Basic fermentation

The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, while in terrestrial plants the content of cellulose is quite hard to use.[1] By contrast, cellulose in macroalgae has looser texture and lower proportion, which makes it an ideal material for biofuel production.[2] As a coastal university, we have a profound theoretical foundation of the sea and of course, algae. Therefore, it is natural that we think of algae as our substrate.

However, we soon meet with another problem---the main sugar used by trains of Saccharomyces cerevisiae currently in bioethanol production is glucose. Other saccharides such as xylose and cellobiose are relatively harder to ferment. It means a lot more energy and cost we need to donate to pretreatment. Therefore, if we can design a recombinant strain capable of using xylose or cellobiose, it will be a big step further since the yeast can take the place of chemicals and fulfill the task itself.

Before the construction of the plasmid, however, we need to deal with glucose repression.[3] We all know that even yeasts engineered to ferment xylose cannot utilize xylose until glucose is completely consumed, which makes the process of fermentation rather slow when in present of glucose. To overcome these bottlenecks, we decide to separate the fermentation of xylose and cellobiose into two yeast strains, which means substrates can be transported into the cell thus isolate glucose from their shared environment and avoid their mutual interference. In other words, intracellular hydrolysis of cellobiose minimizes glucose repression of xylose fermentation. What's more, we can also lighten their metabolic burden using this strategy.[4]

Now we can begin our project.

After grinding our Enteromorpha into powder and extract polysaccharide we get the remains of algae which has the same content as Enteromorpha waste. Because in industrial manufacture, the former one can be well utilized, we mainly focus on the residue of algae containing cellulose and hemicellulose majorly. For a higher reaction efficiency and better utilization of cellulose and hemicellulose, we treat the residue with H₂O₂ to remove the lignin.[5] H₂O₂ is clean pollution free and low cost, which makes it a perfect reagent for pretreatment. After that, the lignin is mostly removed and the microstructure of cellulose will break down, exposing more binding site, which makes it easier to use.

As for the utilization of cellulose there are three main stages: dense fiber with a high content of lignin, cellodextrin, monosaccharide. Since the process from dense fiber to cellodextrin has been a proven technique, we use enzymes to treat cellulose and hemicellulose into cellobios and xylose. And our major work will be based on these two substrates.

As for cellobiose and xylose, we designed two recombinant yeast strains. For yeast A, we subcloned cellobiose transporter and β-glucosidase genes into a plasmid and introduce it to S.cerevisiae. The cellobiose transporter can help transport cellobiose from the environment into the cell and β-glucosidase will turn cellobiose into glucose, which can then be transformed into ethanol through its own pathway.[6] In the same way, we introduced xylitol dehydrogenase and xylose reductase gene to yeast B. S.cerevisiae naturally has the ability to transport xylose. With xylosereductase (XR), xylose can be reduced to xylitol, which will then be oxidized to xylulose in present of xylitol dehydrogenase. Xylulokinase of wild type S.cerevisiae can then phosphorylate xylulose to xylulose-5-phosphate. Finally, it will join Pentose Phosphate Pathway (PPP) and produce ethanol.[7]

Now we have two strains that can live on either cellobiose or xylose and can produce ethanol through their own metabolic pathways, which means they can ferment cellobiose and xylose synergistically.

Adhesion platform

Through a Adhesion platform between heterogeneous cells, we make E.coli the surface display system of S.cerevisiae to enrich its biofunctions and enhance their synergistic effect.

We select the biotin-streptavidin system to accomplish our adhesion because it is known as the strongest covalent binding system in nature. By introducing ice nucleation protein (INP) and monomer streptavidin adhesion (mSA) fusion protein to E.coli, we express mSA on its surface with INP, which is intergrated into the out membrane itself.[8][9][10] Meanwhile, we express biotin acceptor peptide (BAP) and biotin ligase (BirA) in the S.cerevisiae strain EBY100.[11] This strain of yeast contains pYD1, which has another surface-display system called Aga1p-Aga2p adhesion system. By using this strain, we will be able to express biotinylated BAP on the yeast surface. Ultimately, E.coli and S.cerevisiae can connect firmly with each other through the covalent binding of biotin and streptavidin.

The following question may better elucidate our designing details:

Click here to see Q&A

Q1. Why choose to establish an adhesion platform?

At the beginning of our project, we want to express cellulosome in Saccharomyces cerevisiae, where cellulase can located. Then the cellulase can degrade Enteromorpha into cellobiose and xylose. The latter two can be used by S.cerevisiae as the source of fermentation. Although cellulosome is a method to degrade cellulose with high efficiency, it also have a complex structure combined with multiple scaffold protein and cellulase domain which will accrete the difficulty of experiment design and give the S.cerevisiae a heavier burden.[13] Obviously, too much burden can harm the ferment efficiency.

Fig 2.1

Thus, we think of E.coli to do the part of cellulosome. Because E.coli is a model strain, we can change its feature and function easily, and its plasmid has both proper copy number and size that can lead to a lighter burden than that in Saccharomyces cerevisiae. Moreover, we can separate degradation step and fermentation step through this method, which leads to higher efficiency.

Q2:What’s the preponderance of our platform?

This platform consists of two parts, monomer streptavidin surface display system and biotin surface display system. It is known that avidin can bind with biotin firmly in nature, so we choose the biotin-avidin system to achieve the adhesion between heterogenous cells. An important advantage of this platform is that it is not a traditional co-culture system. Through the exploration of express condition, we break the obstacle of the pollution between heterogenous cells. At the same time, we can use the adhesion platform to build a CBP (consolidated bioprocessing ) degradation and fermentation system.

Q3: Why choose ice nuclear protein as the surface display system of E.coli?

INP is a kind of secretive outer membrane protein that exists in gram-negative bacteria like Pseudomonas syringae. The N-terminator domain contains abundant aspartic acid and hydrophilic serine and threonine, and can be fixed on the surface of cells through glycosyl phosphatidyl inositol(GPI), which is good for the display of macro heterogenous protein. Compared to the Lpp-OmpA fused protein, this system will not lead to an unstable growth of cell, thus, the choice of anchor unit is the key to construct the surface display system. Furthermore, INP surface display system has the following advantages: 1) it can be expressed in many kinds of gram-negative bacteria with a steady expression level. 2) INP can display large molecular weight protein, so it has a larger applicate range. 3) INP naturally has the ability to secrete, locate, and anchor a cell, and can display interest protein without accessory protein. It has been confirmed useful in many reports..

Fig 2.2

Fig 2.3

Q4: Why choose The Yeast Display System with the a-agglutinin receptor?

To stabilize cell-cell interactions during mating and to facilitate fusion between a and α haploid yeast cells, we choose this receptor. The Yeast Display System uses the a-agglutinin receptor of S. cerevisiae to display foreign proteins on the cell surface. The a-agglutinin receptor consists of two subunits encoded by the AGA1 and AGA2 genes. The Aga1 protein (Aga1p, 725 amino acids) is secreted from the cell and becomes covalently attached to β-glucan in the extracellular matrix of the yeast cell wall. The Aga2 protein (Aga2p, 69 amino acids) binds to Aga1p through two disulfide bonds and after secretion remains attached to the cell through its contact with Aga1p. The N-terminal portion of Aga2p is required for attachment to Aga1p, while proteins and peptides can fuse to the C-terminus for presentation on the yeast cell surface. A yeast display system is particularly useful for mammalian cell surface and secreted proteins (e.g., receptors, cytokines) that require endoplasmic reticulum-specific posttranslational processing for efficient folding and activity.

Fig 2.4

Q5: How to establish a functional adhesion platform?

At first, we consider using INP surface display system in E.coli to display mSA thus bind with biotin in S.cerevisiae and use OmpA surface display system to display cellulase and hemicellulase, so that cellulose and hemicellulose can be degraded and utilized by S.cerevisiae. However, we found that OmpA-CenA (a kind of cellulase in part registry) did not have the correct function as we expect. Therefore, we decide to construct the adhesion platform.

We use INP from BBa_K523013, BBa_J23106 and mSA from team Peking to build our part J23106-INP-mSA with a 4X linker. Similarly we acquire BirA from E.coli genome that can biotinylate biotin accepted peptide (BAP). In addition, we use pYD1 to display BAP on the surface of S.cerevisiae.

We co-culture these two strains and explore the proper condition to culture them together. To analyze this system with mathematical model, we also characterize the growth curve of these two strains and the only consumption rate of the corresponding carbon source. With these data, we simulate a co-culture model, feed back to our wet lab and optimize the experiment condition so that we can get the best ethanol production.

MINI Combination

To our understanding, the native promoters and terminators for Eukaryote are usually lengthy, with quite a few unnecessary regions. The big size of fungal promoters and terminators limits large-scale synthetic biology efforts in yeast. So this year, we work on a promoter-terminator pair called MINI combination.

For promoters: In the past, this problem of bulky yeast promoters was scarcely addressed in synthetic biology. Yeast promoter engineering mainly focused on building from larger, native scaffolds. Therefore, it is likely that there are some endogenous cellular interactions and homologous recombination among them. To decrease this kind of interference and provide more general potential for artificial gene management, it is necessary to synthesize a series of synthetic promoters with minimal size. For convenience, we call them MINI promoters. The core element in MINI promoter is a TATA box with consensus sequence of TATAWAWR24 followed by a transcription start site (TSS) with consensus sequence of A(Arich)5NYAWNN(Arich)6.The UAS elements contain transcription factor-binding sites (TFBS) thought to aid in RNAP stabilization and enhanced transcription rates. For Neutral AT-rich spacer, Hal S. Alper draw a conclusion that 30bp may be the minimal spacing required between the TATA box and TSS for S. cerevisiae.[14]

Fig 3.1 Structure of mini promoters.

For terminators: Not until 2011, when Yamanishi et al. (2011) demonstrated that fluorescent protein expression can be increased 50% by using the terminator from the TPS1 gene in place of the commonly used CYC1 terminator, did people pay attention to the influence of terminators in term of gene expression.As a matter of fact, 3’ UTR can raise the expression level by stabilizing mRNA. Research has proved that [1] there exists a linear correlation between the mRNA abundance and protein expression. Just the same as in promoters, for Eukaryote terminators there are always some core regions that play a crucial role in their functions, the existence of which makes it possible to optimize those lengthy terminators into shorter but stronger ones. As suggested in his paper, we chose an expression-enhancing terminator with low leakage for standardization. We call it MINI terminator.

ZIJIAN GUO et al. have identified that the yeast terminator has three domains，suggested that 39-end formation of yeast mRNAs involves signals having the following three degenerate elements: (i) the efficiency element, which functions by enhancing the efficiency of positioning elements; (ii) the positioning element, which positions the poly(A) site; and (iii) the actual site of polyadenylation.

Different terminator will affect the stability of the mRNA, thus affecting the amount of protein translation。Hal S. Alper et al. Changed the length and GC ratios of each region and made a combination of qPCR and fluorescence intensity measurements，they found a strong correlation between transcript and fluorescence which suggests these synthetic terminators are affecting protein expression at the transcript level.[15]

combination: What’s more, we noticed that different combinations of promoters and terminators can have various effect on promoter strength. An appreciate terminator can even increase the expression level by 11 times. [15].
So what will happen if we place MINI promoters and MINI terminators together? For circuit one, we pair promoter CYC1 with terminator CYC1, which are among the most commonly used native promoters and terminators in Eukaryon. For circuit two, pCYC1, is paired with tMINI. pMINI-tCYC1 and pMINI-tMINI, respectively, serves as the chosen pair for circuit three and four.

Fig 3.2 Structure of mini terminator.

To explore the strength of MINI promoters as well as the combination effect mentioned above, we designed four promoter-terminator pairs, and constructed four different circuits

For each circuit, the strength of promoters was characterized by yECitrine, a kind of yellow fluorescent protein. And red fluorescent protein mStrawberry was used to represent the leakage of terminators. After monitoring the growth rate of four strains containing different expression regulatory devices, we characterized the expression strength of the promoter-terminator pairs by detecting the fluorescence intensity of both yECitrine and mStrawberry in each circuit, to further describe the expression level of circuits at particular living stages.

One step further, we will also test the expression of these genes on post-transcriptional level to illustrate the difference from another perspectives and to provide another firm proof of its behaviour.

Fig 3.3

Reference