Trehalose is a disaccharide consisting of two subunits of glucose bound by an alpha:1-1
linkage (alpha-D-glucopyranosil alpha-D-glucopyranoside.
Trehalose biosynthesis ability is present in eubacteria, archaea, plants, fungi
and animals. In bacteria there are five different biosynthetic routes, whereas in
fungi, plants and animals there is only one.
There are at least five biosynthetic pathways known for trehalose.

Interestingly, several eubacterial species have multiple pathways, while eukaryotes
have only the TPS/TPP pathway. Vertebrates have lost the capacity to synthesise trehalose
but can break it down to glucose with trehalase.
Eubacteria was the only group where the five pathways were found although not all
together in a single species, most of them having from two to four pathways. The
presence of several biosynthetic pathways in the same organism may be due to the
strict requirement to accumulate trehalose under changeable environmental conditions,
which could limit substrate availability for each pathway. A number of eubacterial
species have multiple copies of some of these pathways. For example, Mesorhizobium
loti, Mycobacterium tuberculosis, Ralstonia solanacearum and Xanthomonas campestris
have two TPS genes, while Thermoanaerobacter tengcongensis has two genes for trehalose
phosphorylase (treP). Phylogenetic analyses suggest that paralogous TPS genes are
the product of lateral gene transfer events instead of recent gene duplications.
The TS pathway might be exclusive to eubacteria, while TreT was only observed in
Archaea and Thermotoga maritima, a thermophilic eubacteria which inherited a substantial
part of its genome from Archaea by lateral gene transfer. In fungi, plants and invertebrates
only the TPS/TPP pathway is present; no homologues to other trehalose biosynthetic
genes were found.

*******FUNCTIONAL VARIANTS:********

1.1 – The most conserved TPS/TPP pathway is present;
1.2 - TS (Trehalose synthase) pathway is present;
1.3 - TreY/TreZ (conversion of maltodextrines to trehalose) pathway;
1.4 - Trehalose phosphorylase (TP) pathway is present;
1.123 – all listed above pathways are present;
1.1x – TPS/TPP pathway is present, and one more is possible

========= TPS/TPP pathway:=================

The first pathway was discovered about 50 years ago, is the most widely distributed, and it has been reported in eubacteria, archaea, fungi, insects, and plants. It involves two enzymatic steps catalyzed by trehalose-6-phosphate synthase (TPS) and trehalose-phosphatase (TPP). TPS catalyzes the transfer of glucose from UDP-glucose to glucose 6-phosphate forming trehalose 6-phosphate (T6P) and UDP, while TPP dephosphorylates T6P to trehalose and inorganic phosphate.
The enzymes for the biosynthesis of trehalose in E. coli, trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, are encoded by the genes otsA and otsB. These genes are similar to the yeast genes TPS1 and TPS2. However, in S. cerevisiae the two enzymes are combined into a single complex, called the trehalose synthase complex, which also includes the regulatory subunits TSL1 and TPS3.
Many studies showed a high conservation of the genomic linkage of the TPS and TPP proteins in eubacteria and in archaea, very likely forming part of a single operon. The only exceptions are bacteria of the genus Mycobacterium, cyanobacteria and S. meliloti in which the TPS and TPP coding genes are not clustered in the genome.

The TPS protein is formed by a single phosphatase domain in prokaryotes while in almost all eukaryotes and in P. aerophylum the TPS proteins are fused to the TPP domain. This organization suggests that all the eukaryotic TPS and TPP fused proteins descend from a common ancestor. C. elegans_1 and C. elegans_2 proteins are not fused to TPP and are clustered with Streptomyces apart from the rest of the eukaryotic proteins.
Some putative TPS/TPP fusion proteins lack both enzymatic activities and are only regulatory subunits, such as ScTPS3 and ScTSL1. There are only three proteins were identified in P. aerophilum, D. melanogaster and A. gambiae species, conserving the residues and active sites of both TPS and TPP domains. These are the first putative TPS/TPP fusion proteins with the two domains found so far.

======== TS pathway:===============

The second biosynthetic pathway, the enzyme trehalose synthase (TS) isomerises the alpha1-alpha4 bond of maltose to a alpha1-alpha1 bond, forming trehalose. This enzyme was first reported in Pimelobacter sp. and orthologs of this protein have been found in other eubacteria
The enzymes for the biosynthesis of trehalose in E. coli, trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, are encoded by the genes otsA and otsB. These genes are similar to the yeast genes TPS1 and TPS2. However, in S. cerevisiae the two enzymes are combined into a single complex, called the trehalose synthase complex, which also includes the regulatory subunits TSL1 and TPS3.

======== TreY/ TreZ pathway:===============

The third pathway involves the conversion of maltodextrines (maltooligosaccharides, glycogen and starch) to trehalose. This pathway was reported in thermophilic archaea of the genus Sulfolobus. These organisms synthesize trehalose in two enzymatic steps catalyzed by maltooligosyl trehalose synthase (TreY), coded by the treY gene, which promotes the transglycosylation of the last glucose moiety at the reduced end of maltodextrins from a alpha1-alpha4 to a alpha1-alpha1 bond leading to maltooligosyltrehalose, which contains a trehalose moiety at the end of the polymer. Next, maltooligosyl trehalose trehalohydrolase (TreZ), coded by the treZ gene, catalyses the hydrolytic release of trehalose.

TreX -Isoamylase (also known as glycogen- or starch-debranching enzyme) hydrolyzes the alpha -1,6-glucosidic linkages in glycogen, or the alpha -1,4-glucosidic linkages found in other polysaccharides such as starch to produce maltodextrin.
In Corynebacterium glutamicum, which possess three different pathways for the biosynthesis of trehalose, this is the main route for trehalose biosynthesis. Recently it has been shown that this pathway is present in the bacterium Brevibacterium helvolum and is prevalent in Rhizobiaceae , including Bradyrhizobium japonicum , Bradyrhizobium elkanii , Rhizobium sp. NGR234 , Sinorhizobium meliloti Rm2011 , Rhizobium tropici , Rhizobium leguminosarum bv. viciae , Rhizobium leguminosarum bv. trifolii , and Azorhizobium caulinodans.

======== TreT pathway:===============

A new biosynthetic pathway for trehalose was found in the hyperthermophilic archaeon Thermococcus litoralis, and involves the trehalose glycosyltransferring synthase (TreT), which catalyses the reversible formation of trehalose from ADP-glucose and glucose. It can also use UDP-glucose and GDP-glucose, although it is less efficient with these substrates. The TreT enzyme transfers the glucose moiety from ADP-glucose, and joins it at position 1 of another glucose molecule to form trehalose (Ref.2).

=======REFERENCES:================

1. Avonce N, Mendoza-Vargas A, Morett E, Iturriaga G. Insights on the evolution of trehalose biosynthesis. BMC Evol Biol. 2006 Dec 19;6:109. PMID: 17178000

2. Cardoso FS, Castro RF, Borges N, Santos H. Biochemical and genetic characterization of the pathways for trehalose metabolism in Propionibacterium freudenreichii, and their role in stress response. Microbiology. 2007 Jan;153(Pt 1):270-80. PMID: 17185556