Team:DTU-Denmark/Results

• The reference strain
• The new strains
• Comparison
  • Simulations
  • Growth
  • Protein secretion
  • Summary
• Signal Peptides
• Back to top

The reference strain

The ATCC 1015 strain of Aspergillus niger was used as our reference strain. It has no known mutations from the wild type A. niger.

ATCC 1015 and the nine A. niger mutant strains were inoculated and grown on four different growth media. The morphology of a fungus differs depending on the environmental conditions it is grown under, which is the reasoning behind choosing to grow the A. niger strains on four different media. We have described the four media types further in the Measurement page.

The strains were grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).

Microscope pictures and Simulation model

Left: Confocal microscope picture of ATCC 1015 at 10X magnification. Right: Growth simulation for 12h performed using the Mycemulator.

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of ATCC 1015 growing for 12 hours (using the experimental growth rate from our BioLector data) is seen above.

Parameters specific for simulating ATCC 1015:

• Branching frequency: 0.0169257
• Gamma distribution parameters used for curvature angles: (1.6114694, 2.0238222)
• Beta distribution parameters used for branching angles: (4.2886834, 0.9839464)
• Experimental growth rate: 0.2757333

The new morphology strains

We made nine new strains with the tools of synthetic biology. Here you will find the characterization of them.

Click on one to learn more!

ΔchsC

Theoretical expectation: chsC encodes a chitin synthase. It is involved in hyphal wall integrity and in conidiophore development. Deletion of the gene was expected to give more dense pelleted growth and an increased citric acid production.

Summary: The strain presents a more hyperbranched morphology, but protein secretion is slightly decreased, and the growth rate is lower than the reference strain. By itself it’s not the best candidate, however, it has potential when combined with other genes.

Radar chart showing 6 different parameters of ΔchsC normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the Summary section.

Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).

Microscope pictures and Simulation model

Left: Confocal microscope picture of ΔchsC at 10X magnification after app. 24h growth. Right: Growth simulation for 12h performed using the Mycemulator.

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of ΔchsC growing for 12 hours (using experimental growth rate from our BioLector data) is seen above.

Parameters specific for simulating ΔchsC:

• Branching frequency: 0.0251938
• Gamma distribution parameters used for curvature angles: (1.5962901, 4.3710747)
• Beta distribution parameters used for branching angles: (2.294856826, 0.9438161)
• Experimental growth rate: 0.21145

BioLector

Comparing the growth kinetics of the ΔchsC mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a longer lag phase followed by an equivalently long exponential growth phase. The growth rate for the mutant is slightly lower than for the reference strain, at μ_Max0.21h^-1 versus μ_Max0.28h^-1.

Growth profile of ΔchsC over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.

Bioreactor

Looking at the growth of the ΔchsC mutant in the bioreactors, it showed a shorter lag phase followed by a longer exponential growth phase when comparing it with the reference strain ATCC 1015. The growth rate is lower than the reference strain, at μ_Max0.23h^-1 and μ_Max0.22h^-1 versus μ_Max0.42h^-1 for both reference duplicates. The fermentation was accidentally run with overpressure and is therefore only directly comparable with the ΔspaA mutant strain and with one run of the ATCC 1015 fermentations, due to these bioreactors also being run with overpressure. Glucose consumption starts slowly but increases quickly after the mutant stops growing.

Growth curve obtained from the off-gas analysis (CO2-44 (%)) of ΔchsC plotted against the reference strain (ATCC 1015) in a logarithmic scale. Right axis: Glucose consumption by ΔchsC during the fermentation in g/L (dark green).

Light microscope picture of ΔchsC after 22 hours of fermentation with a 20X magnification.

Microscope pictures of the bioreactor sample for ΔchsC had a very different morphology from the brightfield microscopy pictures seen above. It showed an increase in agglomeration along with a more hyperbranched phenotype.

Protein secretion

As seen in the figure below glucoamylase activity increases over time during the fermentation. The mutant exhibits lower glucoamylase activity compared to the reference strain.

Glucoamylase activity in UA/mL of ΔchsC from eight samples taken during the fermentation. Plotted against the reference strain (ATCC 1015) shown in yellow.

The final protein concentration is also significantly lower for the mutant strain than for reference strain, which is consistent with the reduced glucoamylase activity level. This is shown in the bar chart below.

ΔchsC bioreactor duplicates compared to reference strain (ATCC 1015) of the last time-point samples from the fermentations. Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.

ΔracA

Theoretical expectation: racA encodes a Rho GTPase which controls actin dynamics and it is thereby also involved in polarity regulation. The knockout mutant was expected to have a hyperbranched morphology.

Summary: The strain showed a slight increase in protein secretion and a hyperbranching pelleted morphology when run in the bioreactor. While it closely follow the behavior of the reference strain, its morphology on solid media was very different. As it had a decreased sporulation it made the strain hard to work with.

Radar chart showing 6 different parameters of ΔracA normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the Summary section.

Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).

Microscope pictures and Simulation model

Left: Confocal microscope picture of ΔracA at 10X magnification after app. 24h growth. Right: Growth simulation for 12h performed using the Mycemulator.

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of ΔracA growing for 12 hours (using experimental growth rate from our BioLector data) is seen above.

Parameters specific for simulating ΔracA:

• Branching frequency: 0.0426064
• Gamma distribution parameters used for curvature angles: (0.8423059, 16.2365375)
• Beta distribution parameters used for branching angles: (1599535.5508791, 2.3452173)
• Experimental growth rate: 0.15865

BioLector

Comparing the growth kinetics of the ΔracA mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a longer lag phase and an almost double as long exponential growth phase. The growth rate for the mutant is lower than for the reference strain, at μ_Max0.16h^-1 versus μ_Max0.28h^-1.

Growth profile of ΔracA over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.

Bioreactor

Looking at the growth of the ΔracA mutant in bioreactors, it showed an equivalently long lag phase as the reference strain ATCC 1015 followed by a slightly shorter exponential growth phase. ΔracA also has a growth rate almost equivalent to the growth rate of the reference strain, at μ_Max0.36h^-1 and μ_Max0.32h^-1 versus μ_Max0.37h^-1 and μ_Max0.33h^-1. Glucose levels start decreasing two thirds of the way through growth but are stil not completely depleted at the time of the last sample.

Left axis: Growth curve obtained from the off-gas analysis (CO2-44 (%)) of ΔracA plotted against the reference strain (ATCC 1015) in a logarithmic scale. Right axis: Glucose consumption by ΔracA during the fermentation in g/L (dark green).

Light microscope picture of ΔracA after 23 hours of fermentation with a 20X magnification.

The microscope picture from the bioreactor sample for ΔracA showed a pelleted morphology making it hard to compare with the brightfield microscopy pictures seen above. It showed a very hyperbranched pelleted morphology. When run in the bioreactor, the pelleted morphology was observable on a macroscopic level.

Protein secretion

The glucoamylase activity increases over time during the fermentation as seen in the figure below. The two duplicates showed a large difference in glucoamylase activity, one being equal to the reference strain whereas the other had an increased activity.

Glucoamylase activity in UA/mL of ΔracA from 8 samples taken during the fermentation. Plotted against the reference strain (ATCC 1015) shown in yellow.

A difference were also seen between the duplicates for the total protein secretion. Therefore, when calculating the specific activity both showed similar result with a value slightly higher than the reference strain. The difference the duplicates might be explained by a deviation in setting up the technical duplicates.

ΔracA bioreactor duplicates compared to reference strain (ATCC 1015) of the last time-point samples from the fermentations. Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.

ΔchsC_Δgul-1

Theoretical expectation: This is a double knock-out mutant, where both the genes chsC, which encodes a chitin synthase, and gul-1, which encodes an mRNA binding protein involved in cell wall remodeling, are knocked out. There does not exist any prior knowledge in the literature about knocking both of these genes simultaneously out in A. niger.

Summary: The strain presents a very hyperbranched morphology, caused by an additive effect of both genes. Protein secretion is higher than in the reference strain. It showed a macroscopic pelleted morphology when run in the bioreactor.

Radar chart showing 6 different parameters of ΔchsC_Δgul-1 normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the Summary section.

Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).

Microscope pictures and Simulation model

Left: Confocal microscope picture of ΔchsC_Δgul-1 at 10X magnification after app. 24h growth. Right: Growth simulation for 12h performed using the Mycemulator.

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of ΔchsC_Δgul-1 growing for 12 hours (using experimental growth rate from our BioLector data) is seen above.

Parameters specific for simulating ΔchsC_Δgul-1:

• Branching frequency: 0.0280344
• Gamma distribution parameters used for curvature angles: (1.272886, 6.5509939)
• Beta distribution parameters used for branching angles: (2.9856469, 0.9208045)
• Experimental growth rate: 0.1381667

BioLector

Comparing the growth kinetics of the ΔchsC_Δgul-1 mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits an equivalently long lag phase followed by a twice as long exponential growth phase. The growth rate for the mutant is lower than for the reference strain, at μ_Max0.14h^-1 versus μ_Max0.28h^-1.

Growth profile of ΔchsC_Δgul-1 over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.

Bioreactor

Looking at the growth of the ΔchsC_Δgul-1 mutant in bioreactors, it showed a slightly shorter lag phase and a shorter exponential growth phase compared to the reference strain ATCC 1015. ΔchsC_Δgul-1 also has a higher growth rate than the reference strain, at μ_Max0.42h^-1 for both duplicates versus μ_Max0.37h^-1 and μ_Max0.33h^-1. Glucose levels start decreasing during growth but are first depleted over 12 hours after the strain enters the stationary phase.

Left axis: Growth curve obtained from the off-gas analysis (CO2-44 (%)) of ΔchsC_Δgul-1 plotted against the reference strain (ATCC 1015) in a logarithmic scale. Right axis: Glucose consumption by ΔchsC_Δgul-1 during the fermentation in g/L (dark green).

Light microscope picture of ΔchsC_Δgul-1 after 23 hours of fermentation with a 20X magnification.

Microscope picture from the bioreactor sample for ΔchsC_Δgul-1 has a similar morphology to that of the brightfield microscopy pictures seen above. It showed a hyperbranched pelleted morphology. When run in the bioreactor, the pelleted morphology was observable on a macroscopic level.

Protein secretion

As seen in the figure below, glucoamylase activity increases during the fermentation and reaches a higher level than the reference strain.

Glucoamylase activity in UA/mL of ΔchsC_Δgul-1 from 8 samples taken during the fermentation. Plotted against the reference strain (ATCC 1015) shown in yellow.

This mutant presents similar tendency in the results as the single knock-out strain Δgul-1, with similar number in total protein secretion but increased activity per total protein when compared to the reference strain. As commented in Δgul-1 results, this might be due to a different protein secretion profile that has a similar amount of protein being secreted but where the family of glucoamylases are preferentially produced. However, these results are lower when compared directly to Δgul-1, but significantly higher than when compared to ΔchsC, showing more properties of the Δgul-1 strain.

ΔchsC_Δgul-1 bioreactor duplicates compared to reference strain (ATCC 1015) of the last time-point samples from the fermentations. Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.

ΔspaA

Theoretical expectation: It has been suggested that spaA encodes a molecule which is involved in polarity maintenance and thereby polar growth. The mutant was expected to have a dense hyperbranched morphology.

Summary: The strain presents a shorter lag phase than the reference strain and the highest growth rate in the BioLector, although this is not seen in the bioreactor. It does not have a great increase in hyperbranching and protein secretion is very similar to the reference strain.

Radar chart showing 6 different parameters of ΔspaA normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the Summary section.

Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).

Microscope pictures and Simulation model

Left: Confocal microscope picture of ΔspaA at 10X magnification af app. 24h growth. Right: Growth simulation for 12h performed using the Mycemulator.

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of ΔspaA growing for 12 hours (using experimental growth rate from ou BioLector data) is seen above.
Parameters specific for simulating ΔspaA:

• Branching frequency: 0.0182251
• Gamma distribution parameters used for curvature angles: (1.6948611, 2.8805343)
• Beta distribution parameters used for branching angles: (5.1349854, 1.2365410)
• Experimental growth rate: 0.345225

BioLector

Comparing the growth kinetics of the ΔspaA mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a shorter lag phase and a shorter exponential growth phase. The growth rate for the mutant is higher than for the reference strain, at μ_Max0.35h^-1 versus μ_Max0.28h^-1.

Growth profile of ΔspaA over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.

Bioreactor

Looking at the growth of the ΔspaA mutant in bioreactors, it showed a short lag phase followed by a long exponential growth phase compared with the reference strain ATCC 1015. The ΔspaA mutant has a lower growth rate than the reference, at μ_Max0.19h^-1 and μ_Max0.20h^-1 versus μ_Max0.42h^-1 for both duplicates. The fermentation was accidentally run with overpressure and is therefore only directly comparable with the ΔchsC mutant strain and with one run of the ATCC 1015 fermentations, due to these bioreactors also being run with overpressure. Glucose levels decrease slowly in the beginning and then decrease fast after the mutant enters stationary phase.

Left axis: Growth curve obtained from the off gas analysis (CO2-44 (%)) of ΔspaA plotted against the reference strain (ATCC 1015) in a logarithmic scale. Right axis: Glucose consumption by ΔspaA during the fermentation in g/L (dark green).

Light microscope picture of ΔspaA after 22 hours of fermentation with a 20X magnification.

The microscope picture from the bioreactor sample for ΔspaA has a similar morphology to that of the brightfield microscopy pictures seen above.

Protein secretion

Glucoamylase activity for the mutant strain shows a similar tendency to the reference strain, with one of the duplicates having lower final value.

Glucoamylase activity in UA/mL of ΔspaA from 8 samples taken during the fermentation. Plotted against the reference strain (ATCC 1015) shown in yellow.

Protein secretion is not consistent but when computing the specific activity per mg of protein, it can be seen that the mutant again presents similar values to the reference strain.

ΔspaA bioreactor duplicates compared to reference strain (ATCC 1015) of the last time-point samples from the fermentations. Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.

ΔpkaR

Theoretical expectation: pkaR encodes a regulatory subunit in Protein Kinase A (PKA) which is involved in stress response and regulation of morphology and growth. The gene has been characterized in Neurospora crassa and a homolog was found in A. niger.

Summary: The strain shoed lower growth rate in the BioLector along with an increase in branching frequency. Protein secretion could not be studied as it was not run in the bioreactor due to time limitations.

Radar chart showing 6 different parameters of ΔpkaR normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the Summary section. *Indicates that not all data was obtained to fill out the chart.

Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).

Microscope pictures and Simulation model

Left: Confocal microscope picture of ΔpkaR at 10X magnification after app. 24h growth. Right: Growth simulation for 12h performed using the Mycemulator.

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of ΔpkaR growing for 12 hours (using the experimental growth rate from our BioLector data) is seen above.

Parameters specific for simulating ΔpkaR:

• Branching frequency: 0.0312627
• Gamma distribution parameters used for curvature angles: (1.6546373, 8.6411386)
• Beta distribution parameters used for branching angles: (1.4343473, 0.5666034)
• Experimental growth rate: 0.2090333

BioLector

Comparing the growth kinetics of the ΔpkaR mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a similar lag phase and a longer exponential growth phase. The growth rate for the mutant is lower than for the reference strain, at μ_Max0.21h^-1 versus &mu_Max0.28h^-1.

Growth profile of ΔpkaR over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.

Bioreactor and Protein secretion

ΔpkaR was not run in the bioreactor due to time limitations. Data for growth in bioreactor and protein secretion for this strain were therefore not obtained.

ΔchsC_ΔspaA

Theoretical expectation: This is a double knock-out mutant, where both chsC, which encodes a chitin synthase, and spaA, which might be involved in polarity maintenance and polar growth, are knocked out. There does not exist any prior knowledge in the literature about knocking both of these genes out simultaneously in A. niger.

Summary: The strain presents the highest branching frequency when compared to the rest of the mutants, even higher than what would be expected by an additive effect of both genes. However, growth rate and protein secretion are slightly lower than the reference strain.

Radar chart showing 6 different parameters of ΔchsC_ΔspaA normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the Summary section.

Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).

Microscope pictures and Simulation model

Left: Confocal microscope picture of ΔchsC_ΔspaA at 10X magnification after app. 24h growth. Right: Growth simulation for 12h performed using the Mycemulator.

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of ΔchsC_ΔspaA growing for 12 hours (using the experimental growth rate from our BioLector data) is seen above.

Parameters specific for simulating ΔchsC_ΔspaA:

• Branching frequency: 0.0476127
• Gamma distribution parameters used for curvature angles: (1.5398610, 5.8823202)
• Beta distribution parameters used for branching angles: (5.6843956, 1.2075437)
• Experimental growth rate: 0.264

BioLector

Comparing the growth kinetics of the ΔchsC_ΔspaA mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a slightly shorter lag phase followed by a slightly longer exponential growth phase. The growth rate for the mutant is almost equivalent to the growth rate for the reference strain, at μ_Max0.26h^-1 versus μ_Max0.28h^-1.

Growth profile of ΔchsC_ΔspaA over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.

Bioreactor

Looking at the growth of the ΔchsC_ΔspaA mutant in bioreactors, it showed a eqvuivalently long lag phase as the reference strain ATCC 1015, followed by a longer exponential growth phase. ΔchsC_ΔspaA has a lower growth rate than the reference strain, at μ_Max0.29h^-1 and μ_Max0.27h^-1 versus μ_Max0.37h^-1 and μ_Max0.33h^-1. Glucose levels start decreasing half way through growth and are depleted shortly after the strain enters the stationary phase.

Left axis: Growth curve obtained from the off-gas analysis (CO2-44 (%)) of ΔchsC_ΔspaA plotted against the reference strain (ATCC 1015) in a logarithmic scale. Right axis: Glucose consumption by ΔchsC_ΔspaA during the fermentation in g/L (dark green).

Light microscope picture of ΔchsC_ΔspaA after 24 hours of fermentation with a 20X magnification.

The microscope picture from the bioreactor sample for ΔchsC_ΔspaA has a very similar morphology to that of the brightfield microscopy pictures seen above. It showed a hyperbranched morphology with a slight increase in agglomeration.

Protein secretion

Glucoamylase activity follows that reference strain with one duplicates higher level of activity.

Glucoamylase activity in UA/mL of ΔchsC_ΔspaA from eight samples taken during the fermentation. Plotted against the reference strain (ATCC 1015) shown in yellow.

Total protein secretion presents a 2-fold increase compared to the reference strain. However, specific activity per total protein is higher in the reference strain, suggesting that protein secretion profile has changed with a decrease in the glucoamylase family.

ΔchsC_ΔspaA bioreactor duplicates compared to reference strain (ATCC 1015) of the last time-point samples from the fermentations. Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.

Δgul-1

Theoretical expectation: gul-1 was found in literature about the fungi Neurospora crassa where it encodes an mRNA binding protein involved in remodeling of the cell wall. A homolog was found in A. niger. The expected morphology of the Δgul-1 mutant was hyperbranched pellets.

Summary: Protein secretion in the strain was highly increased and had a slightly lower growth rate in the bioreactor when compared to the reference. It presents an almost 2-fold increase in branching frequency, making this mutant a very good candidate for an improved cell factory for protein production.

Radar chart showing 6 different parameters of Δgul-1 normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the Summary section.

Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).

Microscope pictures and Simulation model

Left: Confocal microscope picture of Δgul-1 at 10X magnification after app. 24h growth. Right: Growth simulation for 12h performed using the Mycemulator.

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of Δgul-1 growing for 12 hours (using experimental growth rate from our BioLector data) is seen above.

Parameters specific for simulating Δgul-1:

• Branching frequency: 0.0300301
• Gamma distribution parameters used for curvature angles: (0.6220764, 8.0102895)
• Beta distribution parameters used for branching angles: (3.2414641, 0.8869618)
• Experimental growth rate: 0,314625

BioLector

Comparing the growth kinetics of the Δgul-1 mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a shorter lag phase followed by a slightly shorter exponential growth phase. The growth rate for the mutant is higher than for the reference strain, at μ_Max0.32h^-1 versus μ_Max0.28h^-1.

Growth profile of Δgul-1 over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.

Bioreactor

Looking at the growth of the Δgul-1 mutant in bioreactors, it showed a lag phase equivalent to that of the reference strain ATCC 1015 followed by a longer exponential growth phase. Δgul-1 has a slightly lower growth rate than the reference strain, at μ_Max0.31h^-1 and μ_Max0.32h^-1 versus μ_Max0.37h^-1 and μ_Max0.33h^-1. Glucose levels start decreasing two thirds of the way through growth and keep decreasing for over 10 hours after the strain enters the stationary phase.

Left axis: Growth curve obtained from the off-gas analysis (CO2-44 (%)) of Δgul-1 plotted against the reference strain (ATCC 1015) in a logarithmic scale. Right axis: Glucose consumption by Δgul-1 during the fermentation in g/L (dark green).

Light microscope picture of Δgul-1 after 23 hours of fermentation with a 20X magnification.

The microscope picture from the bioreactor sample for Δgul-1 has a similar morphology to that of the brightfield microscopy pictures seen above. It showed a hyperbranched phenotype in a more dispersed network.

Protein secretion

Glucoamylase activity has a significant 2-fold increase in this mutant compared with the reference strain. Such high glucoamylase activities were reach that the total activity was not measured. From the flat curve it could be expected that the glucoamylase activity would have reached even higher levels have it been measured.

Glucoamylase activity in UA/mL of Δgul-1 from 8 samples taken during the fermentation. Plotted against the reference strain (ATCC 1015) shown in yellow.

The total protein secretion was to the reference strain and thereby the specific activity was substantially higher. This might be due to a different protein secretion profile that has a similar amount of protein being secreted but where the family of glucoamylases are preferentially produced.

Δgul-1 bioreactor duplicates compared to reference strain (ATCC 1015) of the last time-point samples from the fermentations. Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.

ΔaplD

Theoretical expectation: aplD encodes a gamma-adaptin involved in endosomal cargo transport and affects filamentous growth and pellet formation. Deletion of the gene was expected to result in a hyperbranched morphology.

Summary: From the different experiments it can be assumed that ΔaplD plays an important role in proper growth. Therefore, the ΔaplD mutant is not viable.

Radar chart showing 6 different parameters of ΔaplD normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the Summary section. *Indicates that not all data was obtained to fill out the chart.

Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).

Microscope pictures and Simulation model

Unfortunately, we did not get a microscopy picture of ΔaplD where it was possible to see branching angles, curvature of mycelia, etc. Thus we could not analyze the images properly and simulate the growth.

BioLector

Comparing the growth kinetics of the ΔaplD mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a much longer lag phase followed by a three times longer exponential growth phase. The growth rate for the mutant is also much lower than for the reference strain, at μ_Max0.10h^-1 versus μ_Max0.28h^-1. Additionally, only one out of the three replicates showed any growth at all.

Growth profile of ΔaplD over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.

Bioreactor and Protein secretion

ΔaplD was not run in the bioreactor due to time limitations. Data for growth in bioreactor and protein secretion for this strain were therefore not obtained.

ΔspaA_Δgul-1

Theoretical expectation: This is a double knock-out mutant, where both spaA, which might be involved in polarity maintenance and polar growth, and gul-1, which encodes an mRNA binding protein involved in cell wall remodeling, are knocked out. There does not exist any prior knowledge in the literature about simultaneously knocking out both of these genes in A. niger.

Summary: The strain presents higher branching frequency and protein secretion than the reference strain. Growth rate and lag phase are very similar to the reference. On solid media it presented a very slow growth making it harder to work with.

Radar chart showing 6 different parameters of ΔspaA_Δgul-1 normalized to the reference values from ATCC 1015 (shown in yellow). Read about the axis in the Summary section.

Plates

The strain was grown on Yeast Extract Peptone Dextrose (YPD), Transformation Media (TM), Creatine Sucrose Agar (CREA) and Czepek Yeast Extract Agar (CYA).

Microscope pictures and Simulation model

Left: Confocal microscope picture of ΔspaA_Δgul-1 at 10X magnification after app. 24h growth. Right: Growth simulation for 12h performed using the Mycemulator.

Microscopic images were analyzed by the image analysis tool extracting growth parameters which were then fed to the Mycemulator. A simulation of ΔspaA_Δgul-1 growing for 12 hours (using the experimental growth rate from our BioLector data) is seen above.
Parameters specific for simulating ΔspaA_Δgul-1:

• Branching frequency: 0.0298207
• Gamma distribution parameters used for curvature angles: (1.6563537, 6.1423278)
• Beta distribution parameters used for branching angles: (3.0350945, 0.9824538)
• Experimental growth rate: 0.3264333

BioLector

Comparing the growth kinetics of the ΔspaA_Δgul-1 mutant with the reference strain ATCC 1015 in the BioLector, the mutant exhibits a shorter lag phase followed by an equivalently long exponential growth phase. The growth rate for the mutant is higher than for the reference strain, at μ_Max0.33h^-1 versus μ_Max0.28h^-1.

Growth profile of ΔspaA_Δgul-1 over 72 hours, measuring absorbance at 620 nm. Plotted against the reference strain (ATCC 1015) shown in yellow.

Bioreactor

Looking at the growth of the ΔspaA_Δgul-1 mutant in bioreactors, it showed an equivalently long lag phase as the reference strain ATCC 1015 followed by a longer exponential growth phase. ΔspaA_Δgul-1 has a growth rate very close to that of the reference strain, at μ_Max0.32h^-1 and μ_Max0.33h^-1 versus μ_Max0.37h^-1 and μ_Max0.33h^-1. Glucose levels start decreasing half way through growth and are depleted simultaneously with the strain entering the stationary phase.

Left axis: Growth curve obtained from the off-gas analysis (CO2-44 (%)) of ΔspaA_Δgul-1 plotted against the reference strain (ATCC 1015) in a logarithmic scale. Right axis: Glucose consumption by ΔspaA_Δgul-1 during the fermentation in g/L (dark green).

Light microscope picture of ΔspaA_Δgul-1 after 26 hours of fermentation with a 20X magnification.

The microscope picture from the bioreactor sample for ΔspaA_Δgul-1 has a very similar morphology to that of the brightfield microscopy pictures seen above. It showed a hyperbranched morphology along with a more pelleted network.

Protein secretion

Both duplicates display the same profile, with a rapid increase in glucoamylase activity during the last 10 hours of the fermentation.

Glucoamylase activity in UA/mL of ΔspaA_Δgul-1 from 8 samples taken during the fermentation. Plotted against the reference strain (ATCC 1015) shown in yellow.

The final protein secretion varies considerably, with one of them being very low, resulting in the specific activity being very high. Considering this is probably caused by some type of experimental error when measuring the protein concentration, we can assume that the specific activity is overestimated. However it is clear that the strain produces more glucoamylase compared with the reference strain.

ΔspaA_Δgul-1 bioreactor duplicates compared to reference strain (ATCC 1015) of the last time-point samples from the fermentations. Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.

Simulations

As we see in the microscope the growth pattern of our strains are very different. This is also clear to see in our growth simulations from the Mycemultor. Take your time to enjoy these amazing simulations.

ATCC 1015

Δgul-1

ΔchsC_Δgul-1

ΔchsC

ΔracA

ΔchsC_ΔspaA

ΔspaA

ΔpkaR

ΔspaA_Δgul-1

Growth

The growth rates of all the mutants were determined from BioLector growth data. Seven of mutants were also run in 1L bioreactors and growth rates were determined from these as well. Unfortunately, we were not able to run the last two, ΔaplD and ΔpkaR, in the bioreactor due to time limitations.
Overall the growth rates obtained from the bioreactor runs were higher than the ones from the BioLector, with two exception ΔchsC and ΔspaA. The growth rates determined from the BioLector are for some of the mutants not comparable to the growth rates obtained from bioreactor runs. An example of this is ΔspaA which shows the highest value in the BioLector whereas its growth rate in the bioreactor is half of the one for the reference strain. These differences might be due to differences in fermentation conditions in the two setups. The bioreactor is in general better as it allows you to control more parameters including pH, agitation and oxygen level.
For growth rates obtained from the bioreactor, the duplicates show consistent results. All the mutants had similar growth rates to the reference strain with the exception of ΔchsC and ΔspaA.

Bar chart of the mean growth rate with their corresponding standard deviation for all the mutant strains compared to the reference strain (ATCC 1015) obtained from the BioLector.

Bar chart of the growth rate for both duplicates for seven of the mutant strains compared to the reference strain (ATCC 1015) obtained from the bioreactor. The black line represents the conditions for the first three strains separated from the others due to them being with overpressure. The strains are therefore only comparable within the first three and within the last six strains.

From the BioLector and bioreactor growth data, the length of the lag phases and exponential phases were determined. From the plot it can be observed that the replicates follow each other very closely. For all mutants it is seen that the lag phase is longer in the BioLector than in the bioreactors. This could be due to bioreactors having more optimal growth conditions compared with the BioLector, thereby resulting in a shorter adaptation phase.
From the bioreactor most strains show a similar lag phase to that of the reference strain. In industry a short lag phase would be favorable as it shorten the fermentation time without affecting the time of efficient production (in the exponential and stationary phases). In this sense, ΔchsC and ΔspaA show great promise as they have a significant shorter lag phases than the reference strain. So although they have a smaller growth rate, their total fermentation time is shorter than that of the reference strain (before entering stationary phase).
ΔchsC_Δgul-1 presents a slightly shorter lag phase and significantly shorter fermentation time (before entering stationary phase) due to its higer growth rate. This makes it a favorable strain.

Stacked bar chart of the duration in hours of lag and exponential phases with their corresponding standard deviation from all the mutant strains compared to the reference strain (ATCC 1015) obtained from the BioLector.

Stacked bar chart of the duration in hours of lag and exponential phases from all the mutant strains that were run in the bioreactor compared to the reference strain (ATCC 1015). The black line represents the conditions for the first three strains separated from the others due to them being run with overpressure. The strains are therefore only comparable within the first three and within the last six strains.

Protein secretion

From the figure below it is seen that protein secretion and glucoamylase activity varies significantly between the strains. The strains with a knock out in the gul-1 gene show significantly higher specific activity than the reference strain. Δgul-1 showed a 3-fold increase where ΔchsC_Δgul-1 and ΔspaA_Δgul-1 showed an approximately 2-fold increase indicating that they are significantly better protein producers.
In this project we have chosen to assess protein production as an indicator of succesful engineering. However in industry it depends on the product of choice. A high protein producing strain might not be the best for production of secondary metabolites.

Protein secretion and glucoamylase activity for all strains run in the bioreactor for the last time-point samples. Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL. The black line represents the conditions for the first three strains separated from the others due to them being with overpressure. The strains are therefore only comparable within the first three and within the last six strains.

Summary

All our new strains are summarized in the radar charts seen below. The strains are evaluated on six criteria, all normalized to the reference strain:

• RESHAPE score: This is a subjective score given by the wetlab team. It is based on how the strain was to work with compared to the reference strain. Things taken into consideration were:
• Sporulation, was it hard to make spore stocks?
• How long did it take for it to grow on solid media?
• How promising did we find it as a good cell factory for protein production?
• Branching frequency: Value analyzed by the Morphologizer from the microscopic pictures. High branching frequency is linked to hyperbranching.
• Space fillingness:
Value analyzed by the Morphologizer from the microscopic pictures. This value is an indicator of agglomeration in mycelium.
• Growth rate (BioLector): Growth rates obtain from the BioLector.
• Growth rate (Bioreactor): Growth rates obtain from the bioreactor.
• Protein secretion (specific activity): Glucoamylase activity per mg secreted protein.

As seen from the radar charts all our strains behave differently due to their gene knock out. In this project we have seen the selected genes effect on morphology and how they affect A. niger performance as a cell factory. We have proven that the tools of synthetic biology can be a key player in controlling and improving the cell factories of the future.

Comparison of the nine radar charts from all the mutant strains normalized to the reference values from ATCC 1015 (shown in yellow). *Indicates that not all data was obtained to fill out the chart.

Signal Peptides

Eleven mutants with signal peptide glucoamylase insertions were constructed. Eight with natural signal peptides found in literature and three with synthetic signal peptides predicted by the SignalPrepper tool. To learn more about the way these strains were designed please see the Design page.We wanted to test the secretion levels for the different signal peptide strains through the glucoamylase assay. Unfortunately we did not reach this point due to time limitations. Here you see the eight of the native signal peptide strains plated on minimal media starch plates. Iodine was added to the plates to show were the starch was consumed. From these pictures it can not be concluded how well the different strains secret glucoamylase.