Bacterial cellulose: Strategies for its production in the context of bioeconomy.

Bacterial cellulose has advantages over plant-derived cellulose, which make its use for industrial applications easier and more profitable. Its intrinsic properties have been stimulating the global biopolymer market, with strong growth expectations in the coming years. Several bacterial species are capable of producing bacterial cellulose under different culture conditions; in this context, strategies aimed at metabolic engineering and several possibilities of carbon sources have provided opportunities for the bacterial cellulose's biotechnological exploration. In this article, an overview of biosynthesis pathways in different carbon sources for the main producing microorganisms, metabolic flux under different growth conditions, and their influence on the structural and functional characteristics of bacterial cellulose is provided. In addition, the main industrial applications and ways to reduce costs and optimize its production using alternative sources are discussed, contributing to new insights on the exploitation of this biomaterial in the context of the bioeconomy.

Influence of glucose on xylose metabolization by Spathaspora passalidarum.

The yeast Spathaspora passalidarum is able to produce ethanol from D-xylose and D-glucose. However, it is not clear how xylose metabolism is affected by D-glucose when both sugars are available in the culture medium. The aims of this work were to evaluate the influence of D-glucose on D-xylose consumption, ethanol production, gene expression, and the activity of key xylose-metabolism enzymes under both aerobic and oxygen-limited conditions. Ethanol yields and productivities were increased in culture media containing D-xylose as the sole carbon source or a mixture of D-xylose and D-glucose. S. passalidarum preferentially consumed D-glucose in the co-fermentations, which is consistent with the reduction in expression of genes encoding the key xylose-metabolism enzymes. In the presence of D-glucose, the specific activities of xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK) were lower. Interestingly, in accordance with other studies, the presence of 2-deoxyglucose (2DG) did not inhibit the growth of S. passalidarum in culture medium containing D-xylose as the sole carbon source. This indicates that a non-canonical repression pathway is acting in S. passalidarum. In conclusion, the results suggest that D-glucose inhibits D-xylose consumption and prevents the D-xylose-mediated induction of the genes encoding XR, XDH, and XK.

Sugar transport systems in Kluyveromyces marxianus CCT 7735.

The pattern of glucose repression in most Kluyveromyces marxianus strains does not correlate with fermentative behaviour; however, glucose repression and fermentative metabolism appear to be linked to the kinetics of sugar uptake. In this work, we show that lactose transport in K. marxianus CCT 7735 by lactose-grown cells is mediated by a low-affinity H+-sugar symporter. This system is glucose repressed and able to transport galactose with low affinity. We also observed the activity of a distinct lactose transporter in response to raffinose. Regarding glucose uptake, specificities of at least three low-affinity systems rely on the carbon source available in a given growth medium. Interestingly, it was observed only one high-affinity system is able to transport both glucose and galactose. We also showed that K. marxianus CCT 7735 regulates the expression of sugar transport systems in response to glucose availability.

Ploidy Variation in Kluyveromyces marxianus Separates Dairy and Non-dairy Isolates.

Kluyveromyces marxianus is traditionally associated with fermented dairy products, but can also be isolated from diverse non-dairy environments. Because of thermotolerance, rapid growth and other traits, many different strains are being developed for food and industrial applications but there is, as yet, little understanding of the genetic diversity or population genetics of this species. K. marxianus shows a high level of phenotypic variation but the only phenotype that has been clearly linked to a genetic polymorphism is lactose utilisation, which is controlled by variation in the LAC12 gene. The genomes of several strains have been sequenced in recent years and, in this study, we sequenced a further nine strains from different origins. Analysis of the Single Nucleotide Polymorphisms (SNPs) in 14 strains was carried out to examine genome structure and genetic diversity. SNP diversity in K. marxianus is relatively high, with up to 3% DNA sequence divergence between alleles. It was found that the isolates include haploid, diploid, and triploid strains, as shown by both SNP analysis and flow cytometry. Diploids and triploids contain long genomic tracts showing loss of heterozygosity (LOH). All six isolates from dairy environments were diploid or triploid, whereas 6 out 7 isolates from non-dairy environment were haploid. This also correlated with the presence of functional LAC12 alleles only in dairy haplotypes. The diploids were hybrids between a non-dairy and a dairy haplotype, whereas triploids included three copies of a dairy haplotype.

Genomic Sequence of the Yeast Kluyveromyces marxianus CCT 7735 (UFV-3), a Highly Lactose-Fermenting Yeast Isolated from the Brazilian Dairy Industry.

Here, we present the draft genome sequence of Kluyveromyces marxianus CCT 7735 (UFV-3), including the eight chromosomes and the mitochondrial genomic sequences.

Physiological characterization of thermotolerant yeast for cellulosic ethanol production.

The conversion of lignocellulose into fermentable sugars is considered a promising alternative for increasing ethanol production. Higher fermentation yield has been achieved through the process of simultaneous saccharification and fermentation (SSF). In this study, a comparison was performed between the yeast species Saccharomyces cerevisiae and Kluyveromyces marxianus for their potential use in SSF process. Three strains of S. cerevisiae were evaluated: two are widely used in the Brazilian ethanol industry (CAT-1 and PE-2), and one has been isolated based on its capacity to grow and ferment at 42 °C (LBM-1). In addition, we used thermotolerant strains of K. marxianus. Two strains were obtained from biological collections, ATCC 8554 and CCT 4086, and one strain was isolated based on its fermentative capacity (UFV-3). SSF experiments revealed that S. cerevisiae industrial strains (CAT-1 and PE-2) have the potential to produce cellulosic ethanol once ethanol had presented yields similar to yields from thermotolerant strains. The industrial strains are more tolerant to ethanol and had already been adapted to industrial conditions. Moreover, the study shows that although the K. marxianus strains have fermentative capacities similar to strains of S. cerevisiae, they have low tolerance to ethanol. This characteristic is an important target for enhancing the performance of this yeast in ethanol production.

Production and characterization of ß-glucanase secreted by the yeast Kluyveromyces marxianus.

An extracellular ß-glucanase secreted by Kluyveromyces marxianus was identified for the first time. The optimal conditions for the production of this enzyme were evaluated by response surface methodology. The optimal conditions to produce ß-glucanase were a glucose concentration of 4% (w/v), a pH of 5.5, and an incubation temperature of 35 °C. Response surface methodology was also used to determine the pH and temperature required for the optimal enzymatic activity. The highest enzyme activity was obtained at a pH of 5.5 and a temperature of 55 °C. Furthermore, the enzyme was partially purified and sequenced, and its specificity for different substrates was evaluated. The results suggest that the enzyme is an endo-ß-1,3(4)-glucanase. After optimizing the conditions for ß-glucanase production, the culture supernatant was found to be effective in digesting the cell wall of the yeast Saccharomyces cerevisiae, showing the great potential of ß-glucanase in the biotechnological production of soluble ß-glucan.

Construction of a Kluyveromyces lactis ku80⁻ host strain for recombinant protein production: extracellular secretion of pectin lyase and a streptavidin-pectin lyase chimera.

In several organisms used for recombinant protein production, integration of the expression cassette into the genome depends on site-specific recombination. In general, the yeast Kluyveromyces lactis shows low gene-targeting efficiency. In this work, two K. lactis ku80⁻ strains defective in the non-homologous end-joining pathway (NHEJ) were constructed using a split-marker strategy and tested as hosts for heterologous gene expression. The NHEJ pathway mediates random integration of exogenous DNA into the genome, and its function depends on the KU80 gene. KU80-defective mutants were constructed using a split-marker strategy. The vectors pKLAC1/Plg1 and pKLAC1/cStpPlg1 were used to evaluate the recovered mutants as hosts for expression of pectin lyase (PNL) and the fusion protein streptavidin-PNL, respectively. The transformation efficiency of the ku80⁻ mutants was higher than the respective parental strains (HP108 and JA6). In addition, PNL secretion was detected by PNL assay in both of the K. lactis ku80⁻ strains. In HP108ku80⁻/cStpPlg1 and JA6ku80⁻/Plg1 cultures, the PNL extracellular specific activity was 551.48 (±38.66) and 369.04 (±66.33) U/mg protein. This study shows that disruption of the KU80 gene is an effective strategy to increase the efficiency of homologous recombination with pKLAC1 vectors and the production and secretion of recombinant proteins in K. lactis transformants.

Cloning and expression of a functional core streptavidin in Pichia pastoris: strategies to increase yield.

Streptavidin is widely used as an analytical tool and affinity tag together with biotinylated surfaces or molecules. We report for the first time a simple strategy that yields high biomass of a Pichia pastoris strain containing a methanol induced core streptavidin (cStp) gene. Three factors were evaluated for biomass production: glycerol concentration, aeration, and feed flow rates in a bioreactor. Recycling of recombinant cells, either free or immobilized, was investigated during induction. Concentration of 2.0 M glycerol, feeding flow rate of 0.11 mL min(-1) , and aeration by air injection dispersed with a porous stone combined with agitation at 500 rpm were the set of conditions resulting into maximum biomass yield (150 g L(-1) ). These parameters yielded 4.0 g L(-1) of cStp, after 96 h of induction. Recombinant biomass was recycled twice before being discarded, which can reduce production costs and simplify the process. Immobilized P. pastoris biomass produced 2.94 and 1.70 g L(-1) of cStp in the first and second induction cycle, respectively. Immobilization and recycling of recombinant P. pastoris biomass opens new possibilities as a potential strategy to improve volumetric productivity for heterologous protein expression.

The high fermentative metabolism of Kluyveromyces marxianus UFV-3 relies on the increased expression of key lactose metabolic enzymes.

The aim of this work was to obtain insights about the factors that determine the lactose fermentative metabolism of Kluyveromyces marxianus UFV-3. K. marxianus UFV-3 and Kluyveromyces lactis JA6 were cultured in a minimal medium containing different lactose concentrations (ranging from 0.25 to 64 mmol l(-1)) under aerobic and hypoxic conditions to evaluate their growth kinetics, gene expression and enzymatic activity. The increase in lactose concentration and the decrease in oxygen level favoured ethanol yield for both yeasts but in K. marxianus UFV-3 the effect was more pronounced. Under hypoxic conditions, the activities of ß-galactosidase and pyruvate decarboxylase from K. marxianus UFV-3 were significantly higher than those in K. lactis JA6. The expression of the LAC4 (ß-galactosidase), RAG6 (pyruvate decarboxylase), GAL7 (galactose-1-phosphate uridylyltransferase) and GAL10 (epimerase) genes in K. marxianus UFV-3 was higher under hypoxic conditions than under aerobic conditions. The high expression of genes of the Leloir pathway, LAC4 and RAG6, associated with the high activity of ß-galactosidase and pyruvate decarboxylase contribute to the high fermentative flux in K. marxianus UFV-3. These data on the fermentative metabolism of K. marxianus UFV-3 will be useful for optimising the conversion of cheese whey lactose to ethanol.

Restricted sugar uptake by sugar-induced internalization of the yeast lactose/galactose permease Lac12.

Kluyveromyces lactis Lac12 permease mediates lactose and low-affinity galactose transports. In this study we investigated the effects of carbon sources on internalization of Lac12 using a LAC12-GFP fusion construct. When galactose- or lactose-grown cells are shifted to a fresh sugar medium, Lac12-GFP is removed from the plasma membrane and is localized intracellularly. Surprisingly, either galactose or lactose in the new media caused the internalization, and cells responded differently to these two sugars. Our results reveal that this process is dependent on sugar species and also sugar concentration. Lac12-GFP internalization causes reduction of [C(14) ]lactose uptake rates and also occurs in a Klsnf1 mutant strain; it is therefore independent of KlSnf1 activity. We suggest that glucose-6-phosphate is the intracellular signal, as internalization was induced by 2-deoxyglucose, and inhibition of phosphoglucomutase by lithium prevented galactose- but not lactose- or glucose-induced internalization. Lac12-GFP internalization was not triggered by 6-deoxyglucose, and was irreversible in the absence of protein synthesis.

Association of a CB1 cannabinoid receptor gene (CNR1) polymorphism with severe alcohol dependence.

Due to the involvement of the endogenous cannabinoid system in brain reward mechanisms a silent polymorphism (1359G/A; Thr453Thr) in the single coding exon of the CB1 human cannabinoid receptor gene (CNR1) was analysed in 121 severely affected Caucasian alcoholics and 136 most likely non-alcoholic controls. The observed frequency of the A allele was 31.2% for controls and 42.1% for alcoholics with severe withdrawal syndromes (P=0.010). Post-hoc exploration indicated that this allelic association resulted from an excess of the homozygous A/A genotype in patients with a history of alcohol delirium (P=0.031, DF 2), suggesting s an increased risk of delirium (OR=2.45, 95% CI 1.14--5.25). This finding suggests that the homozygous genotype CNR1 1359A/A confers vulnerability to alcohol withdrawal delirium.

Sequence variability and candidate gene analysis in complex disease: association of mu opioid receptor gene variation with substance dependence.

To analyze candidate genes and establish complex genotype-phenotype relationships against a background of high natural genome sequence variability, we have developed approaches to (i) compare candidate gene sequence information in multiple individuals; (ii) predict haplotypes from numerous variants; and (iii) classify haplotypes and identify specific sequence variants, or combinations of variants (pattern), associated with the phenotype. Using the human mu opioid receptor gene (OPRM1) as a model system, we have combined these approaches to test a potential role of OPRM1 in substance (heroin/cocaine) dependence. All known functionally relevant regions of this prime candidate gene were analyzed by multiplex sequence comparison in 250 cases and controls; 43 variants were identified and 52 different haplotypes predicted in the subgroup of 172 African-Americans. These haplotypes were classified by similarity clustering into two functionally related categories, one of which was significantly more frequent in substance-dependent individuals. Common to this category was a characteristic pattern of sequence variants [-1793T-->A, -1699Tins, -1320A-->G, -111C-->T, +17C-->T (A6V)], which was associated with substance dependence. This study provides an example of approaches that have been successfully applied to the establishment of complex genotype-phenotype relationships in the presence of abundant DNA sequence variation.

The human beta-myosin heavy chain gene: sequence diversity and functional characteristics of the protein.

The beta-myosin heavy chain gene (MYH7) encodes the motor protein that drives myocardial contraction. It has been proven to be a disease gene for hypertrophic cardiomyopathy (HCM). We analyzed the DNA sequence variation of MYH7 (about 16 kb) of eight individuals: six patients with HCM and two healthy controls. The overall DNA sequence identity was up to 97.2% compared to Jaenicke and coworkers (Jaenicke et al. [1990] Genomics 8:194-206), while the corresponding amino acid sequences revealed 100% identity. In HCM patients, eleven nucleotide substitutions were identified but no causative disease mutation was found: six were detected in coding, four in intronic, and one in 5' regulatory regions. The average nucleotide diversity across this locus was 0.015% with an average of 0.02% in the coding and 0.012% in the noncoding sequence. Analysis of the kinetic behaviour of beta-MHC in the intact contractile structure of normal individuals and HCM patients revealed apparent rate constants of tension development ranging between 1.58 s(-1) and 1.48 s(-1).

Five exon 1 variants of mu opioid receptor and vulnerability to alcohol dependence.

The human mu opioid receptor (hMOR) gene is a prime candidate gene responsible for addictive disorders. The present association study tested the hypothesis that hMOR exon 1 variants elicit susceptibility to alcohol dependence. We have analyzed five nucleotide changes in exon 1 of the hMOR gene. Three of them are in the 5'untranslated region of exon 1 at positions -172G/T,-111C/T and -3 8C/A, the remaining two variants cause amino acid substitutions: +17C/T (Ala6Val) and +118A/G (Asn40Asp). Our population-based association study included 327 German alcohol-dependent subjects and 340 ethnically matched controls. The lack of an allelic association suggests that the analyzed hMOR exon 1 variants do not contribute a common and substantial effect to the genetically determined vulnerability of alcohol dependence.

Genetic variation of the human mu-opioid receptor and susceptibility to idiopathic absence epilepsy.

Pharmacological and autoradiological studies suggest that mu-opioid receptor (OPRM) mediated neurotransmission is involved in the generation of absence seizures. Mutation screening of the human OPRM gene identified a common amino acid substitution polymorphism (Asn40Asp) that differentially modulates the binding affinity of beta-endorphin and signal transduction of the receptor. The present association study tested the candidate gene hypothesis that the Asn40Asp substitution polymorphism in the N-terminal OPRM domain confers genetic susceptibility to idiopathic absence epilepsy (IAE). The genotypes of the Asn40Asp polymorphism were assessed by allele-specific polymerase chain reaction in 72 German IAE patients and in 340 ethnically matched control subjects. The frequency of the Asp40 allele was significantly increased in the IAE patients [f(Asp40) = 0.139] compared to the controls [f(Asp40) = 0.078; chi2 = 5.467, df = 1, P = 0.019; OR = 2.03; 95%-CI: 1.12-3.68]. This allelic association suggests that the functional Asp40 variant of OPRM modulates neuronal excitability underlying the epileptogenesis of IAE.

mu-opioid receptor variants and dopaminergic sensitivity in alcohol withdrawal.

OBJECT: The endogenous opioid system plays an important role in the reinforcing properties of alcohol by an interconnected activation of the mesolimbic dopamine system. The Asn40Asp substitution polymorphism of the human mu-opioid-receptor (OPRM) influences binding of opioids and signal transduction and may, thereby, contribute to the development of alcoholism. The present study tested whether the Asn40Asp substitution polymorphism of the OPRM gene is associated with a variation in central dopaminergic sensitivity during alcohol withdrawal in alcoholics. METHOD: Sensitivity of central dopamine receptors was assessed by apomorphine-induced growth hormone (GH) secretion in 97 alcohol-dependent patients before and 1 week after alcohol cessation, and in a subgroup of 19 alcoholics after 3 months of abstinence. GH response was defined as area under the hormone/time curve. Comparisons of the GH response were conducted between alcoholics carrying the Asn40Asp genotype versus those with the Asn40Asn genotype using U-test statistics. RESULTS: Marginal differences in apomorphine-induced GH response were found between both genotype groups before detoxification (P = 0.799 (n = 97)/P = 0.459 (n = 19)) and after 3 months of abstinence (P = 0.331 (n = 19)). In contrast, the GH response measured seven days after alcohol withdrawal was significantly increased in alcoholics with the Asn40Asp genotype compared with those carrying the Asn40Asn genotype (P = 0.013 (n = 97)/P = 0.026 (n = 19)). CONCLUSION: Our results suggest that genetic variation of the mu-opioid receptor modulates the central dopaminergic sensitivity during acute alcohol withdrawal.

The human mu opioid receptor gene: 5' regulatory and intronic sequences.

The human mu opioid receptor (hMOR) interacts with endogenous and exogenous ligands to mediate its characteristic effects, reward, dependence, and analgesia. Specifically binding morphine, it represents the target of the most valuable pain killer in contemporary medicine. Analysis of its structure, regulation, and expression will elucidate molecular processes involved in opioid/morphine-induced actions. Thus we have contributed significant information on the genomic organization of hMOR, extending the previously known cDNA sequence information (2162 bp) up to a total of 6968 bp: we have determined 2412 bp of 5' regulatory region, identified one major and three minor transcription initiation sites 216, 285, 358, and 373 bp upstream from the translation start codon, as well as potential binding sites for transcriptional regulatory factors, including putative cis-acting enhancer motifs for a glucocorticoid response element, cAMP response elements, activator proteins 1, and Yin Yang-1 boxes. Moreover, we have analyzed the 5' and 3' nucleotide sequences of introns 1 and 3 and the complete sequence of intron 2. In addition to the classical consensus sequences involved in RNA splicing, we have identified intronic repeats (A/T GGG) found to regulate alternative splicing, mutations of which cause human disease. A similar genetic variant is observed in the hMOR gene. Taken together, the sequence information presented will allow comprehensive analysis of this gene for allelic variations associated with vulnerability to drug abuse or individual differences in opiate mediated analgesia.

Serotonin transporter (5-HTT) gene polymorphisms are not associated with susceptibility to mood disorders.

In a population-based association study, we tested the hypothesis that allelic variants of the human serotonin transporter (5-HTT) gene confer susceptibility to mood disorders. Both a biallelic repeat polymorphism in the 5' promotor region that differentially modulates gene expression and a second intron variable-number-tandem-repeat (VNTR) marker were genotyped in 294 controls and 115 patients with mood disorders. Subjects were of West European descent and included 36 patients with major depressive disorder (MDD) and 79 patients with bipolar I disorder (BD). No significant differences in genotype or allele frequencies were found at either locus between controls and combined patients, nor between controls and MDD or BD patients separately. Thus, our data do not support the association between depressive disorder and a nine-repeat allelic variant of the 5-HTT VNTR marker recently reported by Ogilvie et al. (Lancet 347:731-733, 1996). More importantly, no association between alleles conveying functional differences in 5-HTT gene expression and MDD or BD could be found. Taken together, our data suggest that the 5-HTT gene is not commonly involved in the susceptibility to mood disorders.