1.Gene cloning from Arabidopsis thaliana

Starch consists of amylopectin and amylose. Amylopectin is a branched glucan polymer with α-1,6 glucosidic bonds that connect linear chains, which is synthesized by the catalytic action of several enzymes, such as ADP glucose pyrophosphorylase (AGPase), starch synthases (SSs), starch branching enzymes (BEs), and starch debranching enzymes (DBEs). Differently, amylose is essentially linear and composed of 1,4-linked α-D-glucan chains; AGPase and granule-bound starch synthase (GBSS) are involved in amylose production.

To realize the production of amylose synthesis in E. coli, we synthesized 2 genes related to the synthesis of amylose, which were ADP glucose pyrophosphorylase (AGPase) containing 2 subunits ADG1 and APL1, and granule-bound starch synthase 1 (GBSS1) based on the sequence from Arabidopsis thaliana.

1.1 ADP glucose pyrophosphorylase (AGPase)

AGPase catalyzes Glucose-6-phosphate (Glu-6-P) converted to adenosine diphosphate glucose (ADPG), which is a key enzyme in amylose synthesis and catalyzes the first, rate limiting step in amylose biosynthesis. The ADP glucose pyrophosphorylase gene contains 2 subunits, ADG1 and APL1. ADG1encodes the small subunit and APL1 encodes the large subunit of ADP-glucose pyrophosphorylase. The large subunit plays a regulatory role whereas the small subunit (ADG1) is the catalytic isoform responsible for ADP-glucose pyrophosphorylase activity.

In order to insert ADG1(1563bp) and APL1(1569bp) genes to the bi-expressing vector pETDuet-1, the ADG1 and APL1 genes were synthesized by adding restriction enzyme sites sequences at the 5’ and 3’ ends, which were EcoRI and PstI on flanking sequence of ADG1 gene, NdeI and Xhol on flanking sequence of APL1 gene, respectively.

First, we constructed the plasmid pETDuet-1-ADG1. The ADG1gene and vector pETDuet-1were digested by EcoRI and PstI. Gel extraction of digestion production of the ADG1gene and vector pETDuet-1 were performed, and were ligated together to construct the plasmid pETDuet-1-ADG1. Figure 1 shows the identification result for the plasmid pETDuet-1-ADG1 construction.

Fig.1. The identification result of plasmid pETDuet-1-ADG1construction.

M. Marker; 1. Plasmid pETDuet-1-ADG1 digested by EcoRI and PstI; 2. Plasmid pETDuet-1-ADG1.

Second, the APL1 gene was inserted to the plasmid pETDuet-1-ADG1 by restriction enzyme (NdeI and Xhol) digestion method, similar to those mentioned above. Figure 2 shows the identification result of the plasmid pETDuet-1-ADG-APL1 construction.

Fig.2. The identification result of plasmid pETDuet-1-ADG1-APL1construction. M. Marker; 1.

Plasmid pETDuet-1-ADG1-APL1; 2. Plasmid pETDuet-1-ADG1-APL1 digested by NdeI and Xhol.

1.2 Granule-bound starch synthase 1 (GBSS1)

Another enzyme for the amylose synthesis is Granule-bound starch synthase 1 (GBSS1), which belongs to UDP-Glycosyl transferase super family protein.

GBSS1 is an enzyme that catalyzes the transfer of glucose to glucose-containing polysaccharides with α1,4-linkage, which is the main gene controlling amylose synthesis.

In order to insert GBSS1(1833bp) gene to the vector pET-28a (+), the GBSS1 gene was synthesized by adding restriction enzyme sites sequences at the 5’ and 3’ ends, which were EcoRI and Xhol on flanking sequence of GBSS1 gene, respectively.

The GBSS1 gene was inserted to the vector pET-28a (+) by restriction enzyme digestion method. Figure 3 shows the identification result for the plasmid pET-28a (+)-GBSS1 construction.

Fig.3. The identification result of plasmid pET-28a (+)-GBSS1 construction.

M. Marker; 1. Plasmid pET-28a (+)-GBSS1; 2. Plasmid pET-28a (+)-GBSS1 digested by EcoRI and Xhol.

2.The expression of ADG1, APL1 and GBSS1 in E. coli

In order to synthesize amylose in E. coli, the two plasmids of pETDuet-1-ADG1-APL1 and pET-28a (+)-GBSS1 were co-transformed into E. coli (BL21) cells. This is a key step to realize the production of disposable and biodegradable straw made from amylose.

2.1 The expression of ADP glucose pyrophosphorylase (AGPase)

The ADG1 and APL1 were cloned into the EcoRI/PstI and NdeI/Xhol sites of pETDuet-1. Overnight cultures of E. coli (BL21) cells transformed with plasmid pETDuet-1-ADG1-APL1 were cultured in fresh LB medium containing ampicillin. Soluble cytoplasmic proteins were prepared from BL21 cells with and without IPTG induction, respectively. The purification of recombinant protein ADG1 with a 6xHis was performed by His-tag, and APL1 with fused S-tag in its C-terminus by S-tag according to the instruction. The SDS-PAGE result shows the ADG1 and APL1 expression in E. coli in figure 4 and 5, respectively.

Fig.4. The expression of ADG1 in transformed E. coli.

M. Marker; 1. Purified ADG1 protein (57kD) with IPTG induction; 2. Whole proteins of E. coli with IPTG induction; 3. Whole proteins of E. coli without IPTG induction.

Fig.5. The expression of APL1 in transformed E. coli.

M. Marker; 1. Whole proteins of E. coli without IPTG induction; 2. Purified APL1 protein (57.5kD) with IPTG induction; 3. Whole proteins of E. coli with IPTG induction.

2.2 The expression of Granule-bound starch synthase 1 (GBSS1)

The GBSS1 was cloned into the EcoRI/Xhol sites of pET-28a (+). Overnight cultures of E. coli (BL21) cells transformed with plasmid pET-28a (+)-GBSS1 were cultured in fresh LB medium containing kanamycin. Soluble cytoplasmic proteins were prepared from transformed BL21 cells containing pET-28a (+)-GBSS1 with and without IPTG induction, respectively. The purification of GBSS1 protein was performed by His-tag according to the instruction. The SDS-PAGE result shows the GBSS1 expression in E. coli in figure 6.

Fig.6. The expression of GBSS1 in transformed E. coli.

M. Marker; 1. Whole proteins of E. coli without IPTG induction; 2. Whole proteins of E. coli with IPTG induction; 3. Purified GBSS1 protein (67kD) with IPTG induction.

3.The optimization of culture media conditions of E. coli

After the identification of the recombinant genes construction, we hoped to culture the E. coli to optimize some conditions for high amylose production yield. We searched some references, and figure out that the iodine binding e assay in a 96-well plate format for measuring amylose at both OD₆₂₀ nm and OD₅₁₀ nm. The initial testing to produce a standard curve, utilizing amylose as a standard, was scaled to a 96-well plate format. The dual wavelength method exhibited a very high coefficient of determination (R²= 0.993) with plate to plate repeatability on slope and intercept values. The level of accuracy and repeatability of the standard curves allows for quantification of amylose in samples.

We found that the temperature, pH value, fermentation time, inoculum size, and the carbon sources are the important conditions. So, we tried to culture the E. coli for amylose production with different conditions. And the results are showed in the following figures (Fig. 7 - Fig. 11).

Fig.7. The effect of temperature on amylose production.

Fig.8. The effect of pH value on amylose production.

Fig.9. The effect of fermentation time on amylose production.

Fig.10. The effect of inoculum size on amylose production.

Fig.11. The effect of carbon sources on amylose production.

Glu, Glucose; Man, Mannitol; Suc, Sucrose; Fru, Fructose; GluA, glucosamine; 2-DOG, 2-deoxyglucose; 3-OMG, 3-O-methylglucose.

The optimization experiment results indicated that 37°C, pH7.6, fermentation 22h, 14% inoculum size, and glucose serving as the carbon source are the best culture conditions for high amylose production yield in E. coli. And we know that for the amylose production in E. coli, it still need further experiments to realize disposable and biodegradable straw made from amylose produced by E. coli.