With external help we expressed two HPPD enzymes for the production of HGA.
We compared the yield of the two recombinant strains.
We validated the results through Mass Spectrometry.
We outlined future steps for next year’s team.
COVID Impact
Due to the COVID-19 pandemic and the lockdown and distancing measures imposed as a result in the UK, we have been unable to work in the laboratory ourselves. As discussed above this has made it difficult to follow the DBTL model as we would have done under different circumstances.
In order to gain some laboratory results in the face of this
challenging situation we looked for alternative ways to carry out the experimental work.
We first attempted to perform the laboratory work ourselves with Dr Daniel Schindler, our supervisor, in his laboratory at the Max Planck Institute of Terrestrial Microbiology, in Marburg, Germany. However, this was impossible due to time constraints and the potential need to quarantine upon arrival in Germany and upon return to the UK.
We have been lucky in that our host institution, the Manchester Institute of Biotechnology (MIB) of the University of Manchester, and some of its members have been able to continue carrying out laboratory work during the pandemic and thus we were able to collaborate to produce the following results.
The Solution
We have worked closely with Dr Kris Nino Valdehuesa to design our
constructs, and with Dr Mark Dunstan and Dr Andrew Curin from SYNBIOCHEM, the Synthetic Biology Research Centre for Fine and Speciality Chemicals in the Manchester Institute of Biotechnology, who built and expressed them for us.
As mentioned, due to the COVID-19 lockdown measures in place between March and July, our ability to carry out experimental research in the laboratory had to be outsourced to our collaborators at MIB. Aditionally, we were under a time-constraint due to it being July by the time the laboratory reopened and even later when our experiments were able to be started. As a result of these constraints we had to narrow down our list of desired experiments and thus prioritised the production and validation of HGA, our predicted precursor of Hipposudoric Acid.
The enzymes cloned and expressed are two variants of 4-Hydroxyphenylpyruvate dioxygenase chosen from the output from our Pathway Design enzyme selection tool, Selenzyme. One of them is the variant from Pseudomonas aeruginosa (paHPPD) and the other the variant from Streptomyces averimilits (saHPPD).
The chassis used for expression was Escherichia coli strain DH5alpha grown in the following growth conditions. Overnight seed cultures were grown from freshly transformed colonies in the desired production media at 37 °C with shaking at 180 rpm. Seed cultures were inoculated 1/50 into fresh media (1.2 mL in 96-deepwell blocks with breathable seals) and grown at 30 °C, 950 rpm shaking, 75% humidity. When cultures reached OD600 = 1.5 they were induced with the addition of 100 µM IPTG, 3 mM substrate (tyrosine) and Ascorbic acid, as appropriate, then transferred back to the 30 °C shaker-incubator for 24 h. Cultures were processed for mass spectrometry analysis using a Hamilton Star robotic platform.
Lysogeny broth or Terrific Phosphate Broth phosphate buffered (TBP)
0.4 % Glycerol
Kanamycin
IPTG in the induced cultures
Table 1. Production of HGA in uninduced and induced cultures.
As seen in Table 1, the induced recombinant DH5alpha with the saHPPD gene produced the highest yields of HGA, when grown in the TBP media. The validation of the chemical identity of the product was achieved through Mass Spectrometry by comparing the compound produced by the bacteria with a standard, as seen in Figure 1.
Figure 1. Mass spectrometry data for the three replicates from the DH5alpha saHPPD recombinant strain, and the standard of HGA.
Samples were analysed on a triple quadrupole tandem mass spectrometer (Xevo TQ-S; Waters MS Technologies) connected to an Acquity Ultra Performance Liquid Chromatography system (Acquity UPLC; H-Class, Waters). HGA was stabilised by addition of ascorbic acid (reducing agent), but if the culture is left exposed to oxygen it starts turning red at 24h, and progresses through various shades of red to brown in the following days and weeks as seen on Figure 2.
Figure 2. (A) negative control; (B) paHPPD expression; (C) saHPPD expression after 2 weeks storage at 4 degrees; initially, the cultures turned red, indicating the formation of hipposudoric acid. Further polymerization proceeds slowly, and reduces the environmental harm caused by our product.
Future Steps
The experimental results outlined on this page and our modelling work come together to support our hypothesis that Homogentisic Acid is a promising precursor compound for the production of Hipposudoric Acid.
This year’s special circumstances have forced us to reduce our experimental assays, and have pushed us to become a Two-Phase Project. The experiments presented here were not carried out by our team members, but are due to the generous help of the researchers at our host institution. The following steps are clear:
Expressing other HPPD enzymes, and HPPD analogues found through our modelling to find the most efficient ones.
Purifying Hipposudoric Acid.
Characterizing the basic chemical properties of the compound.
Testing UV protective and anti-microbial properties of the produced compound.
Furthermore, a UV filter is only one component of a sunscreen, great consideration must go into choosing all the additional ingredients that will make up the sunscreen matrix included in the final product. Thorough testing must be carried out to ensure that the excipients do not undermine the UV-filtering effect as well as being non-toxic to aquatic life.
Additionally, sunscreens need to undergo several rounds of testing to be in accordance with both European and UK Laws and Regulations, and next year’s team will explore the necessary experimental protocols.
