Improved production of arachidonic acid by combined pathway

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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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1 ACS Paragon Plus Environment


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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

Improved production of arachidonic acid by combined pathway

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