Re-configuring Yarrowia lipolytica lipogenesis platform towards free fatty acid and biofuel production

Transcription

1 Re-configuring Yarrowia lipolytica lipogenesis platform towards free fatty acid and biofuel production Yonghong Meng, Ali Abghari, Rishikesh Ghogare, Xiaochao Xiong, GuokunWang, and Shulin Chen Bioprocessing and Bioproducts Engineering Laboratory (BBEL) Department of Biological Systems Engineering Washington State University, Pullman, WA, USA

2 Outline Introudction Cellulosic oleochemical and biofuel Yarrowia lipolytica Synthetic biology of Y. lipolytica Biological parts, devices and systems Pathway assembly Metabolic engineering Free fatty acid production Fatty alcohol production

3 Goal: build a versatile platform for producing renewable oleochemical and biofuel Biomass Fatty alcohol (Acyl-CoA reductase) Wax esters (Wax synthase/dgat) Sugars Microbial conversion Fatty acid Triacylglyceride (TAG) (Microbial lipid) Dicarboxylic acid (ω-oxidation) Olefins (Reduction) Fatty acid methyl esters (Esterification) Ricinoleic acid (Castor oil) (Hydroxylation) Alkane or alkene (Hydrotreating or biosynthesis)

4 Yarrowia lipolytica as a platform organism Six chromosomes, 20 Mb of DNA GRAS, Yarrowia lipolytica (formerly Candida, Endomycopsis, or Saccharomyces lipolytica) is one of the more intensively studied nonconventional yeast species. Academic study: Yeast evolution Dimorphism and cell stress Protein secretion: Proteases, phosphatases & lipases Lipid metabolism Hydrophobic substrate utilization Peroxisome biogenesis Technology development: Single cell protein, organic acid and flavors Protein expression and secretion: less hyperglycosylation, high secretion capacity and yield and less degradation after deletion of protease genes (more than 60 genes were expressed.) Lipid production: 61.7% of CDW (0.195 g/g glucose, g/l/h) (MIT), Eicosapentaenoic acid (EPA) (DuPont)

5 Capitalize on the capability of oleaginous yeast in produce lipids Structure in LD Core LDs and Phospholipid Turnover LDs frequently localize adjacent to the ER, mitochondrion, and peroxisome, which likely reflects close functional interactions between LDs and these organelles. It is a organelle also. Ohsaki Y, Suzuki M, Fujimoto T. Open questions in lipid droplet biology. Chem Biol Jan 16;21(1): doi: /j.chembiol Epub 2013 Nov 14. Review. PubMed PMID:

6 Needs for engineering Yarrowia lipolytica Goal: Developing expression platform for expression of recombinant proteins and for the precise control over the expression level and timing of the genes for metabolic engineering. Overcoming various technical barriers, promoters for example: The levels of even strong endogenous promoters in Y. lipolytica are too low for metabolic engineering purposes. Lipid (oleic acid) inducible promoters require long time for the full induction. It also changes the culture conditions. Repressible promoters, which activities can be down regulated by either chemical or physical factors, were widely used for metabolic engineering and construction of genetic circuits, but rare for Y. lipolytica.

7 (a) Parts Synthetic biology in Y. lipolytica Synthetic biology hierarchy DNA RNA Protein Tools development 1). More than 10 constitutive promoters; 2). 6 Cu 2+ -inducible promoters; 3). 8 Repressible promoters; 4). Terminators, markers and replicons. (b) Devices (c) Systems Enhancers P 5 -UTR Gene 3 -UTR 1). 15 Engineered promoters; 2). Marker reusable knockout system; 3). Expression vectors. 1). Assembly of multiple-gene pathway; 2). Promoter replacement; 3). Knock-in strategy. (d) Chassis modification NOT explored. Genome editing technology, genome engineering, genome minimization Genome

8 Biological parts (promoter, reporter, terminator) and assembly UAS (upstream activated sequence) for enhancement of gene expression Reporter gene: lacz (easy detection) and florescence protein (single cell) UAS UAS UAS Promoter UAS UAS Engineered hybrid promoter UAS UAS UAS UAS UAS Promoter Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach. Appl Environ Microbiol (22):

9 Assembly of standard biological parts

10 Fine regulation of promoter activity hp4d promoter- wild applications

11 Device: expression vectors for Yarrowia lipolytica SalI hyg R HindIII HpaI NdeI Centromeric and integrative expression vectors

12 Application 1: Metabolic engineering of Yarrowia lipolytica for accumulation of carotenoid in lipid body Three steps of insertion of the genes into genome accompanying marker removal Ovexression of native GGS1, synthesized crtb and crti from Pantoea ananatis. Production of Lycopene in the Non-Carotenoid-Producing Yeast Yarrowia lipolytica Appl. Environ. Microbiol vol. 80 no

13 System: assembly of multiple-gene pathway in a single plasmid Sequential insertion of multiple gene expression cassettes for lycopene biosynthesis in a single plasmid by the iterative insertion of XbaI/SpeI fragments into vectors digested with XbaI or SpeI. In the new vector, there is a new XbaI/SpeI, and an uncleavable scar (XS*).

14 Application 2: Rapid development of strains for producing lipid Targeted genes ACC1, SCD, DGA1 and Gut2 have been reported. Metabolic Engineering, Vol 13, 2011, Pages Metabolic Engineering, Vol 15, 2013, Pages 1 9 Metabolic Engineering, Vol 29, 2015, Pages 56 65

15 System: gene knock-in and promoter replacement Step1: Insert three expression gene cassettes to locus of Gut2 XbaI/SpeI DNA fragments Step2: Replace promoter of ACC1 with FBA

16 Mechanism for fatty acid formation, activation and transport Sugars FFA FFA Efflux pump?? FFA De novo fatty acid biosynthesis pax1 and pax2: acyl-coa fatty acid transporter Acyl-CoA Acyl-CoA binding protein Lipid biosynthesis TAG FFA FAA Acyl-CoA AOX Peroxisome Chain shortened acyl-coa PEX10: peroxisomal biogenesis factor

17 Identification of faa genes from Yarrowia lipolytica Cerulenin Inhibit Fatty acid Acetyl-CoA ACC FAS Fatty acid Cell components FAA Acyl-CoA 1. Mechanism for screening faa candidate genes by using S. cerevisiae faa mutant Ylfaa1, Ylfaa2, Ylfaa3, Ylfaa4 2. Expression of candidate genes under galactose-inducible promoter in S. cerevisiae 3. Complementation of growth of S. cerevisiae faa mutant on fatty acid 4. Enzyme assay and metabolism analysis

18 Application 3: recombinants for overproduction of free fatty acid ATCC MYA-2613 (po1f) Leu-, Ura- Ku70 (Enables homologous integration Leu-, Urausing short flanking fragments with high frequencies) FAA1 (Block of fatty acid activation) Pxa2 (Block of acyl CoA peroxisomal uptake) Promoter replacement of acc1 (Improvement of fatty acid biosynthesis) FAA1 Pxa2 FAA1-4 Pxa2 expressing tesa (high yield of free fatty acid) FAA1 Pxa2 Pex10 FAA1 Pxa2 ACBP (Grow slowly on synthetic media)

19 +

20 Application 4: Metabolic engineering of Yarrowia lipolytica for oleochemicals (fatty alcohol) production Overexpression of fatty alcohol reductase with 2 copies or 5 copies in strains with different genetic backgrounds. Trace products were detected in the supernatants of media.

21 Products Triglyceride (TAG) Free fatty acid (FFA) Fatty alcohol Carotenoids Summary of technology status Achievements and development plan Current yield of 4.1 g/l (wild-type strain, 0.32 g/l) in shaking flasks; Ready for testing the technology in larger-scale fermentation. Current yield of 2.3 g/l (wild-type, g/l) in shaking flasks; Can be scaled up a platform technology. Current yield of 630 mg/l; Further improvement by eliminating pathway for non-essential lipid accumulation. Strains capable for producing lycopene; Further extending pathway for biosynthesis of astaxanthin; Further transcriptional factor and enzyme engineering. Dicarboxylic acid (DCA) Wax ester (WE) and fatty acid alkyl ester (FAAE) Detected the targeted product by feeding plant oils; Further developing strains for producing DCA from sugars. Cloned and expressed WE synthase; Further developing strains for producing FAAE.

22 Acknowledgement Washington State University Dr. Falk Matthäus from Dresden University of Technology, Germany for providing the genes, crtb and crti

23 Thank you for your attention