Team:Worldshaper-Nanjing/Design

Worldshaper-nanjing Design

Summary

For the last two decades, the biodiesel attracted increasing attention as a promising biofuel to replace fossil diesel. In this project, our main goal is to use raw starch from the substandard grains as the fermentation substrate, to produce biodiesel. In this way, it is expected to achieve sustainable development and save finance in the different field. Details as follows.

    • Yarrowia lipolytica

      1. To achieve the goal of turning waste (substandard grains) into treasure (biodiesel), the first thing to consider is to choose a suitable model host. Y. lipolytica is an oleaginous yeast (Figure 1a), which is rapidly emerging as a promising host for the production of biodiesel due to the fast growth rate, high precursor acetyl CoA content and oil production capacity. In addition, another main reason we selected this host is that Y. lipolytica is considered non‐pathogenic and has been classified as Generally Regarded As Safe (GRAS) by the American Food and Drug Administration (FDA)[1]. Based on these factors, the oil biosynthesis genetic information of Y. lipolytica was deeply mined (Figure 1b) and the related efficient genetic tools including TALEN, URA Blaster and CRISPR/Cas9 have been developed fast, which significantly accelerate the rate of the strain construction by metabolic engineering strategies[2-4].

        Technological developments have made Y. lipolytica the cell factories for high-level production of biodiesel. For example, overexpression of acetyl-CoA carboxylase and diacylglycerol acyltransferase1 can significantly improve the oil content. Combined the disruption of lipid degradation pathway with the optimization of fermentation process, Y. lipolytica can accumulate up to 90% of the lipid in the cell, far exceeding other microorganisms[5].


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          Figure 1 Cell morphologies (a) and lipid biosynthesis pathway (b) in Y. lipolytica

        However, Y. lipolytica lacks the capacity to degrade raw starch (the main content of substandard grains), and thus pre‑treatments and expensive enzymatic are needed, including steaming, ɑ-amylase liquefying, and then acid hydrolysis or glucoamylase treatment, etc. The fermentation process is complicated and the energy consumption is large. Thus, we want to construct engineered Y. lipolytica to use directly raw starch as the fermentation substrate, which was expected to simplify procedures and reduce energy consumption.


    • Carbon Source Utilization Capacity Predication of Y. lipolytica

      1. In the first half of 2020, due to the epidemic, we were unable to carry out offline experiments, so we first used mathematical modeling to predict the feasibility of our ideas, i.e. make Y. lipolytica directly utilize starch.

        The enzyme-constrained model of Y. lipolytica ec_iYLI647 was constructed to predict the carbon source utilization capacity. There were 36 kinds of carbon sources, which were estimated respectively, by setting the lower bound value of the corresponding exchange reaction as -1000 mmol·gDW·h-1. According to the simulation results, 29 kinds of carbon sources could be used by Y. lipolytica, including 13 kinds of amino acids. When using glucose as a carbon source, the growth rate of Y. lipolytica was 0.1610 h-1. The growth rate value was the maximum value, which means that glucose was the optimum carbon source of Y. lipolytica. In addition, when using starch as a carbon source, the growth value was 0.1508 h-1 (Figure 2), which was only 6.34% lower than the value cultured by glucose. These results provided the theoretical evidence for further modification of Y. lipolytica to utilize starch.


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          Figure 2 The predict results of the carbon source utilization capacity of Y. lipolytica of ec_iYLI647 model.

    • Biobricks design for raw starch degradation

        1. Based on the predication results of the above model, the α-amylase and glucoamylase genes from Aspergillus niger were introduced into Y. lipolytica genome to meet the requirement for the complete hydrolysis of raw starch.

          The α-Amylase (AnAmyA) is an endoglycosidase that randomly acts on the α-1,4 glycosidic bond to cut the glucose unit. Comparatively, glucoamylase (AnGlu) can not only cleavage α-1,4 glycosidic bonds but also slowly cleavage α-1,6 glycosidic bonds. Thereby the starch is crushed into glucose, which can be used by Y. lipolytica. As shown in Figure 3, the starch degradation system was designed. These are the gene expression elements in Y. lipolytica, including EXP promoter, glucoamylase gene, α-amylase genes, XPR2t terminator as well as Leu selection marker. We first separately constructed the α-amylase and glucoamylase expression units to test their ability to degrade starch. Finally, the combined expression of α-amylase and glucoamylase will accelerate the utilization of starch by Y. lipolytica.


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            Figure 3 Schematic diagram of the starch decomposition system design

          Starch decomposition 1.0

          AnGlu Expression Unit (BBa_K3578010)

          Our AnGlu expression unit has four parts: EXP promoter, glucoamylase gene, XPR2t terminator, and Leu selection marker (Figure 4). EXP (BBa_K3578000), XPR2t (BBa_K3578002) were amplified from Y. lipolytica genome. Glucoamylase gene (AnGlu, BBa_K3578001) was derived from A. niger to degrade the starch. Leu (BBa_K3578004) selection marker is a β-isopropylmalate dehydrogenase, which can rescue the leucine auxotroph in Y. lipolytica Po1g and therefore serve as the selection marker. This cassette can be transformed into the Y. lipolytica protoplast and integrated into the genome for efficient AnGlu expression to improve the degradation ability of starch.


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            Figure 4 Schematic diagram of AnGlu expression unit.

          AnAmyA Expression Unit (BBa_K3578011)

          As shown in Figure 5, the main parts involved in the AnAmyA expression unit are the same as those in the AnGlu expression unit, with the exception of the α-amylase gene (AnAmyA, BBa_K3578003), which is also derived from A. niger. Similarly, this cassette can also be separately transformed into the Y. lipolytica protoplast and integrated into the genome for AnAmyA efficient expression to improve the degradation ability of starch.


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            Figure 5 Schematic diagram of AnAmyA expression unit.

          Starch decomposition 2.0

          Combined Expression system

          Considering that the experiment cleaved by glucoamylase and alpha-amylase are significantly different, and there are both amylose and amylopectin in actual grains, we have designed a co-expression system for these two enzymes (Figure 6). It is expected that the co-expression system can further improve the ability and efficiency of Y. lipolytica to degrade starch.


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            Figure 6 Schematic diagram of Combined Expression Unit.


References

  1. Nicaud JM. Yarrowia lipolytica. Yeast, 2012, 29(10): 409-418.
  2. Schwartz, CM., Hussain, MS., Blenner, M., Wheeldon, I. Synthetic RNA polymerase III promoters facilitate high-efficiency CRISPR/Cas9-mediated genome editing in Yarrowia lipolytica. ACS Synthetic Biology, 2016, 5: 356−359.
  3. Gao, S., Tong, Y., Zhu, L., Ge, M., Zhang, Y., Chen, DJ., Jiang Y., Yang S. Iterative integration of multiple-copy pathway genes in Yarrowia lipolytica for heterologous β-carotene production. Metabolic Engineering, 2017, 41: 192-201.
  4. Rigouin, C., Guéroult, Marc., Croux, C., Dubois, G., Borsenberger, V., Barbe, S., Marty, A., Daboussi,F., Andre, I., Bordes, F. Production of medium chain fatty acids by Yarrowia lipolytica: combining molecular design and TALEN to engineer the fatty acid synthase. ACS Synthetic Biology, 2017, 6(10):1870-1879.
  5. Blazeck, J., Hill, A., Liu, L., Knight, R., Miller, J., Pan, A., Otoupal, P., Alper, HS. Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production. Nature Communications, 2014,5: 3131.

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