**6.1 Metabolic engineering for modifying starch content**

**Figure 6** and **Table 5** highlights the information on important candidate genes used in the modification of starch content in transgenic potato tubers. ADP-glucose pyrophosphorylase (AGPase), starch synthase (SS) and starch branching enzyme (SBE) are involved in the process of starch synthesis while amylases (AMY) and starch phosphorylase (SP) are responsible for its breakdown [152]. Amylose is synthesized by the granule bound starch synthase (GBSS), whereas soluble starch synthases (isoforms) and SBEI/SBEII, with various debranching enzymes (DBE), kinases and

#### **Figure 6.**

*Simplified schematic overview of starch and sugar metabolism. The figure explains the simplified pathway of starch and sugar metabolism in potato tubers. Only the key enzymes involved in the pathway are shown in the figure. Starch synthesis involves AGPase, ADP-glucose pyrophosphorylase; SS, starch synthase; GBSS, granule bound starch synthase; and SBE, starch branching enzyme. This process involves more than one SS and SBE (not shown in figure). Further starch is degraded into Glucose-1-Phosphate (Glu-1-P) by Amy, amylase; SP, starch phosphorylase which forms Glucose-6-Phosphate (Glu-6-P) via PGM, phosphoglucomutase. Fructose-6- Phosphate (Fru-6-P) is formed from PHI, Glc-6-P via phosphohexoisomerase. Sucrose is formed in the cytoplasm from UDP-Glucose. SPS and S6S are sucrose phosphate synthase and sucrose 6 phosphate phosphatase. UGPase, UDP-glucose pyrophosphorylase; SPS, sucrose phosphate synthase; and S6P, sucrose 6-phosphate phosphatase are involved in the process. Sucrose is transported into the vacuole and converted by AI, acid invertase leading to the formation of RS glucose and fructose. AI is inhibited by vacuolar invertase inhibitor (INH) in the vacuole. NI, neutral invertase converts sucrose into glucose and fructose in the cytoplasm. The figure is adapted and modified from Sowokinos [139].*


#### **Table 5.**

*Genes used in the modification of carbohydrate metabolism and enzymatic browning in transgenic potatoes.*

other enzymes are also involved in amylopectin synthesis [153, 154]. The modification of starch properties is important to food processing industries and the demand for amylose free potatoes wherein higher amylopectin content is desired [122, 155]. Food products containing high amylose content and long chains of amylopectin contribute to the formation of resistant starch that is responsible for a lower glycemic index after intake with enhanced health benefits by promoting the growth of healthy gut fora and lowering both the caloric intake and cholesterol levels in the blood [156–158].

Transgenic studies focused on the modification of starch content and its properties using the genes involved starch biosynthesis in potatoes [140, 141, 143–145, 159–163]. AGPase is the key enzyme involved in the synthesis of starch in amyloplasts which consists of two regulatory subunits and two slightly smaller catalytic subunits. The function of the large subunit of AGPase is to modulate the regulatory properties of the small subunit (sAGP) and the function of sAGP is primarily catalysis [152, 159, 160]. An antisense inhibition of sAGP under the control of CaMV 35S promoter resulted in starch reduction and a lower amylose content in transgenic tubers [143].

Nutritional quality enrichment by starch modification can be efficiently performed using TALENs technology [164] as well as CRISPR/Cas9 system [142]. Site-specific mutations induced in *GBSS* gene (CaMv35S promoter) using Emerald-Gateway TALEN system resulted in 63 nucleotide deletion suggesting that the system can be utilized for nutritional enhancement in potato [164]. All four *GBSS* alleles were knockout by CRISPR/Cas9 technology in cv. 'Kuras' protoplasts resulted in complete loss of GBSS activity and amylose-free high amylopectin starch in regenerated potato microtubers [142]. In another study, overexpression of SBEII using hybrid cDNA/ gDNA intragene construct containing a single intron increased short-chain branching of amylopectin and altered the physicochemical properties of starch in potato tuber [165]. CRISPR-Cas9 was used to crete mutations in the two *SBEs* (*SBE1* or *SBE2* alone or in combination) in potato. Results revealed that lines mutated in SBE1 did not have an altered starch structure, while tuber cells from SBE2 mutated lines displayed an increased number of starch granules. One line had a strong reduction in both SBEs, resulting in starch with an altered granule phenotype, longer amylopectin chains and

reduction in a degree of branching [146]. The quality characteristics of potatoes for table and processing purposes are largely dependent on the starch, dry matter, sugar concentration and tubers free from any deformities such as enzymatic discoloration. Overall studies revealed that, transgenic, intragenic as well as CRISPR tools proved their usefulness in modification of various starch synthesizing enzymes using gene silencing or gene knockout approaches. Alteration in starch synthesis genes can lead to modification in starch properties without affecting the yield and dry matter content of the transgenic tubers.
