Team:Jiangnan China/Experiments
To alleviate the coral bleaching situation caused by chemical sunscreen pollution, we planned to produce an eco-friendly bio-sunscreen this year. First, in the part of Design, we will systematically explain the ideas of experimental design, including the choice of chassis organism, and how we engineered the metabolic pathway to transform the chassis into a cell factory at the molecular level. Second, we sorted out a file of all general steps we did, including molecular biology experiments, fermentation and product quantification & qualification analysis in the part of Protocol. Next, the Results of our project are demonstrated, and we will also discuss the Future Works at the end of this page.
Since the natural origin of the substance Gadusol, what we wanted to produce, was zebrafish eggs, we first excluded prokaryotic chassis and chose eukaryotic chassis, and because we wanted to use it for cosmetics production, we chose Saccharomyces cerevisiae as the starting strain after inquiring the Measures for Genetic Engineering Safety. In addition to the heterologous expression ability of vertebrates' substance and safety issues, yeast also has irreplaceable advantage in cosmetics production. It not only reduces the pollution from chemical reagent extraction, but also reduces the production costs of purification since cosmetics ingredients produced by Saccharomyces cerevisiae could be added into cosmetics in the form of yeast lysate.
Gadusol is bio-synthesized by two enzymes: EEVS and MT-Ox with sedoheptose 7-phosphate (S7P) as the substrate (Fig.1). However, its natural production is quite low: On average, 1000 eggs could only extract about 2 mg Gadusol. Moreover, the output of Gadusol is strictly limited by the time of fertilization, but the zebrafish eggs will usually hatch into juveniles in 2~3 days in the appropriate temperature and the output of Gadusol will decrease dramatically due to the decreasing enzymatic activity in this progress, which means that producing the Gadusol sunscreen in a large-scale by natural extraction is not only limited by its low natural production, but also inhumane and will bring some negative effects on the zebrafish population. Therefore, using engineered S. cerevisiae to produce Gadusol is a feasible and relatively low-cost industrialization pathway.
Fig.1 Biosynthesis Pathway of Gadusol
First, the Gadusol synthetic pathway was needed to be introduced into S. cerevisiae. In our wet lab experiments, we first performed codon optimization of the EEVS and MT-Ox genes for better expression in S. cerevisiae. Afterwards, the recombinant EEVS and MT-Ox genes were cloned into the high-copy plasmid pYEP352 for efficient expression (Fig.2). Finally, the pYEP352 vector containing the EEVS and MT-Ox genes was transferred into the S. cerevisiae BY4743 strain for the production of Gadusol.
Fig.2 The Expression Cassette of Gadusol Synthetic Pathway
However, the precursor of Gadusol we mentioned above, S7P, has a low accumulation in S. cerevisiae. Therefore, to increase Gadusol production, the S7P pool must be expanded by appropriate regulatory strategies.
One of the most direct method is to regulate the important metabolic enzyme expression in the HMP pathway, but this way needs to both upregulate the expression of S7P synthesis enzymes and downregulate the expression of S7P consumption enzymes, or even knockout those related genes to cut off its consumption. However, it may cause the imbalance of co-factors in yeast, and affect the generation of aromatic amino acids. In order to eliminate the above negative effects, the expression regulative intensity of those genes needs to be very precise. However, S. cerevisiae itself has a low dependence on the HMP pathway and a low proportion of glucose entering into the HMP pathway, which means no matter how we regulate genes' expression, the amount of S7P will not significantly improved because of its limitation by glucose flux.
Therefore, we propose to introduce a more efficient carbon source for the HMP pathway in S. cerevisiae. After comparison, we finally chose a cheaper carbon source, which can not only effectively improve S7P supplement, but also has economic potential: xylose.
Saccharomyces cerevisiae can obtain the ability to utilize xylose by introducing Xylose Isomerase (XI) or Xylose Reductase / Xylitol Dehydrogenase (XR/XDH) ways. Among them, XI is an enzyme presenting in most xylose-consuming bacteria with the advantage of eliminating co-factor imbalance during xylose fermentation. However, due to the low enzymatic activity of XI expression in yeast, the XR/XDH pathway is more suitable to be introduced in S. cerevisiae.
In the XR/XDH pathway, xylose reductase (XR) first transforms xylose to xylitol, and xylitol dehydrogenase (XDH) further converts xylitol to xylulose. Xylulose will be converted to xylulose 5-phosphate (X5P) by the native xylulose kinase (Xks) of S. cerevisiae. X5P is also an intermediate in the HMP pathway and will eventually be converted into S7P by Transketolase (Tkl1).
Therefore, the introduction of XR/XDH pathway is an effective strategy to improve the metabolic flux of HMP pathway and improve S7P accumulation.
However, given the low promoter intensity and low enzyme expression of native xylulose kinase (Xks), after introducing the XR/XDH pathway, it is still not likely to obtain sufficient X5P and that will further result in low gadusol production and metabolic disorders in S. cerevisiae (due to the xylitol has certain cytotoxicity). Thus, we decided to perform heterologous expression of XK simultaneously (you can see the modified metabolic pathway in Fig. 3).
Fig.3 The Metabolic Network After Introducing Gadusol Synthetic and Xylose Utilization Pathway (Xyl1, Xyl2 and Xyl3 Respresent XR, XDH and XK, Respectively)
Considered that XR and XDH will use different cofactors (NADPh and NAD+ respectively), it is likely to aggravate the imbalance of NADP+ and NAD+ in Saccharomyces cerevisiae and lead to growth disorders if we express those 3 genes with high copy and high initiation intensity vector. Therefore, to obtain a yeast using xylose without affecting its own growth, we first chose low copy plasmid pY15TEF1 as the vector of xylose utilization genes (Fig.4).
Fig.4 The Expression Cassette of Xylose Utilization Pathway
This year, our experiment includes Molecular Biology Experiments, fermentation and production quantification & qualification analysis, you can obtain the protocol file below:
1.The Production Quantification & Qualification Analysis
First, under the help of Laboratory of Functional Molecule and Health, a laboratory focusing on production and application development of value-added natural products, we addressed the problem that there are no standard products available currently. Through the method proposed by Andrew R Osborn et al in De novo synthesis of a sunscreen compound in vertebrates, we obtained some Gadusol sample with high purity (Fig.5), and we also tested the analysis methods used in the above publication.
Fig.5 The HPLC Result of Gadusol Sample We Obtained
Currently, we have completed the construction of all the S. cerevisiae strains required for Gadusol biosynthesis. Fermented by different YNB media respectively (Fig.6), we found that the Gadusol production of S. cerevisiae strains only containing the EEVS and MT-Ox genes was extremely low, about 40 mg/L (Fig.7), confirming our conjecture that the accumulation of precursor S7P was extremely low that cannot meet the requirement for large amounts of synthesis during the experimental design stage. The Gadusol production increased after the import of XR and XDH (about 110 mg/L), and after introducing three xylose-related genes involved in XR, XDH and XK, the production further reached at 170 mg/L. It's about 4.25-fold more than the strain only has Gadusol synthetic pathway (Fig.8), and equal to the Gadusol production from 85,000 zebrafish eggs.
Fig.6 All YNB Media Used in Experiments
Fig.7 The HPLC Result of Gadusol Obtained from the S. cerevisiae only Contains the Gadusol Synthetic Pathway
Fig.8 The HPLC Result of Gadusol Obtained from the S. cerevisiae Introduced Xylose Utilization Pathway
2.The Growth Curves of Strains
At the same time, we found that the OD600 value of S. cerevisiae increased significantly after the Gadusol synthetic pathway was introduced into (Fig.9), while that of the starting strain and strain containing empty plasmid were around 1.8 in YNB medium. It is probably due to the influence of MT-Ox that may affect some other methylation reactions in yeast or the influence of Gadusol that may have some substrate facilitation. But this phenomenon is greatly conductive to the subsequent large-scale fermentation production.
Fig.9 The Growth Curves of Three Yeast Strains
In addition to this, we have already achieved highly efficient fermentation (The OD600 value was up to 23 at the stationary phase) using xylose as the only carbon source, which is beneficial for production improvement and costs reduction (Fig.10).
Fig.10 The Growth Curves of Two Strains Using Xylose as the Only Carbon Source
Influenced by the COVID-19 epidemic out-broke in Jiangsu Province this year, our wet lab experiments had been suspended for nearly 3 months. Although we have now achieved considerable results, we still plan to accomplish other works to obtain higher production and make a complete sunscreen formula in future.
1.Future Plan for Production Improvement
In the completed experiments, we found that strains containing both the Gadusol synthetic pathway and the xylose utilization pathway consisted of 3 genes not only achieved the highest Gadusol yield on xylose YNB medium, but also the highest yeast concentration (OD600max=23). It proves that this strain has potential to be further engineered, and the production is likely to be further increased if we use fermentation medium with richer nutrition and growing factors in a lager fermentation system.
To further engineer the strain in molecular level, we plan to upregulate the expression of transketolase (Tkl1), and downregulate the expression of or knockdown the transaldolase (Tal1). As we mentioned in Design part, this strategy cannot improve Gadusol production before we introduced the xylose utilization pathway because the glucose flux in HMP pathway of S. cerevisiae is too low. However, since we have obtained an efficient xylose utilization strain that has higher metabolic flux in HMP pathway, it could be a promising strategy to further increase the production (Fig.11).
Fig.11 Biosynthesis Pathway of Gadusol
It was verified by our Metabolic Flux Model that we used to predict some possible effects of molecular engineering on the final production: in the condition of high Tkl1 expression intensity, it could further increase the production of Gadusol by knocking down Tal1. So, we will next knockout the Tal1 gene and find a suitable promoter to replace the native one for upregulating the expression of Tkl1.
However, to achieve the molecular engineering success above, fermentation optimization must be done as well. First, we need to find an optimum proportion of glucose and xylose. This is because, although this strain's ability to utilize xylose has slightly exceeded glucose, and xylose is cheaper than glucose, which may contribute to a lot of potential applications. For example, ethanol large-scale production is more willing to use xylose as the only carbon source. However, in our project, we prefer that xylose can be converted into HMP pathway to produce more valuable Gadusol, than to provide energy for strain growth. Besides, Gadusol is mainly generated during the stationary phase in yeast, which starts almost 20 hours earlier in glucose media than xylose. According to previous report, the ability of utilizing glucose will not affect by xylose. Thus, glucose can be used to provide energy for strain growth and shorten logarithmic phase while xylose can be used to produce Gadusol. We hope to calculate the lowest addition of glucose with a fixed total amount of carbohydrates by wet lab and modeling.
After the optimum proportion was confirmed, we will change YNB medium into fermentation medium that is no growth factor restriction, and enlarge the fermentation system into 5 L fermentor.
By doing this, we can achieve reducing industrial costs, improving productivity and output of Gadusol and finally bringing great returns for the Gadusol sunscreen producer simultaneously.
2.Future Plan for Sunscreen Formula Completion
We also learned that the current SPF/PA indicators used in sunscreen evaluation are not reasonable. However, the film formation of sunscreen is the essential factor in deciding whether the sunscreen can effectively protect us against UV rays. Film formation is closely related to the sunscreen formula. We will ask Prof. Wang from National Medical Products Administration (NMPA) Key Lab for Cosmetics Monitoring & Evaluation to customize a suitable procedure for our Gadusol bio-sunscreen in the future to achieve a better performance.
