Team:USTC

Gene transform and tag recycle

Design1

    The construction of MVA pathway in Yarrowia lipolytica requires multiple plasmids. In the case of very limited selection of tags, we must reuse the recyclable tag URA3. The original strain po1f lacked orotidine 5-phosphate decarboxylase ( encoded by URA3 ). After co-transforming URA3 with the functional gene into yeast, successful transformants will be allowed to grow on URA-deficient medium ( SD-URA ). After the target strain was identified, the yeast was transformed again using a knockout box (BBa_K4231011) based on homologous recombination mechanism, and the URA3 tag was deleted. The FOA plate will allow URA3-deficient yeast to grow, whereas the yeast with URA3 will be killed by 5-fluoroorotic acid ( FOA ). In this way, we will be able to obtain URA3-deficient strains with transferred foreign genes on FOA plates.

Figure.1 URA3 Label Recycling Solution

Test 1-learn 1

    However, due to the low natural homologous recombination ability of Y.lipolytica, our knockout box cannot work well, and the knockout efficiency is only about 2 %. This requires a lot of manual screening (e.g. Colony PCR) to obtain a true knockout. Extending the length of homologous arms can increase the knockout efficiency, but the length of URA3 gene is very limited. Multiple knockouts can increase the probability of successful in the screening of strains. In summary, the knockout box strategy has a lower ability to recycle URA3 when po1f is the starting strain.

Figure.2 Artificial screening of strains that really knock out URA3. (A) pick a single colony, streak inoculated on FOA plate ; (B) After re-scratching the colonies on the FOA plate, single colonies were picked and streaked on the SD-URA medium, numbered as it on FOA plate. As shown in the red circle above, the colony at 4-D can grow on the FOA plate, but cannot grow on the SD-URA plate, which is preliminarily determined to be a complete knockout of the URA3 gene. The other colonies that could grow on both plates may be caused by knockdown of the URA3 gene.

Design2

    According to the existing commonly used design, the addition of a 1kb-sized hisG homologous arm on both sides will cause the URA3 selection tag to be spontaneously and efficiently deleted after insertion into the yeast genome, while avoiding the need to transform the knockout box again. We tried to transform the URA3 selection tag with hisG homologous arm of Kluyveromyces marxianus and Candida tropicalis (provided by the instructor) into Yarrowia lipolytica. But because of promoter mismatch, the expression efficiency of these heterologous selection tags are poor, only a few colonies grow on SD-URA medium. NNU provided us with hisG-URA3-hisG designed for Yarrowia lipolytica (BBa_K4343027). It has been verified that this tag is credible in ensuring the transfer of the target gene.

Figure.3 hisG-URA3-hisG Label Recycling

Figure.4 ( A ) Y.lipolytica transformed with hisG-URA3-hisG selection tag provided by NNU could grow normally on SD-URA plate ; ( B ) The hisG-URA3-hisG selection tag of K. marxianus ( pKmU1 ) cannot induce colonies on SD-URA plates ; ( C ) positive control, the yeast transformed plasmid was spread on YPD plate, plasmid transformation does not affect the normal growth of yeast. After verification, the plasmid provided by NNU is available in proving the transfer of the target gene.

Test2-learn2-design3

    Unfortunately, since the spontaneous deletion of URA3 caused by hisG arm still depends on homologous recombination, the application of this strategy can only slightly increase the knockout rate of URA3 to 14 %, which is difficult to solve the demand for manual screening. The traditional method to increase the homologous recombination ability of Yarrowia lipolytica is to knock out the ku70 gene, but we abandoned this method due to the disruption of the stability of the yeast genome and the extremely low knock-out efficiency that we have verified. In view of the high homologous recombination efficiency of Saccharomyces cerevisiae, we plan to introduce the homologous recombination enzyme ( ScRad52 ) of S. cerevisiae into Y. lipolytica.

Test3-learn3

    Sustained high expression of ScRad52 also poses the same problem as knocking out ku70. Most seriously, the recombinase initiates the recombination of identical parts ( such as promoters ) between different transcription units of the foreign gene, starting with the transformation of the second plasmid. Simply put, the transformed second plasmid will become the ' knockout box ' of the first plasmid. The transfer of the next target gene often results in the loss of the gene transferred in the previous step. Therefore, we need to control the insertion of exogenous DNA in Yarrowia lipolytica, so that it mainly relies on non-homologous end joining when inserting the target gene, and mainly relies on homologous recombination when transferring into the knockout box.

Figure.5 Since the promoters and terminators on different plasmids are identical, under the action of ScRad52, the promoter and terminator of the new transcription unit ( gene2 ) act as homologous arms to knock out the previously transformed gene ( gene1 ), resulting in the invalidation of the previous transformation.

Figure.6 It can be seen in the figure that in the identification of colony PCR, the four colonies we selected mostly resulted in the previously transformed genes (ERG10 and ERG13) being knocked out by the subsequent transformed genes (ERG8 and ERG19) due to the strong homologous recombination ability caused by ScRad52.

Design4

    A UAS sequence induced by oleic acid will be added before the promoter TEFin (BBa_K4231016) that initiates ScRad52 expression, composing an improved inducible promoter A1-R1-A3-TEFin. In the expanded culture before transformation, we used glucose as the sole carbon source to prepare the medium. The expression of the recombinant enzyme ScRad52 in Saccharomyces Cerevisiae was shut down, and the homologous recombination efficiency was greatly reduced. The gene was mainly integrated into the genome by NHEJ method. Oleic acid will be used as the sole carbon source to induce the expression of ScRad52 before transforming the knockout cassette of URA3 or to induce the spontaneous recombination of hisG, resulting in a 90 % homologous recombination efficiency and the recovery of the URA3 tag.

Figure.7 Linking A1-R1-A3 fragments by overlap-PCR

    SCUT provides us with the sequence of the blue light-controlled transcription factor of Pichia pastoris. We can apply the light control mechanism to regulate the expression of recombinant enzymes after verifying that the transcription factor can also be used in closely related Yarrowia lipolytica.

Figure.8 Regulated homologous recombination

    Due to the limitation of competition time, we have not transfer the controlled expression of recombinase in yeast; however, according to the induction ability of the reported regulatory sequence, it must be effective for the expression control of ScRad52. We will continue to test and improve our efficient gene transformation - tag recycle mechanism after the competition.

fermentation condition optimization

Design1

    In order to apply our fermented borneol to the medical field, high purity and high yield are necessary. Changing fermentation conditions is the best way to meet the demand without complex genetic manipulation. At first, in order to maintain the single strain and the controllable fermentation conditions, we chose the synthetic SC-LEU-URA medium ( based on the synthetic SC complete medium, leucine and uracil were deficient ). Only the successfully constructed engineering yeast can grow normally in this medium.

Test1

    The culture after 3 days of fermentation was extracted with 1-fold volume of ethyl acetate and centrifuged at 12,000 rpm for 20 min. The extracted organic phase was analyzed by GC-MS. Unfortunately, there seems to be no or very little borneol produced in the fermentation broth.

Figure.9 In the red box of the GC image above ( the possible elution time section of borneol ), there is no chromatographic peak that can be identified.

Learn1-design2.1-design2.2

    Since the SC medium can only meet the basic needs of yeast growth, the maximum number of engineered yeast is low, resulting in a fermentation level that is far from meeting the needs of factory drug production. In order to meet the needs of large-scale fermentation after the correct yeast population is expanded to a certain scale, we resuscitated the preserved glycerol yeast in the SC-URA-LEU medium to OD600 = 0.2, and then inoculated the yeast solution into the more nutritious YPD medium. Shake flask fermentation. Considering the close relationship between leucine and acetyl-CoA metabolism, we transferred the resuscitated yeast solution to a larger SC medium and supplemented the leucine solution before fermentation.

Test2-learn2

Figure.10 Figure ( A ) : 72 hours of PYB1 strain cultured in different medium in shake flasks ( 1 ) SC medium + glucose ( 2 ) YPD medium + oleic acid ( 3 ) SC medium + oleic acid ; Figure ( B ) : Turbidity after 72 hours of fermentation in different medium

Figure.11 At 13.23 minutes, the elution peak of suspected borneol appeared. Analysis of mass spectrometry images showed that there was a quantitative ion peak of m / z ≈ 95 in the corresponding time period, which may be related to the presence of borneol.

    According to the turbidity of the fermentation broth shown above, our nutrient-rich strategy has played a certain role in increasing the production capacity of borneol; in the GC-MS result graph, we also received positive feedback. But this was far from the intended purpose.

Design3

    According to the metabolic characteristics of Yarrowia lipolytica, we replaced the carbon source in the medium from glucose to oleic acid, according to the carbon content. Fatty acids can intercept part of the volatile borneol ; at the same time, the use of fatty acids as carbon source can increase the metabolic flux of acetyl-CoA in yeast peroxisomes, thereby promoting the MVA pathway. We tried to replace carbon sources in SC medium and YPD medium.

Test3

    Carbon source substitution did bring metabolic benefits ( the figure in test2 ). The improvement effect of oleic acid as carbon source on SC medium was very obvious. For YPD medium, it has no obvious effect on promoting cell proliferation, and even it is not as good as of YPD with glucose. On the one hand, glucose is a more convenient carbon source for microbial utilization; on the other hand, in the case of sufficient nutrition, oleic acid will initiate the production of more secondary metabolites ( such as borneol ), and its metabolic pressure leads to the delay of strain growth.

    We found very serious problems when using YPD medium. In some shake flasks, borneol cannot be produced at all.

Learn3

    We measured the growth curves of the original strain po1f and the modified strain pyB1, and found that the production of borneol brought a series of metabolic pressures to the engineering yeast, resulting in its inability to effectively expand the population at the initial proliferation stage, and eventually replaced by strains that did not produce borneol. The YPD medium allows the growth of untransformed strains, so we need pure culture before fermentation. However, due to the scale of fermentation and spontaneous degradation of strains, it is almost impossible to obtain a large number of pure culture of high yield borneol strains.

Figure.12 As can be seen from the growth curve in the figure above, po1f, which does not produce borneol, has a distinct advantage over PYB1, which can produce borneol, in terms of growth rate in YPD medium. When the strain isolation is impure or the strain itself degenerates, the wrong strain will dominate the population during the large-scale fermentation phase, resulting in abnormal production of borneol.

Design4

    In order to ensure that the modified pyB1 can occupy the advantage in the fermentation broth, we chose the SC medium with relatively controllable components. In the preparation of SC medium, we reduced the composition of calcium pantothenate. By removing pantothenic acid during the population expansion phase, the non-essential acetyl-CoA metabolic pathway in engineered yeast is closed, thereby reducing its metabolic disadvantage. In this stage, glucose, which requires less coenzyme A in the metabolic process, is used as the carbon source. After the yeast was amplified, the medium was removed by centrifugation, and the cells were resuspended in SC medium with new oleic acid as carbon source, and pantothenic acid was supplemented to initiate the metabolism of MVA pathway. Pantothenic acid as a metabolic switch greatly ensures the advantages of engineered yeast in the population and the adequate supply of acetyl-CoA in the subsequent fermentation process.

Figure.13 In the logarithmic growth phase of the expanded cell number, the production of borneol was closed by limiting the supply of pantothenic acid, thereby ensuring that the PYB1 engineered yeast was dominant in the final fermentation population; after completion of cell proliferation into the stationary phase, adding pantothenic acid to start the production of borneol.

    Since one fermentation takes too long ( 2-3 days ), we are limited to partially validate all our fermentation strategies. And the use of more controllable culture conditions bioreactor can better enhance the yield of borneol. We will further complete the exploration of fermentation conditions later.

Summary

    In order to construct a strain for producing borneol and improve its production capacity, we have achieved engineering success in the gene operation and fermentation conditions of Yarrowia lipolytica. After many times of design, testing, learning and improvement, we have explored a set of production schemes for high-purity terpenoids. This will inspire future work.

    In addition, we have also achieved engineering success in yeast colony PCR and the construction of large fragment plasmid. See it in the result. (Results)