Yeast transporter with affinity substrate ratios and expression regulation.
Yeast organisms are widely explored by humans for different biotechnological applications. During their growth, they need to adapt and interact themselves with the environment medium. For this purpose, organisms uptake nutrients and at the same time secrete different molecules include proteins to extracellular medium. This phenomenon requires the use of specialized structures to regulate entry and exit of molecules called transporters. Two transporters, namely Proteins and Vesicles, are specialized in translocating molecules in and out across the wall. The knowledge of these systems is important and served to bring novel applications of yeast. Taking together, this book chapter is divided into two parts: at first, it primarily accounts on few examples of protein (carbohydrates and peroxisome proteins) and vesicle (intracellular and extracellular vesicles) transporters of yeasts. Second, it deals with the recent advances of yeast applications in diverse area of science.
2. Hydrocarbons transporters of yeast
There is a great diversity of yeasts and they all require a carbon source to maintain metabolic, physiological and cell growth processes. One of the main nutrients is glucose because it plays a key role regulating the expression of sugar-carrying genes. Yeasts can also consume different types of sugars like xylose, arabinose and under very specific conditions glycerol. These nutrients need to be introduced into the cell whereby yeasts have developed numerous transporters proteins, with similar structures, but with very specific substrate functions and affinities.
2.1. Glucose regulation
Glucose is a substrate of easy metabolism and can act as a signaling molecule depending of its extracellular/intracellular concentration to adjust diverse cellular activities. In
2.1.1. Pathway of glucose induction Rgt2/Snf3, responsible for its consumption.
Snf3, and Rgt2 are important sugar sensors (not glucose transport) in the
2.1.2. Pathway of glucose repression Snf1/Mth1 negatively regulates genes involved in glucose oxidation and the use of alternative sugars.
Under glucose limitation, there is transcriptional inhibition of hexose transporter genes (HXT) by blocking of their promoter by a repressor complex conformed with Snf1, the complex Cyc8-Tup1 and the Mth1/Std1 [1, 5]. This mechanism is required for the yeast to adapt to glucoses limitation medium.
2.2. Hexoses transporters
The vast majority of yeast carbohydrate transporters belong to the Major Facilitator Superfamily (MFS) and the hexoses transporters (HXT) of
All transporters are expressed in several specific conditions. Hxt1 has a low affinity and is expressed in high glucose levels to control carbon flux; in some cases the affinity of the transporters can be modulated to adapt to consumption needs, as Gal2 and Hxt2, they switch affinity to regulate specific transport. Some transporters (Hxt 8–17) are transcribed at low levels and cannot support the demand of nutrients by themselves and in the particular case of Hxt12 does not transport glucose. HXT family has the ability to translocate other sugars such as fructose, which also covers a role of expression regulation [9, 10]. In Schizosaccharomyces pompe, the symporter Ght2 has better affinity for fructose instead of glucose .
Specific fructose symporter (Fsy1) has been described to function as a proton symporter; this transporter is able to discriminate between fructose and other hexoses in
2.3. Pentoses transporters
Glucose is preferentially transported into the cell due to a 100-fold lower affinity of xylose for the transporters. In
2.4. α-glucosides transporters
MAL loci contains genes necessary for the transport and consumption of maltose as MALx1 which encodes a maltose permease with low affinity and MALx3 encoding a positive regulatory protein of these genes in the presence of maltose, a clear example would be maltotriose/maltose: symporter Mal61 encoded by MAL61 and a positive regulatory protein encoded by MAL63 . In yeast there are maltose transporters with high and low affinity, for example MAL11, MAL21 and Mal 61 have high affinities to maltose (Michaelis constant (Km): 2–4 mM) and can carry other sugars as turanose but cannot convey maltotriose. Atg1:H+ is a symporter transporter capable of transporting a wide variety of α-glucosides (trehalose, sucrose, maltose, α-methyl-glucoside, maltotriose) in
2.5. Glycerol transporters
Polyols like glycerol are used as osmoprotectants by many organisms; yeasts accumulate glycerol under high osmolality conditions. Fps1 glycerol efflux facilitator in
Another glycerol:H+ proton symport transporters like Stl1 are expressed transitorily and activated when all sugar is consumed and the yeast enters into diauxic shift, during this, major changes in gene expression alter the fermentative to oxidative metabolism, allowing to utilize the produced ethanol and glycerol before entry into the stationary phase. Stl1 was inactive in the presence of glucose . By homology analysis with Stl1 from
2.6. Inositol transporter
Inositol transporters ITR1 and ITR2 (from
All transporters mentioned have the transport of carbohydrates in common, but they present variation on substrate affinity that can be classified in low (Km: >40 mM) and high (Km <40 mM) affinity, this feature leads to control carbon flux; therefore at high substrate concentrations the expression of low affinity transporters is induced. One way to measure carbohydrate transport rates (uptake) is by scintillation assay, where studied strains that express the transporter of interest. It is harvested and transferred to a substrate-free buffer to subsequently expose them to a solution of known concentration of the radioactively labeled carbohydrate of interest for a defined period of time, then, the cells are filtered and washed with the same buffer, after, the remanent is analized by a liquid scintillation counter. The difference between the radioactivity data of the initial substrate and the remaining concentrations, allows substrate consumption quantification per unit time; this information can also be integrated into an enzymatic modeling or nonlinear regression analysis to obtain kinetic parameters of Km and maximal initial uptake speed (Vmax). Table 1 presents a list of diverse characterized yeast and transporters.
|Hxt1||Glu > Fru||Low||Xyl||Low||Induced by high glucose level|
Does not transport xylose as unique carbon source
|Hxt2||Glu||Mod||Induced by low glucose levels|
|Repressed by high glucose levels|
|Hxt3||Glu > Fru||Low||Induced by high glucose levels|
|Hxt4||Glu > Fru||Low||Xyl||Low||As(OH)3||NR||Induced by low glucose levels|
|Repressed by high glucose levels|
|Hxt5||Glu > Fru||Low||Xyl||Low||Regulated by cellular growth|
|Hxt6||Glu > Fru > Man||High||Xyl||Maltose||NR||Induced slightly at low glucose concentrations Highly induced in non-fermentable substrates|
|Hxt7||Glu||High||Xyl||Low||Repression by high glucose levels. It varies only in 2 amino acids with Hxt6|||
|Atg1||Treha/Sucr:H+ > Malt/ α-met-gluc: H*|
|High levels of expression of this gene during wort fermentation|||
|Gal2||Glu > Gal||Mod||Xyl/Ara||low||Induces by presence of galactose.|
Repressed at high glucose concentrations
|Mal11, 61||Maltose > Turanose||High||MAL loci of constitutive expression and induced by presence of maltose.|||
|Mph2, 3||Maltose/ Maltotriose|||
|Irt1–2||Inositol||Low||Repression by the presence of glucose|||
|Stl1: H+||Glycerol||Active when found in a system with nonfermentable carbon sources, inducible in saline conditions, transient expression, inactive in the presence of glucose|||
|Fps1||Glycerol efflux||Form protein channel, essential in maintaining the balance in changes hypoosmotic|||
|Repressed by high concentrations of glucose or fructose and was highly expressed on ethanol as the sole carbon source|||
|Gup1 / 2||Glycerol||They allow medium growth with glycerol as the sole carbon source and stabilize cell under salinity conditions, Membrane-bound O-acyl transferases family.|||
|Xut1/3||Glu/Fru||NR||Xyl||High||Preference for xylose over glucose but moderate transport efficiency|||
|Arby||Ara||Repression by the presence of glucose|||
|Gt1||Glycerol||NR||Is active when the medium contains ethanol and absence of sugars|||
|Sut1||Glu > Fru||High||Xyl||Low||Induced by glucose presence|||
|Sut2||Glu||High||Xyl||Low||Constitutive expression under aerobic conditions and are independent of carbon source|
|Xyl > Gal||Low||Constitutive expression under aerobic conditions and are independent of carbon source|
|Gxs1||Glu: H+||Xyl: H+||High||Repression by the presence of glucose|||
|Hgt1/Hgt2||Glu||High||Repression by high glucose levels|||
|Hgt7||Glu||NR||Induced by low glucose levels|
|Hgt12||Glu||NR||Induced by low glucose levels|
|Stl1||Glycerol: H+||High||Induced by salt stress|||
|Yht1||Glu/Fru/Man||NR||Xyl||NR||Main hexose transporters|
Induced by presence of glucose and galactose
|Yht2||Fru||NR||Detected in stationary phase of growth|
|Yht3||Fru||NR||Detected in stationary phase of growth|
|Yht4||Glu/Fru/Man||NR||Main hexose transporters|
Induced by presence of glucose and galactose
|Yht6||Xyl||NR||Detected in stationary phase of growth|
|Ght1||Glu > Fru: H+ / Fru||High|||
|Ght3||Glu: H+||High||Gluconate||Transitory expression|
Gluconate transport inhibited by glucose presence
|Constitutive expression in different carbon sources|
|Ght6||Fru > Glu: H+||High||Fru||High||Constitutive expression in different carbon sources|
|Sut1||Mal > Suc: H+||High||No specific induction. Glucose repression|||
|Hgt1||Glu > Gal||High||Constitutive expression with 26–31% identity with Hxt in |||
|Ftr1||Fru: H+||High||Induced by presence of glucose, fructose and galactose|||
|Rag1||Glu||Low||Fru||NR||Induced by high levels of glucose, fructose and other sugars.|
Repressed by absence of glucose
|Kht1||Glu||Low||Induced by high levels of glucose, fructose and other sugars|
|Kht2||Glu||High||Induced by low glucose levels|
|Repressed by high glucose levels|
|Xylh||Xyl: H+||High||Induced by the presence of xylose|||
|High||Sorbose||Low||Induced by low Fru levels|||
|Ffz1||Fru: H+||High||Induced by High levels of Fru|||
|Induced by low Fru levels|||
3. Protein transport
Membranous and non-membranous proteins are the indispensable machinery for the cells life. Membranous proteins are integral and peripheral membrane proteins that include transporters (sugars, ions), GTP binding proteins, cell wall synthesizing proteins. While, non-membranous proteins are metabolic proteins, transcription factors and so on. Most proteins are usually encoded in the nucleus and synthesized in the free ribosomes of cytoplasm . Once synthesized, they must have to be transported to different compartmentalized organelles such as Nucleus, Endoplasmic Reticulum (ER), Mitochondria (Mt), Golgi bodies, Vacuoles and Peroxisomes [39, 40, 41]. These compartmentalized organelles are constituted by multiple sites like outer membrane, intermembrane space, inner membrane and matrix as shown in Figure 3. The proteins should be transported to all specified sites of organelle(s) and across the wall (cell wall) to the extracellular medium [39, 40, 41]. The order of events that leads the protein to get transported are protein recognition and its subsequent translocation into the organelle. Despite the organelle specific transport, multiple steps of protein transport are briefly generalized here.
3.1. Signal sequence
Most proteins synthesized in the cytosol are mostly precursors or preproteins carrying signal sequences . The signal sequences, present in each protein molecule, are organelle specific. They can be found either at the N-terminal or C-terminal ends of proteins [39, 40, 41]. The signal sequence has three conserved general domains: A N-terminal region that varies widely in length, but typically, contains amino acids which contribute a net positive charge: a central hydrophobic region made up of seven to 16 amino acids; followed by a signal cleavage site (Figure 4). For instance, Mt. preproteins are rich in positively charged amino acids, arginine and lysine, and hydroxyl bearing ones, serine and threonine. In nuclear preproteins, the sequence region of first 10–90 N-terminal residues, exhibiting a high composition of arginine and near absence of negatively charged residues, is considered as signal peptide . Regarding membrane proteins, the targeting signals have so far only been identified for a small subset of proteins . In general, non-membranous proteins carry signal peptides at N-terminal, whereas signal peptides are located at the carboxyl termini of membranous proteins . Additional signal sequences found in the proteins conceive multiple entries across the membrane layers of organelles. The example is shown in Figure 4, where Mt. luminal proteins contain three signal sequences as follows: (1). A N-terminal protein signal required to gain access into organelle, (2). A stretch of amino acids signalizing the intermembrane space and (3). The mature part of the precursor protein signal that allows the protein to locate themselves into the Mt. lumen [44, 45].
As proteins contain unique signals to each organelle, various bioinformatics databases are developed to facilitate the search process of signals in the proteins. The databases are listed at the end of the book chapter. The enlisted bioinformatic databases will assist the researchers to study and explore the signal peptides appropriate for the organelles of interest.
3.2. Protein recognition and entry into organelle
The signal sequences present in the protein molecules are recognized by signal receptors or signal recognition particles and outer-membrane translocases [42, 44, 45]. They are usually found either in the cytosol or on the membrane of the organelles’. Pex5p is a remarkable example of cytoplasmic receptor protein . Some examples of membranous receptors are exportins and importins (nuclei), translocase outer-membrane complex (Tom70; Mt) [44, 45]. The receptor always function by coupling with other accessory proteins to import and export proteins. For instance, Tom 70 binds to a subset of mitochondrial precursor proteins, with Tom70, are Tom22, Tom5, Tom6, Tom7, Tom20 and Tom70 [44, 45]. These binding partners cooperate and facilitate the targeting of mitochondria proteins. Usually, receptors contain binding sites for signal sequence in the precursor proteins. After gaining access to the organelle specific receptors, precursor proteins are either further processed and deposited into the respective compartments, or translocated directly through the membrane pore complexes [39, 40, 41]. In the case of processing precursors, the function of multiple peptidases locating in the respective compartment is required. Especially, in the transport of Mt. luminal proteins, three peptidases: Mitochondrial processing peptidase, Mitochondrial intermediate peptidase, Mitochondrial inner membrane peptidase and their complex proteins are involved to translocate protein from the outer - membrane to the matrix [44, 45] (Figure 3).
In the following section, we account on the examples of two typical protein transport systems based on the presence (peroxisome protein) and absence of signal sequences (vesicle-associated protein).
3.3. Transport of peroxisome proteins
Peroxisomes are ubiquitous eukaryotic cell organelles that compartmentalize a large variety of oxidative metabolic reactions. Peroxisome proteins play essential roles in glycolate recycling, amino acid biosynthesis and in fatty acid degradation. Since, it does not contain any genetic material, all the peroxisome proteins are encoded in the nuclear genome. Two types of Peroxisome transport sequence (PTS) have been discovered: type I (PTS1) and type II (PTS2) to translocate proteins from cytoplasm [46, 47]. Some of the identified peroxisome signal peptides are listed in Table 2. The PTS1 is found in most of the peroxisome matrix proteins and is located at the C-terminus as a tripeptide SKL20. It generally fits the consensus sequence (S/A/C)-(K/R/H)-(L/M). The PTS2 is a conserved sequence which is located near the N-terminus of a protein and is comprised in some species within a pre-sequence that is cleaved off after import into the peroxisomal matrix. Sequence comparisons showed the conserved nonapeptide of PTS2 as (R/K)-(L/V/I)-X5-(H/Q)-(L/A/F). Some proteins which do not contain neither a PTS1 nor a PTS2 have been identified and well known examples are acyl-CoA oxidase, catalase from
Pex5p protein, the cytoplasmic receptor, shuttles between a soluble form and an integral membrane-bound form [46, 49, 50]. They guide free-ribosomal-synthesized peroxisome proteins to translocate across the peroxisome membrane to matrix. It has been characterized that this protein has the capacity to translocate folded, and even oligomeric proteins. The C-terminal domain comprises of seven tetratricopeptide (TPR) repeats, in which 1–3 and 5–7 TPRs adopt extended conformation to link other three TPRs . This conformation produces a funnel shaped binding site for the proteins containing PTS1 signal sequence. Once the receptor recognizes the cargo in the cytosol, a set of proteins Pex13p, Pex14p, Pex17p associate to it forming a docking complex [46, 50]. This establishes a possible link to cargo-receptor complex with peroxisome membrane. At the peroxisome membrane, Pex5p would act as intrinsic membrane protein forming a stable complex with the docking proteins. This complex is shown to exhibit the main conductance of a pore with 3.8 nm in diameter [46, 50]. Also, they can transiently expand to more than 9 nm, when they are importing large oligomeric cargo proteins. The formed pore might at some stage import and translocate the proteins to the lumen [46, 50]. After the luminal protein is released, Pex5p is recycled and translocated to the cytosol by an ATP dependent ubiquitination machinery [46, 49, 50]. In summary, in the cytosol, Pex5p functions as PTS1-receptor in cargo recognition and at the peroxisome membrane where it contributes to pore formation and presumably translocation (Figure 5).
3.4. Transport mechanism of a transmembrane protein, Snc1p/2p
Here, we give an example of transport of a transmembrane protein associated to vesicles (discussed below in the following section). Synaptobrevin (Snc1p/Snc2p) is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) transmembrane protein. These proteins bind onto vesicles and interact with t-SNARE proteins on the plasma membrane, by which they provide specificity for the targeting and fusion of vesicles with the plasma membrane . It consists of a variable N-terminal domain, a central coiled-coil domain, and, in most cases, of a single C-terminal transmembrane domain (TMD) that is thought to be α-helical. The conserved region in the SNARE proteins was predicted to contain two amphipathic alpha helices . Helix 1, from 39 to 53, is unusually hydrophobic and Helix 2, from 60 to 88, predicted to be interacted with other hydrophobic segments of membrane proteins t-SNAREs (Syntaxin) during the fusion of vesicles. Other than helices, it carries a variable domain in the N-terminal, a carboxy trans-membrane domain (TMD) region of 96–110 amino acids is usually hydrophobic and some amino acids present intravesicular in vesicles .
Just like the other class of membrane proteins, it lacks a signal sequence and contains a single hydrophobic segment close to their C-terminus, leaving most of the polypeptide chain in the cytoplasm (tail-anchored) . The initial targeting of these proteins to the ER is mediated by hydrophobic signal sequences, which are recognized during translation by the signal recognition particle. This hydrophobic stretch near the C termini of membranous protein do not bind to signal recognition particles and are inserted into membranes post-translationally. Once after getting entry into ER, it wasn’t clear about the regions responsible in targeting them to secretory vesicles. Deletion and mutational studies were made in the SNARE proteins to investigate the region possessing the ability to target it (Table 3). From the targeting studies of Grote et al.  and Gerst , it was clear that in the absence of helical loops, it is not possible to target the Snc proteins onto secretory vesicles. Thus, deletion or gross substitutions in either of the predicted H1or H2 segments result either in the loss of targeting or in a complete loss of functions. This shows that conserved amphipathic alpha helical region (32–85) is essential for the confinement of snare proteins.
In the other hand, deletions of both variable domain and transmembrane domain do not produce a more deleterious effect in the fusion of vesicles. That is, their localization onto vesicles is not affected by these mutations [52, 53, 54]. These results substantiate that the TMD of Snc protein is tuned to conduct its delivery into ER, while the helices take it over from ER to Golgi. Besides, the targeting of SNAREs to vesicles, TMD plays a key role in their sorting and fine tunes their distribution within the secretory pathway. That is, TMD sorts Sncp proteins and let them to undergo a dynamic cycle of transport to and retrieval from the plasma membrane to vesicles. Thus, it is understood that TMD serves both, as a key factor in the membrane distribution and as the targeting signal for initial insertion of protein to ER domain. Taking together, it was concluded that the sequence-specific information present in the membrane proteins is important for the respective localization to specific organelles and its subsequent protein function.
4. Vesicular transport
Despite the appreciable functionality of various transporters and protein machinery, there is another existing sophisticated source to transport materials across the walls. They are “naturally existing liposomes” which are made up of an outer hydrophobic lipid bi-layer and an inner aqueous hydrophilic core. Two vesicle types depending on their localization: intracellular and extracellular vesicles are identified and extensively studied in the literature. This section briefly describes the role of such vesicles in the transport of biological materials in yeast organisms.
4.1. Intracellular vesicles
The PSVs move vectorially towards sites of polarized growth (the bud and mother/daughter neck). They move to arrive at the target membrane dock and subsequently fuse to transfer their contents to extracellular medium [39, 55]. This complete process is termed as polarized exocytosis. It consists of at least three stages. First, PSVs are targeted to the vicinity of designated plasma membrane domains via microtubule- and/or actin-based transport systems [55, 56]. Second, after vesicles arrive at their sites of active exocytosis, where a exocyst complex consisting of eight components: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 mediate the targeting and tethering of PSVs for subsequent membrane fusion [56, 57, 58]. Two proteins Sec15p and Sec10p bridge Sec4p, a Rab Gtpase, to other exocyst components. On the plasma membrane, Sec3 and Exo70 interact with PIP2 and with other family members of Rho Gtpases (Cdc42, Rho1p) . Finally, the fusion between PSVs and plasma membrane takes place allowing the secretion of vesicle contents and the incorporation of membrane proteins at specific plasma membrane domains. This specific fusion event is mediated by interaction of proteins present in PSVs membrane (v-SNAREs, snc1p/2p) (SNARE, soluble N-ethylmaleimide-sensitive fusion attachment protein receptors) and plasma-membrane (t-SNAREs; sso1p/2p) .
4.2. Exosomes or extracellular vesicles
In all the three kingdoms of life, Exosomes or Extracellular Vesicles (EVs) are one of the most protective sources of conducting trans-cell wall transfer of macromolecules to the recipient cells. EVs (Ø 50–120 nm) are secreted from cells as membranous vesicular organelles by a wide variety of cells, from lower to higher eukaryotic organisms, i.e., from fungi to mammals [60, 61]. Unlike intracellular vesicles, they act as extracellular carriers of proteins and/or nucleic acids, particularly microRNAs and mRNAs, between cells and serve as shuttle vectors and mediators of intercellular communication, immune responses, and antigen presentation . The biogenesis of exosomes begins in the last stage of endocytosis, during which the endocytic membrane undergoes budding to form intraluminal vesicles (ILVs). The accumulated ILVs within the original endocytic membrane, at this stage, is named the multi-vesicular body. These bodies, then fuse with either lysosomes for degradation or the plasma membrane for extracellular release of ILVs, i.e., exosomes or EVs. EVs are released from cells, either constitutively or upon activation of a secretory pathway . The machinery involved in the biogenesis of exosomes varies in different cell types [60, 61]; however, in most cells, the ESCRT (endosomal sorting complex required for transport) machinery plays a major role in EVs biogenesis [60, 61]. The roles of both the ESCRT-dependent and -independent mechanisms in exosome biogenesis remain largely unknown and are yet to be fully elucidated. Similarly, the mechanisms underlying the packaging of cargo into exosomes and the transport of these exosomes across cellular membranes have been described both
EVs have been conserved and distributed widely in many different fungal species, including yeast cells and hyphae . Pathogenic fungus and opportunistic fungus are the well-recognized candidates for the release of EVs. Some of the examples are as follows:
4.2.1. Diverse roles of transport cargoes of EVs
The EVs derived from pathogenic fungus are natural born carriers of cargo responsible for fungal pathogenesis. Several components of fungal EVs are potent elicitors of immunological activities . For instance, the very common protein HSP60 carried by EVs acts as immunogen and induces protective antibodies . The main virulence factor of EVs derived from
5. Methods to determine the secretion-proteins across yeast wall
Numerous established techniques are already available in literature to detect, characterize and demonstrate the phenomenon of secreting proteins, towards the extracellular medium, across yeasts wall. At cellular level, usually, the proteins destined to secretion are always preserved intact into the secretory vesicles of yeasts. Taking advantage of this nature, many fluorescent methods detect the proteins presence througth the secretion route in cells by fluorescence. The availability of several fluorescent proteins (FPs): Green-FP, Red-FP, Yellow-FP and Blue-FP has made the detection process simple and effective . To this end, tagging proteins of interest with FPs, using genetic engineering techniques, will come handy and serve the purpose of locating them into the cells. In the other hand, immunofluorescent technique makes use of antibodies to demonstrate the integrity of secretion proteins inside vesicles . For this purpose, various temperature sensitive sec-mutant strains, with the ability to accumulate vesicles, are highly recommended .
Once the proteins are secreted outside, they can be characterized by molecular techniques like SDS-PAGE and Western blotting to identify specifically the proteins of interest in the extracellular medium [53, 74]. By other hand enzyme activity studies are suitably advantageous to determine the proper functioning of the secreted protein The design of such experiments generally varies with respect to the enzymes and must be handled appropriately and the experiments can be performed either by using the whole extracellular medium containing secreted proteins or by using the purified proteins of interest (see protein purification section in applications below). Combining all together, we conclude that one of the abovementioned techniques could be suitable for realizing adequate studies on the proteins secretion.
6. Applications in biotechnology
This is an overview of the main trends reported within the last years in current research on applications related to transport proteins in some yeast, which has not yet discussed in detail. Major advances, of the role of different biological transporters in
Some other works involves the study of trafficking mechanisms of small and large compounds to regulate biosynthesis of appreciated biochemical products. Also, mitochondrial transport mechanisms are relevant due to its use in future comparative studies aiding explorations of human mitochondrial diseases and to improve biochemical process. Because energy is a fundamental enabler of the economy, energy security and environmental safety are two major issues in the current world that have boosted the demand for an alternative and eco-friendly energy source.
6.1. Protein purification mediated by heterologous expression
Using genetic engineering techniques, recombinant proteins can be synthesized in anyone of three compartments of heterologous hosts: cytoplasm, periplasm and the extracellular medium. The natural ability of secreting proteins is captivated by many researchers as a medium for the large-scale industrial production of foreign proteins and simplifying downstream processes . The secretory expression requires a simple tagging of recombinant proteins of interest with three essential components: (1). A signal peptide sequence targeting secretion, followed by (2) a purification tag and (3) a protease cleavage site [78, 79, 80]. Some of the examples of these three essential components are enlisted and the recommendable design of a gene fusion cassette for recombinant protein secretion is shown in Figure 6. The expression of this gene fusion cassette in the following hosts enables the secretion of protein towards extracellular medium. The purification tag serves as an anchor and allows the recombinant protein to separate from rest of the media culture, which is subsequently recovered by using protease enzyme . Some of the valuable hosts as recommended by Food and Drug Administration are
|Human β-defensin-2, (hBD2)||MFα1 (mating factor alpha) leader||Antimicrobial activity|||
|Beta glucosidase||Sed1, glucoamylase, alpha mating leader||Cellulolytic activity|||
|Cel3A||Native secretion signal||Lignocellulosic|||
|Trx-HPV16-L2 immunogen||alpha-factor signal peptide||Vaccine|||
|Horseradish peroxidase||MATα prepro secretion signal|||
|Candida antartica lipase|
|Human Pro-relaxin L2||alpha-factor signal peptide||Therapeutic applications|||
|FSL2, Lipase||Lipolytic activity|||
|Endo-polygalacturonase||alpha-factor signal peptide||Textile scouring|||
|Camel Hepcidin||Antimicrobial activity, Hormone|||
|Human anti-αIIbβ3 antibody||alpha-factor signal peptide||Atheroma Targeting|||
|Subtilisin QK||alpha-factor signal peptide||Thrombolytic activity|||
|Glucoamylase||preLip2, preXpr2, and preSuc2||Starch degradation|||
|Fructosyltransferase||alpha-factor signal peptide||Hypocaloric sweeteners|||
|Arylsulfatase||alpha-factor signal peptide||Milk processing|||
|Interferon-Beta||Glucoamylase signal sequence||Therapeutic applications|||
6.2. Peroxisome production of valuable bioproducts
Here, we highlight the use of signal peptides and transporter system of Peroxisome for the synthesis of valuable bioproducts. Mostly, researchers took advantage of the active fatty acid pathways and PTS1 signals to generate polyhydroxyalkanoates (bioplastics) and biofuels (fatty-acid-derived fatty alcohols, alkanes and olefins) [99, 100, 101, 102, 103, 104, 105, 106]. From literature, a simple modification of polyhydroxyalkanoate synthase with PTS was sufficient for targeting and synthesizing PHAs in peroxisome of
Another effective exploration is targeting synthetic pathways to peroxisomes to produce medium fatty alcohols and long fatty alcohols [103, 104, 105, 106]. The targeted expression of fatty acyl-CoA reductase TaFAR to the peroxisome of
6.3. Vesicles in therapeutic applications
The prime role of intercellular communication has motivated researchers to conceive EVs as potential nano-vehicles for biodelivery applications. Recently in 2016, A patent entitled ¨Compositions and Methods for Yeast Extracellular Vesicles as Delivery Systems, US 20160331686¨ was filed and published . The authors have proposed the use of native and modified EVs from yeasts cells as practical drug delivery vehicles. In the case of modified EVs, an exosomal transmembrane peptide of mammalian origin is immobilized onto the outer membrane of EVs for targeted biodelivery applications. Using these yeast EVs, various therapeutic sources of cargoes: therapeutic RNAs (circular RNAs), autonomously replicating cytoplasmic linear mammalian plasmid (express either therapeutic RNAs or proteins), therapeutic peptides, have been tested for delivery applications. Once the cargo loaded EVs are released from cells, they have been isolated from culture supernatants by either centrifugation or ultra/micro filtration. Authors conducted
Subcellular localization program
NNPSL (neural network-based predictor)
SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/)
PTS1 Predictor - http://mendel.imp.univie.ac.at/mendeljsp/sat/pts1/PTS1predictor.jsp
The authors acknowledge Juan Corona-Hernandez and Alonso Cid Cervantes for their artwork contribution. GKM thank CONACyT (CB-2014-01; 236285 and Fronteras de la Ciencia 2015-1: 016) for financial support. López-vargas acknowledge to scholarship of PNPC-CONACyT program 5353.