Improved γ-linolenic acid production in Mucor circinelloides by homologous overexpressing of delta-12 and delta-6 desaturases

Abstract

Backgroundγ-Linolenic acid (GLA) is important because of its nutritional value and medicinal applications. Although the biosynthetic pathways of some plant and microbial GLA have been deciphered, current understanding of the correlation between desaturases and GLA synthesis in oleaginous fungi is incomplete. In previous work, we found that a large amount of oleic acid (OA) had not been converted to linoleic acid (LA) or GLA in Mucor circinelloides CBS 277.49, which may be due to inadequate activities of the delta-12 or delta-6 desaturases, and thus leading to the accumulation of OA and LA. Thus, it is necessary to explore the main contributing factor during the process of GLA biosynthesis in M. circinelloides.ResultsTo enhance GLA production in M. circinelloides, homologous overexpression of delta-12 and two delta-6 desaturases (named delta-6-1 and delta-6-2, respectively) were analyzed. When delta-6 desaturase were overexpressed in M. circinelloides, up to 43% GLA was produced in the total fatty acids, and the yield of GLA reached 180 mg/l, which were, respectively, 38 and 33% higher than the control strain.ConclusionThese findings revealed that delta-6 desaturase (especially for delta-6-1 desaturase) plays an important role in GLA synthesis by M. circinelloides. The strain overexpressing delta-6-1 desaturase may have potential application in microbial GLA production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0723-8) contains supplementary material, which is available to authorized users.

Increased Accumulation of Medium-Chain Fatty Acids by Dynamic Degradation of Long-Chain Fatty Acids in Mucor circinelloides

“…To select mutants that impulsively eliminate the pyrF marker gene (encodes for uracil biosynthesis), 5-fluoroorotic acid (2.5 mg/mL) was added to the YPG medium [ 49 ]. All the mutant (i.e., Mu-TE+, the strain harboring the TE-over-expressing plasmid, hereafter referred to as M-1; Mu-TE+, [ acoxΔ ], the strain harboring the TE-over-expressing plasmid and exhibiting the disrupted allele for the ACOX gene, hereafter referred to as M-2; and Mu-TE+ [ acoxΔ , acotΔ ], the strain harboring the TE-over-expressing plasmid and exhibiting the disrupted allele for ACOX and ACOT genes, hereafter referred to as M-3) and the wild-type (used as the control, hereafter referred to as WT) strains of M. circinelloides (MU758) were initially cultivated by using 80 μL spore suspension (~10 7 spores/mL) in 1.0-L flasks containing 150 mL of Kendrick and Ratledge medium (K and R); 30 g/L glucose, 3.3 g/L diammonium tartrate, 7.0 g/L KH 2 PO 4 , 2.0 g/L Na 2 HPO 4 , 1.5 g/L MgSO 4 ·7H 2 O, 1.5 g/L yeast extract, 0.1 mg/L CaC1 2 ·2H 2 O, 8 mg/L FeC1 3 ·6H 2 O, 1 mg/L ZnSO 4 ·7H 2 O, 0.1 mg/L CuSO 4 ·5H 2 O, 0.1 mg/L CO (NO 3 )·6H 2 O and 0.1 mg/L MnSO 4 ·5H 2 O, pH 6.0) [ 4 , 9 , 16 , 17 , 51 , 52 , 53 ]. Subsequently, these flasks were equipped with baffles to improve aeration and finally placed in a shaker at 150 rpm at a temperature of 26 °C for 24 h. the resultant seed culture was used for inoculation at 10% ( v / v ) into a 5-L fermenter (BioFlo/CelliGen 120, New Brunswick Scientific, Edison, NJ, USA) containing 1.5 L of modified K and R medium (i.e., 80.0 g glucose/L plus inorganic salts, 2.0 g diammonium tartrate).…”

“…The acyl-ACP thioesterase ( TE ) gene was cloned into vector pMAT1552- LeuA [ 4 , 9 ] for expression in the M . circinelloides strain (MU758).The aforesaid vector carried the LeuA gene from M .…”

“…Mucor circinelloides Wj11 has an innate ability to accumulate oils up to 36% of the cell dry weight (CDW) [ 7 ]. Owing to its high-oil-generating ability, it offers a reliable option for genetic modulation to produce functional lipids and green energy fuels [ 8 , 9 ]. Previously, these fungal strains have been engineered to produce biofuels and microbial lipids consisting mostly of long alkyl-chains (C14 or more carbons), ultimately making them less attractive for diverse industrial applications [ 8 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ].…”

Improved SDA Production in High Lipid Accumulating Strain of Mucor circinelloides WJ11 by Genetic Modification

“…The strong promoter zrt1 worked for fad3 gene overexpression. 1-kb sequences upstream and downstream of the carotenogenic carRP gene flanked the previous elements to permit chromosomal integration by the whole DNA fragment construct through homologous recombination (Zhang et al, 2017) (Fig. 2).…”

Both Determinants of Allosteric and Active Sites Responsible for Catalytic Activity of Delta 12 Fatty Acid Desaturase by Domain Swapping

“…FADS12 has been studied as an important desaturase in fatty acid transformation for many years, with experiments based on gene cloning, heterologous expression, and analysis of its catalytic mechanism (Brandstetter & Ruther, 2016; Kaye et al, 2015; Kazuhiro et al, 2004; Kikukawa et al, 2013; Lamers et al, 2019; Rodríguez-Rodríguez, 2016; Sakamoto et al, 2017; ShanlinYu et al, 2008; Sun et al, 2016; Watanabe et al, 2004). These studies were mostly focused on the preparation of PUFAs, the regulation of the proportion of omega-3/omega-6 fatty acids and heterologous expression from EFAs, which could not be synthesized in the human body (Wang et al, 2016; Yan et al, 2013; Zhang et al, 2013; Zhang et al, 2017). However, heterologous expression of FADS12 in different host cells often resulted in significant changes in its activity, which has made it difficult to screen for FADS12 with high activity in a target strain.…”