Engineering the oleaginous yeast  Yarrowia lipolytica  for production of α-farnesene

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Abstract

Background

Yarrowia lipolytica, a non-traditional oil yeast, has been widely used as a platform for lipid production. However, the production of other chemicals such as terpenoids in engineered Y. lipolytica is still low. α-Farnesene, a sesquiterpene, can be used in medicine, bioenergy and other fields, and has very high economic value. Here, we used α-farnesene as an example to explore the potential of Y. lipolytica for terpenoid production.

Results

We constructed libraries of strains overexpressing mevalonate pathway and α-farnesene synthase genes by non-homologous end-joining (NHEJ) mediated integration into the Y. lipolytica chromosome. First, a mevalonate overproduction strain was selected by overexpressing relevant genes and changing the cofactor specificity. Based on this strain, the downstream α-farnesene synthesis pathway was overexpressed by iterative integration. Culture conditions were also optimized. A strain that produced 25.55 g/L α-farnesene was obtained. This is the highest terpenoid titer reported in Y. lipolytica.

Conclusions

Yarrowia lipolytica is a potentially valuable species for terpenoid production, and NHEJ-mediated modular integration is effective for expression library construction and screening of high-producer strains.

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Most cited references 42

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Statins and cancer prevention.

Marie-France Demierre, Peter Higgins, Stephen B. Gruber … (2005)

Randomized controlled trials for preventing cardiovascular disease indicated that statins had provocative and unexpected benefits for reducing colorectal cancer and melanoma. These findings have led to the intensive study of statins in cancer prevention, including recent, large population-based studies showing statin-associated reductions in overall, colorectal and prostate cancer. Understanding the complex cellular effects (for example, on angiogenesis and inflammation) and the underlying molecular mechanisms of statins (for example, 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase-dependent processes that involve geranylgeranylation of Rho proteins, and HMG-CoA-independent processes that involve lymphocyte-function-associated antigen 1) will advance the development of molecularly targeted agents for preventing cancer. This understanding might also help the development of drugs for other ageing-related diseases with interrelated molecular pathways.

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Yarrowia lipolytica: safety assessment of an oleaginous yeast with a great industrial potential.

Teun Boekhout, Peter van Lent, Markus Wyss … (2014)

Yarrowia lipolytica has been developed as a production host for a large variety of biotechnological applications. Efficacy and safety studies have demonstrated the safe use of Yarrowia-derived products containing significant proportions of Yarrowia biomass (as for DuPont's eicosapentaenoic acid-rich oil) or with the yeast itself as the final product (as for British Petroleum's single-cell protein product). The natural occurrence of the species in food, particularly cheese, other dairy products and meat, is a further argument supporting its safety. The species causes rare opportunistic infections in severely immunocompromised or otherwise seriously ill people with other underlying diseases or conditions. The infections can be treated effectively by the use of regular antifungal drugs, and in some cases even disappeared spontaneously. Based on our assessment, we conclude that Y. lipolytica is a "safe-to-use" organism.

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Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production.

Yongjin J. Zhou, Wei Gao, Qixian Rong … (2012)

Microbial production can be advantageous over the extraction of phytoterpenoids from natural plant sources, but it remains challenging to rationally and rapidly access efficient pathway variants. Previous engineering attempts mainly focused on the mevalonic acid (MVA) or methyl-d-erythritol phosphate (MEP) pathways responsible for the generation of precursors for terpenoids biosynthesis, and potential interactions between diterpenoids synthases were unexplored. Miltiradiene, the product of the stepwise conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP) catalyzed by diterpene synthases SmCPS and SmKSL, has recently been identified as the precursor to tanshionones, a group of abietane-type norditerpenoids rich in the Chinese medicinal herb Salvia miltiorrhiza . Here, we present the modular pathway engineering (MOPE) strategy and its application for rapid assembling synthetic miltiradiene pathways in the yeast Saccharomyces cerevisiae . We predicted and analyzed the molecular interactions between SmCPS and SmKSL, and engineered their active sites into close proximity for enhanced metabolic flux channeling to miltiradiene biosynthesis by constructing protein fusions. We show that the fusion of SmCPS and SmKSL, as well as the fusion of BTS1 (GGPP synthase) and ERG20 (farnesyl diphosphate synthase), led to significantly improved miltiradiene production and reduced byproduct accumulation. The MOPE strategy facilitated a comprehensive evaluation of pathway variants involving multiple genes, and, as a result, our best pathway with the diploid strain YJ2X reached miltiradiene titer of 365 mg/L in a 15-L bioreactor culture. These results suggest that terpenoids synthases and the precursor supplying enzymes should be engineered systematically to enable an efficient microbial production of phytoterpenoids.

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Author and article information

Contributors

Qingsheng Qi: qiqingsheng@sdu.edu.cn

Jin Hou: houjin@sdu.edu.cn

Journal

Journal ID (nlm-ta): Biotechnol Biofuels

Journal ID (iso-abbrev): Biotechnol Biofuels

Title: Biotechnology for Biofuels

Publisher: BioMed Central (London )

ISSN (Electronic): 1754-6834

Publication date (Electronic): 23 December 2019

Publication date PMC-release: 23 December 2019

Publication date Collection: 2019

Volume: 12

Electronic Location Identifier: 296

Affiliations

[1 ]ISNI 0000 0004 1761 1174, GRID grid.27255.37, State Key Laboratory of Microbial Technology, , Shandong University, ; Binhai Road 72, Qingdao, 266237 People’s Republic of China

[2 ]ISNI 0000000119573309, GRID grid.9227.e, CAS Key Lab of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, , Chinese Academy of Sciences, ; Qingdao, 266101 People’s Republic of China

Article

Publisher ID: 1636

DOI: 10.1186/s13068-019-1636-z

PMC ID: 6927232

PubMed ID: 31890024

SO-VID: be467ed1-a40b-47f0-b37e-20a4bfb19bb1

License:

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

History

Date received : 22 October 2019

Date accepted : 12 December 2019

Funding

Funded by: National Natural Science Foundation of China

Award ID: 31730003

Award ID: 31770101

Award ID: 31970082

Award ID: 31961133014

Funded by: the Key R&D Program of Shandong Province

Award ID: 2017GSF21110

Custom metadata

ScienceOpen disciplines: Biotechnology

Keywords: α-farnesene,mevalonate,yarrowia lipolytica,non-homologous end-joining

Data availability:

ScienceOpen disciplines: Biotechnology

Keywords: α-farnesene, mevalonate, yarrowia lipolytica, non-homologous end-joining

Engineering the oleaginous yeast Yarrowia lipolytica for production of α-farnesene

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Abstract

Background

Results

Conclusions

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Genome Engineering using CRISPR

Most cited references 42

Statins and cancer prevention.

Yarrowia lipolytica: safety assessment of an oleaginous yeast with a great industrial potential.

Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production.

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