Improvement of fatty acid productivity of thraustochytrid, Aurantiochytrium sp. by genome editing

Watanabe, Kenshi; Perez, Charose Marie Ting; Kitahori, Tomoki; Hata, Kunihiko; Aoi, Masato; Takahashi, Hirokazu; Sakuma, Tetsushi; Okamura, Yoshiko; Nakashimada, Yutaka; Yamamoto, Takashi; Matsuyama, Kiichi; Mayuzumi, Shinzo; Aki, Tsunehiro

doi:10.1016/j.jbiosc.2020.11.013

Cited by 16 publications

(10 citation statements)

References 54 publications

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“…It was reported that disrupting the gene associated with β-oxidation ( Acox ) could reduce the degradation of fatty acids, and thus increased the accumulation of total lipids in Aurantiochytrium sp. ( 10 ). Acox catalyzes the transformation of acyl-CoA into trans-2, 3-dehydroacyl-CoA ( 35 ).…”

Section: Resultsmentioning

confidence: 99%

Development of an Efficient Gene Editing Tool in Schizochytrium sp. and Improving Its Lipid and Terpenoid Biosynthesis

Huang

Sun

et al. 2021

Front. Nutr.

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Schizochytrium sp. HX-308 is a marine microalga with fast growth and high lipid content, which has potential as microbial cell factories for lipid compound biosynthesis. It is significant to develop efficient genetic editing tool and discover molecular target in Schizochytrium sp. HX-308 for lipid compound biosynthesis. In this study, we developed an efficient gene editing tool in HX-308 which was mediated by Agrobacterium tumefaciens AGL-1. Results showed that the random integration efficiency reached 100%, and the homologous recombination efficiency reached about 30%. Furthermore, the metabolic pathway of lipid and terpenoid biosynthesis were engineered. Firstly, the acetyl-CoA c-acetyltransferase was overexpressed in HX-308 with a strong constitutive promoter. With the overexpression of acetyl-CoA c-acetyltransferase, more acetyl-CoA was used to synthesize terpenoids, and the production of squalene, β-carotene and astaxanthin was increased 5.4, 1.8, and 2.4 times, respectively. Interestingly, the production of saturated fatty acids and polyunsaturated fatty acids also changed. Moreover, three Acyl-CoA oxidase genes which catalyze the first step of β-oxidation were knocked out using homologous recombination. Results showed that the production of lipids increased in the three knock-out strains. Our results demonstrated that the A. tumefaciens-mediated transformation method will be of great use for the study of function genes, as well as developing Schizochytrium sp. as a strong cell factory for producing high value products.

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Section: Resultsmentioning

confidence: 99%

Development of an Efficient Gene Editing Tool in Schizochytrium sp. and Improving Its Lipid and Terpenoid Biosynthesis

Huang

Sun

et al. 2021

Front. Nutr.

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“…Recently, zeocin resistance cassettes were shown to be more efficiently integrated into the genome of Aurantiochytrium sp. RH-7A and A. limacinum SR21 with assistance from the CRISPR-Cas9 system using HDR [ 36 ]…”

Section: The Properties Of the Dna Affect The Outcome Of The Transformationmentioning

confidence: 99%

“…In the recently established CRISPR-Cas9 method in thraustochytrids, the Cas9-gRNA Ribonucleoprotein (RNP) complex was directly electroporated into the cell to execute its function [ 36 ]. As Cas9-gRNA RNP is assembled in vitro by relatively costly gRNA and Cas9 proteins, an alternative strategy is to express both gRNA and the endonuclease after their genes are integrated into the genome, as already established in the diatoms T. pseudonana and P. tricornutum [ 109 , 110 ].…”

Section: The Properties Of the Dna Affect The Outcome Of The Transformationmentioning

confidence: 99%

“… ( a ) The number of successfully engineered thraustochytrid strains by one or more methods (electroporation [ 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 ], biolistic transformation [ 5 , 27 , 53 , 54 , 55 ], and others [ 56 , 57 , 58 , 59 ]) ( Tables S1 and S2 ). The doughnut pie charts indicate the proportion of different genera.…”

Section: Figurementioning

confidence: 99%

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Method Development Progress in Genetic Engineering of Thraustochytrids

Rau

Ertesvåg

2021

Marine Drugs

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Thraustochytrids are unicellular, heterotrophic marine eukaryotes. Some species are known to store surplus carbon as intracellular lipids, and these also contain the long-chain polyunsaturated fatty acid docosahexaenoic acid (DHA). Most vertebrates are unable to synthesize sufficient amounts of DHA, and this fatty acid is essential for, e.g., marine fish, domesticated animals, and humans. Thraustochytrids may also produce other commercially valuable fatty acids and isoprenoids. Due to the great potential of thraustochytrids as producers of DHA and other lipid-related molecules, a need for more knowledge on this group of organisms is needed. This necessitates the ability to do genetic manipulation of the different strains. Thus far, this has been obtained for a few strains, while it has failed for other strains. Here, we systematically review the genetic transformation methods used for different thraustochytrid strains, with the aim of aiding studies on strains not yet successfully transformed. The designs of transformation cassettes are also described and compared. Moreover, the potential problems when trying to establish transformation protocols in new thraustochytrid species/strains are discussed, along with suggestions utilized in other organisms to overcome similar challenges. The approaches discussed in this review could be a starting point when designing protocols for other non-model organisms.

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“…To our knowledge, there is, so far, only one publication exploring the catabolic side of FA metabolism in Aurantiochytrium. The authors showed that disrupting the genes coding for acyl-CoA oxidases resulted in a higher FA productivity [29].…”

Section: Introductionmentioning

confidence: 99%

An Oil Hyper-Accumulator Mutant Highlights Peroxisomal ATP Import as a Regulatory Step for Fatty Acid Metabolism in Aurantiochytrium limacinum

Deragon

Schüler

Cigliano

et al. 2021

Cells

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Thraustochytrids are marine protists that naturally accumulate triacylglycerol with long chains of polyunsaturated fatty acids, such as ω3-docosahexaenoic acid (DHA). They represent a sustainable response to the increasing demand for these “essential” fatty acids (FAs). Following an attempt to transform a strain of Aurantiochytrium limacinum, we serendipitously isolated a clone that did not incorporate any recombinant DNA but contained two to three times more DHA than the original strain. Metabolic analyses indicated a deficit in FA catabolism. However, whole transcriptome analysis did not show down-regulation of genes involved in FA catabolism. Genome sequencing revealed extensive DNA deletion in one allele encoding a putative peroxisomal adenylate transporter. Phylogenetic analyses and yeast complementation experiments confirmed the gene as a peroxisomal adenylate nucleotide transporter (AlANT1), homologous to yeast ScANT1 and plant peroxisomal adenylate nucleotide carrier AtPNC genes. In yeast and plants, a deletion of the peroxisomal adenylate transporter inhibits FA breakdown and induces FA accumulation, a phenotype similar to that described here. In response to this metabolic event, several compensatory mechanisms were observed. In particular, genes involved in FA biosynthesis were upregulated, also contributing to the high FA accumulation. These results support AlANT1 as a promising target for enhancing DHA production in Thraustochytrids.

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Improvement of fatty acid productivity of thraustochytrid, Aurantiochytrium sp. by genome editing

Cited by 16 publications

References 54 publications

Development of an Efficient Gene Editing Tool in Schizochytrium sp. and Improving Its Lipid and Terpenoid Biosynthesis

Development of an Efficient Gene Editing Tool in Schizochytrium sp. and Improving Its Lipid and Terpenoid Biosynthesis

Method Development Progress in Genetic Engineering of Thraustochytrids

An Oil Hyper-Accumulator Mutant Highlights Peroxisomal ATP Import as a Regulatory Step for Fatty Acid Metabolism in Aurantiochytrium limacinum

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