Journal of Energy Bioscience 2025, Vol.16, No.3, 151-162 http://bioscipublisher.com/index.php/jeb 155 5 Biotechnology and Genetic Engineering in Maize Biofuel Breeding 5.1 Controlling lignin and cellulose content with genetic engineering Lignin is an important part of the cell wall. It helps plants stay strong and resist stress. But if there is too much lignin, it makes it harder for enzymes to break down the plant material, which also makes it tough for cellulose and hemicellulose to be broken down. To solve this, scientists use genetic engineering to control how lignin is made. They target key enzyme genes in the process, like using antisense RNA or RNA interference to reduce the expression of the Caffeoyl-CoA O-methyltransferase gene (Abdel-Rhman, 2015). One method involves using a particle bombardment technique to insert an antisense fragment of the 4CL gene into maize. This reduces lignin content and increases the amount of bioethanol produced from the straw (Abdel-Rhman, 2015). Some natural brown midrib (bm) mutants have also been used to breed maize varieties with lower lignin content (Choudhary et al., 2020). Besides controlling lignin, scientists are also trying to adjust the cellulose content and its crystallization using genetic engineering. They’ve even introduced the genes for hydrolytic enzymes into maize, so the maize can produce its own cellulase. This reduces the need for added enzymes during the biomass conversion process (Abdel-Rhman, 2015). 5.2 Improving biofuel traits with CRISPR/Cas9 gene editing Compared to traditional genetic modification methods, CRISPR/Cas9 is faster and more accurate. It can directly modify specific parts of DNA, like knocking out, inserting, or adjusting genes. In controlling lignin and cellulose content in maize straw, this technology can directly target the key genes involved in lignin synthesis (Barriere et al., 2009). Scientists have also used CRISPR/Cas9 to adjust genes related to starch synthesis, making the starch structure and ratio in maize kernels more suitable. This improves fermentation efficiency and increases ethanol production (Niu et al., 2023) (Figure 1). Figure 1 Starch biosynthesis in maize endosperm. Black arrows show the conventional biosynthesis of starch: glucose-1-P is converted to ADPG, which is transported into the amyloplast and polymerized into starch. Red arrows show the oxidative pentose phosphate pathway, which provides NADPH and pentose sugars for starch synthesis. 1, sucrose synthase; 2, UDP-glucose pyrophosphorylase; 3, hexokinase; 4, AGPase; 5, phosphoglucomutase; 6, complexes including AGPase, starch synthase IIa (SSIIa), SSIII, starch branching enzyme IIb (SBEIIb), and SBEIIa; 7, glucose-6-phosphate dehydrogenase; 8, 6-phosphogluconate dehydrogenase; 9, malic enzyme; 10, transaldolase; 11, transketolase; 12, triose-P/P translocator and P/phosphoenolpyruvate translocator. (Adopted from Niu et al., 2023)
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