Improved production of arachidonic acid by combined pathway

To date, several recent reviews have covered advances. 54 in the frontier ..... potential possibility may be related to the lipid extraction process. ...
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Biotechnology and Biological Transformations

Improved production of arachidonic acid by combined pathway engineering and synthetic enzyme fusion in Yarrowia lipolytica Hu-Hu Liu, Chong Wang, Xiang-Yang Lu, He Huang, Yun Tian, and Xiao-Jun Ji J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03727 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 18, 2019

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Journal of Agricultural and Food Chemistry

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Improved production of arachidonic acid by combined pathway

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engineering and synthetic enzyme fusion in Yarrowia lipolytica

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Hu-Hu Liu1, Chong Wang1, Xiang-Yang Lu1, He Huang1,2, Yun Tian1*, Xiao-Jun Ji 2*

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1. College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128,

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People’s Republic of China

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2. College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30

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South Puzhu Road, Nanjing 211816, People’s Republic of China

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*Corresponding authors: Xiao-Jun Ji (E-mail: [email protected], Tel: +86 25 58139942);

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Yun Tian (E-mail: [email protected], Tel: +86 731 84635292)

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Abstract

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Arachidonic acid (ARA, C20:4) is a typical ω-6 polyunsaturated fatty acid with

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special functions. Using Yarrowia lipolytica as an unconventional chassis, we

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previously showed the performance of the Δ-6 pathway in ARA production. However,

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a significant increase in the Δ-9 pathway has rarely been reported. Herein, the Δ-9

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pathway from Isochrysis galbana was constructed via pathway engineering, allowing

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us to synthesize ARA at 91.5 mg L-1. To further improve the ARA titer, novel enzyme

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fusions of Δ-9 elongase and Δ-8 desaturase were redesigned in special combinations

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containing different linkers. Finally, with the integrated pathway engineering and

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synthetic enzyme fusion, a 29% increase in the ARA titer, up to 118.1 mg/L, was

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achieved using the reconstructed strain RH-4 that harbors the rigid linker (GGGGS).

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The results show that the combined pathway and protein engineering can significantly

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facilitate applications of Y. lipolytica.

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Keywords: arachidonic acid; Yarrowia lipolytica; pathway engineering; protein

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engineering

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1. Introduction

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Yarrowia lipolytica is emerging as a preferred nonconventional yeast due to its

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special characteristics1-2. Generally, native Y. lipolytica strains are used for producing

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organic acids, lipid-based biofuels and endogenous functional enzymes3-6. In

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particular, as a promising oleaginous yeast, the genetic mechanisms of lipid

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biosynthesis and accumulation in Y. lipolytica have become popular7-9. Meanwhile,

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genetically engineered strains of Y. lipolytica have been used as suitable chasses for

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the biosynthesis of various value-added products (e.g., nonnative fatty acids, sugar

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products and terpenoids)10-12. To date, several recent reviews have covered advances

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in the frontier applications of Y. lipolytica13-16.

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Arachidonic acid (ARA, C20:4) is a typical ω-6 polyunsaturated fatty acid with

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special functions in human health17. ARA can be synthesized by either the Δ-6

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pathway or the Δ-9 pathway18. Currently, ARA is produced on a large scale by

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microbial fermentation using Mortierella alpina harboring the Δ-6 pathway19-20.

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Meanwhile, different ARA biosynthetic pathways have been heterologously

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constructed in various chassis cells, including Brassica napus, Arabidopsis thaliana

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and Saccharomyces cerevisiae21-22. With Y. lipolytica as a cell factory, we

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successfully developed an in vivo assembly method for rapidly constructing the Δ-6

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pathway23. Moreover, the performance of the Δ-6 pathway on ARA production was

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comprehensively examined24. However, there are a few studies performing significant

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stepwise increases in ARA production using the novel Δ-9 pathway in Y. lipolytica.

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Due to the substrate channeling of enzymes catalyzing sequential reactions,

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heterologous multienzymatic pathways often trigger flux imbalances in the chassis. In

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fact, spatial clustering of enzymes has been proved as an efficient approach to

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optimize metabolite transfers in multienzymatic pathways for the synthesis of high

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value

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multienzymatic synthesis by substrate channeling have been reviewed in detail27.

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Among the multiple methods used to improve biocatalytic cascades, previous studies

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have shown that the end-to-end multiple protein fusion strategy is an effective and

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easy approach to bring different enzymes into close proximity control for improved

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performance. Previously, Zhou et al. showed that the fusion enzyme of copalyl

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diphosphate synthase and kaurene synthase-like from Salvia miltiorrhiza, harboring a

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GGGS linker, is able to significantly improve miltiradiene production in engineered S.

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cerevisiae28. To improve fumarate production, Chen et al. made a fusion enzyme of

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KGD2-SUCLG2 with a GGGS linker, which resulted in a 13.7% increase in the

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engineered S. cerevisiae strain TGFA091-729. Recently, Nogueira et al. created

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enzyme fusions of the astaxanthin biosynthetic pathway using three different flexible

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size linkers. Obviously, the production levels of astaxanthin in the engineered E. coli

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were improved when the ketocarotenogenic enzymes were fused, compared with the

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individual enzymes in this research30.

chemicals25-26.

Currently,

a

variety

of

approaches

for

engineering

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In the present study, the production of ARA was gradually improved by

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combined pathway engineering and synthetic enzyme fusion in Y. lipolytica. Using

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the pathway engineering approach, the Δ-9 pathway from Isochrysis galbana was

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constructed in Y. lipolytica. Then, an ARA titer of 91.5 mg L-1 was produced using the

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engineered strain YL 8-1. Furthermore, in order to improve ARA production,

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synthetic protein fusions were performed with either no linker or linker peptides with

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different properties. Finally, a 29% increase in the ARA titer, up to 118.1 mg/L, was

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achieved using the reconstructed strain RH-4, harboring the rigid linker (GGGGS).

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The results show that a combined engineering approach can significantly facilitate the

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biotechnological applications of Y. lipolytica in many fields.

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2. Materials and methods

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2.1. Strains and media

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Y. lipolytica Po1f was used as the parental strain31. The engineered strain YL 6-1

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has been previously constructed23. Escherichia coli DH5α was grown in an LB

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medium (10 g L-1 tryptone, 5 g L-1 yeast extract and 10 g L-1 NaCl) with 100 μg mL-1

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ampicillin for cloning and plasmid amplification. The constructed strains and

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plasmids are listed in Table 1.

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YPD medium (10 g L-1 yeast extract, 20 g L-1 glucose and 20 g L-1 tryptone) was

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used for the cultivation of Y. lipolytica. Selective YNB-Ura medium (20 g L-1 glucose,

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6.7 g L-1 yeast nitrogen base without amino acids, 0.77 g L-1 CSM-Ura and 20 g L-1

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agar) was used for the selection of Y. lipolytica. Feed medium (9 g L-1 yeast extract,

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50 g L-1 glucose and 8 g L-1 YNB without amino acids) was used for the fermentation.

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2.2. Construction of engineered strains

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Promoters (TEF1p and FBA1p), terminators (LIP2t and XPR2t), URA3 and 28S

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rDNA sequences were synthesized in our previous research23. The functional

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encoding genes from Isochrysis galbana, including Δ-9 elongase, Δ-8 desaturase and

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Δ-5 desaturase, were codon-optimized (Genscript, Nanjing, China). Each functional

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expression cassette was constructed using different gene parts, by overlap extension

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PCR32. The artificially designed Δ-9 pathway, containing four functional gene

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cassettes, and the redesigned Δ-9 pathway, containing three functional gene cassettes,

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were transformed into Y. lipolytica Po1f strain, following our previous research23.

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Specifically, for the redesigned Δ-9 pathway, the fusion protein module in the first

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gene cassette was constructed using different linkers. The positive transformants were

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obtained using the selective media and confirmed by PCR analysis. The

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codon-optimized genes and primers are listed in Supplementary Table S1 and Table

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S2, respectively.

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2.3. Flask fermentation

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For the seed cultures, a single Y. lipolytica colony was transferred to 5 mL of

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YPD medium (180 rpm, 28 °C). Next, the 12-h cultures were inoculated into 50 mL

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of YPD medium with culturing for one day. They were then reinoculated under the

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same conditions with an inoculation rate of 5% with an OD600 of 0.1 for two days.

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Finally, the 48-h cultures were inoculated into fermentation medium (100 mL in a

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500-mL flask) with an inoculation rate of 10% with culturing for three days. Samples

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were taken every 24 h during fermentation.

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2.4. Determination of biomass, pH and glucose concentration

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The broth pH was measured using a pH meter (Mettler-Toledo, Urdorf,

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Switzerland). The biomass was measured using the previously described dry weight

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method23. The glucose concentration was measured using a glucose oxidase electrode

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(SBA-40C, Institute of Biology, Shandong Academy of Sciences, China).

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2.5. Determination of fatty acid profiles

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To measure fatty acid profiles, methylation of the fatty acid composition from

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the extracted lipids was performed, according to a previously described method23.

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2.6. Statistics

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The results of the different performances between the parental and engineered Y.

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lipolytica strains were analyzed using Student’s t-test. P