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

ß-Carotene is a terpenoid molecule with high hydrophobicity that is often used as an additive in foods and feed. Previous work has demonstrated the heterologous biosynthesis of ß-carotene from an intrinsic high flux of acetyl-CoA in 12 steps through 11 genes in Yarrowia lipolytica. Here, an efficient biosynthetic pathway capable of producing 100-fold more ß-carotene than the baseline construct was generated using strong promoters and multiple gene copies for each of the 12 steps. Using fed-batch fermentation with an optimized medium, the engineered pathway could produce 4g/L ß-carotene, which was stored in lipid droplets within engineered Y. lipolytica cells. Expansion of these cells for squalene production also demonstrated that Y. lipolytica could be an industrially relevant platform for hydrophobic terpenoid production.

Production of ß-carotene by expressing a heterologous multifunctional carotene synthase in Yarrowia lipolytica.

OBJECTIVES: To obtain functional expression of a heterologous multifunctional carotene synthase containing phytoene synthase, phytoene dehydrogenase, and lycopene ß-cyclase activities encoded by carS from Schizochytrium sp. in order to allow Yarrowia lipolytica to produce ß-carotene. RESULTS: To increase the integration efficiency of a 3.8 kb carS under the control of P GPD promoter with a 2 kb selection marker, ura3, along with a geranylgeranyl diphosphate synthase (GGS1) expression cassette (~10 kb in total), was inserted into the Y. lipolytica chromosome, and the DNA assembler method was combined with double chromosomal deletions of ku70 and ku80. This method resulted in a 13.4-fold increase in integration efficiency compared with the original method, reaching 63% (10/16). The resulting recombinant Y. lipolytica produced 0.41 mg ß-carotene per g dry cell weight, while the wild type did not produce any indicating the functionality of the multifunctional carotene synthase in Y. lipolytica. CONCLUSION: Expression of GGS1 and a multifunctional carotene synthase from Schizochytrium sp. in Y. lipolytica led to ß-carotene production. DNA assembler efficiency was greatly increased by the deletion of ku70 and ku80, which resulted in decreased in vivo nonhomologous end-joining (NHEJ) in Y. lipolytica.

Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system.

Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6 % for the wild-type strain and 94.1 % for the ku70/ku80 double-deficient strain, within 4 days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3 %, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica.

Autotrophic denitrification by sulfur-based immobilized electron donor for enhanced nitrogen removal: Denitrification performance, microbial interspecific interaction and functional traits.

Sulfur-driven autotrophic denitrification (SdAD) is a promising nitrogen removing process, but its applications were generally constrained by conventional electron donors (i.e., thiosulfate (Na2S2O3)) with high valence and limited bioavailability. Herein, an immobilized electron donor by loading elemental sulfur on the surface of polyurethane foam (PFSF) was developed, and its feasibility for SdAD was investigated. The denitrification efficiency of PFSF was 97.3%, higher than that of Na2S2O3 (91.1%). Functional microorganisms (i.e., Thiobacillus and Sulfurimonas) and their metabolic activities (i.e., nir and nor) were substantially enhanced by PFSF. PFSF resulted in the enrichment of sulfate-reducing bacteria, which can reduce sulfate (SO42-). It attenuated the inhibitory effect of SO42-, whereas the generated product (hydrogen sulfide) also served as an electron donor for SdAD. According to the economic evaluation, PFSF exhibited strong market potential. This study proposes an efficient and low-cost immobilized electron donor for SdAD and provides theoretical support to its practical applications.

Engineering the oleaginous yeast Yarrowia lipolytica for ß-farnesene overproduction.

ß-farnesene is a sesquiterpenoid with various industrial applications which is now commercially produced by a Saccharomyces cerevisiae strain obtained by random mutagenesis and genetic engineering. We rationally designed a genetically defined Yarrowia lipolytica through recovery of L-leucine biosynthetic route, gene dosage optimization of ß-farnesene synthase and disruption of the competition pathway. The resulting ß-farnesene titer was improved from 8 to 345 mg L-1 . Finally, the strategy for decreasing the lipid accumulation by individually and iteratively knocking out four acyltransferases encoding genes was adopted. The result displayed that ß-farnesene titer in the engineered strain CIBT6304 in which acyltransferases (DGA1 and DGA2) were deleted increased by 45% and reached 539 mg L-1 (88 mg g-1 DCW). Using fed-batch fermentation, CIBT6304 could produce the highest ß-farnesene titer (22.8 g L-1 ) among the genetically defined strains. This study will provide the foundation of engineering Y. lipolytica to produce other terpenoids more cost-efficiently.