Metabolic engineering of Pichia pastoris to produce ricinoleic acid, a hydroxy fatty acid of industrial importance

Meesapyodsuk, Dauenpen; Chen, Yan; Ng, Siew Hon; Chen, Jianan; Qiu, Xiao

doi:10.1194/jlr.m060954

Cited by 34 publications

(21 citation statements)

References 32 publications

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“…The co‐expression mutant strain at the highest time point had a maximum of 495 μg mL −1 ricinoleic acid. Inside yeast cell, the hydroxyl fatty acid was predominantly localized in the neutral lipid fractions in the free fatty acid form …”

Section: Metabolic Engineering Of P Pastorismentioning

confidence: 99%

Heterologous Protein Expression in Pichia pastoris: Latest Research Progress and Applications

Juturu

2017

ChemBioChem

159

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Pichia pastoris is a well‐known platform strain for heterologous protein expression. Over the past five years, different strategies to improve the efficiency of recombinant protein expression by this yeast strain have been developed; these include a patent‐free protein expression kit, construction of the P. pastoris CBS7435Ku70 platform strain with its high efficiency in site‐specific recombination of plasmid DNA into the genomic DNA, the design of synthetic promoters and their variants by combining different core promoters with multiple putative transcription factors, the generation of mutant GAP promoter variants with various promoter strengths, codon optimization, engineering the α‐factor signal sequence by replacing the native glutamic acid at the Kex2 cleavage site with the other 19 natural amino acids and the addition of mammalian signal sequence to the yeast signal sequence, and the co‐expression of single chaperones, multiple chaperones or helper proteins that aid in recombinant protein folding. Publically available high‐quality genome data from multiple strains of P. pastoris GS115, DSMZ 70382, and CBS7435 and the continuous development of yeast expression kits have successfully promoted the metabolic engineering of this strain to produce carotenoids, xanthophylls, nootkatone, ricinoleic acid, dammarenediol‐II, and hyaluronic acid. The cell‐surface display of enzymes has obviously increased enzyme stability, and high‐level intracellular expression of acyl‐CoA and ethanol O‐acyltransferase, lipase and d‐amino acid oxidase has opened up applications in whole‐cell biocatalysis for producing flavor molecules and biodiesel, as well as the deracemization of racemic amino acids. High‐level expression of various food‐grade enzymes, cellulases, and hemicellulases for applications in the food, feed and biorefinery industries is in its infancy and needs strengthening.

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Section: Metabolic Engineering Of P Pastorismentioning

confidence: 99%

Heterologous Protein Expression in Pichia pastoris: Latest Research Progress and Applications

Juturu

2017

ChemBioChem

159

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“…Pichia pastoris is a highly successful candidate for the production of heterologous protein (for review, see [ 21 , 22 ]), however, few studies have been performed using the metabolic engineering of this microorganism [ 23 – 25 ]. Our study shows for the first time that genetically engineered P. pastoris strains containing LDH activity are able to produce lactic acid from glycerol.…”

Section: Discussionmentioning

confidence: 99%

Novel homologous lactate transporter improves l-lactic acid production from glycerol in recombinant strains of Pichia pastoris

Lima

Mulder²,

Melo

et al. 2016

Microb Cell Fact

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BackgroundCrude glycerol is the main byproduct of the biodiesel industry. Although it can have different applications, its purification is costly. Therefore, in this study a biotechnological route has been proposed for further utilization of crude glycerol in the fermentative production of lactic acid. This acid is largely utilized in food, pharmaceutical, textile, and chemical industries, making it the hydroxycarboxylic acid with the highest market potential worldwide. Currently, industrial production of lactic acid is done mainly using sugar as the substrate. Thus here, for the first time, Pichia pastoris has been engineered for heterologous l-lactic acid production using glycerol as a single carbon source. For that, the Bos taurus lactate dehydrogenase gene was introduced into P. pastoris. Moreover, a heterologous and a novel homologous lactate transporter have been evaluated for l-lactic acid production.ResultsBatch fermentation of the P. pastoris X-33 strain producing LDHb allowed for lactic acid production in this yeast. Although P. pastoris is known for its respiratory metabolism, batch fermentations were performed with different oxygenation levels, indicating that lower oxygen availability increased lactic acid production by 20 %, pushing the yeast towards a fermentative metabolism. Furthermore, a newly putative lactate transporter from P. pastoris named PAS has been identified by search similarity with the lactate transporter from Saccharomyces cerevisiae Jen1p. Both heterologous and homologous transporters, Jen1p and PAS, were evaluated in one strain already containing LDH activity. Fed-batch experiments of P. pastoris strains carrying the lactate transporter were performed with the batch phase at aerobic conditions followed by an aerobic oxygen-limited phase where production of lactic acid was favored. The results showed that the strain containing PAS presented the highest lactic acid titer, reaching a yield of approximately 0.7 g/g.ConclusionsWe showed that P. pastoris has a great potential as a fermentative organism for producing l-lactic acid using glycerol as the carbon source at limited oxygenation conditions (below 0.05 % DO in the bioreactor). The best strain had both the LDHb and the homologous lactate transporter encoding genes expressed, and reached a titer 1.5 times higher than the strain with the S. cerevisiae transporter. Finally, it was also shown that increased lactic acid production was concomitant to reduction of acetic acid formation by half.

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“…Compared to non-hydroxylated fatty acids, HFAs have higher reactivity, solvent miscibility, stability, and viscosity. Therefore, HFAs have been widely used in food, chemical, pharmaceutical, cosmetic industries, and as synthetic precursors (Yang et al, 2014 ; Meesapyodsuk et al, 2015 ; Bowen et al, 2016 ). Omega HFAs (ω-HFAs) are fatty acids with a hydroxyl group near the methyl end, or omega end.…”

Section: Introductionmentioning

confidence: 99%

“…Although considerable effort has been made in engineering of oil crops or microbes for producing HFAs, there has been limited success in the production of medium chain fatty acid (MCFA) sub-terminal HFAs (Meesapyodsuk et al, 2015 ; Bowen et al, 2016 ). Different strategies have been applied to increase MCFA sub-terminal or terminal HFAs production.…”

Section: Introductionmentioning

confidence: 99%

Metabolic Engineering for Enhanced Medium Chain Omega Hydroxy Fatty Acid Production in Escherichia coli

et al. 2018

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Medium chain hydroxy fatty acids (HFAs) at ω-1, 2, or 3 positions (ω-1/2/3) are rare in nature but are attractive due to their potential applications in industry. They can be metabolically engineered in Escherichia coli, however, the current yield is low. In this study, metabolic engineering with P450BM3 monooxygenase was applied to regulate both the chain length and sub-terminal position of HFA products in E. coli, leading to increased yield. Five acyl-acyl carrier protein thioesterases from plants and bacteria were first evaluated for regulating the chain length of fatty acids. Co-expression of the selected thioesterase gene CcFatB1 with a fatty acid metabolism regulator fadR and monooxygenase P450BM3 boosted the production of HFAs especially ω-3-OH-C14:1, in both the wild type and fadD deficient strain. Supplementing renewable glycerol to reduce the usage of glucose as a carbon source further increased the HFAs production to 144 mg/L, representing the highest titer of such HFAs obtained in E. coli under the comparable conditions. This study illustrated an improved metabolic strategy for medium chain ω-1/2/3 HFAs production in E. coli. In addition, the produced HFAs were mostly secreted into culture media, which eased its recovery.

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Metabolic engineering of Pichia pastoris to produce ricinoleic acid, a hydroxy fatty acid of industrial importance

Cited by 34 publications

References 32 publications

Heterologous Protein Expression in Pichia pastoris: Latest Research Progress and Applications

Heterologous Protein Expression in Pichia pastoris: Latest Research Progress and Applications

Novel homologous lactate transporter improves l-lactic acid production from glycerol in recombinant strains of Pichia pastoris

Metabolic Engineering for Enhanced Medium Chain Omega Hydroxy Fatty Acid Production in Escherichia coli

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