Model

    To determine the ability of Y. lipolytica in producing 2-PE, Y. lipolytica po1g harboring empty plasmid pYXLP’ (stain po1g pYXLP’) was performed shaking flask in the fermentation medium CSM with adding different concentrations of L-phenylalanine (0, 2, 4, 6, 8, and 10 g/L). Shaking flask results (Fig. 2a, b, c) indicated that the highest titer of 2-PE reached to 720.43 mg/L with 0.342 g/g L-phenylalanine yield by adding 10 g/L L-phenylalanine, simultaneously generating 197.59 mg/L phenylacetate. However, consideration of mediocre bioconversion performances of Y. lipolytica and economic efficiency, 4 g/L was determined as the incipient concentration of L-phenylalanine in the following shaking flask cultivations (641.59 mg/L of 2-PE with 0.339 g/g L-phenylalanine yield). However, commercialization depends on lowering the feedstock cost, thus it requires improving the overall yield and productivity.

    Therefore, to systematically assess the Ehrlich pathway, we analyzed the stoichiometrics of 2-PE biosynthesis using L-phenylalanine as substrate (Dugar & Stephanopoulos, 2011; Gu et al., 2019; Xu, 2021), listed below:

    L-Phenylalanine + 2-Oxoglutarate + NADH.c -> 2-PE + Glutamate + NAD+.c + CO2 (1) Here, 2-oxoglutarate is synthesized by the central carbon metabolism with the glucose as substrate (Fig 1b):

    Glucose + 2ADP.c + 2NAD+.c -> 2-Oxoglutarate + 2NADH.c + +2ATP.c + CO2 (2) Thus, the overall stoichiometry of 2-PE biosynthesis is:

    L-Phenylalanine + Glucose + 2ADP.c + NAD+.c 2-PE + Glutamate + NADH.c +2ATP.c + 2CO2 (3) On the basis of the stoichiometry, producing 1 mol 2-PE will consume 1 mol of L-phenylalanine and glucose, with the formation of 1 mol cytosolic NADH, 1 mol of glutamate, 2 mol of cytosolic ATP and 2 mol of CO2. This overall stoichiometrics suggests that NADH is not the limiting factor of 2-PE production.

    Next, to rationally predict the engineering targets, a global stoichiometric model was established. Herein, for production of 1 mol of 2-PE, x mol of L-phe and y mol of glucose will be consumed. The final outcome of nitrogen metabolism in microbes should be some proteins or metabolisms with N elements. Herein, we assumed that the final outcome of nitrogen metabolism is proteins and the formula is (RC2H4O2N)n, the general linear formula of proteins (R represents the side chain). For the convenience of the following deduction, we further assumed that the R group is H. Thus, the overall stoichiometrics will be

    x L-Phe (C9H11NO2) + y Glucose (C6H12O6) + (8.5x+6y-10) O2 -> 2-PE (C8H10O) + x Proteins (C2H5O2N)n + (7x +6y-8) CO2 + (3x+6y-5) H2O (4)

    The yield of 2-PE (g/g L-phenylalanine) is

    Y2-PE=122/165x (5)

    Furthermore, introduction of the respiratory quotient (RQ):

    RQ=(7x+6y-8)/(8.5x+6y-10) (6)

    Solve the above equation (6) and substitute x into equation (5), we will get a general yield which depends on both RQ and the amount of glucose (y) consumed.

    Y2-PE=122/165 *(8.5RQ-7)/((6-6RQ)y+10RQ-8) (7)

    However, the above mathematical model with three unknown variables is obscure and esoteric. Thus, we decided to reduce the degree of freedom of the above model and divided the yield into two parts, namely L-phe consumption and 2-oxoglutarate supplementation.

    To deduce the boundary of above model (Eqn.7), we assumed that production of 1 mol of 2-PE will only require m mol of L-phe without glucose (y=0), which means L-phe is used as both N and C sources for cell maintenance and product production. Thus, the metabolic model was

    m L-Phenylalanine (C9H11NO2) + (8.5m-10) O2 -> 2-PE (C8H10O) + m proteins (C2H5O2N)n + (7m-8) CO2 + (3m-5) H2O (8)

    As a result, the yield of 2-PE (g/g L-phenylalanine) and the respiratory quotient (RQ) were Y2-PE=122/165m and RQ=(7m-8)/(8.5m-10), respectively. And, the yield of 2-PE could be solved as

    Y2-PE=122/165 *(8.5RQ-7)/(10RQ-8) (9)

    As Shown in the metabolic model (Fig. S1), Y2-PE increases as RQ increases, whereas RQ increases as L-phe consumption (m) decreases. High RQ value represents less L-phe is oxidized to maintain cell metabolism, and more L-phe is used to synthesize 2-PE through the Ehrlich pathway. In addition, the stoichiometrics (Eqn. 9) suggest that the theoretically maximum Y2-PE is 0.739 g/g L-phenylalanine. On the other hand, to account for the cofactor aKG, we assumed that RQ value is a certain value (β) and production of 1 mol of 2-PE will require m mol of aKG. Thus, the metabolic model was

((8+βa-5m)/7)L-Phenylalanine (C9H11NO2) + m 2-oxoglutarate (C5H6O5) + aO2 -> 2-PE (C8H10O) + ((8+βa-5m)/7) proteins (C2H5O2N)n +βa CO2 + ((3βa+6m-11)/7) H2O (10)

    According to mass balance of element O, ‘a’ could be further simplified, which is (29m+4)/(17β-7). As a result, the yield of 2-PE (g/g L-phenylalanine) is

    Y2-PE=122/(165*(8-5m+β*(29m+4)/(17β-7))/7) (11)

    If β=1, Y2-PE=122/(165*(1.2-0.3m)) (12)

    If β=2, Y2-PE=122/(165*(224+53m)/189) (13)

    If β=10, Y2-PE=122/(165*(1344-525m)/1141) (14)

    Analysis of the above equation (Fig. 2) indicates that increasing 2-oxoglutarate supplementation will significantly improve 2PE yield Y2-PE under all different β values. Thus, as suggested by the mathematical model, 2-PE yield is more sensitive to the supply of akG.

    Dugar, D., Stephanopoulos, G. 2011. Relative potential of biosynthetic pathways for biofuels and bio-based products. Nature Biotechnology, 29(12), 1074-1078.

    Gu, Y., Lv, X., Liu, Y., Li, J., Du, G., Chen, J., Rodrigo, L.A., Liu, L. 2019. Synthetic redesign of central carbon and redox metabolism for high yield production of N-acetylglucosamine in Bacillus subtilis. Metab Eng, 51, 59-69.

    Xu, P. 2021. Dynamics of microbial competition, commensalism, and cooperation and its implications for coculture and microbiome engineering. Biotechnol Bioeng, 118(1), 199-209.