Synthetic Biology and Genome-Editing Tools for Improving PHA Metabolic Engineering

The bioplastic PHA, which features biodegradability, biocompatibility, and thermoprocessibility, is moving toward low-cost microbial production to replace nondegradable petrochemical plastics. Wild-type or weakly engineered bacteria are insufficient to meet demands for improved PHA structures and low production cost. Synthetic biology and genome-editing approaches can promote PHA synthesis, enlarge cells for more PHA storage, control shape changes, accelerate growth, aid the co-production of multiple products, direct flux toward final products, and make product recovery more convenient. Optimized promoters and RBSs increase the expression of PHA synthesis genes. CRISPR interference and CRISPR/Cas9 are useful for downregulating the expression of multiple genes simultaneously, allowing more flux to be directed to PHA synthesis in an optimized strain. Polyhydroxyalkanoates (PHAs) are a diverse family of biopolyesters synthesized by many natural or engineered bacteria. Synthetic biology and DNA-editing approaches have been adopted to engineer cells for more efficient PHA production. Recent advances in synthetic biology applied to improve PHA biosynthesis include ribosome-binding site (RBS) optimization, promoter engineering, chromosomal integration, cell morphology engineering, cell growth behavior reprograming, and downstream processing. More importantly, the genome-editing tool clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been applied to optimize the PHA synthetic pathway, regulate PHA synthesis-related metabolic flux, and control cell shapes in model organisms, such as Escherichia coli, and non-model organisms, such as Halomonas. These synthetic biology methods and genome-editing tools contribute to controllable PHA molecular weights and compositions, enhanced PHA accumulation, and easy downstream processing. Polyhydroxyalkanoates (PHAs) are a diverse family of biopolyesters synthesized by many natural or engineered bacteria. Synthetic biology and DNA-editing approaches have been adopted to engineer cells for more efficient PHA production. Recent advances in synthetic biology applied to improve PHA biosynthesis include ribosome-binding site (RBS) optimization, promoter engineering, chromosomal integration, cell morphology engineering, cell growth behavior reprograming, and downstream processing. More importantly, the genome-editing tool clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been applied to optimize the PHA synthetic pathway, regulate PHA synthesis-related metabolic flux, and control cell shapes in model organisms, such as Escherichia coli, and non-model organisms, such as Halomonas. These synthetic biology methods and genome-editing tools contribute to controllable PHA molecular weights and compositions, enhanced PHA accumulation, and easy downstream processing. developed by mutating both the nuclease (RuvC and HNH) domains of Cas9. dCas9 lacks DNA-cleavage activity but retains RNA-directed DNA-binding activity. a two-component genome-editing system including a single guide RNA (sgRNA) and a Cas9 nuclease that can achieve simple and precise genetic manipulation. A predesigned sgRNA directs Cas9 to bind to and cut a DNA sequence upstream. genetic interference technique that allows for sequence-specific repression of gene expression in different organisms. This is a two-component genome-editing system including a sgRNA and a dCas9 nuclease with mutated nuclease (RuvC and HNH) domains. changing DNA sequences in genomes of organisms. changing the shape of an organism using molecular engineering approaches. two forms of NAD; the reduced and oxidized forms are abbreviated as NADH and NAD+, respectively. They have important roles in enzyme-catalyzed metabolic reactions as electron carriers. family of diverse intracellular biopolyesters synthesized by many microorganisms. PHAs serve as a source of energy and a carbon store; they also provide stress resistance for bacterial cells. changing DNA sequences of a promoter, which is a region of DNA located near the transcription start sites of a gene, to improve gene expression in organisms. changing DNA sequences of ribosome-binding sites to improve gene expression in organisms. T7-like RNA polymerase-promoter pairs, obtained by mining phage genomes. There are three expression systems, namely MmP1, VP4 and K1F, which can be induced to different gene expression in the presence of an IPTG inducer.