Iterative integration of multiple-copy pathway genes in Yarrowia lipolytica for heterologous β-carotene production

Gao, Shuliang; Tong, Yongliang; Zhu, Li; Ge, Mei; Zhang, Yian; Chen, Daijie; Jiang, Yu; Yang, Sheng

doi:10.1016/j.ymben.2017.04.004

Cited by 200 publications

(221 citation statements)

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“…In a representative fed batch fermentation with a 2 L bioreactor, the cells cultured in YPD medium with glucose supplements achieved a titer close to ~ 2.5 g/L at 160 h (Fig. 2d), similar to previously reported efforts [23]. …”

Section: Resultssupporting

confidence: 88%

“…Particularly, Y. lipolytica has a rich acetyl-CoA pool due to its cytosolic ATP citrate lyase (ACL) [20, 21]. Genetic tools have been established in Y. lipolytica [22], and several groups have successfully engineered the species to produce diverse carotenoids [23–25]. Another industrial advantage of utilizing Y. lipolytica is its capacity to grow on a large range of carbon sources (including but not limited to: glucose, glycerol, alcohols, and fatty acids), allowing for the use of diverse and cheap feedstock [26].…”

Section: Introductionmentioning

confidence: 99%

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Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone

et al. 2018

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Backgroundβ-Ionone is a fragrant terpenoid that generates a pleasant floral scent and is used in diverse applications as a cosmetic and flavoring ingredient. A growing consumer desire for natural products has increased the market demand for natural β-ionone. To date, chemical extraction from plants remains the main approach for commercial natural β-ionone production. Unfortunately, changing climate and geopolitical issues can cause instability in the β-ionone supply chain. Microbial fermentation using generally recognized as safe (GRAS) yeast offers an alternative method for producing natural β-ionone. Yarrowia lipolytica is an attractive host due to its oleaginous nature, established genetic tools, and large intercellular pool size of acetyl-CoA (the terpenoid backbone precursor).ResultsA push–pull strategy via genome engineering was applied to a Y. lipolytica PO1f derived strain. Heterologous and native genes in the mevalonate pathway were overexpressed to push production to the terpenoid backbone geranylgeranyl pyrophosphate, while the carB and biofunction carRP genes from Mucor circinelloides were introduced to pull flux towards β-carotene (i.e., ionone precursor). Medium tests combined with machine learning based data analysis and 13C metabolite labeling investigated influential nutrients for the β-carotene strain that achieved > 2.5 g/L β-carotene in a rich medium. Further introduction of the carotenoid cleavage dioxygenase 1 (CCD1) from Osmanthus fragrans resulted in the β-ionone production. Utilization of in situ dodecane trapping avoided ionone loss from vaporization (with recovery efficiencies of ~ 76%) during fermentation operations, which resulted in titers of 68 mg/L β-ionone in shaking flasks and 380 mg/L in a 2 L fermenter. Both β-carotene medium tests and β-ionone fermentation outcomes indicated the last enzymatic step CCD1 (rather than acetyl-CoA supply) as the key bottleneck.ConclusionsWe engineered a GRAS Y. lipolytica platform for sustainable and economical production of the natural aroma β-ionone. Although β-carotene could be produced at high titers by Y. lipolytica, the synthesis of β-ionone was relatively poor, possibly due to low CCD1 activity and non-specific CCD1 cleavage of β-carotene. In addition, both β-carotene and β-ionone strains showed decreased performances after successive sub-cultures. For industrial application, β-ionone fermentation efforts should focus on both CCD enzyme engineering and strain stability improvement.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0984-x) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone

et al. 2018

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“…The generated strain (ob‐CH) increased β‐carotene content up to 17.4 mg/gDCW and 121.6 mg/L, 2.0 and 3.4 times more than the parental strain ob‐C (Figure ). Again, this results are higher than the ones recently obtained from a strain overexpressing the same genes but using different parental strain and set of promoters (4.36 mg/gDCW and 64.6 mg/L) (Gao et al, ).…”

Section: Resultscontrasting

confidence: 59%

“…In addition, during the preparation of this manuscript, Gao et al () engineered Y. lipolytica to produce up to 4 g/L of β‐carotene in fed‐batch fermentation; the highest production titer so far described in a heterologous microorganism. The authors engineered one strain after 12 steps where 11 genes were modified and they found that the integration of multiple copies of some of the genes was essential to increase β‐carotene production.…”

Section: Introductionmentioning

confidence: 99%

A synthetic biology approach to transform Yarrowia lipolytica into a competitive biotechnological producer of β‐carotene

Larroude

Celińska

Back

et al. 2017

Biotech & Bioengineering

257

237

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The increasing market demands of β-carotene as colorant, antioxidant and vitamin precursor, requires novel biotechnological production platforms. Yarrowia lipolytica, is an industrial organism unable to naturally synthesize carotenoids but with the ability to produce high amounts of the precursor Acetyl-CoA. We first found that a lipid overproducer strain was capable of producing more β-carotene than a wild type after expressing the heterologous pathway. Thereafter, we developed a combinatorial synthetic biology approach base on Golden Gate DNA assembly to screen the optimum promoter-gene pairs for each transcriptional unit expressed. The best strain reached a production titer of 1.5 g/L and a maximum yield of 0.048 g/g of glucose in flask. β-carotene production was further increased in controlled conditions using a fed-batch fermentation. A total production of β-carotene of 6.5 g/L and 90 mg/g DCW with a concomitant production of 42.6 g/L of lipids was achieved. Such high titers suggest that engineered Y. lipolytica is a competitive producer organism of β-carotene.

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“…Previous study has demonstrated the heterologous biosynthesis of β‐carotene in Y. lipolytica by introducing the intrinsic high carbon flux of Acetyl‐CoA into the pathway via 12 steps after manipulating eleven homogenous and heterogeneous genes . High levels of β‐carotene (4 g/L) stored in lipid droplets within engineered Y. lipolytica was achieved under optimized fed‐batch fermentation conditions . To further improve the gene expression levels, promoter shuffling strategy via the Golden gate approach was explored to find the best assembly of the promoters and maximize the β‐carotene production .…”

Section: Y Lipolytica As the Biomanufacturing Platformmentioning

confidence: 99%

Cellular and metabolic engineering of oleaginous yeast Yarrowia lipolytica for bioconversion of hydrophobic substrates into high‐value products

Soong

Liu

Yoon

et al. 2019

Engineering in Life Sciences

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The non‐conventional oleaginous yeast Yarrowia lipolytica is able to utilize both hydrophilic and hydrophobic carbon sources as substrates and convert them into value‐added bioproducts such as organic acids, extracellular proteins, wax esters, long‐chain diacids, fatty acid ethyl esters, carotenoids and omega‐3 fatty acids. Metabolic pathway analysis and previous research results show that hydrophobic substrates are potentially more preferred by Y. lipolytica than hydrophilic substrates to make high‐value products at higher productivity, titer, rate, and yield. Hence, Y. lipolytica is becoming an efficient and promising biomanufacturing platform due to its capabilities in biosynthesis of extracellular lipases and directly converting the extracellular triacylglycerol oils and fats into high‐value products. It is believed that the cell size and morphology of the Y. lipolytica is related to the cell growth, nutrient uptake, and product formation. Dimorphic Y. lipolytica demonstrates the yeast‐to‐hypha transition in response to the extracellular environments and genetic background. Yeast‐to‐hyphal transition regulating genes, such as YlBEM1, YlMHY1 and YlZNC1 and so forth, have been identified to involve as major transcriptional factors that control morphology transition in Y. lipolytica. The connection of the cell polarization including cell cycle and the dimorphic transition with the cell size and morphology in Y. lipolytica adapting to new growth are reviewed and discussed. This review also summarizes the general and advanced genetic tools that are used to build a Y. lipolytica biomanufacturing platform.

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Iterative integration of multiple-copy pathway genes in Yarrowia lipolytica for heterologous β-carotene production

Cited by 200 publications

References 67 publications

Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone

Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone

A synthetic biology approach to transform Yarrowia lipolytica into a competitive biotechnological producer of β‐carotene

Cellular and metabolic engineering of oleaginous yeast Yarrowia lipolytica for bioconversion of hydrophobic substrates into high‐value products

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