Enhanced productivity of extracellular free fatty acids by gene disruptions of acyl-ACP synthetase and S-layer protein in Synechocystis sp. PCC 6803

Abstract

Background Based on known metabolic response to excess free fatty acid (FFA) products, cyanobacterium Synechocystis sp. PCC 6803 preferentially both recycles via FFA recycling process and secrets them into medium. Engineered cyanobacteria with well growth and highly secreted FFA capability are considered best resources for biofuel production and sustainable biotechnology. In this study, to achieve the higher FFA secretion goal, we successfully constructs Synechocystis sp. PCC 6803 mutants disrupting genes related to FFA recycling reaction (aas gene encoding acyl–acyl carrier protein synthetase), and surface layer protein (encoded by sll1951). Results Three Synechocystis sp. PCC 6803 engineered strains, including two single mutants lacking aas (KA) and sll1951 (KS), and one double mutant lacking both aas and sll1951 (KAS), significantly secreted FFAs higher than that of wild type (WT). Certain increase of secreted FFAs was noted when cells were exposed to nitrogen-deficient conditions, BG11-half N and BG11-N conditions, with the exception of strain KS. Under BG11-N condition at day 10, strain KAS strikingly secreted FFAs products up to 40%w/DCW or 238.1 mg/L, with trace amounts of PHB. Unexpectedly, strain KS, with S-layer disruption, appeared to have endured longer in BG11-N growth medium. This strain KS significantly acclimated to the BG11-N environment by accumulating a greater glycogen pool with lower FFA production, whereas strain KA favored higher PHB and intracellular lipid accumulations with moderate FFA secretion. Conclusions Mutations of both aas and sll1951 genes in Synechocystis sp. PCC 6803 significantly improved the productivity of secreted FFAs, especially under nitrogen deprivation.

Incorporation, fate, and turnover of free fatty acids in cyanobacteria

“…In those double and triple mutants, lipA transcription was observed to be increased (Eungrasamee et al 2020 ). Building up from these results, the same authors reported that the highest level of secreted FFAs, along with the accumulation of neutral lipid droplet in the vicinity of thylakoid membranes was reached after overexpressing lipA in an aas -deleted strain (Eungrasamee et al 2022 ). Physiologically, LipA was very recently shown to participate in PSII repair through deacylation of galactolipids present in PSII dimers (Jimbo and Wada 2023 ).…”

“…Not only did deletion of rndA1B1 in Synechococcus 7942 result in impairment of FFA secretion, but also overexpression of these genes in a high FFA-producing Synechococcus 7942-based strain enhanced FFA accumulation extracellularly (Kato et al 2015 ). Another interesting observation is that deletion of the gene encoding the S-layer protein in Synechocystis 6803 resulted in increased FFA-secretion in comparison to the parental strain (Liu et al 2011 , Eungrasamee et al 2022 ). The S-layer (or surface layer) is a cellular structure located outside of the OM of some bacteria, composed mainly of a glycoprotein; in Synechocystis 6803 that protein is Sll1951 (Trautner and Vermaas 2013 ).…”

“…For example, E. coli TesA (TE I) was introduced into Synechocystis 6803 to enhance FFA production (Liu et al 2011 ). To date, deletion of the Aas enzyme in Synechocystis 6803 and Synechococcus 7942, overexpression of the lipA, or both, are among the described modifications performed to significantly increase the production of FFAs (Kaczmarzyk 2008 , Kaczmarzyk and Fulda 2010 , Kato et al 2015 , Eungrasamee et al 2019 , 2020 , 2022 , Kojima et al 2022 ). The FASII was also targeted as for pathway engineering (Santos-Merino et al 2018 ): for instance, overexpression of the acc genes encoding for acetyl CoA carboxylase (Kawahara and Hihara 2021 ) as well as deletion of lexA (Kizawa et al 2017 ), encoding a key transcription regulator of carbon assimilation pathways (Domain et al 2004 ) were reported to improve FFA production.…”

The adc1 knockout with proC overexpression in Synechocystis sp. PCC 6803 induces a diversion of acetyl-CoA to produce more polyhydroxybutyrate

“…Then, dissolved sample (0.2 mL) was mixed with 10% H 2 SO 4 (0.2 mL) and anthrone reagent (0.8 mL) before boiling for 10 min. After cooling down the samples to room temperature, the absorbance of samples was measured at 625 nm by spectrophotometer (modified from [ 4 , 7 ]). A commercial oyster glycogen was used as the standard which was prepared as similar as the sample.…”

Increased Biomass and Polyhydroxybutyrate Production by Synechocystis sp. PCC 6803 Overexpressing RuBisCO Genes

“…To obtain the glycogen pellets, the sample mixture was centrifuged at 12,000 rpm (14,489× g ), at 4 °C for 30 min, and dried at 60 °C for overnight. The anthrone assay was used for determining the glycogen content, and commercial oyster glycogen (Sigma-Aldrich, St. Louis, MO, USA) served as the standard (modified from [ 56 , 57 ]). Glycogen pellets were dissolved with 1 mL of 10% H 2 SO 4 .…”