Production of hydroxy fatty acids and its effects on photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803
Introduction
Recently, vegetable oils have become an important feedstock for chemicals as they contain lipids and fatty acids (FAs), which are not present in petroleum oil, and large amount of vegetable oils are produced worldwide [1] and used in the oleochemical industry for various applications. To expand the use of lipids and FAs from vegetable oils as raw materials, the synthesis of functionalized FAs by chemical and biotechnological engineering is essential. The hydroxy group is one of the most effective functional groups in the chemical reactivity of FAs, and its positions in the acyl chain play a vital role in its physiological mechanisms and chemical applications [2]. In particular, FAs that have a hydroxy group at the proximity of the ω-position of the acyl chain possess a high availability for various kinds of chemical feedstock, such as adhesives, lubricants, cosmetic intermediates [3], potential anticancer agents [4], and building blocks for the synthesis of polyesters, which exhibit similar or superior physicochemical properties to polyethylene and other bioplastics [5]. FAs with a hydroxy group near the ω-position are naturally produced in a wide variety of organisms [[6], [7], [8]], most of which synthesize these hydroxylated FAs through ω-oxidation by cytochrome P450 monooxygenase. Whole-cell biocatalysis of the enzyme achieves a high conversion ratio of FA hydroxylation and shows great potential for large-scale production of industrial applications [9].
Microalgal oil is attracting attention as a resource of FA because microalgae have the potential to achieve high productivity, cultivate on non-arable land, grow in wastewater, and their cells can be modified by genetic engineering. Despite these advantages, FA from microalgae is a controversial technology because several issues remain before achieving economic competitiveness in the production process, i.e., collection of cells, drying of the cells, and FA extraction. The production of high-value compounds by microalgae is a potential solution to achieve economic competitiveness. As high-value products from microalgal oils, hydroxy FAs might be a possible candidate that could be applied for the feedstock of unique compounds. Recently, the genetically modified diatom Chaetoceros gracilis, with the FA dehydrogenase gene from ergot fungus Claviceps purpurea, produces ricinoleic acid (12-hydroxy-9-cis-octadecanoic acid) [10]. Despite the growth inhibition of yeast or other organisms owing to the production of hydroxy FAs, this diatom can produce ricinoleic acid without growth inhibition because detoxification of hydroxy FA naturally occurs by esterification of the hydroxy group with a carboxyl group on the other FAs. In this diatom, The detoxification mechanism and the effect of hydroxy FA on photosynthesis are unclear [11]. However, there are no studies either to produce FAs that contain a hydroxy group at the proximity of the ω-position by genetic engineering of microalgae or to investigate the effect of hydroxy FAs on photosynthesis.
Cyanobacteria are utilized as a model to investigate the function and mechanism of photosynthesis. A study on the cyanobacteria FAs is significant in terms of FA production and physiological analysis. For free FA production, overproduction and secretion of free FAs are achieved in cyanobacterial strains by genetic manipulations [[12], [13], [14]]. In addition, to obtain cyanobacterial cells that are oxidation-tolerant and maintain the fluidity of membrane lipids, we succeeded in producing FAs such as cyclopropane FAs and 10-methyl stearic acid in transformants of Synechocystis sp. PCC 6803 (hereafter Synechocystis) [15,16]. These results indicate that cyanobacteria are a promising organism for application in the modification to produce unnatural FAs. A thorough study on the relationship between FA and photosynthetic pigment in Synechocystis can help understand the effects of hydroxy FA production on photosynthesis, which has never been investigated. Regarding biochemical synthesis, cyanobacteria mainly produce glycolipids, i.e., monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) as the major lipid components, while Escherichia coli and Rhodospirillum rubrum, which are host organisms for the production of hydroxy FAs in this study, mainly synthesize phospholipids. Glycolipids with hydroxy FAs have the potential to be utilized as novel glycolipid biosurfactants [17].
In this study, we attempted to synthesize (ω−1)-hydroxy fatty acids (ω1HFAs) in the cyanobacterium Synechocystis by genetic engineering. Synechocystis synthesizes 3-hydroxybutyryl-CoA (3HB-CoA) as a metabolic intermediate in the synthesis of polyhydroxybutyrate (PHB). If 3HB-CoA is incorporated into the de novo synthesis pathway of FA instead of acetyl-CoA, it might produce ω1HFAs. To achieve this, the substrate flexibility of β-ketoacyl-ACP-synthase III (KASIII), which is the enzyme for the first reaction of the fatty acid synthesis pathway, is attractive. The reaction by KASIII defines the chemical structure of the ω-end of FAs by the structure of the acyl-CoA moiety and substrate specificity of KASIII. In general, KASIII uses acetyl-CoA as a substrate. KASIII from Alicyclobacillus (aaKASIII) uses alicyclic acyl-CoA instead of acetyl-CoA, and its specific activity for the reaction between 3HB-CoA and malonyl-ACP is 3-fold higher than that for the reaction between acetyl-CoA and malonyl-ACP [18]. This feature of aaKASIII is good for 3HB-CoA as a substrate. For 3HB-CoA biosynthesis, PhaAB from Cupriavidus necator H16 (CPhaAB) is one of the best enzymes in Synechocystis. In previous studies, CphaAB showed much higher activity than other enzymes for overproduction of PHB or 3-hydroxybutyrate in engineered cyanobacteria and did not show growth inhibition [19,20]. An engineered E. coli strain, with the promiscuous KASIII and 3HB-CoA synthesis pathway, produces ω1HFAs [18]. Thus, we adapted this strategy for the production of ω1HFAs in Synechocystis.
Section snippets
Organisms and culture conditions
In this study, a glucose-tolerant strain of Synechocystis [21] was used as the wild-type strain. Synechocystis cells were grown in BG11 medium [22] buffered with 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–NaOH (pH 7.5) at 34 °C under continuous illumination by white fluorescent lamps and aerated with 1% (v/v) CO2-enriched air [23]. Fifty milliliters of cultures were used for measurement of growth, FA compositions, and mixotrophic culture, and 500 mL of cultures were used
Construction of the hydroxy FA synthetic pathway and analysis of the FAs in transformants
To introduce 3HB-CoA into the FA synthesis pathway in Synechocystis cells, we utilized a substrate-variable β-keto-acyl-(acyl carrier protein) synthase III (KASIII), which catalyzes the condensation of acetyl-CoA and malonyl-ACP at the first reaction of the FA synthesis pathway. KASIII from A. acidocalderius (aaKASIII) has a very wide substrate specificity [18], which can react with many types of acyl-CoA, e.g., propionyl-CoA, isobutyryl-CoA, and benzoyl-CoA, as substrates instead of
Conclusions
We succeeded in constructing the ω1HFA-producing cyanobacterial strain, which expresses the promiscuous KASIII from A. acidocalderius by substitution of the native KASIII gene and the exogenous PhaAB from C. necator H16 by substitution of the PHB synthetase gene. Under 70 μmol photons m−2 s−1, this transformant produced 2.1 mol% of ω1HFAs in the total FAs and showed growth inhibition. In contrast, under 35 μmol photons m−2 s−1, the mutant strain growth recovered to almost the same level as that
CRediT authorship contribution statement
Takashi Inada: Conceptualization, methodology, validation, formal analysis, investigation, writing - original draft, visualization. Shuntaro Machida: Methodology, investigation, resources. Koichiro Awai: Investigation resources. Iwane Suzuki: Conceptualization, methodology, resources, writing-review & editing, visualization, supervision, project administration, funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
The strains of A. acidocalderius subsp. acidocalderius JCM 5260T and C. necator H16 JCM 20644 were obtained from RIKEN BRC, which is a participant in the National BioResources Project of MEXT, Japan. This work was supported by the Japanese Society for the Promotion of Science (JSPS) KAKENHI [grant numbers JP17H00800].
Statement of informed consent, human/animal rights
We did not apply any materials from human and animal in this study.
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Production of Fatty Acids and Derivatives Using Cyanobacteria
2023, Advances in Biochemical Engineering/Biotechnology
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