration of GC-MS in identification of possible final metabolites from phytase production in Pichia Pastoris based on sorbitol induction optimization

The isolation of phytase using Pichia Pastoris under methanol/sorbitol co-feeding induction technique was investigated. The biological activity of extracellular phytase after optimization with co-substrates induction in 4 liters fermentor (NBS) increased to 13250 U/ml. This led to a 509 fold increases in comparison to the other type of phytase. This effect was studied via induction with sorbitol/methanol in fermentation by Pichia Pastoris GS115 (Mut+) at 20 °C. The interference of by products; methylal, hexamine and (S)-(+)-1,2-propanediol with release of phytase in Pichia Pastoris under methanol induction were detected and cannot be repressed by methanol induction alone. The TLC was used for glycerin analysis under methanol/sorbitol induction and the results were lesser compare to that obtained during phytase production under methanol induction alone. This work showed the higher expression of heterologous proteins and by fed batch fermentation; the expression identified an advantage of producing a significant activity of phytase. Practical applications Plant derived products including sorbitol have been used as alternative medicines for the therapeutic treatment of various diseases, food supplements and could be used in many manufacturing processes. It serves as a culture media for bacteria, and helps to distinguish the pathogenic E. coli O157:H7 from its most other strains. Cells growing on methanol require high oxygen consumption. Sorbitol was used as an alternative cheap co-feeding for the production of proteins and is a non-repressing carbon source for AOX1 promoter with no effect on the level of r-protein at its induction phase. This report describes the isolation of phytase using Pichia Pastoris under methanol/sorbitol co-feeding induction techniques, and sorbitol showed to be a promising co-substrate, as it could enhance both cell growth and targeted protein productivity. This co-feeding and fed-batch induction technique was used for recombinant phytase production in a small and large scale production and the metabolites were analyzed.

Catabolism of phenylacetic acid in Penicillium rubens. Proteome-wide analysis in response to the benzylpenicillin side chain precursor

Biosynthesis of benzylpenicillin in filamentous fungi (e.g. Penicillium chrysogenum - renamed as Penicillium rubens- and Aspergillus nidulans) depends on the addition of CoA-activated forms of phenylacetic acid to isopenicillin N. Phenylacetic acid is also detoxified by means of the homogentisate pathway, which begins with the hydroxylation of phenylacetic acid to 2-hydroxyphenylacetate in a reaction catalysed by the pahA-encoded phenylacetate hydroxylase. This catabolic step has been tested in three different penicillin-producing strains of P. rubens (P. notatum, P. chrysogenum NRRL 1951 and P. chrysogenum Wisconsin 54–1255) in the presence of sucrose and lactose as non-repressing carbon sources. P. chrysogenum Wisconsin 54–1255 was able to accumulate 2-hydroxyphenylacetate at late culture times. Analysis of the P. rubens genome showed the presence of several PahA homologs, but only Pc16g01770 was transcribed under penicillin production conditions. Gene knock-down experiments indicated that the protein encoded by Pc16g01770 seems to have residual activity in phenylacetic acid degradation, this catabolic activity having no effect on benzylpenicillin biosynthesis. Proteome-wide analysis of the Wisconsin 54–1255 strain in response to phenylacetic acid revealed that this molecule has a positive effect on some proteins directly related to the benzylpenicillin biosynthetic pathway, the synthesis of amino acid precursors and other important metabolic processes. SignificanceThe adaptive response of Penicillium rubens to benzylpenicillin production conditions remains to be fully elucidated. This article provides important information about the molecular mechanisms interconnected with phenylacetate (benzylpenicillin side chain precursor) utilization and penicillin biosynthesis, and will contribute to the understanding of the complex physiology and adaptation mechanisms triggered by P. rubens (P. chrysogenum Wisconsin 54–1255) under benzylpenicillin production conditions.

Induction studies of a novel α-glucosidase purified from Lactobacillus fermentum grown on resistant starch

Aim: The aim of this study is to investigate the induction studies of resistant starch (RS)-degrading enzyme purified from Lactobacillus fermentum. Background: RS is one of the components of dietary fiber and considered as a good prebiotic. Earlier studies from our laboratory reporting L. fermentum to be the most efficient in degrading RS among several lactic acid bacteria tested. L. fermentum produced α-glucosidase when grown on RS, which was purified biochemically and characterized. Results and Discussion: The alpha-glucosidase was synthesized de novo and was repressed by glucose. Known protein synthesis inhibitors such as potassium cyanide, 2,4-dinitrophenol, and tetracycline were found to inhibit α-glucosidase synthesis. Cyclic adenosine 3',5'-monophosphate was found to be a stimulator of α-glucosidase synthesis; however, it did not have any impact on the lag phase. Glutamate acted as an excellent nonrepressing carbon source. Maltooligosaccharides, dextrins, and soluble starch had varied influence on the induction of α-glucosidase synthesis. Conclusion: In the present communication, possible factors regulating α-glucosidase synthesis in L. fermentum were examined and discussed in terms of catabolite and apparent temperature-sensitive repression, culture age, induction, inhibitors, and various carbon sources. Abbreviation used: RS: Resistant starch.

Selection of suitably non-repressing carbon sources for expression of alcohol oxidase isozyme promoters in the methylotrophic yeast Pichia methanolica

In this work, we aimed to select suitable non-repressing carbon sources for the expression of promoters derived from the alcohol oxidase (AOD) isozyme genes, PMOD1 and PMOD2, during the growth of Pichia methanolica. Our results revealed that xylose is the best non-repressing carbon source for heterologous gene expression using both PMOD1 and PMOD2, and that glycerol is also a suitable carbon source with by which the on/off of PMOD2 expression can be controlled.

Enhanced expression of an industry applicable CotA laccase from Bacillus subtilis in Pichia pastoris by non-repressing carbon sources together with pH adjustment: Recombinant enzyme characterization and dye decolorization

In this study, protease-deficient Pichia pastoris strain SMD1168H was selected for heterologous expression of the CotA laccase from Bacillus subtilis. Four non-repressing carbon sources were individually applied to facilitate the recombinant laccase production. An up to 76-fold increase in laccase activity was achieved through sorbitol addition in combination with pH adjustment. Recombinant CotA (rCotA) demonstrated a remarkable stability under alkaline conditions, the enzyme retained 637% and 94.37% of its initial activity after 10-day incubation at pH 9.0 and 10.0, respectively. The rCotA laccase showed an outstanding thermostability and exhibited higher tolerance towards organic solvents than the spore laccase. The Km and kcat values of rCotA laccase for ABTS were of 146.4±2.7μM and 14.4±0.1s−1 and for SGZ of 12.7±2.6μM and 6.9±0.6s−1, respectively. A repeated-batch decolorization experiment was carried out at pH 10.0, 40°C to evaluate the capacity of rCotA for repetitive decolorization of indigo carmine under alkaline conditions. During the first six repeated cycles, more than 95% decolorization was observed in 10min and total color was removed within one hour. The decolorization efficiency stayed above 90% after twenty decolorization cycles.

Systematic Unraveling of the Unsolved Pathway of Nicotine Degradation in Pseudomonas

Microorganisms such as Pseudomonas putida play important roles in the mineralization of organic wastes and toxic compounds. To comprehensively and accurately elucidate key processes of nicotine degradation in Pseudomonas putida, we measured differential protein abundance levels with MS-based spectral counting in P. putida S16 grown on nicotine or glycerol, a non-repressive carbon source. In silico analyses highlighted significant clustering of proteins involved in a functional pathway in nicotine degradation. The transcriptional regulation of differentially expressed genes was analyzed by using quantitative reverse transcription-PCR. We observed the following key results: (i) The proteomes, containing 1,292 observed proteins, provide a detailed view of enzymes involved in nicotine metabolism. These proteins could be assigned to the functional groups of transport, detoxification, and amino acid metabolism. There were significant differences in the cytosolic protein patterns of cells growing in a nicotine medium and those in a glycerol medium. (ii) The key step in the conversion of 3-succinoylpyridine to 6-hydroxy-3-succinoylpyridine was catalyzed by a multi-enzyme reaction consisting of a molybdopeterin binding oxidase (spmA), molybdopterin dehydrogenase (spmB), and a (2Fe-2S)-binding ferredoxin (spmC) with molybdenum molybdopterin cytosine dinucleotide as a cofactor. (iii) The gene of a novel nicotine oxidoreductase (nicA2) was cloned, and the recombinant protein was characterized. The proteins and functional pathway identified in the current study represent attractive targets for degradation of environmental toxic compounds.

Isolation, characterization and evaluation of the Pichia pastoris sorbitol dehydrogenase promoter for expression of heterologous proteins

Sorbitol is used as a non-repressive carbon source to develop fermentation process for Muts recombinant clones obtained using the AOX1 promoter in Pichia pastoris. Sorbitol dehydrogenase is an enzyme in the carbohydrate metabolism that catalyzes reduction of d-fructose into d-sorbitol in the presence of NADH. The small stretch of 211bps upstream region of sorbitol dehydrogenase coding gene has all the promoter elements like CAAT box, GC box, etc. It is able to promote protein production under repressive as well as non-repressive carbon sources. In this study, the strength of the sorbitol dehydrogenase promoter was evaluated by expression of two heterologous proteins: human serum albumin and erythrina trypsin inhibitor. Sorbitol dehydrogenase promoter allowed constitutive expression of recombinant proteins in all carbon sources that were tested to grow P. pastoris and showed activity similar to GAP promoter. The sorbitol dehydrogenase promoter was active in all the growth phases of the P. pastoris.

Catabolite repression of SOS-dependent and SOS-independent spontaneous mutagenesis in stationary-phase Escherichia coli

Previous work in our laboratory established that a spontaneous mutagenesis process operating in stationary-phase Escherichia coli cells undergoing selection is subject to regulation by the global regulatory mechanism known as catabolite repression (formerly also called glucose-repression). Here, we set out to determine the identity of this hitherto unknown catabolite-repressible spontaneous mutation generation mechanism(s). We used two different spontaneous mutation detection assays, reversion of a Lac(-) (lacI33OmegalacZ) frameshift marker and forward mutation to valine-resistance, and tested the effects of varying the nature of the carbon source(s) present in the selective plating medium on the mutability of bacterial cells carrying known defects in the recA, umuDC and dinB genes, three well-known SOS response genes, whose products are important for mutagenesis in E. coli. Consistent with the results of our previous Lac(-)-->Lac(+) assay using otherwise SOS-proficient bacterial cells, we found that the overall numbers of spontaneous Lac(+)E. coli revertants were highest when the selective medium contained lactose and lowest when it contained lactose plus the non-utilizable but strongly catabolite-repressing glucose analogue, methyl-alpha-d-glucopyranoside (alphaMG). In contrast, we found that the numbers of Lac(+) revertants appearing on the lactose and lactose+alphaMG selection plates were greatly diminished and not significantly different when the bacterial cells concerned carried either a DeltarecA or DeltadinB mutation. Furthermore, introducing the DeltadinB mutant allele into bacterial cells over-expressing the recA gene reduced the numbers of Lac(+) mutations to those being recovered with the DeltadinB cells. These results appear to suggest that (i) the DinB-dependent mutation generation pathway is alone responsible for spontaneous reversion of the lacI33OmegalacZ frameshift marker, and (ii) the varying numbers of Lac(+) colonies that we recover on the lactose and lactose+alphaMG plates provide a direct measure of the differential effects of these particular carbon compounds on the overall expression of the dinB gene. Interestingly, the yields of spontaneous Val mutations arising in wild-type, DeltarecA, DeltadinB and DeltaumuDC cells were found to be similar, but always tended to be highest when the medium contained only a non-repressing carbon source (glycerol) and lowest when it had been supplemented with a strong catabolite repressor such as glucose or alphaMG. Together, our results would seem to establish that stationary-phase E. coli cells exposed to strong selection pressures can accumulate spontaneous mutations via SOS-dependent and SOS-independent mutation generation pathways whose levels of expression are regulated by catabolite repression.

13C-metabolic flux ratio and novel carbon path analyses confirmed that Trichoderma reesei uses primarily the respirative pathway also on the preferred carbon source glucose

BackgroundThe filamentous fungus Trichoderma reesei is an important host organism for industrial enzyme production. It is adapted to nutrient poor environments where it is capable of producing large amounts of hydrolytic enzymes. In its natural environment T. reesei is expected to benefit from high energy yield from utilization of respirative metabolic pathway. However, T. reesei lacks metabolic pathway reconstructions and the utilization of the respirative pathway has not been investigated on the level of in vivo fluxes.ResultsThe biosynthetic pathways of amino acids in T. reesei supported by genome-level evidence were reconstructed with computational carbon path analysis. The pathway reconstructions were a prerequisite for analysis of in vivo fluxes. The distribution of in vivo fluxes in both wild type strain and cre1, a key regulator of carbon catabolite repression, deletion strain were quantitatively studied by performing 13C-labeling on both repressive carbon source glucose and non-repressive carbon source sorbitol. In addition, the 13C-labeling on sorbitol was performed both in the presence and absence of sophorose that induces the expression of cellulase genes. Carbon path analyses and the 13C-labeling patterns of proteinogenic amino acids indicated high similarity between biosynthetic pathways of amino acids in T. reesei and yeast Saccharomyces cerevisiae. In contrast to S. cerevisiae, however, mitochondrial rather than cytosolic biosynthesis of Asp was observed under all studied conditions. The relative anaplerotic flux to the TCA cycle was low and thus characteristic to respiratory metabolism in both strains and independent of the carbon source. Only minor differences were observed in the flux distributions of the wild type and cre1 deletion strain. Furthermore, the induction of the hydrolytic gene expression did not show altered flux distributions and did not affect the relative amino acid requirements or relative anabolic and respirative activities of the TCA cycle.ConclusionHigh similarity between the biosynthetic pathways of amino acids in T. reesei and yeast S. cerevisiae was concluded. In vivo flux distributions confirmed that T. reesei uses primarily the respirative pathway also when growing on the repressive carbon source glucose in contrast to Saccharomyces cerevisiae, which substantially diminishes the respirative pathway flux under glucose repression.

Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation

Alkaline polygalacturonate lyase (PGL) production by Pichia pastoris GS115 was used as a model to study the mechanism and strategy for enhancing heterologous protein production. In order to enhance cell viability and volumetric recombinant protein productivity, sorbitol, which had been confirmed to be a non-repressive carbon source, was added together with methanol during the induction phase. The resultant PGL activity was up to 1593 U mL −1, which was enhanced 1.85-fold compared to the control (863 U mL −1) cultured with sorbitol added at a constant rate of 3.6 g h −1 L −1 after an induction period of 100 h. Further results revealed that an appropriate sorbitol co-feeding strategy not only decreased the cell mortality to 8.8% (the control is about 23.1%) in the end of fermentation, but also reduced the proteolytic degradation of PGL.

Microarray analysis of gene regulation by oxygen, nitrate, nitrite, FNR, NarL and NarP during anaerobic growth of Escherichia coli: new insights into microbial physiology

RNA was isolated from cultures of Escherichia coli strain MG1655 and derivatives defective in fnr, narXL, or narXL with narP, during aerobic growth, or anaerobic growth in the presence or absence of nitrate or nitrite, in non-repressing media in which both strain MG1655 and an fnr deletion mutant grew at similar rates. Glycerol was used as the non-repressing carbon source and both trimethylamine-N-oxide and fumarate were added as terminal electron acceptors. Microarray data supplemented with bioinformatic data revealed that the FNR (fumarate and nitrate reductase regulator) regulon includes at least 104, and possibly as many as 115, operons, 68 of which are activated and 36 are repressed during anaerobic growth. A total of 51 operons were directly or indirectly activated by NarL in response to nitrate; a further 41 operons were repressed. Four subgroups of genes implicated in management of reactive nitrogen compounds, NO and products of NO metabolism, were identified; they included proteins of previously unknown function. Global repression by the nitrate- and nitrite-responsive two-component system, NarQ-NarP, was shown for the first time. In contrast with the frdABCD, aspA and ansB operons that are repressed only by NarL, the dcuB-fumB operon was among 37 operons that are repressed by NarP.

Genome-wide analysis of differentially expressed genes from Penicillium chrysogenum grown with a repressing or a non-repressing carbon source

Penicillium chrysogenum is an economically important ascomycete used as industrial producer of penicillin. However, with the exception of penicillin biosynthesis genes, little attention has been paid to the genetics of other aspects of the metabolism of this fungus. In this article we describe the first attempt of systematic analysis of expressed genes in P. chrysogenum, using a suppression subtractive hybridization approach to clone and identify sequences of genes differentially expressed in media with glucose or lactose as carbon source (penicillin-repressing or non-repressing conditions). A total of 167 clones were analysed, 95 from the glucose condition and 72 from the lactose condition. Genes differentially expressed in the glucose condition encode mainly proteins involved in the mitochondrial electron transport chain and primary metabolism. Genes expressed differentially in lactose-containing medium include genes for secondary metabolism (pcbC, isopenicillin N synthase), different hydrolases and a gene encoding a putative hexose transporter or sensor. The results provided information on how the metabolism of this fungus adapts to different carbon sources. The expression patterns of some of the genes support the hypothesis that glucose induces higher rates of respiration in P. chrysogenum while repressing secondary metabolism.

Use of different carbon sources in cultivation of recombinant Pichia pastoris for angiostatin production

To improve the growth of recombinant Pichia pastoris with a phenotype of Mut S and expression of angiostatin, the effects of glycerol, sorbitol, acetate and lactic acid which were, respectively, added together with methanol in the expression phase, were studied in a 5-l fermentor. Methanol concentration was automatically controlled at 5 g/l by a methanol monitor and control system, while the feeding of the other carbon source was manually adjusted. The angiostatin production level was 108 mg/l when glycerol was added at an initial rate of 2.3 g/h and gradually increased to 9.9 g/h within an induction period of 96 h. The angiostatin concentration was 141 mg/l as sorbitol was used, while only 52 mg/l were obtained on acetate. The highest angiostatin production of 191 mg/l was achieved as lactic acid was used; whose feeding rate was gradually increased from 2.6 to 11.3 g/h. Lactic acid accumulated during the induction phase and reached 6.3 g/l at the end of fermentation. However, the accumulation of lactic acid did not interfere with angiostatin production, indicating that lactic acid to be a non-repressive carbon source. The average productivity and specific productivity of angiostatin obtained on lactic acid and methanol were, respectively, 2.96 and 0.044 mg/(g h), 1.7- and 2.5-fold of those obtained in the fermentation fed with glycerol and methanol.

Saccharomyces cerevisiae JEN1 promoter activity is inversely related to concentration of repressing sugar.

When carbon sources are changed, Saccharomyces cerevisiae transcriptional patterns drastically change. To identify genes whose transcription can be used to quantitatively measure sugar concentrations, we searched genomic expression databases for a set of genes that are highly induced during the diauxic shift, and we used the promoters from these genes to drive expression of green fluorescent protein (GFP). Certain sugars, including glucose, fructose, and mannose, repress the promoter of JEN1, which encodes a lactate-pyruvate transporter, in a dose-dependent manner. Nonrepressing carbon sources include galactose, raffinose, ethanol, lactate, and glycerol. JEN1 promoter activity is a linear function of glucose concentration when organisms are grown at a steady-state glucose concentration below 1 g/liter. JEN1 promoter repression is specific to carbon source; heat or cold shock, osmotic stress, DNA damage, and nitrogen starvation do not significantly affect promoter activity. Activation of the JEN1 promoter requires the Snf1 protein kinase, but multiple regulatory elements most likely combine to provide the linear relationship between JEN1 promoter activity and sugar concentration. Thus, a JEN1 promoter-reporter system appears to provide a good living cell biosensor for the concentration of certain sugars. The JEN1 promoter also permits quantitative regulation of cellular functions not normally controlled by sugar concentrations. For example, a strain expressing FLO1 under control of the JEN1 promoter flocculates at a low glucose concentration.

The cyo operon of Pseudomonas putida is involved in carbon catabolite repression of phenol degradation.

A bicistronic reporter consisting of the promoterless genes aacC1 (conferring gentamycin resistance) and lacZ fused to the catabolic promoter of the phenol degradation genes was used to identify and analyse mutants of Pseudomonas putida with altered carbon catabolite repression (CR) of phenol degradation. Out of approximately 2500 mini-Tn5 mutants analysed so far, 12 mutants that were resistant to gentamycin during growth on succinate were identified. In eight of these mutants mini-Tn5 was inserted into one of the genes of the cyo operon. The cyo operon encodes the cytochrome o ubiquinol oxidase, the terminal oxidase of the cyanide-sensitive branch of the respiratory chain. In these mutants the activity of the PphlA promoter was significantly increased during growth on succinate and reached 15-20% of that found during growth with the non-repressing carbon source pyruvate. During growth on glucose the reduction of CR was less obvious, during growth on lactate CR was unchanged. The possible significance of the cyo operon for the generation of signal(s) for carbon catabolite repression is discussed.

Physiological aspects involved in production of xylanolytic enzymes by deep-sea hyperthermophilic archaeon Pyrodictium abyssi.

Xylanases (EC 3.2.1.8) catalyze the hydrolysis of xylan, the major constituent of hemicellulose. The use of these enzymes could greatly improve the overall economics of processing lignocellulosic materials for the generation of liquid fuels and chemicals. The hyperthermophilic archaeon Pyrodictium abyssi, which was originally isolated from marine hot abyssal sites, grows optimally at 97 degrees C and is a prospective source of highly thermostable xylanase. Its endoxylanase was shown to be highly thermostable (over 100 min at 105 degrees C) and active even at 110 degrees C. The growth of the deep-sea archaeon P. abyssi was investigated using different culture techniques. Among the carbohydrates used, beech wood xylan, birch wood glucuronoxylan and the arabinoxylan from oats pelt appeared to be good inducers for endoxylanase and beta-xylosidase production. The highest production of arabinofuranosidase, however, was detected in the cell extracts after growth on xylose and pyruvate, indicating that the intermediate of the tricarboxylic acid cycle acted as a nonrepressing carbon source for the production of this enzyme. Electron microscopic studies did not show a significant difference in the cell surface (e.g., xylanosomes) when P. abyssi cells were grown on different carbohydrates. The main kinetic parameters of the organism have been determined. The cell yield was shown to be very low owing to incomplete substrate utilization, but a very high maximal specific growth rate was determined (micromax = 0.0195) at 90 degrees C and pH 6.0. We also give information on the problems that arise during the fermentation of this hyperthermophilic archaeon at elevated temperatures.

Non-repressing carbon sources for alcohol oxidase ( AOX1) promoter of Pichia pastoris

The growth of Pichia pastoris in a mixture of either glycerol or glucose and methanol follows a diauxic growth, with C 1 utilizing enzymes being repressed. Therefore, these carbon sources can not be used as a mixture with methanol to simultaneously grow P. pastoris and induce C 1 utilizing enzymes, especially in a shake flask cultures of AOX-deficient P. pastoris. Among the alternative carbon sources tested, alanine, sorbitol, mannitol and trehalose, did not repress β-gal production when methanol was used as an inducer in mut − strain of P. pastoris. Our results show that either one of alanine, sorbitol, mannitol or trehalose can be used as a sole carbon and energy source for P. pastoris, although the doubling time on trehalose was very long. Mut − strains growing in media containing trehalose, alanine, sorbitol and mannitol with methanol (0.5%) as an inducing agent expressed as much or higher amount of β-gal as compared to the mut + growing in methanol containing media.

Carbon catabolite repression in Lactobacillus pentosus: analysis of the ccpA region.

The catabolite control protein CcpA is a central regulator in low-G+C-content gram-positive bacteria. It confers carbon catabolite repression to numerous genes required for carbon utilization. It also operates as a transcriptional activator of genes involved in diverse phenomena, such as glycolysis and ammonium fixation. We have cloned the ccpA region of Lactobacillus pentosus. ccpA encodes a protein of 336 amino acids exhibiting similarity to CcpA proteins of other bacteria and to proteins of the LacI/GalR family of transcriptional regulators. Upstream of ccpA was found an open reading frame with similarity to the pepQ gene, encoding a prolidase. Primer extension experiments revealed two start sites of transcription for ccpA. In wild-type cells grown on glucose, mRNA synthesis occurred only from the promoter proximal to ccpA. In a ccpA mutant strain, both promoters were used, with increased transcription from the distant promoter, which overlaps a presumptive CcpA binding site called cre (for catabolite responsive element). This suggests that expression of ccpA is autoregulated. Determination of the expression levels of CcpA in cells grown on repressing and nonrepressing carbon sources revealed that the amounts of CcpA produced did not change significantly, leading to the conclusion that the arrangement of two promoters may ensure constant expression of ccpA under various environmental conditions. A comparison of the genetic structures of ccpA regions revealed that lactic acid bacteria possess the gene order pepQ-ccpA-variable while the genetic structure in bacilli and Staphylococcus xylosus is aroA-ccpA-variable-acuC.

Sorbitol as a non-repressing carbon source for fed-batch fermentation of recombinant Pichia pastoris

Glycerol/methanol and sorbitol/methanol mixed-feed fermentation strategies for the production of recombinant proteins by Pichia pastoris were compared in order to examine sorbitol's potential as a carbon source. Although P. pastoris does have a lower cell yield on sorbitol than on glycerol, the specific rate of product formation is higher (60 μg protein g−1 dry wt ⋅ h for sorbitol/methanol, vs 45 μg protein g−1 dry wt ⋅ h for glycerol/methanol), resulting in comparable final recombinant expression levels. Importantly, the presence of residual sorbitol in the growth medium appears to be less repressive to the alcohol oxidase promoter in this organism, providing a more forgiving means of operating mixed-feed fed-batch recombinant P. pastoris fermentations.