Team:Tartu TUIT/Description

Inspiration

The use of genetically modified organisms in industrial biotechnology is increasing at a high rate. It is becoming a strong competitor against chemical production. Is the bioproduction of chemicals an advantage for the modern world? Well, it is not a secret that chemical production can harm the environment as in most cases it produces toxic waste [1]. Besides that, the cost of chemical production might be higher than bioproduction, especially if it requires special conditions such as high pressure or high temperature. For example, 37 steps are needed for the chemical synthesis of cortisone. Many of those reactions are conducted under extreme conditions. Until the bioproduction method in bacteria was developed, a gram of synthetic cortisone was worth 260$. Using microbial hydroxylation it was possible to decrease the number of steps to 11 and bring down the price per one gram of cortisone to about 9.70$ [2].

However, the production of compounds using microorganisms has some complications: purification of the product often takes 85% of total production costs in a biomanufacturing process. The complexity of downstream processing is one of the aspects that makes bioproduction less competitive in comparison to chemical synthesis [2]. Besides that, it is not always possible to synthesize the final product in microorganisms because of its complexity or toxicity to the producer strain. Another concern is associated with potential troubles that might arise from incorrect handling or misuse of genetically modified organisms (for example, potential generation of new viruses and pathogens). It rises the need for strict regulation and control over the synthetic biology field.

In our project, we are particularly interested in yeasts as bioproducers. These organisms, which can be used to make not only day-to-day things like bread, wine, and beer but also probiotic drinks with yeast as a supplement, are of great importance. Also, yeasts, particularly Saccharomyces cerevisiae have been exploited for the production of various metabolites over the years [3]. Besides that, Saccharomyces cerevisiae cells synthesize a vast number of aroma compounds. Indeed, many steps have been taken to unlock the key elements in yeast aroma compound metabolism and find a way to use them in other biosynthetic pathways [4]. Taking everything into account it can be concluded that after certain genetic manipulations yeast can be used as a little factory to produce numerous substances. For example, yeast strains have been engineered to synthesize vanilla extract and many pharmaceuticals [5].

Therefore, the rearrangement of yeast cellular metabolism according to the needs of the industry is of great interest. However, the extraction of the majority of valuable bioactive compounds from yeast cells requires cell wall disruption. Considering the fact yeasts possess a complex cell wall composed of a layered meshwork of β-glucans, chitin, and mannoproteins, the cell wall disruption is one of the most challenging and therefore expensive parts of extraction of the desired substance from yeast [6].

To overcome this issue, many cell wall disruption methods including physical, chemical and enzymatic have been developed. However, the cell wall disruption remains to be either expensive (enzymatic digestion), time-consuming or environmentally unfriendly (physical and chemical approaches).

Based on these concerns, the goal of our project is to develop an autolytic yeast strain. The use of such strain as a platform for a yeast cell factory will ease the extraction of the valuable compounds from the cells. Besides that, the employment of this strain into scientific research will simplify laboratory protocols involving yeast lysis (for example, cell wall disruption for plasmid rescue from yeast cells).
To achieve our goal, we want to introduce the genes encoding for yeast cell wall degrading enzymes - glucanases - and modify some enzymes involved in the cell wall biosynthesis. At the initial stage, we will induce the production of glucanases combined with the downregulation of cell-wall-synthesizing enzymes to make the cell wall weaker and to promote the release of the cellular content into the media. As the next step, a fully automated system to control the lysis of the cells will be developed.(Fig. 1) The lysis can be tuned to be activated at a certain point in the bioproduction process, for example at the end of the exponential phase when bioproduction becomes less efficient.


Figure 1. Comparison of regular strain and "Pop-culture" strain

Description

Yeast cell wall structure, synthesis, and degradation

As yeast can be widely used for the production of certain compounds and its cell wall is hard to digest, our team decided to take a step in creating an autolytic yeast strain. Thinking of which way should be taken to achieve the goal, we decided to investigate the cell wall structure alone with its degradation pathway naturally occurring in yeast.

Saccharomyces cerevisiae cell wall mostly consists of β-glucans, chitin, and mannoproteins. Mannoproteins form the outer layer of the cell wall, whereas β-glucans and chitin form the inner layer. This layered ultrastructure makes yeast cell wall rigid and hard to digest [6].

During normal yeast growth, there is a periodical need for cell wall degradation upon the mating of yeast cells. While mating, yeast of two opposite types - a and α – start to produce special molecules – a and α pheromones, respectively. Cells of opposite mating types sense each other due to the presence of receptors for partner pheromone on their cell surface. After finding a mating partner, two haploid cells are fused to form a single diploid cell. When two cells of different mating types have “recognized” each other, they start to grow towards each other forming an outgrowth, so-called shmoo, in the direction of the highest pheromone concentration. While making the cell-to-cell contacts and fusing, yeast cells of opposite mating types have to dissolve their cell walls at the shmoo tip. This process must be highly regulated as the mistake - cell wall disruption at the wrong time or place - will lead to cell death. Cell wall dissolution is catalyzed by enzymes degrading ß-glucans, which are called ß-glucanases. They catalyze the hydrolysis of β-1,3-glucans consisting of β-1,3-linked glucose monomers [7] [8].

Hence, the yeast cell wall is a dynamic structure, which is constantly changing during cell growth, cell division and mating. Cell wall remodeling enzymes are encoded by numerous genes, which are regulated as a very well-balanced system.

Our first approach at the remodeling of the yeast cell wall was aimed to unbalance this system by introducing extra copies of S. cerevisiae genes responsible for cell wall destruction. Initially, we have chosen four genes: EXG1, EXG2, SCW4, and SCW11.According to the Carbohydrate-Active enZymes (CAZy) database, enzymes encoded by these four genes have glycoside hydrolase activity. Exg1 and Exg2 belong to Glycoside Hydrolase Family 5. In CAZy, they are classified as exo-1,3-ß-glucanases. Exg1 non-covalently binds to the cell wall and is partly secreted into the growth medium, while Exg2 is anchored to the plasma membrane via glycosylphosphatidylinositol anchor. It has been shown that overexpression of EXG1 results in the reduction in cell wall ß-1,6-glucan [9] [6].The other two enzymes, Scw4 and Scw11, belong to the GH17 family and their amino acid sequences are similar to glucanases. Scw4 non-covalently binds to the cell wall, but can also be covalently linked by an alkali-labile linkage to β-1,3-glucan. It has been shown that SCW4 expression is constitutive, while SCW11 is expressed only in young daughter cells. Deletions of either gene led to cell separation defects, indicating a role in cell wall degradation during cytokinesis [9] [6] [10].

In parallel with the overexpression of the endogenous genes encoding for yeast cell wall degradation enzymes, we decided to introduce heterologous genes from bacteria. As the first step, we screened the registry and found the BBa_K2711000 part added by UiOslo_Norway 2018 team. This is an endo-1,3-β-glucanase from Cellulosimicrobium cellulans(or Arthrobacter luteus) whose catalytic domain belongs to the GH16 family. After further investigation, we found that that it is not the only glucanase present in this bacterial species. Arthrobacter luteus is also a producer of β-1,3-glucan laminaripentao-hydrolase, encoded by GLC1 gene. This enzyme is one of the main components of Zymolyase, a widely used commercial enzyme mixture for yeast cell wall lysis. Glc1 possesses a catalytic domain that belongs to another GH family (GH64) and catalyzes the hydrolysis of glucans in the yeast cell wall with predominant formation of pentoses, instead of a mixture of biose and glucose that was shown for BBa_K2711000. High catalytic activity against yeast cell wall glucans was reported for Glc1 [11]. Taking into account all the features of Arthrobacter luteus glucanases listed above, BBa_K2711000 and GLC1 were used for overexpression in yeast cells.

However, the main concern with bacterial enzymes was their potential mislocalization in S. cerevisiae cells. Arthrobacter sp. GLC1 and BBa_K2711000 genes contain an N-terminal Tat-type secretion signals. In the bacterial cell, these enzymes are secreted out of the cells to the environment. However, Tat-type secretion signals are not used in eukaryotes, suggesting that Glc1 and BBa_K2711000 may not be secreted when expressed in yeast and, therefore, these bacterial proteins might stay in the cytoplasm. To achieve efficient lysis of yeast cell wall, it was important to get those bacterial enzymes out of the cells so they could reach the cell wall. Therefore, it was decided to fuse bacterial genes with yeast secretion signals to make sure that the enzymes reach yeast cell wall.

To facilitate protein secretion out of the yeast cells, we decided to add eukaryotic secretion signals to the target proteins. After reviewing the literature, we have found various secretion signals from different proteins. Taking into consideration that, most likely, not all of them would be efficient in yeast cells, we chose 2 different secretion signals for future work:

1) α-factor (α) pre-pro signal

2) Pre-Ost1-pro-α-factor (Ost1) signal

α signal is the most widely used secretion signal in yeast synthetic biology, which contains two regions: a 19-amino acid N-terminal signal sequence (pre-region) that directs translocation into the endoplasmic reticulum (ER), followed by a 66-amino acid pro region that mediates packaging into ER-derived transport vesicles. The α signal sequence is removed by a signal peptidase in the ER lumen, and the pro region is cleaved by the Kex2 processing protease in the Golgi apparatus (Berrero et al., 2018). The α signal works in a way that after translation, the protein to be secreted is transported to ER in an unfolded form. Thus, in the case, if Glc1 protein folding takes place in the cytoplasm right after translation, this secretion signal might be inefficient. For this reason, we also tried Ost1 signal, which promotes translocation to the ER during translation [12]. We tried both approaches assuming that at least one of them will ensure efficient secretion of Glc1 or BBa_K2711000 proteins outside the yeast cell wall.

To be able to initiate yeast cell wall degradation when we need it, all target genes were cloned into yeast cells under synthetic promoter (pLexA). This promoter binds synthetic transcription factor LexA-ER-B112, where LexA is a DNA binding domain of bacterial inhibitor protein LexA, ER is a β-estradiol receptor and B112 is a transcription activator. This synthetic transcriptional factor binds to the promoter with embedded LexA binding sites and activates transcription only after the conformational change caused by β-estradiol bound to ER domain. The advantage of this inducible synthetic system is that it does not interfere with yeast cell metabolism. Moreover, the level of gene expression depends on the concentration of estradiol, which provides additional possibilities for control over target enzyme expression [13].

As it was mentioned before, yeast cell wall remodeling is a complex process. In this balanced system, cell wall degradation can be counteracted by parallel cell wall synthesis. That means that our approach of overexpression of cell was degrading enzymes may not work due to the activity of cell wall synthetases. For this reason, we decided to combine glucosidase overexpression with knock-out of the gene encoding for the enzyme responsible for cell wall biosynthesis. We have chosen the Gas1 gene as its deletion does not affect the growth of the cell but makes it more sensitive to zymolyase treatment. Enzyme GAS1 (1,3-beta-glucanosyltransferase) is involved in cell wall biosynthesis, catalyzing elongation of 1,3-ß-glucan chains in the cell wall [14].

References:

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