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Revision as of 03:27, 27 October 2020



OVERVIEW

Yarrowia lipolytica is an up and coming yeast species gaining interest in the molecular biology community. Its oleaginous nature and its GRAS status make it a suitable chassis in food and nutrition applications. While there are synthetic biology tools for Y. lipolytica emerging in the greater scientific community, only a few iGEM teams have worked with this chassis, resulting in a limited number of Y. lipolytica parts available in the iGEM Registry of Standard Biological Parts.

While we are providing our Y. lipolytica collection to the registry, two of our parts (BBa_K3629013 and BBa_K3629016) in particular have been modelled and designed for optimal activity in Y. lipolytica. Once we have greater access to the lab, we plan to characterize these two parts (along with our other parts), and add characterization to an existing Y. lipolytica promoter in the registry (BBa_K2117000) that is important to our experimental design. Read more about our plans below!


OUR FEATURED PARTS

Two of our cellulase enzymes: CBHI and EGI blah blah


Figure 1. Homology model of Modified Trichoderma reesei EGI (BBa_K3629008) on the left and Modified Penicillium funiculosum CBHI (BBa_K3629005) on the right.

Modified PfCBHI

BBa_ is a modified CBHI (Cellobiohydrolase 1)  optimized for efficiency and function in moderate pH using modelling. As a CBH, it functions to cleave two-glucose cellobiose units from the ends of frayed cellulose chains, therefore functioning as the second step in cellulose degradation. BBa_ is a chimeric protein derived from a combination of the catalytic domains of CBH found in T. reesei and P. funiculosum. This hybrid protein was found to be more effective than CBH from T. reesei, and more resilient than the CBH from P. funiculosum.

 

Due to the nature of cellobiose as a reducing sugar, future experiments to characterize the efficiency of BBa_ will utilize DNS assays in order to determine the rate of conversion of cellulose into cellobiose. We intend to characterize its capacity to degrade both refined cellulose and crude plant matter.

 

WITH CELLULOSE:

  1. Dissolve 1g of cellulose in 100mL of water (it likely won’t dissolve= 1% w/v)
  2. Dissolve 1g of enzyme in 100mL of water (10mg/mL)
    1. Make 4 serial dilutions of 10x: 
      1. 1mL +9mL = 1mg/mL or 1000µgmL
      2. 1mL + 9mL= 100µg/mL
      3. 1mL + 9mL= 10µg/mL
      4. 1mL + 9mL= 1µg/mL or 1000ng/mL
    2. Dilute another range of 5 samples 1mg/mL - 5mg/mL
  3. Mix 1mL cellulose solution and 1mL of each enzyme solution
  4. Incubate at 30º for 1 hour 
  5. Add 2mL DNS reagent 
  6. Boil for 5min  
  7. Cool to room temperature under cool water 
  8. Measure A540

 

WITH CRUDE PLANT BIOMASS: 

  1. Take dried plant material (Cymbopogon citratus leaf and Cicer arietinum seed pod) and rip or grind into small pieces. 
  2. Add 0.2g of ground plant matter to 2mL water and boil for 10 mins
  3. Mix 1mL of 1% cellulose solution and 1mL of enzyme solution. Repeat for each dilution of enzyme above.
  4. Incubate at 30ºC for 1 hour s
  5. Add 2mL DNS reagent 
  6. Boil for 5min 
  7. Cool to room temperature under cool water 
  8. Measure A540 

Modified TrEGI

As an EGI (Endoglucanase 1), SEGI8 functions as the essential first step in the cellulose degradation pathway. It works by cutting cellulose strands internally, exposing new ends for further degradation by cellobiohydrolase. In order to ensure optimal effectiveness in our conditions, we have used a CBH already mutated to improve activity. We then used modelling to further increase stability, and chose the linker segment ApCel5A best suited to improve reducing sugar production. This linker was also chosen due to its ability to operate effectively in the presence of lignin, which may be found in certain potential feedstocks in communities, such as woody plant tissue.


CHARACTERIZING BBa_K2117000

Literature-reviewed characterization

Molecular elements for Y. lipolytica such as strong constitutive promoters have not been well established in the iGEM registry. In an effort to add to the limited library of Y. lipolytica parts in the iGEM registry, we characterized the important TEF1 promoterBBa_K2117000 using the information available in the literature. TEF1 is a native promoter of the Translation Elongation Factor 1 (TEF1) gene in Y. lipolytica . TEF1 is considered to be one of the strongest native promoters found inY. lipolytica . This was confirmed when expression of the reporter gene, hrGFP, under TEF1 promoter and several other strong endogenous Y. lipolytica promoters were tested and TEF1 was shown to have one of the highest expression levels among the native promoters.

To further characterize this commonly used Y. lipolytica promoter, we gathered data on the performance of individual components of the TEF1 promoter such as the proximal promoter element (PPE), the TATA box, and the upstream activation sequence (UAS). Upon the removal of PEE, TEF1 was shown to remain functional but its strength was significantly reduced. Removal of the TATA box, however, completely diminished promoter function. Both the TEF1 TATA box and the PPE were shown to have one of the highest affinities for transcription factors in Y. lipolytica . Thus their inclusion in building hybrid promoter significantly increased promoter strength. We also found that the addition of TEF1 UAS upstream of expression construct was shown to significantly boost expression levels.

We hope that our in-depth characterization of the Y. lipolytica TEF1 promoter can make it easier for future iGEM teams to work this new exciting chassis. Using the information we provided, teams can make a more informed decision on their choice of promoter. Moreover, based on the characterization information provided on individual promoter components, teams can decide on how to play around with promoter composition to obtain their desired expression levels.

Future Characterization

We used the TEF1 promoter in a few of our genetic constructs (BBa_K3629015 and BBa_K3629018) as we needed a strong constitutive promoter to express our cellulase genes. However, we are unsure if the activity of the TEF1 promoter varies depending on the growth stage of the yeast, which would affect all recombinant protein production. Therefore, we decided to characterize the growth-phase dependent activity of the TEF1 promoter BBa_K2117000.

PART DESIGN

This part was based on the design of BBa_2117005 with the hrGFP, and was ordered from IDT with an XRP2 terminator added at the end.

EXPERIMENTAL DESIGN

Figure 2 shows the experimental workflow that will be followed to characterize this part in the future upon greater lab access

Figure 2. Experimental workflow of how BBa_K2117000 will be experimentally characterized in the future. The activity of the TEF1 promoter in Yarrowia lipolytica will be characterized based on growth state to determine how growth phase may impact promoter activity.

  1. Make a 4mL overnight culture with YPD media and a wild-type Y. lipolytica colony and grow overnight at 30ºC
  2. Make a 3 replicate 1:50 dilutions with YPD media with 1mL each of the overnight culture. Grow shaking at 28ºC
  3. Sample under fluorescence plate reader with 488 nm laser excitation and 510 nm emission, as well as OD 650, after 6, 12, 18, 24, and 30 hours
    • Samples at 6, 12, and 18 hours are expected to correspond with log phase. Samples at 24 and 30 hours are expected to correspond with stationary phase.

REFERENCES

Next Steps

In order to provide a sustainable, community-based solution, we plan to genetically modify Rhodosporidium toruloides, an oleaginous yeast that naturally produces beta-carotene and lipids, to be more robust and resource-efficient. By modifying the yeast to produce cellulase, it can then use common agricultural waste products as an energy source for synthesizing its oil. It can then be eaten as a vitamin A supplement. The yeast strain, while naturally safe and non-pathogenic, will also be genetically modified to include a kill switch for bio-containment, and optimized for oil production.