Team:UNIPV-Pavia/Project/Solution

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Solution


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.


Pyruvate decarboxylase (PDC)

Pyruvate decarboxylase is a homotetrameric enzyme (EC 4.1.1.1) that catalyses the decarboxylation of pyruvic acid to acetaldehyde and carbon dioxide. It is also called 2-oxo-acid carboxylase, alpha-ketoacid carboxylase, and pyruvic decarboxylase. In anaerobic conditions, this enzyme is part of an alternative pathway for fermentation,known as Entner-Dourdoff pathway, which is characterized by the bypass of Acetyl-CoA formation, and determines an irreversible loss of a CO2 molecule, in order o produce acetaldehyde. Pyruvate decarboxylase starts this process by converting pyruvate into acetaldehyde and carbon dioxide. To do this, two thiamine pyrophosphate (TPP) and two magnesium ions are required as cofactors. This enzyme should not be mistaken for the unrelated enzyme pyruvate dehydrogenase, an oxidoreductase (EC 1.2.4.1), that catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA.


Alcohol dehydrogenase (ADH)

They are a class of enzymes that catalyse the conversion between alcohols and aldehydes or ketons, in order to decrease the toxicity of alcoholic byproducts. Though, in the glycolytic process it is used in the opposite direction, by converting aldehydes into alcohols. All the reactions involved are reduction or oxidations, with the contribution of nicotinamide dinucleotide (NAD), as a cofactor.

ADH2 (gene name adhB) catalyses the crucial step into ethanol conversion: acetaldehyde comes from the last steps of glycolysis and the bacteria converts it into an ethanol molecule in order to gain a NAD+, as a reducing power, when in anaerobic, or microaerobial environment.

Structurally, the alcohol dehydrogenase family is compose by multimeric proteins, about 300 amino acids long (ADH2 of Z.mobilis is by 383 amino acids and 40,145 KDa weight). The ratio between isoforms of each monomer may vary. Moreover, each unit has two zinc binding domains, where a zinc ion is the core of the active site: it settles an hydrogen bond with NAD, helped by Cys-146, Cys-174, and His-67; while the lateral chains of Phe-319, Ala-317, His-51, Ile-269 and Val-292 bind aldehyde.

Deprotonation of NAD is catalysed by Ser-48, which is deprotonated by His-51. When NAD+ loses its positive charge, it can reduce acetaldehyde to ethanol and CO2 and become NADH.

We selected Zymomonas mobilis as the source of the adhB transgene for implementing the conversion efficiency in the glucose-to-ethanol process, because it has been found that this microbiota follows a special glucose degradation pathway (Entner-Dourdoff pathway), that hass as only final byproducts, two ethanol molecules by each glucose, while two ATP molecules are produced.

Moreover, Z.mobilis has another adh gene (adhA), which does not seem an isoform even if it accomplishes the same function. In addition, adhB seems more efficient in ethanol conversion than its homologue.

Actually, our final goal should be to integrate even adhA into an E. coli strain genome, disrupting genes related to aerobial respiration, if possible.