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Our aim is to merge and optimize the main features of naturally occurring lactose-cleaving and ethanol-producing microorganisms to build up an engineered biological system that can metabolize lactose and can ferment it in ethanol with high yield. Here we provide an overview of the main enzymes and biochemical pathways involved in these two processes.

MICROBIAL SOURCES OF LACTASE

The complete lactose to ethanol transformation pathway is summarized in the following figure.

Lactose to ethanol transformation pathway

The first task of conversion is provided by β-D-Galactosidase. It can be obtained from a wide variety of sources such as microorganisms, plants and animals; however, according to its source, its properties differ markedly.

β-galactosidase

The products of lactose hydrolysis may inhibit β-galactosidase action. Galactose is often a competitive inhibitor but glucose is usually ineffective, except at higher concentrations when it is usually non-competitive. Complete hydrolysis is therefore very difficult to achieve unless high concentrations of the enzyme are used. Yeast enzymes are generally inhibited by galactose (competitively) and glucose (non-competitively). Aspergillus niger enzyme is strongly inhibited by galactose; however, enzyme from A. oryzae is less subject to galactose inhibition.

Fungal enzymes

These enzymes have a pH optimum in the acidic range 2.5–5.4, which makes them suitable for processing of acid whey and its ultrafiltration permeate. These have a relatively high temperature optimum and are typically used at temperatures up to 50°C, where they are reasonably stable. The production and purification of β-D-galactosidase from different fungal sources have been carried out using a variety of purification techniques. Fungal sources are: Aspergillus niger, Beauveria bassiana, Aspergillus fonsecaeus, Rhizomucor sp., Penicillium chrysogenum, Aspergillus carbonarius.

Yeast enzymes

Yeast has been considered an important source of β-D-galactosidase from an industrial viewpoint. Yeast sources are: Kluyveromyces fragilis, Kluyveromyces lactis, Saccharomyces lactis, Saccharomyces anamensis, Kluyveromyces marxianus, recombinant yeast Saccharomyces cerevisiae.

Bacterial enzymes

A large number of bacteria produce β-D-galactosidase, but relatively few bacterial species are regarded as safe sources. However, Streptococcus thermophilus and Bacillus stearothermophilus can be considered as potential bacterial sources. The former is eminently suitable as a source organism from the safety viewpoint because it is used as a starter culture in yoghurt and some cheeses. Other bacterial sources are: Escherichia coli, Propionibacterium shermanii, Streptococcus salivarius, Pseudoalteromonas sp., S. thermophilus, Thermoanaerobacter, Bacillus coagulans, Lactobacillus delbrueckii subsp. Bulgaricus.

Beta Galactosidase in E. coli (β-gal / gene name: LacZ)

It is a tetrameric hydrolytic enzyme, coded by the Lac operon and able to catalyse the hydrolysis of a disaccharide (β-galacotisedes) into two monosaccharides: it is, then, related to the reaction that injects new glucose into glycolysis.

The E. coli isoform is 464K-Da weight and 1021 amino acid long. Each monomer is composed by five domains, the third of which hosts the active site. The protein can be split into two peptides, which are unable to catalyse reaction when separated.

This peculiarity gives β.galactosidase a primary importance in synthetic and molecular biology, for it is used as a reporter gene, by fusing protein into the β-gal chain, disrupting the first peptide’s sequence, in a test known as “blue white screening”. A well known galactose homologue, X-Gal, turns blue when hydrolysed by this enzyme and shows whether colonies have incorporated the fusion genes or not.

The active site is associated to Glu-537, that is the main actor of a nucleophil substitution between the carboxyl group of the side chain of Glu and the ketonic oxygen that links the two mono-saccharides. Result of this reaction is the substitution of an alcoholic group to the ketonic group and the following liberation of the other monosaccharide. Thus β-gal is the gateway for glucose into the glycolytic process.

ENGINEERING FERMENTATION USING Z. MOBILIS METABOLISM

Zymomonas mobilis

Zymomonas mobilis is a Gram-negative bacteria of the soil that has several appealing features for its use in the industrial production of ethanol. Zymomonas is the only known microorganism capable of oxidizing glucose anaerobically, via the Entner-Doudoroff pathway (see figure), as opposed to the classical glicolytic pathway.

Unfortunately Zymomonas can only catabolize simple sugars as glucose, fructose and sucrose. Since our project relies on lactose catabolism, we decided to insert two Zymomonas key fermenting enzymes: the Pyruvate Decarboxylase (pdc) and the Alcohol Dehydrogenase II (adhB) in E. coli, which is, instead, capable of fermenting lactose.

E. coli does not possess a native Pyruvate Decarboxylase: although, it uses Pyruvate Formate Lyase. This enzyme has an unbalanced consumption of NADH, and the microorganism balances it by producing acetic acid and succinic acid rather than ethanol. The insertion of Zymomonas pdc gene should determine the production of ethanol as only final product of the fermentation pathway.

Although E. coli strains have Alcohol Dehydrogenase, the activity of this native enzyme is insufficient to achieve this high yield of ethanol, while Zymomonas adhB enzyme has a higher efficiency.