Lactose utilization by Saccharomyces cerevisiae strains expressing Kluyveromyces lactis LAC genes.

Whey generated in cheese manufacture continues being an industrial problem without a satisfactory solution. Genetic modification of the yeast S. cerevisiae to obtain strains able to utilize lactose, is a prerequisite for the utilization of this yeast to convert cheese whey into useful fermentation products (i.e. biomass, heterologous protein and other recombinant products). Although the construction of S. cerevisiae Lac(+) strains has been achieved by different strategies, most of these strains have unsuitable characteristics, such as genetic instability of the Lac phenotype or diauxic growth. In previous communications we have described the construction of genetically stable strains of S. cerevisiae that assimilate lactose with a high efficiency. These strains carry multiple copies of Kluyveromyces lactis LAC4 and LAC12 genes, which code for a beta-galactosidase and a lactose permease, respectively. In this work we report additional results about the effect of gene dosage, and analyze the performance of a selected strain in the bioconversion of cheese whey. Additionally, we describe the construction of a new strain, which combines the Lac(+) phenotype with additional properties of biotechnological interest: flocculence, and the ability to hydrolyze starch.

The ribosomal DNA of the Zygomycete Mucor miehei.

The ribosomal DNA-from the Zygomycete Mucor miehei has been characterised. The complete rDNA unit was cloned by heterologous PCR using primers whose sequence matched conserved regions of the rDNA from related fungal species. The sequence of the overlapping PCR products revealed the existence of a repeated unit of 9574 bp. The genes encoding the different rRNA species were identified by their homology to the corresponding sequences from other fungi. We estimate that the rDNA unit is present in the genome of M. miehei in about 100 copies. This estimation was made by comparing the intensity of its hybridisation signal in a Southern blot with that of the mmp gene coding for aspartyl protease, which was assumed to be contained in single copy. The size and structure of the M. miehei rDNA unit was similar to that of other fungi. The genes encoding the 25S, 18S and 5.8S RNAs are closely linked within the repeated unit which also contains the 5S gene. This latter gene appears to be transcribed in the opposite direction. The 25S, 18S and 5.8S genes showed 70-80% homology to the corresponding genes from other fungi, whereas the degree of homology for the 5S gene was much lower. The highest homology (about 80%) corresponded to the few available sequences from other Mucor species. Homology to genes from other Zygomycota was no higher than that observed for genes from the Ascomycota or Basidiomycota fungi.

Increased thermal resistance and modification of the catalytic properties of a beta-glucosidase by random mutagenesis and in vitro recombination.

The bglB gene from Paenibacillus polymyxa was subjected to random mutagenesis mediated by error prone polymerase chain reaction amplification and DNA shuffling. After this treatment, mutant variants of the encoded beta-glucosidase with enhanced thermal resistance were selected. We identified five amino acid substitutions at four different positions of the sequence that increased the resistance of the enzyme to heat denaturation. Four of the mutations, H62R, M319V, M319I, and M361I, did not change the kinetic parameters of the enzyme. However, mutant N223Y, which caused only a marginal increase in thermoresistance, showed an 8-fold decrease in K(m). Copies of the bglB gene carrying each one of the individual mutations were recombined in vitro by DNA shuffling. As a result, we obtained an enzyme that simultaneously exhibited a 20-fold increase in heat resistance and an 8-fold increase in the catalytic efficiency. The structural basis of the properties conferred by the mutations was analyzed using homology-based structural models. The four mutations causing a more pronounced effect on thermoresistance were located in loops, on the periphery of the (alpha/beta)(8) barrel that conforms the structure of the protein. Mutation N223Y, which modifies the catalytic properties of the enzyme, was on one of the barrel beta-strands that shape the active center.

Directed evolution of beta -glucosidase A from Paenibacillus polymyxa to thermal resistance.

The beta-glucosidase encoded by the bglA gene from Paenibacillus polymyxa has a half-life time of 15 min at 35 degrees C and no detectable activity at 55 degrees C. We have isolated random mutations that enhance the thermoresistance of the enzyme. Following a directed evolution strategy, we have combined some of the isolated mutations to obtain a beta-glucosidase with a half-life of 12 min at 65 degrees C, in the range of resistance of thermophilic enzymes. No significant alteration of the kinetic parameters of the enzyme was observed. One of the mutants isolated in the screening for thermoresistant beta-glucosidase had the same resistance to denaturation as the wild type. This mutation caused the accumulation of enzyme in E. coli, probably due to its lower turnover. The structural changes responsible for the properties of the mutant enzymes have been analyzed. The putative causes increasing thermoresistance are as follows: the formation of an extra salt bridge, the replacement of an Asn residue exposed to the solvent, stabilization of the hydrophobic core, and stabilization of the quaternary structure of the protein.

Transformation of Escherichia coli with DNA from Saccharomyces cerevisiae cell lysates.

We developed a system to monitor the transfer of heterologous DNA from a genetically manipulated strain of Saccharomyces cerevisiae to Escherichia coli. This system is based on a yeast strain that carries multiple integrated copies of a pUC-derived plasmid. The bacterial sequences are maintained in the yeast genome by selectable markers for lactose utilization. Lysates of the yeast strain were used to transform E. coli. Transfer of DNA was measured by determining the number of ampicillin-resistant E. coli clones. Our results show that transmission of the Amp(r) gene to E. coli by genetic transformation, caused by DNA released from the yeast, occurs at a very low frequency (about 50 transformants per microg of DNA) under optimal conditions (a highly competent host strain and a highly efficient transformation procedure). These results suggest that under natural conditions, spontaneous transmission of chromosomal genes from genetically modified organisms is likely to be rare.

Construction of a lactose-assimilating strain of baker's yeast.

A recombinant strain of baker's yeast has been constructed which can assimilate lactose efficiently. This strain has been designed to allow its propagation in whey, the byproduct resulting from cheese-making. The ability to metabolize lactose is conferred by the functional expression of two genes from Kluyveromyces lactis, LAC12 and LAC4, which encode a lactose permease and a beta-galactosidase, respectively. To make the recombinant strain more acceptable for its use in bread-making, the genetic transformation of the host baker's yeast was carried out with linear fragments of DNA of defined sequence, carrying as the only heterologous material the coding regions of the two K. lactis genes. Growth of the new strain on cheese whey affected neither the quality of bread nor the yeast gassing power. The significance of the newly developed strain is two-fold: it affords a cheap alternative to the procedure generally used for the propagation of baker's yeast, and it offers a profitable use for cheese whey.

Structural basis of increased resistance to thermal denaturation induced by single amino acid substitution in the sequence of beta-glucosidase A from Bacillus polymyxa.

The increasing development of the biotechnology industry demands the design of enzymes suitable to be used in conditions that often require broad resistance against adverse conditions. beta-glucosidase A from Bacillus polymyxa is an interesting model for studies of protein engineering. This is a well-characterized enzyme, belonging to glycosyl hydrolase family 1. Its natural substrate is cellobiose, but is also active against various artificial substrates. In its native state has an octameric structure. Its subunit conserves the general (alpha/beta)8 barrel topology of its family, with the active site being in a cavity defined along the axis of the barrel. Using random-mutagenesis, we have identified several mutations enhancing its stability and it was found that one them, the E96K substitution, involved structural changes. The crystal structure of this mutant has been determined by X-ray diffraction and compared with the native structure. The only difference founded between both structures is a new ion pair linking Lys96 introduced at the N-terminus of helix alpha2, to Asp28, located in one of the loops surrounding the active-site cavity. The new ion pair binds two segments of the chain that are distant in sequence and, therefore, this favorable interaction must exert a determinant influence in stabilizing the tertiary structure. Furthermore, analysis of the crystallographic isotropic temperature factors reveals that, as a direct consequence of the introduced ion pair, an unexpected decreased mobility of secondary structure units of the barrel which are proximal to the site of mutation is observed. However, this effect is observed only in the surrounding of one of the partners forming the salt bridge and not around the other. These results show that far-reaching effects can be achieved by a single amino acid replacement within the protein structure. Consequently, the identification and combination of a few single substitutions affecting stability may be sufficient to obtain a highly resistant enzyme, suitable to be used under extreme conditions.

Highly efficient assimilation of lactose by a metabolically engineered strain of Saccharomyces cerevisiae.

A diploid strain of Saccharomyces cerevisiae able to metabolize lactose with high efficiency has been obtained. Haploid strains of Saccharomyces able to grow on lactose were constructed by cotransformation with two genes of Kluyveromyces lactis required for the utilization of the sugar, LAC4 and LAC12, encoding beta-galactosidase and lactose permease respectively. Both genes were placed under the control of a galactose-inducible promoter and targeted to the rDNA encoding region (RDN1 locus) of the Saccharomyces genome. Lac+ transformants were selected on medium with lactose as the only carbon source. These transformants were mitotically stable, they maintained the Lac+ phenotype after growing in non-selective medium for more than 60 generations, but their growth was slow. We found that this lack of vigour was caused by their genetic background and not by a deficient expression of the heterologous genes. Therefore, their performance could be improved by crossing with a wild-type strain. Among the offspring of the crosses, two strains of opposite mating type were selected and mated to obtain a fast-growing Lac+ diploid. This diploid strain showed the typical fermentative behaviour of S. cerevisiae when it was grown in aerated liquid medium with glucose. In lactose medium, it exhibited a respiro-fermentative metabolism similar to that of K. lactis, with low ethanol production and high biomass yield.

Crystal structure of beta-glucosidase A from Bacillus polymyxa: insights into the catalytic activity in family 1 glycosyl hydrolases.

Family 1 glycosyl hydrolases are a very relevant group of enzymes because of the diversity of biological roles in which they are involved, and their generalized occurrence in all sorts of living organisms. The biological plasticity of these enzymes is a consequence of the variety of beta-glycosidic substrates that they can hydrolyze: disaccharides such as cellobiose and lactose, phosphorylated disaccharides, cyanogenic glycosides, etc. The crystal structure of BglA, a member of the family, has been determined in the native state and complexed with gluconate ligand, at 2.4 A and 2.3 A resolution, respectively. The subunits of the octameric enzyme display the (alpha/beta)8 barrel structural fold previously reported for other family 1 enzymes. However, significant structural differences have been encountered in the loops surrounding the active-center cavity. These differences make a wide and extended cavity in BglA, which seems to be able to accommodate substrates longer than cellobiose, its natural substrate. Furthermore, a third sub-site is encountered, which might have some connection with the transglycosylating activity associated to this enzyme and its certain activity against beta-1,4 oligosaccharides composed of more than two units of glucose. The particular geometry of the cavity which contains the active center of BglA must therefore account for both, hydrolytic and transglycosylating activities. A potent and well known inhibitor of different glycosidases, D-glucono-1,5-lactone, was used in an attempt to define interactions of the substrate with specific protein residues. Although the lactone has transformed into gluconate under crystallizing conditions, the open species still binds the enzyme, the conformation of its chain mimicking the true inhibitor. From the analysis of the enzyme-ligand hydrogen bonding interactions, a detailed picture of the active center can be drawn, for a family 1 enzyme. In this way, Gln20, His121, Tyr296, Glu405 and Trp406 are identified as determinant residues in the recognition of the substrate. In particular, two bidentate hydrogen bonds made by Gln20 and Glu405, could conform the structural explanation for the ability of most members of the family for displaying both, glucosidase and galactosidase activity.

Amino acid substitutions enhancing thermostability of Bacillus polymyxa beta-glucosidase A.

Mutations enhancing the thermostability of beta-glucosidase A of Bacillus polymyxa, a family 1 glycosyl hydrolase, have been obtained after hydroxylamine mutagenesis of a plasmid containing the bglA gene, transformation of Escherichia coli with the mutagenized plasmid, and identification of transformant colonies that showed beta-glucosidase activity after a thermal treatment that inactivated the wild-type enzyme. Two additive mutations have been characterized that cause replacement of glutamate at position 96 by lysine and of methionine at position 416 by isoleucine respectively. The thermoresistant mutant enzymes showed increased resistance to other denaturing agents, such as pH and urea, while their kinetic parameters did not change. CD spectra indicated that the E96K replacement caused an increase in alpha-helix content. The observed effect of the M416I mutation is consistent with the lower content of cysteine and methionine found in family 1 enzymes of thermophilic species compared with similar ones from mesophilic organisms.

Induced expression of bacterial beta-glucosidase activity in Saccharomyces.

The bglA gene which encodes a beta-glucosidase from Bacillus polymyxa, has been expressed in Saccharomyces cerevisiae under control of the yeast CYC-GAL promoter. Strains have been constructed which carry the gene in different locations: in a multicopy plasmid, a single integration at the URA3 locus, or multiple integrations at the RDN1 locus. Integrative transformation at RDN1 yielded genetically stable clones with a high level of beta-glucosidase activity. Coordinated overexpression of the GAL4 inducer protein further increased the level of enzyme activity, although eventually caused the lysis of the cultures. Diploid, triploid and tetraploid strains derived from the transformants with multiple integrations were constructed and expression of beta-glucosidase activity in different conditions of growth was assayed. While per-cell activity increased with ploidy, specific activity was about the same in strains of equivalent genotype regardless of ploidy. Genetically stable and regulated expression in Saccharomyces of beta-glucosidase activity is interesting for the development of strains able to ferment beta-glycosidic sugars (i.e. cellobiose and lactose). From another point of view, the bglA product proved to be a convenient reporter enzyme to monitor heterologous gene expression.

Crystallization and preliminary X-ray diffraction analysis of a type I beta-glucosidase encoded by the bgIA gene of Bacillus polymyxa.

The enzyme encoded by the bgIA gene of Bacillus polymyxa, a type I beta-glucosidase belonging to family I of glycosyl hydrolases, has been purified to homogeneity from an Escherichia coli culture which overexpressed the gene, and crystallized. The crystals, which diffract to 3.0 A resolution, belong to the orthorhombic space group C222(1). The cell dimensions are a = 155.4 A, b = 209.4 A, c = 209.7 A.

Self-diploidization in Saccharomyces cerevisiae kar2 heterokaryons.

Zygotes isolated by micromanipulation from crosses of Saccharomyces cerevisiae strains, one of which carries a kar mutation, give rise most frequently to cytoductant colonies showing the nuclear constitution of either one of the two haploid parental strains. In crosses of kar2-1 strains to wild-type, about 10% of the cytoductants of both mating types are homozygous autodiploids. There is evidence indicating that self-diploidization occurs by fusion between sibling nuclei in the heterokaryotic zygote. Here we describe this phenomenon and propose to take advantage of it for the construction of genotypically-defined diploids able to mate, and of polyploid strains, which are useful tools in genetic and cytological studies.

Random mutagenesis of a plasmid-borne glycosidase gene and phenotypic selection of mutants in Escherichia coli.

The bglA gene from Bacillus polymyxa encodes a beta-glucosidase able to hydrolyze p-nitrophenyl-beta-D-glucopyranoside (PNPG), and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal), chromogenic substrates for beta-glucosidases and beta-galactosidases respectively. A plasmid carrying the blgA gene inserted in vector pUC18 was mutagenized in vitro with hydroxylamine, and subsequently used to transform E. coli selecting for the ampicillin resistance conferred by the cloning vector. The transformants were screened on petri dishes for mutations causing inability to hydrolyze either one of the two substrates, and for mutations increasing resistance of the enzyme to thermal inactivation. The isolation of several mutants with such characteristics suggests that the simple procedure used here can be applied to generate modifications of enzymatic properties that fit specific industrial requirements.

Purification and characterization of a Bacillus polymyxa beta-glucosidase expressed in Escherichia coli.

The beta-glucosidase encoded by the bglA gene from Bacillus polymyxa was overproduced in Escherichia coli by using a plasmid in which bglA is under control of the lacI promoter. Induction with isopropyl-beta-D-thiogalactopyranoside allowed an increase in the specific activity of the enzyme of about 100 times the basal level of gene expression. The enzyme was purified by a two-step procedure involving salting out with ammonium sulfate and ion-exchange chromatography with DEAE-cellulose. Fractions of beta-glucosidase activity recovered by this procedure, after electrophoresis in an acrylamide gel and staining with silver nitrate, yielded a single band of protein. This band was shown by a zymogram to correspond to beta-glucosidase activity. The purified protein showed an apparent molecular mass of 50 kDa and an isoelectric point of 4.6, values in agreement with those expected from the nucleotide sequence of the gene. Km values of the enzyme, with either cellobiose or p-nitrophenyl-beta-D-glucoside as the substrate, were determined. It was shown that the enzyme is competitively inhibited by glucose. The effects of different metallic ions and other agents were studied. Hg2+ was strongly inhibitory, while none of the other cations tested had any significant effect. Ethanol did not show the stimulating effect observed with other beta-glucosidases. The mechanism of enzyme action was investigated. High-pressure liquid chromatography analysis with cellobiose as the substrate confirmed previous data revealing the formation of two products, glucose and another, unidentified, compound. Results presented here indicate that this compound is cellotriose formed by transglycosylation.

Construction of a Saccharomyces cerevisiae strain able to ferment cellobiose.

The bglA gene, encoding a beta-glucosidase from Bacillus polymyxa, has been expressed in Saccharomyces cerevisiae under control of the CYC-GAL promoter inducible by galactose. The expression of bglA-encoded activity in the strain used as a host was not sufficient to allow its growth with cellobiose as a carbon source. However, a recessive mutation in a gene designated cem1 has been obtained which, combined with the expression of beta-glucosidase activity, allows the growth of S. cerevisiae on cellobiose. The expression of the blgA gene in a cem1 strain confers on S. cerevisiae the capability for an efficient fermentation of cellobiose, as detected by the formation of CO2.

Sequences and homology analysis of two genes encoding beta-glucosidases from Bacillus polymyxa.

The nucleotide sequences of the bglA and bglB genes encoding beta-glucosidases from Bacillus polymyxa have been determined. Both genes contain coding regions of 1344 bp, corresponding to polypeptides with Mrs of 51,643 and 51,547, respectively. Patterns of codon usage indicate that both genes are expressed at a low frequency. Previous data suggested that the proteins encoded by bglA and bglB were intra- and extracellular enzymes, respectively; however, neither of the two deduced amino acid sequences has N termini with the typical features of a leader peptide. The proteins encoded by bglA and bglB show remarkable homology to each other and to other beta-glucosidases (Bgl) and beta-galactosidases (beta Gal). On the basis of the observed homologies, we can define two groups of microbial Bgl: one of them, type I, including most bacterial Bgl, and type II, including enzymes from different yeast species and one from Clostridium thermocellum. Likewise, at least two groups of beta Gal can be distinguished: type I, including enzymes homologous to type-I Bgl, and type II, showing no homology to any of the previous groups.

Cloning and characterization of two genes from Bacillus polymyxa expressing beta-glucosidase activity in Escherichia coli.

DNA fragments from Bacillus polymyxa which encode beta-glucosidase activity were cloned in Escherichia coli by selection of yellow transformants able to hydrolyze the artificial chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside. Restriction endonuclease maps and Southern analysis of the cloned fragments showed the existence of two different genes. Expression of either one of these genes allowed growth of E. coli in minimal medium with cellobiose as the only carbon source. One of the two enzymes was found in the periplasm of E. coli, hydrolyzed arylglucosides more actively than cellobiose, and rendered glucose as the only product upon cellobiose hydrolysis. The other enzyme was located in the cytoplasm, was more active toward cellobiose, and hydrolyzed this disaccharide, yielding glucose and another, unidentified compound, probably a phosphorylated sugar.

Cloning of the STA2 and SGA genes encoding glucoamylases in yeasts and regulation of their expression by the STA10 gene of Saccharomyces cerevisiae.

The Saccharomyces STA2 and SGA genes, encoding the extracellular and intracellular sporulation-specific glucoamylase respectively, have been cloned and their transcription and regulation studied. The STA2 gene differs from the SGA gene in that it contains an extra piece of DNA, which encodes the domain for exportation of the extracellular glucoamylase. The STA2 gene produces a single 2.85 kb transcript. Transcription of the SGA gene is initiated from two different sites, yielding two transcripts of 1.95 and 2.40 kb. Transcription of both STA2 and SGA genes is repressed by the STA10 gene of Saccharomyces cerevisiae.