Team:Jianhua/Proof Of Concept

iGEM 2021 - Jianhua

Expression of three proteins to produce 2’-FL

As a proof of concept, we have demonstrated that the proteins to produce 2’-FL—so far produced by engineered E. coli—can also be produced by engineered S. cerevisiae. Through experiments, we successfully integrated the three genes FKP(BBa_K4081992), LAC12(BBa_K4081996) and FucT2(BBa_K4081998) into the yeast genome and co-expressed them.

Figure 1. The result of SDS-PAGE. Line 1: Marker; line 2: Control; line 3: Express FKP(106KD), LAC12(65KD), FucT2(35KD).

Improve 2'-FL Efflux

One challenge facing all microbiological production platforms for 2′FL is the lack of known transporters that can export 2′FL across the cell membrane. Bacterial transporters have been identified that are capable of 2′FL transport. For example, the native E. coli sugar transporter SetA has been used to enable 2′FL secretion from bacteria, and an ABC transporter is hypothesized to be responsible for 2′FL uptake by the gut bacterium Brevibacterium longum subsp. Infantis. However, the ABC transporter operates via a unidirectional transport process that consumes ATP and is therefore undesirable for a cell factory, even if it could be reversed to export 2′FL. In addition, it is possible that 2′FL accumulation inside the cell could compromise membrane stability and cause cell rupture. Moreover, effective expression of bacterial integral membrane proteins in eukaryotes is challenging. Therefore, development of an effective yeast biofactory for 2′FL production is expected to require discovery and optimization of transporters that function in those species. To solve this problem, we use CDT2 from Neurospora crassa to export 2′FL across the cell membrane. The N. crassa cellobiose transporter, CDT2, is likely to be a good platform on which to start protein engineering to improve 2′FL transport. CDT2 has already been shown to be tolerant of mutation by directed evolution experiments that improved its ability to transport cellobiose[1]. The result of SDS-PAGE showed that we successfully express CDT2 in BY4741.

Figure 2. The result of SDS-PAGE. Line: Marker; line 2: Express CDT2(59kD); line 3: Control.

Considering numerous benefits of using a GRAS host for mass production, these results have paved a road for more economical and safer production of 2’-FL. After obtaining the result, a more effective and safe way to produce 2’-FL can be approached, or even put into industry. By this, we were hoping to provide a reliable human milk substitute for parents and a safe food for babies, which is a merit that synthesis biology brings to us.

Prediction of Optimum Culture Conditions for Saccharomyces Cerevisiae

1.1 The benefits and reasons for modeling

In the process of promoting iGEM project, there will be many problems that can not be solved through experiments. At this time, it is necessary to simplify the problem to a certain extent, and infer the problem reasonably through the method of mathematical modeling, so as to get the best solution.
In our project, we use this idea to solve the problems encountered in the experiment. At the same time, we sorted out the significance of modeling, and hope that through our modeling work, we can provide some ideas for the modeling direction selection of iGEM team in the future to a certain extent.
1: Reduce project costs and avoid unnecessary waste of resources.
2: According to the existing data, speculation through mathematical modeling and verification with a small number of experiments can ensure the accuracy and shorten the experimental time.

1.2 Abstract

For our project, the output of 2′-FL produced by S.cerevisiae is different under different conditions. Therefore, it is necessary for the external environment to achieve the optimal fermentation conditions for 2′-FL produced by S.cerevisiae. Here, we selected the following main conditions to explore: time, cell density, medium type, carbon source and transporter type are all factors. However, we have a small number of S.cerevisiae, so it is not easy to use S.cerevisiae to conduct a large number of experiments to obtain the optimal fermentation conditions, which has caused difficulties for our project. Therefore, we consult the data, draw images through modeling operations. By doing this, we found out the best culture conditions of S. cerevisiae.

2. 2′-FL inspection method

Extracellular and intracellular 2′-FL concentrations were quantified during fed-batch fermentation of the engineered S. cerevisiae. To measure extracellular 2′-FL concentrations, cells were removed by centrifugation of fermentation broths at 1789×g for 5 min at 4 °C, and the supernatants were analyzed by HPLC to determine the concentrations of 2′-FL . To determine intracellular 2′-FL concentrations, intracellular metabolites containing 2′-FL were enabled to be release outside cells by boiling fermentation broths for 10 min at 100 °C. The boiled fermentation broths were centrifuged at 9447×g for 10 min at 4 °C, and the supernatants were analyzed by HPLC to measure 2′-FL concentrations. The measured 2′-FL concentrations of the supernatants were considered the total concentrations of 2′-FL in the fed-batch flasks of the engineered yeast. To determine the intracellular 2′-FL concentrations, the total 2′-FL concentrations was subtracted by the extracellular 2′-FL concentrations.

3. Selection of culture conditions

3.1 Carbon source

Carbon source is the primary independent variable in yeast production of 2′-FL . Previous experiments have verified that, the deletion of Gal80 enabled 2′-FL production with sucrose as the carbon source. Gal80 was knockout in FL16, resulting strain FL19.
The strains were cultured in YP medium containing glucose 2% (w/v) and 0.12 g/L adenine. Galactose 3% (w/v) or sucrose 3% (w/v) and lactose 0.4% (w/v) were added at 24 h and cultured for another 48 h. FL16 gal and FL19 gal indicate 2′-FL production from galactose while FL16 suc and FL19 suc indicate 2′-FL production from sucrose.

Fig.1. The yield of 2′-FL in intracellular and extracellular

It can be seen from the experimental data that the yield of 2′-FL synthesized by lactose is higher than that by sucrose. Therefore, lactose was selected as the carbon source for 2′-FL production.

3.2 Teansporter

Referring to previous experimental papers, we found that S. cerevisiae and Y. lipolytica lack an effective mechanism to export 2′-FL because the measured concentrations of extracellular 2′FL were always substantially lower than the intracellular concentrations in our experiments, consistent with results reported by previous study[1].

For example, the intracellular 2′FL concentration in strain HS07, grown in a shake flask using FeedBeads™ to limit the glucose concentration, was approximately 20 g/L after forty hours, while the extracellular concentration in the same sample was 0.3 g/L. Therefore, to complete our validation of these strains as potential cell factories, we sought to identify transporters that could effectively move 2′-FL out of the cell.In most cases, we focused on transporters of fungal origin because of the differences in synthesis and membrane insertion of proteins between prokaryotes and eukaryotes (Rutkowski and Lingappa, 2015[4]).

Twenty-seven candidate transporters were selected for evaluation in S. cerevisiae (Table S5). This selection was based on the transporters meeting any of the following three criteria:

1. Any evidence of ability to transport 2′-FL.
2. Fungal transporters able to transport two-, three-, or four-carbon oligosaccharides.
3. Sugar transporters where substrate export was not coupled to ATP hydrolysis, either directly, or indirectly via maintenance of metal ion homeostasis during symport or antiport.

The functionality of each transporter was assessed by growing the resulting strains in SC medium, measuring the concentrations of intracellular and extracellular 2′FL, and using the ratio of external 2′FL to internal 2′FL as an indicator of 2′FL export. Of the candidate transporter genes tested, expression of E. coli SetA and N. crassa CDT2 resulted in a substantial increase in the ratio of extracellular to intracellular 2′-FL compared to the control strain that lacks a transporter gene (Fig. 4(a) and (b); Table S7). For the remaining transporters, no reproducible increase in the extracellular: intracellular 2′-FL ratio was detected compared to the control (Fig. 4(a) and data not shown). Note that although the extracellular: intracellular 2′-FL ratio varied between experiments for a given strain, the increase in extracellular: intracellular 2′-FL for strains expressing SetA or CDT2 compared to the control lacking transporter was consistent across experiments.

Fig. 2. Identification of transporters that enable 2′-FL export by yeast.

A. Ratio of extracellular 2′-FL:intracellular 2′-FL measured after 24h growth of S. cerevisiae strains expressing the indicated transporters, or containing a control plasmid lacking a transporter (strains HS108, HS109, HS110, HS111, HS13 and HS12, from left to right). Strains were grown in SC-Ura-His-Trp with FeedBeads™ as carbon source and 0.5% lactose. Data is shown for a sub-set of the transporters that did not increase the external:internal 2′-FL ratio compared to the control.
B. Ratio of extracellular 2′-FL: intracellular 2′-FL measured after 24h growth of S. cerevisiae strains expressing CDT1 or CDT2 from N. crassa, or containing a control plasmid lacking a transporter (strains HS119, HS14 and HS12, from left to right). Strains were grown in SC-Ura-His-Trp with 2% glucose and 0.5% lactose.
C. Ratio of extracellular 2′-FL: intracellular 2′-FL measured after 72h growth of Y. lipolytica strain HY108, expressing SetA from E. coli, or the control strain with a plasmid lacking a transporter (HY110).
D. Ratio of extracellular 2′-FL: intracellular 2′-FL measured after 72h growth of Y. lipolytica strain HY109, expressing CDT2 from N. crassa, or a control strain with a plasmid lacking a transporter (HY111). From the experimental data above, we can see that the SetA and CDT2 transporters are most efficient, so both transporters are best suitable as transporters of 2′-FL.The CDT2 transporters were the most efficient.

3.3 Medium

The choice of medium is also a very important factor. The composition and content of organic compounds in the medium will affect the yield of 2′-FL production by yeast, so we investigated the influence data of YP medium and Verduyn medium on the yield of 2′-FL production by S. cerevisiae. We synthesize them into curves and find out their functional expressions.

Fig.3. Comparison of 2′-FL content in yeast culture in Verduny medium and YP medium

The abscissa represents time in hours. The vertical axis represents the production of 2′-FL in g/L.
The orange curve represents the yield of 2′-FL over time when S. cerevisiae was cultured in YP medium. The blue curve represents the production of 2′-FL over time when S. cerevisiae was cultured in Verduyn medium.
The solid line is the curve drawn from the experimental data, and the curve is the curve of the fitting function.
The fitting function is as follows:
YP: $$y = -1E-09x^6 + 3E-07x^5 - 4E-05x^4+ 0.0025x^3 - 0.0634x^2 + 0.5123x - 0.0702$$ Verduyn:$$y = 1E-10x^6- 9E-08x^5+ 2E-05x^4- 0.002x^3+ 0.0826x^2- 0.7961x+0.124$$ It can be seen from the figure that although the production rate of 2′-FL in verduyn medium group was faster than that in YP medium 40 hours ago. However, after 40 hours, 2′-FL in verduyn medium group was no longer produced, while 2′-FL production in YP medium continued to increase. Therefore, we chose YP medium to culture Saccharomyces cerevisiae.

3.4 Cell Density

Fig.4. Changes of cell density of Saccharomyces cerevisiae with time

After comparing the effects of different media on 2′-FL yield, we also continued to explore the changes of Saccharomyces cerevisiae cell density with time and its effect on 2′-FL yield.
The orange curve represents the change of Saccharomyces cerevisiae cell density with time in Verduyn medium.The blue curve represents the change of Saccharomyces cerevisiae cell density with time in YP medium.
The abscissa is time and the ordinate is OD at 600nm.
Verduyn:$$y = -1E-08x^6 + 3E-06x^5 - 0.0004x^4 + 0.0182x^3 - 0.3255x^2 + 2.9917x + 1.0266$$ YP: $$y = -2E-08x^5 - 4E-08x^4 + 0.0008x^3 - 0.0956x^2 + 4.1997x - 2.9869$$ After consulting the data, the literature shows that, lactose feeding was initiated at 36 h when a cell density reached OD~90. This allows for higher yeast cell density and possibly higher 2′-FL production.

3.5 Time

Fig.5. Change of product over time

Ethanol produced from the initially added glucose was completely consumed at 36 h, and 20 g/L ethanol was added to the flask two times，and OD600 reached 34.0, and 2′-FL concentration reached 503 mg/L at 120 h. Until 120 h, 270 mg/L of L-fucose and 1.19 g/L of lactose were consumed. Thus, the final yields of 2′-FL were 0.63 mol/mol from L-fucose and 0.3 mol/mol from lactose.

Fig.6. The amount of product under different raw material conditionsFig.5. Change of product over time

In order to verify the change of 2′-FL yield and time, the main measurement of fucose and lactose consumption (because S. cerevisiae needs to absorb both from the culture medium to synthesize 2′-FL in the cell); Data read through the result of the experiment we will after the relationship between production and time of 2′-FL summed up in Saccharomyces cerevisiae within 36 hours after the medium for its breath of glucose and ethanol is depleted, but with Saccharomyces cerevisiae, after will continue to produce ethanol itself from Saccharomyces cerevisiae will continue to use of ethanol from respiration, slow to produce 2′-FL, after 48 hours 2′-FL was no longer produced, but when ethanol was added again to provide organic matter for S. cerevisiae respiration, S. cerevisiae continued to produce 2′-FL , but the production rate decreased significantly compared with the first 48 hours, and tended to be gentle. After a large number of experiments, the yield of the first 48 hours was 0.63, and the yield of 120 hours was 0.3. So we draw the conclusion that the best yield is 48 hours in actual production.

4. Model

In order to comprehensively consider the effects of time and cell density on 2′-FL production, we carried out the following modeling.
Functional equation of time①：
$$y = 0.0506x^2 - 0.3519x - 2.277$$ Functional equation of cell density②: $$y = -2E-08x^5 – 4E-08x^4 + 0.0008x^3 – 0.0956x^2 + 4.1997x – 2.9869$$ In order to maximize the yield and yield of 2′-FL , it is necessary to maximize the y value of ① and the y value of ② is closest to 90.

In order to obtain the optimal solution, we transform equation ① symmetrically along the x-axis into equation ③
$$y = -0.0506x^2+ 0.3519x+ 2.277$$ So we need to find the minimum of y.
Similarly, we can use 90 minus ② to get ④
$$y = 90-(-2E-08x^5 – 4E-08x^4 + 0.0008x^3 – 0.0956x^2 + 4.1997x – 2.9869)$$ Then add the two equations ③ and ④ to get the expression ⑤
$$y = 90-(-2E-08x^5 – 4E-08x^4 + 0.0008x^3 – 0.0956x^2 + 4.1997x – 2.9869)+( -0.0506x^2+ 0.3519x+ 2.277)$$ Draw the image of ⑤ as follows:

Fig.7

The x value corresponding to the minimum value of y in formula (5) is the optimal production time of 2′-FL .

As can be seen from the image, when x=138， y gets the minimum value. So when S. cerevisiae culture lasts for 138 hours, 2′-FL yield are considered to be the highest, and its economic benefit is also the highest, which is suitable for application in practical production.

5. Conclusion

To sum up, the best culture conditions of Saccharomyces cerevisiae are as follows: take lactose as carbon, use YP medium and the CDT2 as transporter for 140 hours.

In the follow-up project, we will also conduct relevant experiments to verify our prediction results and fine-tune them to make them more accurate. The mathematical modeling method greatly reduced our time and economic cost, and created better conditions for the production of 2′-FL by S. cerevisiae. We hope that Saccharomyces cerevisiae 2′-FL production technology will soon be available so that more mothers who are unable to breastfeed will benefit from our program and will not suffer from a lack of breast milk.

6. Improvement

1. Our research on the relevant influencing factors of 2′-FL production is not perfect, such as temperature, pH and other conditions. More influencing factors should be further considered to improve our model.
2. There are few experimental data sources. We will continue to search for more data to make our model more scientific.
3. Due to the limited data we have selected, the specified formula may not be completely in line with the actual situation. We will continue to improve the formula we use to make it more rigorous.

Reference

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[2] Yu, S. , Liu, J. J. , Yun, E. J. , Kwak, S. , Kim, K. H. , & Jin, Y. S. . (2018). Production of a human milk oligosaccharide 2′-fucosyllactose by metabolically engineered saccharomyces cerevisiae. Microbial Cell Factories, 17.
[3] Hollands, K. , CM Baron, Gibson, K. J. , Kelly, K. J. , & Rothman, S. C. . (2018). Engineering two species of yeast as cell factories for 2'-fucosyllactose. Metabolic Engineering, 52.
[4] Lee, J. W. , Kwak, S. , Liu, J. J. , Yu, S. , & Jin, Y. S. . (2020). Enhanced 2′-fucosyllactose production by engineered saccharomyces cerevisiae using xylose as a co-substrate. Metabolic Engineering, 62(2), 322-329.