Modification of Starch Metabolic Pathway

Overview of Chlamydomonas Starch Metabolism

In order to enhance the total yield of starch and the proportion of amylose in Chlamydomonas reinhardtii, our project focuses on modulating the starch metabolism pathway of this green microalga using synthetic biology approaches. The starch metabolism pathway in Chlamydomonas has been elucidated by scientists, providing us with valuable insights. Based on literature research, the starch metabolism process begins with the conversion of glucose-6-phosphate to glucose-1-phosphate catalyzed by an isomerase. Subsequently, glucose-1-phosphate is transformed into the activated form of ADP-glucose by ADP-glucose pyrophosphorylase (AGPase). The activated ADP-glucose is then utilized to synthesize starch. For starch degradation, it can occur through either phosphorylysis or hydrolysis, with one pathway involving the key phosphorylation activation by glucan water dikinase (GWD) . Our strategy utilized the principle of "broaden the source of income and economize on expenditure", aiming to promote starch synthesis while inhibiting starch degradation .

Figure 1 Starch metabolism pathway in Chlamydomonas reinhardtii [1]

Furthermore, during the process of HP, we were inspired by communication with the China Starch Industry Association that changing the composition ratio of starch adds more value and economic significance. Chemically, starch consists of two types of anhydroglucose polymers called amylose and amylopectin. High amylose starch (HAS) is a resistant starch that can ferment and produce various short-chain fatty acids (SCFA) in the colon through the action of intestinal microflora, providing several health benefits. HAS also shows potential for treating obesity, diabetes, and other diseases. Therefore, we regulated the starch composition in Chlamydomonas reinhardtii to produce strains with high production of amylose.


Figure 2 Starch amylose and amylopectin structure [2]

Experiment Design

Enhancing Starch Yield

“Broaden the source of income”-AGPase

AGP4 encodes ADP-glucose pyrophosphorylase, which plays a crucial role as the second and rate-limiting step in glucose-to-starch synthesis. After research, we find the gene glgC encodes a highly efficient homolog from Escherichia coli. According to literature reports, its specific activity is several hundred-fold greater than that of the plant enzyme[3]. Additionally, several residues have been identified as important allosteric regulatory sites. A glycine-336 mutant AGPase (G336D) encoded by E. coli glgC has been shown to have high activity with or without the activator FBP, higher substrate (ATP and glucose-1-phosphate) affinity, and reduced affinity for the inhibitor AMP[4].

Figure 3 Km value comparison of AGPase from Chlamydomonas reinhardtii, Escherichia coli, Zea mays (data from BRENDA) . The glgC exhibits significantly higher Km value than the plant homolog

Therefore, we have aimed to express the G333D site-directed mutant AGPase from E.coli in AGP4 knocked out Chlamydomonas. This substitution of the functional enzyme with a highly efficient homolog will promote starch synthesis.

"Economize on expenditure"-GWD1 & GWD2

GWD1 and GWD2 encode Alpha-glucan water dikinase 1 and Alpha-glucan water dikinase 2, respectively. They are isoenzymes that function by phosphorylating the C-3 and C-6 positions of alpha glucans with the beta phosphate of ATP in the initial step of starch hydrolysis. By knocking out these two genes and inhibiting the synthesis of this enzyme, we can effectively block the major starch hydrolysis pathway, thereby enhancing the overall starch production.

After obtaining individual mutants for GWD1 and GWD2, we further created double knockout mutants through hybridization, enabling both mutations to coexist in the same algal strain.

Gene editing in Chlamydomonas reinhardtii, on a global scale, has proven to be quite challenging. However, we have successfully overcome this obstacle by utilizing a gene editing system developed by the collaborative laboratory of professor JUNMIN PAN. We employed CRISPR/Cas9 technology to perform gene editing on the 21gr or srt2 Chlamydomonas cell lines (both considered wild-type)

Figure 4 Design framework for AGPase and GWD

Utilizing lipid synthesis inhibitor

The starch metabolism pathway and the lipid metabolism pathway have significant crosstalk. Since starch synthesis and lipid synthesis compete with each other, we decided to inhibit lipid synthesis to promote starch synthesis. Due to the uncertainty associated with this indirect approach, we chose not to manipulate it at the genetic level initially. Instead, we cultured Chlamydomonas in the presence of cerulenin , a lipid inhibitor. Cerulenin is a natural compound found in the Cephalosporium caerulensfungus , and it has been reported to bind to b-keto-acyl-ACP synthase, inhibiting fatty acid synthesis[5].

Figure 5 Design framework for using cerulenin sbe2-3 sbe3-5 by carrying out gene knockout in obtained mutant.

Improving Starch Value

SBE2 & SBE3

SBE2 and SBE3 are genes that encode Starch Branching Enzyme 2 and Starch Branching Enzyme 3, respectively. They, along with SBE1 and SBE4 in Chlamydomonas, are isoenzymes responsible for catalyzing the formation of alpha-1,6 linkages within the polymer. Considering their varying transcription levels, we have chosen SBE2 and SBE3, which exhibit higher transcription levels, to achieve more effective gene knockout. By inhibiting the synthesis of branched starch, we aimed to obtain a higher proportion of linear starch.

After obtaining individual mutants for SBE2 and SBE3, we also created double knockout mutants to introduce both mutations into the same algal strain.

Figure 6 Design framework for SBE

Data and Results

Chlamy mutants

First, we utilized CRISPR-Cas9 technology to knock out the GWD and SBE genes.

Additionally, to verify the functionality of glgC in Chlamy, we obtained a mutant strain with AGP4 knocked out, which would allow us to introduce the plasmid containing glgC. We successfully generated five mutant strains with respective gene knockouts.

Figure 7 Gene insertion and sequencing results of the five mutant strains. In the sequencing results, the red, bolded sequences with asterisks represent the inserted stop codons. All five mutant strains successfully inserted stop codons, resulting in disrupted gene expression and successful gene knockout. Also, we got a SBE double mutant sbe2-3 sbe3-5 by carrying out gene knockout in obtained mutant.

Figure 8 gel-shift conformation of corresponding mutants. We conducted PCR and gel electrophoresis to analyze the target sequence of mutant variants. The gel-shift conformational analysis, as shown in the figure, indicates that the PCR products of the mutants are significantly larger than those of the wild type.

Transferring glgC-G336D

We constructed three types of glgC-G336D plasmids that can be expressed in Chlamydomonas reinhardtii . The YFP tag or the HA tag made sure that the expression of glgC-G336D can be verified by performing Western Blot.

< img src=" https://static.igem.wiki/teams/4772/wiki/2023-10-12-6-08-15.png " /> Figure 9 Three glgC-G336D plasmid profiles. (Promoters, resistance elements, etc., not drawn)

By performing Fluorescence intensity detection and Western Blot, we can verify that glgC-G336D was successfully expressed in our Chlamy.

Fig 10. Results of fluorescence intensity detection
Excitation light wavelength: 488nm
Absorption light wavelength range: 500-520nm
The two 24-well plates in the first row show 48 different cell clones introduced into the PsaD CTP-glgC G336D-mVenus vector, and the second row both show 48 different cell clones introduced into the glgC G336D-YFP vector. One positive cell line was obtained from each of the two vectors. Scale of the map is shown on the right.

Growth Curve Determination

In order to assess whether the gene knockout affects the normal growth of Chlamydomonas, we conducted a three-day growth curve experiment with several obtained mutants, using the wild-type strain as a control. The growth curve was measured at 8-hour intervals using optical density (OD) readings as a classic method for characterizing cell density. OD values of the Chlamydomonas culture were measured at 16:00, 24:00, and 8:00, respectively. The growth curves were plotted, and the differences were calculated. By comparing the growth curves and analyzing the differences in OD values, we can evaluate the impact of the gene knockout on the growth of Chlamydomonas and determine if there are any noticeable deviations from the wild-type control.

Figure 12 data for measuring the OD values of Chlamydomonas

It can be seen that the growth of the wild type and each mutant basically conforms to the s-shaped curve of the logistic equation. It can be seen that the growth rate of gwd1 mutant was significantly faster than that of the wild type, and the growth rate of sbe3 was significantly slower.

Starch Content Determination

To test the effectiveness of our strategy, we have developed a method for extracting and quantifying starch content in Chlamydomonas based on literature research and constant trial. This method allows us to measure the starch content in both the wild-type strain and the mutants, thereby validating whether there is an increase in starch content. Additionally, we have utilized a commercial assay kit to determine the proportion of linear starch in the mutants, specifically measuring the increase in linear starch content. Furthermore, we referred to literature and captured scanning electron microscope (SEM) images of starch granules to provide a visual representation of the proportion of linear starch within the cells.

By employing these techniques, we aim to obtain quantitative data on starch content and assess the changes in linear starch proportions in the mutants compared to the wild-type strain.

Total starch content

Starch content standard curve

The standard curve was drawed using starch sample with a series of concentration gradients in the test kit(Starch Content Assay Kit (BOXBIO, China, AKSU015C)). The curve would be used in the concentration of starch produced by the sample we wanted to measure.

Figure 13 Standard curve of starch content

The horizontal axis represents the concentration of the standard starch samples and the vertical axis represents their absorbance at △620nm. The data were analyzed by linear regression. The linear regression formula is △620 = 6.614 Concentration. R2 = 0.9994.

Total starch content of cells cultivated by lipid synthesis inhibitor

According to the previous research using cerulenin in Chlamydomonas[6], we designed a control group and an experimental group. The control group was supplemented with 0.1% methanol, while the experimental group was supplemented with 10 μM cerulenin in 0.1% methanol. After 48 hours of nitrogen-depleted culture, we measured the starch content.

Figure 14 Qualitative and quantitive starch content of control and cerulenin treatment group

It can be seen from the figure that the starch content of cerulenin treatment group was significantly higher than the control group, about 2 times that of wild type.

Total starch content of six mutants

According to previous research[7], we measured the starch content of mutants after 5 days of nitrogen-depleted culture. 5×10^7 cells of wild type and each six mutants were collected for measurement.

Figure 15 Qualitative and quantitive starch content of wild type and all mutants

It can be seen from the figure that the starch content of mutant gwd1-1 and gwd2-11 were higher than wild type, but gwd1-1 was significant while gwd2-1 was not. The starch content of mutant sbe2-3 and sbe2-3; sbe3-5 were significantly higher than wild type, in which sbe2-3 was 7.91 times that of wild type. But sbe3-4 was lower than wild type(not significant). Agp4 was significantly lower than wild type.

Amylose content and perception

We use the Amylose Content Assay Kit ( Solarbio, China, BC4265) to measure amylose content. The starch sample for measurement were from the starch we extracted from cells before.

Amylose content of mutant sbe2-3/sbe3-4/sbe2-3;sbe3-5

Figure 16 Qualitative and quantitive amylose content of wild type and SBE mutants

It can be seen from the figure that the amylose content of 3 kinds of mutants were higher than wild type. sbe2-3 and sbe2-3; sbe3-5 were significantly while sbe3-4 was not. sbe2-3 was 8.82 times that of wild type.

Amylose perception of mutant sbe2-3/sbe3-4/sbe2-3;sbe3-5

Figure17 Percent of Amylose to total starch

It can be seen from the figure that amylose perception of mutant sbe3-4 and sbe2-3; sbe3-5 were significantly higher than wild type, and sbe2-3; sbe3-5 double mutant’s amylose perception was about twice that of wild type. sbe2-3 was also higher but not significant. The result was consistent with the research paper[8].

Phenotypic characterization of starches produced by mutant

Figure18 Phenotypic characterization of starches produced by mutant sbe2-2; sbe3-3

The figures were observed using SEM. It can be seen that in wild type cells, the surface of starch particles were round and smooth like that in the previous article[9]. But in mutant cells the particles were folded and angular, which showed that the phenotypic characterization changed significantly.

Conclusion

  • Gene editing basically did not affect the normal growth of Chlamydomonas.
  • Oil inhibitors can increase starch production, which indicates that further inhibition of oil pathway by knocking down genes can further increase starch production.
  • The sbe2-3 mutant strain was a strain with high starch yield up to 7.91 times that of wild type.
  • The sbe2-3;sbe3-5 mutant strain was a strain producing twice amylose starch of wild type.

    • Discussion

    The growth rate of sbe3-4 and gwd1-1 mutants varied significantly, while the knockout gene basically did not affect the normal growth of several other mutants. The gwd mutant grew significantly faster than the wild type and has increased starch production, making it the very desired mutant. gwd2-2, agp4, and sbe2-3 mutants were not significantly different, indicating that the growth rate is not slower than the wild type and is an ideal mutant. sbe3-4 growth rate slowed significantly and may affect starch yield.
    Western blot result showed a positive bang but with an error size of glgc protein, which probably was caused by proteinase degradation. We could try to use other efficient homologous enzymes in other species later.
    sbe2-3 grows fast and is unexpectedly a motor-defective type , with short flagella and slow movement. We speculate that the reason for its high starch accumulation is that it does not exercise to consume energy, so this energy accumulates in starch or other forms. In the future, we will build movement defect mutant to detect whether starch will accumulate like this one.

    ChlF:Extended Light Harvesting of Chlamy

    Background

    In runway ponds for industrial production and cultivation of Chlamydomonas, Chlamydomonas in the upper layer uses near-red light for photosynthesis. Therefore, a serious problem of near-red light attenuation in the depth of runway ponds exists, which leads to serious decrease of the photosynthetic efficiency of Chlamydomonas in the depth of runway ponds.

    Figure 1 Schematic diagram of different layers of Chlamy in industrial runway pond

    Our Inspiration: Far-Red Light Photoacclimation (FaRLiP)

    In terrestrial cyanobacteria, they live in an environment under near-infrared light(720nm) because of shading by plants or because of their associations with soil crusts, benthic mat communities, or dense cyanobacterial blooms.For evolution pressure, these cyanobacteria have evolved a novel far-red light photoacclimation (FaRLiP) that enable them to use far-red light (FRL) for photosynthesis.

    Figure 2 Schematic diagram of terrestrial cyanobacteria in their living environment

    And for evolution pressure, terrestrial cyanobacteria have evolved a novel far-red light photoacclimation (FaRLiP) that enable them to use far-red light (FRL) for photosynthesis. Chlorophyll f (Chl f) is the key pigment in FaRLiP whose maximal absoption is at around 707nm. And ChlF is the enzyme that produce chlorophyll f by oxidizing chlorophyll a.

    Our Design

    Learning from terrestrial cyanobacteria, we try to express enzyme ChlF from cyanobacteria to introduce FaRLiP into Chlamydomonas, thereby extending their light harvesting into far red (700 to 800 nm) and improving their photosynthetic light use efficiency.

    Figure 3 Introduce of FaRLiP in Chlamy by expressing ChlF

    4. Star-Chlamy with FaRLiP

    We constructed the ChlF plasmid that can be expressed in Chlamydomonas reinhardtii. And the YFP tag made sure that the expression of ChlF can be verified by performing Western Blot. PsaD CTP is a chloroplast transport peptide that helps transport ChlF into chloroplasts to function.

    Figure 4 Construction of PsaD CTP-ChlF-YFP plasmid.

    By performing Fluorescence intensity detection and Western Blot, we can verify that ChlF was successfully expressed in our Chlamy.

    Figure 5 Fluorescence intensity detection experiment. Excitation light wavelength: 488nm. Absorption light wavelength range: 500-520nm. The first and second lines are two repeats. Samples in the same columns but different lines come from one strains of algae. Scale of the heat map is shown on the right. NC: Chlamy that do not express YFP; PC: Chlamy that express YFP; ChlF-YFP: Chlamy that express ChlF-YFP plasmid. ChlF-YFP-1 and ChlF-YFP-2 are two different strains of Chlamy.
    Figure 6 Western Blot result of PsaD CTP-ChlF-YFP. The lane of ChlF-YFP and YFP is indicated. Lane 1: ChlF-YFP-1; Lane 9: ChlF-YFP-2; Lane 2-8,10: Chlamy that failed to express ChlF-YFP plasmid. NC: Chlamy that do not express YFP; PC: Chlamy that express YFP; * The smaller lane of PC might result from degradation of YFP or other unknown reasons.

    According to Roberta Croce's calculations, under special conditions of high far-red light, the total energy output (calculated as the product of total light absorption and photochemical heat production) of FaRLiP PSII is 70% greater than that of white light PSII. Due to the higher total energy output of Chlamydomonas reinhardtii with the introduction of FaRLiP under far-red light conditions, the growth rate of our Star-Chlamy with FaRLiP under far-red light will be higher than that of WT. All in all, introducing FaRLiP to solve the shade problem of Chlamydomonas reinhardtii in industrial production runway pond is a promising and feasible approach.

    Figure 7 Star-Chlamy with FaRLiP

    Broaden Application Scenarios of CCM

    Background

    Chlamydomonas reinhardtii has acquired a series of specialized photosynthetic structures and pathways to achieve high photosynthetic efficiency during long-term evolution. However, due to the limited CO₂ solubility in water, Chlamydomonas reinhardtii has only evolved mechanisms（CCM pathway）under lower CO₂ concentrations than conventional to maintain its photosynthetic rate, but not under abundant（over normal）CO₂ concentrations.

    In today's Chlamydomonas agriculture, regardless of whether the production mode is run tank or fermentation，there are various ways to ensure that the cultured Chlamydomonas receive higher light intensity than the wild environment without damaging the cells. Therefore, the CO2 (or CO3-) concentration in the culture medium has become the main factor limiting the photosynthetic rate and yield of Chlamydomonas.

    Figure 1

    Study showed that the photosynthetic rate of green algae increased from 5.1% to 9.8% by artificially enriching carbon dioxide and bicarbonate at the periphery of the cell wall[1]. This strongly support the feasibility of improving carbon assimilation capacity of Chlamydomonas to increase its photosynthetic rate under high carbon concentration.

    Figure 2

    Inspiration

    CCM, short for Carbon Concentrating Mechanism, represents a pathway that specially enrich carbon dioxide for photosynthesis in green algae such as Chlamydomonas reinhardtii. Through a series of coordinated membrane transporters and carbonic anhydrase, extracellular CO2 is actively transported into the pyrenoid structure within chloroplasts against its concentration gradient.

    CCM pathway is an adaptation of green algae to the changes of carbon dioxide concentration in the water environment. In wild-type Chlamydomonas reinhardtii cells, a shift to limiting CO2 conditions induces expression of more than 50 genes, most of which encodes proteins in CCM pathway, thereby enhancing the CCM process and improving the utilization of inorganic carbon during cellular photosynthesis.

    CIA5, a transcription factor protein found in Chlamydomonas cells, plays a pivotal role in activating downstream gene expression related to CCM[2]. Under limited CO2 condition, the chemical modification of CIA5 changes, thus CIA5 locates at the nucleus and functions as a transcription factor for CCM genes. However, under high CO2 condition, the modification of CIA5 is not favorable to its transcription factor activity, resulting as a low CCM efficiency.

    Figure 3. CCM pathway under limited and ambient CO2 conditions

    It has been reported that the CIA5 protein gene with a deletion of 54 amino acids in the C terminus can complement the CIA5 mutant phenotype[3], which suggests that the C terminus of Cia5 may function as the upstream CO2 sensing domain and regulate its own transcription factor activity. Therefore, we planned to transfer an artificial CIA5 mutant with a C-terminal truncation (CIA5-C-del) into the wild type Chlamydomonas reinhardtii to allow it to function as a transcription factor under any CO2 concentration.

    Besides CIA5, LCR1 is another transcription factor responsible for CCM genes, locating downstream of CIA5 in the CCM regulating pathway. CCM genes CAH1, LCI1 and LCI6 are induced by LCR1, suggesting that LCR1 transmits the low CO2 signal to at least three CO2-responsive genes and then fully induces CCM[4]. Thus it becomes another site where CCM could be regulated artificially.

    Figure 4 LCR1 function in CCM regulation pathway

    Design and Results

    3.1 Star-Chlamy with CIA5-C-del

    We constructed the CIA5-C-del plasmid that can be expressed in Chlamydomonas reinhardtii. The NLS sequences enhanced its transcription factor activity, and the 3x HA tag made sure that the expression of CIA5-C-del can be verified by performing Western Blot.

    Figure 5
    Figure 6 Western blot method was applied to verify the expression of the plasmid. Results showed no HA tag expression was observed in all 96 lines examined, which means that screening for the target phenotype should continue. Lane 1-12: Chlamy that fail to expression CIA5ΔC; PC: positive control; 1st Ab: Rat anti-HA; 2nd Ab: Goat anti-rat.

    3.2 other attempts and ideas

    3.2.1 CIA5 gene edit for confirming its function

    Figure 6

    Before we constructed the CIA5-C-del plasmid, we had tried to knock out the CIA5 gene at 4 different target site as well as reproduce the C-terminus truncation mutant by CRISPR method, aiming at confirming its CO2 responding function and transcription factor activity. A total of 1,008 algae strains after CRISPR operating had been tested by genotyping, with no positive result showing a successful editing on CIA5 gene. This suggests that 1) CIA5 is strongly required for Chlamydomonas life activities and CIA5 knockout may lead to cell death; 2) The chromatin status of CIA5 is not suitable for CIRSPR system for gene editing. Either way, we have provided a good topic for future iGEM teams to pursue Chlamydomonas related research.

    3.2.2 LCR1 expression plasmid’s construction

    Figure 7

    We also proposed to construct an LCR1 gene expression plasmid and insert it into Chlamydomonas genome, and to screen for a line with constitutively high LCR1 gene expression (relative to wild type). We hope that this could further enhance CCM protein expression in Chlamydomonas on the basis of the CIA5-C-del strain to further enhance its CO2 assimilation capacity.Relevant experiments are still in progress.

    Reference

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