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Molecular Biological Approaches for the Improvement of Biofuels Production
Published in Debabrata Das, Jhansi L. Varanasi, Fundamentals of Biofuel Production Processes, 2019
Debabrata Das, Jhansi L. Varanasi
Genes are the fundamental codes in DNA by virtue of which specific functions are carried out by the organisms. The elucidation of DNA sequences using recombinant DNA technologies has enabled the identification of the gene patterns of an organism having similarity with the proteins of known functions. Gene deletion refers to the permanent deletion of the gene function from an organism. In this approach, part of DNA or chromosome is deleted using site specific nucleases such as Zinc fingers, TALENS (transcription activator-like effector nucleases), and CRISPR (clustered regularly interspaced short palindromic repeats) (Figure 5.1). On the other hand, gene knockout refers to the suppression or deactivation of a gene function through genetic engineering. Gene knockout is necessary when the deletion of a particular gene is lethal to the organism. Thus, unlike gene deletion where the gene is permanently removed, in gene knockout the genes remain present in an inactive state. There are three main approaches for gene deletion or gene knockout: (1) replacing the gene with other non-functional sequences, (2) introducing an allele whose encoded protein inhibits functioning of the expressed normal protein, and (3) promoting destruction of mRNA expressed from a gene. The first approach leads to the modification of endogenous gene while in the second and third approach the endogenous gene is unaltered. Gene deletion is often used as a strategy to suppress the production of unwanted metabolites and enhance the production of targeted compounds during biofuel production. For example, deletion of gene encoding the twin-arginine translocation system (TAT) in the wild strain E. coli MC4100 led to enhanced hydrogen productivity (double than the wild type) in the mutant strain E. coli HD701 (Penfold et al. 2003). Similarly, targeted gene knockouts of nitrate reductase gene HygR in Nannochloropsis oceanica led to higher triacylglycerol (TAG) accumulation (Wang et al. 2016). Gene deletion or gene knockout of several genes in a pathway can be used to block the entire pathway. The manipulation of entire metabolic pathway will be discussed in the following metabolic engineering section.
Cadmium contamination in food crops: Risk assessment and control in smart age
Published in Critical Reviews in Environmental Science and Technology, 2023
Yan Huili, Zhang Hezifan, Hao Shuangnan, Wang Luyao, Xu Wenxiu, Ma Mi, Luo Yongming, He Zhenyan
Recently, many innovative gene editing tools have been developed, which allow not only randomly knock-down or knock-out genes, but also accurately deletion, insertion and substitution of one base in genome. Prime editing has come into sight because it can realize artificial variation creation including insertion, deletion and substitution between any base pairs. Using modified prime-editing-guide RNA (pegRNA) which guides the nickase to the proper editing target and serves as template of reverse transcriptase, prime editing can be more accurate with less side-effect and no need of donor DNA (Lin et al., 2020). In plant cells, Zong et al. has created an engineered plant prime editor (ePPE) with 2 modifications on the reverse transcriptase, which provided a 5.8-fold enhancement for editing efficiency with no significant increase of byproduct and miss-target effect when tested in rice (Zong et al., 2022).