Progress 07/01/22 to 02/28/23
Outputs
Target Audience:The principal target audience for this research is commercial producers and commercial consumers of aquaculture feeds. Xylome has been in contact with Green Plains Ethanol in Omaha, Nebraska concerning the formulation of aquaculture feeds from our lipogenic yeasts and with AquaBounty of Maynard, MA, which has production facilities in Albany, Indiana. Changes/Problems:The first problem we encountered was difficulty assembling the plasmid containing PKS subunit A. Our initial design split the ~8500bp gene sequence into eight fragments, which we planned to assemble using our Gibson Assembly based method. Unfortunately, fragment 7 could not be synthesized by Twist Biosciences (California, USA), and so had to be divided into a further 3 fragments. This delayed our attempts to assemble the complete plasmid. Additionally, some of our attempts at assembling the intermediate fragments (e.g., segments 1+2+3 are assembled before being combined with segments 4+5+6) have been unsuccessful. However, we are confident that we can complete assembly of the plasmid with PKS subunit A early in the second half of this project period. PKS subunit Awas ~8500bp long, and difficult to synthesize as a complete fragment. Initially, we divided the sequence into eight fragments (blue and green), each to be synthesized by Twist Biosciences and then assembled by the Xylome team. Twist Biosciences was unable to synthesize fragment 7, necessitating the division of that fragment into a further 3 fragments (orange) which are being assembled by Xylome scientists. The second problem we encountered was integrating the complete FasB & FasC cassette into L. starkeyi. Our method involves transforming linear DNA fragments into L. starkeyi, which are then integrated into the genome randomly via non-homologous recombination. The efficiency of transformation/integration decreases with the length of the linear fragment (Németh et al. 2021) and this ~16,500bp fragment is the longest we have ever attempted to integrate. We selected eight strains with antibiotic resistance to Nourseothricin (indicating at least some portion of the fragment had successfully integrated into the genome) and performed PCR amplification to determine whether the complete sequence was present. Unfortunately, we found the complete sequence was present in only 50% of cases. Although this isn't an issue for Phase I of this SBIR project, it will be important to consider as we move forward with optimizing expression levels of the PKS complex. We now know it will be necessary to screen many more transformants than we initially anticipated finding strains with the complete sequence of both PKS subunits B & C. This may also become an issue when we begin integrating the cassette containing FasA, however, because this fragment is much shorter (~11,500bp), we expect this issue to be less severe for FasA than for FasB & FasC. What opportunities for training and professional development has the project provided?Professional support personnel have learned how to perform DNA minipreps, DNA transformations, Site directed mutagenesis and Q-PCR. The professional staff - including the scientific leadershiip have developed and mastered the synthesis, construction and introduction of large synthetic constructs into a non-conventional lipogenic yeast How have the results been disseminated to communities of interest?No patents have been filed as yet, so discussions with potential end users of Xylome's novel high omega-3 aquaculture feed have been limited to to conceptural discussions. What do you plan to do during the next reporting period to accomplish the goals?In the next period we plan to complete construction of the plasmid with PKS subunit A. We plan to transform this expression cassette into L. starkeyi, and we will use qPCR to identify the strain with the highest expression of PKS subunit A and the strain with the highest level of expression of subunit B & C. We will then cross these two strains so that we have a strain with all three subunits expressed. Next, we will cultivate the mated strain in a shake flask or bioreactor, extract the resulting oil, and use gas chromatography to assay for the production long chain ω3 PUFAs. Going forward we will also assess newer methods for targeted integration, which may help with complete integration of segments and their expression.
Impacts
What was accomplished under these goals?
In this half of the project, we made progress towards most of our objectives. 1) The biosynthesis of long chain omega-3 fatty acids is not completely understood, but the final steps are carried out by a specialized polyketide synthase consisting of three subunits (Fig. 1). We designed, synthesized, and cloned a plasmid with two subunits (B&C) for expression into L. starkeyi (Fig. 2). We have synthesized all the necessary fragments to assemble a plasmid containing subunit A for expression in L. starkeyi (Fig. 3), but synthesis and assembly have been more difficult than expected (discussed below). We anticipate that subunit A will be ready for transformation early in the second half of the Phase I SBIR grant. 2) We transformed L. starkeyi with the cassette containing PKS subunits B & C. After screening transformants, we identified four strains with the complete sequences of both the B and C subunit sequences. To obtain strains with very high levels of expression, we expect to have to screen many more transformants, but to move forward with this project we will begin with this preliminary set of four strains. In the next half of this project, we will transform L. starkeyi with subunit A and cross the highest expressing subunit A strain with the highest expressing subunits B & C strain. 3) We have developed methods for cultivating L. starkeyi in both shake flasks and bioreactors that will enable us to obtain enough oil for lipid profile assessment using gas chromatography. In the next half of this project, we will explore conditions that improve production of long chain omega-3 fatty acids in L. starkeyi. 4) We will begin our assessment of PUFA levels by measuring the gene expression levels of each of the three PKS subunits. Once we have our highest expressing PKS subunit A strain, and our highest expressing B and C strain, we will cross those two strains to create a strain with FasA, FasB and FasC (the complete PKS complex). We will then cultivate the strain and extract oil using methods we developed previously and perform gas chromatography to compare the fatty acid profile of the original strain to our PKS expressing strain. We will look specifically for the increased production of C20:5 (EPA) and C22:6 (DHA) fatty acids. 5) Our goal for the end of the project is to identify a strain of L. starkeyi that produces long chain omega-3 fatty acids. We will do this through a combination of screening for high expression levels of the three subunits of the PKS synthase complex with qPCR, and by measuring production of EPA and DHA fatty acids by gas chromatography.
Publications
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Progress 07/01/22 to 02/28/23
Outputs
Target Audience:The target audience for this work consists of (1) Researchers in the field of omega-3 fatty acid production by yeasts and algae, (2) Aquaculture feed producers, (3) Terrestrial-based aquaculture producers, and (4) the General public. Changes/Problems:Change: In our initial proposal, we planned on expressing 3 PKS subunits. During the course of this project, we identified a fourth subunit essential for producing long chain omega-3 fatty acids. We designed and transformed L. starkeyi with this additional PKS subunit. Problem: One of the PKS subunits essential for PUFA production has a region with many repeated domains, making it difficult to synthesize. We had to split this subunit into several fragments for DNA synthesis, and then assemble the fragments using our Gibson Assembly based method. This delayed our ability to transform our yeast with all the necessary subunits for PUFA production. What opportunities for training and professional development has the project provided?Xylome scientists received training to use two different qPCR thermocyclers and associated software from the University of Wisconsin-Madison Biophysics Instrumentation Facility. We received training to operate a new gas chromatograph and a new GC analysis software system. We learned about interpreting GC/MS and lipidomics data from the UW Madison Biotechnology Center. How have the results been disseminated to communities of interest?We have shared information about our progress and results to potential strategic partners under mutual NDA agreements. What do you plan to do during the next reporting period to accomplish the goals?This is the final reporting period for this grant. In Phase II, we will perform GC analysis of the lipids produced by our best L. starkeyi and perform additional genetic engineering on our yeast to obtain higher omega-3 producing strains.
Impacts
What was accomplished under these goals?
We synthesized and cloned three subunits for polyunsaturated fatty acid (PUFA) polyketide synthase for expression in L. starkeyi. We identified, synthesized, and cloned a fourth subunit that we believe to be essential for PUFA production. We transformed L. starkeyi with four essential proteins comprising the PUFA polyketide synthase. We cultivated the transformed L. starkeyi strains under conditions that allowed us to perform mRNA expression analysis of each of the four subunits and will also allow us to assess PUFA levels in Phase II of this SBIR grant. We established methods that will enable Xylome scientists to assess PUFA levels quickly and efficiently. We identified the L. starkeyi strain with the highest mRNA expression levels of four PKS subunits essential for PUFA production.
Publications
- Type:
Websites
Status:
Published
Year Published:
2022
Citation:
https://www.xylome.com/solutions/aquaculture-feed/
- Type:
Websites
Status:
Published
Year Published:
2022
Citation:
https://www.globalaquachallenge.com/post/the-yield-lab-asia-pacific-global-aquaculture-challenge-xylome
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