Evolutionary Engineering of Cyanobacteria to Enhance the Production of α-Farnesene from CO₂

Abstract

Photosynthetic cyanobacteria can fix CO2 and utilize it as the sole carbon source for cell growth and production of biochemicals. Here, we metabolically engineered Synechococcus elongatus PCC 7942 for an enhanced production of α-farnesene by optimizing the ribosome-binding site (RBS) of the codon-optimized farnesene synthase gene. The production of α-farnesene was found to be enhanced in strains with a low translation initiation rate, resulting in α-farnesene production (0.57 mg/(L day)). Using the RBS variants and random mutations, we performed fluorescence-based analysis of cells grown in 96-well culture plates to screen the α-farnesene-producing strains but could not improve the titers of the RBS-optimized strains. However, evolutionary engineering of the RBS-optimized strains resulted in a twofold increase in α-farnesene production (1.2 mg/(L day)) compared to the previous study. Therefore, combining metabolic and evolutionary engineering might be helpful for enhancing the cellular fitness of cyanobacteria for the production of target chemicals.

Screening and characterization of polyhydroxyalkanoate granules, and phylogenetic analysis of polyhydroxyalkanoate synthase gene PhaC in cyanobacteria

“…2012, Lai and Lan 2015, Pattharaprachayakul et al. 2019, Choi and Woo 2020), and high genetic and metabolic diversity (Bolch et al. 1999, Welker et al.…”

“…Key index words: Carbon storage; Cyanobacteria; Horizontal gene transfer; Polyhydroxyalkanoates; Polyhydroxybutyrate Abbreviations: HGT, horizontal gene transfer; LDs, neutral lipid droplets; NRPs, nonribosomal peptides; PHAs, polyhydroxyalkanoates; PHB, poly-3hydroxybutyrate Cyanobacteria are good candidates for the development and production of bioproducts due to their short life cycles, ease of genetic manipulation (Rosgaard et al 2012, Lai and Lan 2015, Pattharaprachayakul et al 2019, Choi and Woo 2020, and high genetic and metabolic diversity (Bolch et al 1999, Welker et al 2006, Beck et al 2012) resulting in part due to a rich history of horizontal transfer of genes (Matzke et al 2014, Wang et al 2020, and in some cases entire operons (Marin et al 2007, Rae et al 2011. Their ability to fix carbon dioxide (Asada et al 1999, Peramuna and Summers 2014, Velu et al 2020, and, in some cases nitrogen (Do Nascimento et al 2015, Chittapun et al 2018, Godlewska et al 2019, in addition to their tolerance for wide ranges of pH, salinity, and temperature (Hirooka et al 2014, Varshney et al 2015, give cyanobacteria potential cost-saving advantages when used for the production of bioproducts (Georgianna andMayfield 2012, Singh et al 2016).…”

Genetic, Genomics, and Responses to Stresses in Cyanobacteria: Biotechnological Implications

“…In Synechococcus PCC 7942 (Table 2), the most frequently employed chromosomal neutral sites are NSI (GenBank accession n • U30252), NSII (GenBank accession U44761) [253]), and NSIII (GenBank accession ABB56771.1) [254,255]. Photoproduction of isobutyraldehyde [360] Photoproduction of 1-butanol [361,362] Analysis of carboxysomes [363,364] Photoproduction of isopropanol [364] Photoproduction of isobutanol [365] Photoproduction of 3-hydroxypropionic acid [366] Photoproduction of 1,3-propanediol [367] Photoproduction of amorphadiene and squalene [368] Photoproduction of limonene [369] Photoproduction of acetone [370] Photoproduction of isoprene [371] Analysis of gene-expression control systems [372] Photoproduction of 2,3-butanediol [373] Photoproduction of farnesene [374,375] Photoproduction of lactate [376] NSI and NSIII Photoproduction of biomass and sucrose export [377] Photoproduction of a synthetic CO 2 -fixing photorespiratory bypass [378] Photoproduction of 2,3 butanediol [379] Photoproduction of amorphadiene or squalene [380] NSI, NSII, and NSIII Overproduction of transporters to facilitate sugar export [254] Promoter analysis [381] Photoproduction of polyketides [382] Analysis of the influence of pilus biogenesis on the natural transformation [229] psbA1 Photoproduction of ethylene [383] glgc Photoproduction of isobutanol [366]…”

Recent Advances in the Biosynthesis of Farnesene Using Metabolic Engineering

“…61 Later, Pattharaprachayakul et al reported the α-farnesene production in S. elongates PCC 7942, and the titer reached 13.0 mg/L by optimizing the expression components of FS and adaptive evolution of strains. 62 Currently, the research on farnesene production in cyanobacteria remains stuck in the laboratory stage, owing to the bottleneck associated with the low yield. The lack of highly efficient genetic editing tools and genetic components resulted in the MVA pathway not being introduced into cyanobacteria.…”

“…81,82 Moreover, the sequential error-prone polymerase chain reaction (PCR) was selected for screening random mutants of FS with high catalytic activity. 62 In this study, more than 500 FS mutant strains were obtained by measuring the level of intracellular inorganic pyrophosphate (PPi). However, the mutant strains with FS activity exceeding that of the control group were not found, possibly because the lack of a fluoresce-based biosensor has hindered the development of a FACS-assisted AFS system.…”