Team:ShanghaiFLS China/Results

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Results

Results and Analysis

The main indicators of the GFP production efficiency in our experiment shall be the total GFP production per gram methanol during the 108 h incubation period and the measured unit cell GFP production during the short-term methanol induction period, as the former demonstrates the overall conversion rate from the substrate to the desired product over a fairly extended period, while the latter demonstrates the instantaneous expression efficiency of the construct after stabilization in the methanol media.

For the pGMP1-P_AOX1-GFP strain, it is evident from the data that during the 108 h incubation period, the expression efficiency of the construct started roughly at the same level as pAOX1-GFP but eventually surpassed the control. It is rather ambiguous whether the conversion rate from methanol to GFP in the pGMP1-P_AOX1-GFP strain after the 108 h incubation period is higher or lower than the control, yet during the short-term methanol induction period, the pGMP1-P_AOX1-GFP strain clearly has a much higher GFP expression level. This shows that pGMP1-P_AOX1-GFP does have a higher expression efficiency compared to the wildtype overall.

When it comes to the pAPM1-P_AOX1-GFP strain, it is observed that of the two pAPM1-P_AOX1-GFP constructs, pAPM1-P_AOX1-GFP 1 exhibited very strong methanol induced GFP expression and its unit cell GFP expression surpassed that of P_AOX1-GFP at virtually every time point during the 108 h incubation period. In terms of total GFP production per gram methanol during the 108 h incubation period, both pAPM1-P_AOX1-GFP strains are at least at the same level as (pAPM1-P_AOX1-GFP 2) or a much higher level than PAOX1-GFP (pAPM1-P_AOX1-GFP 1). During the short-term methanol induction period, however, both strains have exhibited a unit cell GFP production at the same level as (pAPM1-P_AOX1-GFP2) or a much lower level than P_AOX1-GFP (pAPM1-P_AOX1-GFP1). In an overview, however, pAPM1-P_AOX1-GFP does have higher expression efficiency compared to the wildtype.

We fairly recently obtained our pGMP1-pAPM1-P_AOX1-GFP constructs and need to conduct more experiments to draw more conclusive results. In general, to yield more accurate data, incubation over a further extended period in a more stable system (e.g. week-long chemostat incubation) and the substitution of GFP with a more stable reporter might be necessary.

Conclusions and Discussions

In general, our tested constructs do show a higher efficiency of methanol consumption compared to the wild type construct (P_AOX1-GFP). Of the four constructs tested, a 120% increase in total GFP production per gram methanol is exhibited pAPM1-P_AOX1-GFP in comparison to P_AOX1-GFP.

For pGMP1-P_AOX1-GFP, the reason for the protracted upregulation of P_AOX1 may lie in the modified regulatory pathway itself. During the initial stages of methanol induction, P_PRM1 is the major promoter being upregulated. Given that the homogeneous P_PRM1-PRM1 cassette is unaltered and the introduced PRM1 is not expressed by P_PRM1, it is plausible that during these initial stages of methanol induction, P_AOX1 is activated to approximately the same level in both pGMP1-P_AOX1-GFP and P_AOX1-GFP. In the later stages of methanol media incubation, however, P_MIT1 begins to be activated by Prm1, which means that while the homogeneous Mit1 is upregulated to about the same level as in P_AOX1-GFP, the heterogeneous Prm1 is also upregulated. Moreover, whereas the homogenous expression of Prm1 is later suppressed by Mit1, the heterogeneous expression of Prm1 remains to be self-upregulated since P_MIT1 is not inhibited like P_PRM1. In the end, the expression of both the Prm1 and Mit1 is much upregulated in pGMP1-P_AOX1-GFP compared to that of PAOX1-GFP.

For pAPM1-P_AOX1-GFP, on the other hand, the ostensibly self-contradicting results from the 108h incubation period and the short-term methanol induction period can be explained by the potentially cyclical nature of the modified regulation. In the initial stages of methanol induction, P_PRM1 is activated, leading to an upregulation of both the homogeneous Prm1 and the heterogeneous Mit1, which is further amplified by the upregulation via Prm1 (Prm1 activates BOTH P_PRM1 promoters AND the homogeneous P_MIT1 promoter). This overall leads to a very strong activation of the P_AOX1 promoter, which corresponds to a much higher expression level of GFP compared to P_AOX1-GFP. But later on, due to the accumulation of the over-expressed Mit1, the expression of both the homogenous Prm1 and the heterogeneous Mit1 are strongly suppressed, and consequently the expression of the homogenous Mit1 is also lowered as there's no Prm1 to activate it. This results in a much more suppressed activation of P_AOX1 in pAPM1-P_AOX1-GFP compared to P_AOX1-GFP. Our data does reflect this process: during the 108 h incubation period, Mit1 is much over-expressed and leads to a very strong activation of P_AOX1. Yet later in the short-term methanol induction period, P_AOX1 activation is very suppressed due to the compound effect of the suppression of P_PRM1 by Mit1 and the degradation of Mit1. Nevertheless, after Mit1 is sufficiently degraded to derepress _PPRM1, the over-expression should take place again. If the incubation is extended long enough, we may observe a much higher GFP expression level overall. Taken together, these results suggest that a co-culture of pGMP1-P_AOX1-GFP and pAPM1-P_AOX1-GFP may yield the highest methanol conversion rate throughout the incubation process.

Measured and calculated data during the 108 h incubation period. (a) Total methanol consumption during the 108 h incubation period. (b) Measured cell concentration curves during the 108 h incubation period, where concentration Is expressed by OD600. (c)Measured unit cell GFP production curves during the 108 h incubation period, where the measured unit cell GFP production is the ratio of the measured GFP fluorescence intensity to the measured cell concentration expressed by OD600. (d) Aggregate unit cell GFP production accounts for the degradation of the GFP protein based on its reported half-life of ~7 h in yeast (Mateus & Avery, 2000). This shall be a more accurate indicator of total product production by per unit cell. (e) Total GFP production curves during the incubation period, where total GFP production is the product of the measured cell concentration and the aggregate unit cell GFP production. This shall reflect the total production capacities of an incubation system. (f) Total GFP production per gram methanol of each incubation system, where the total GFP production per gram methanol is the ratio of the total GFP production at the 108 h time point to the measured total methanol consumption. This is the indicator of the conversion rate from methanol to the desired product by each incubation system.

Measured unit GFP production curves during the methanol short-term induction period.

Bibliography

Mateus, C., & Avery, S. V. (2000). Destabilized green fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry. Yeast, 16(14), 1313–1323. https://doi.org/10.1002/1097-0061(200010)16:14<1313::AID-YEA626>3.0.CO;2-O

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