Sustainable production of lipids from cocoa fatty acid distillate fermentation driven by adaptive evolution in Yarrowia lipolytica.

Single cell oil production using oleaginous yeasts is a promising alternative to animal and plant-derived lipids. But substrate costs for microbial fermentation are a major bottleneck. Using side streams as alternative to substrates like glucose, for growing yeast, is a potential cost-effective solution. By combining a previously reported process of growing yeasts on a solid cocoa fatty acid distillate side stream with adaptive evolution techniques, the growth of oleaginous yeast Yarrowia lipolytica was improved by 2-fold. The lipid titre was also boosted by more than 3-fold. Using transcriptomics, key genes were identified that are possibly involved in tailoring of lipid composition, side stream utilisation and enhancement of lipid titres. Candidate genes were also identified that might enable efficient growth and utilization of fatty acids and triacylglycerides found in cocoa fatty acid distillate. In summary, this research has improved the understanding of side stream utilisation for lipid production in oleaginous yeast.

Co-culture of Kluyveromyces marxianus and Meyerozyma guilliermondii with In Situ Product Recovery of 2-Phenylethanol.

Production of 2-phenylethanol (2-PE) via Kluyveromyces marxianus is well-established. However, co-culture with other microbes in combination with in situ product recovery (ISPR) yields improved selectivity and volumetric productivity. Fermentation ofK. marxianus (MUCL 53775) with direct inclusion of absorptive polymer Hytrel3548 achieved ISPR, but accumulation of the byproduct phenylethyl acetate (PEA) was strongly favored. Co-culture of K. marxianus (MUCL 53775) with Meyerozyma guilliermondii (MUCL 28072) with ISPR limited PEA production, thereby improving the 2-PE selectivity from 13 to 90%, compared to a pure culture of K. marxianus (MUCL 53775) under similar conditions. This improved the volumetric productivity by 85% compared to 2-PE ISPR with a pure culture of K. marxianus. This is the first report of co-culture in a two-phase fermentation for 2-PE bioproduction and demonstrates that interactions between co-culture and ISPR techniques can modulate bioproduction between 2-PE and byproduct PEA, and this technique will be explored for other strain combinations and for other high-value molecules of interest.

Production of (R)-mandelic acid from styrene, L-phenylalanine, glycerol, or glucose via cascade biotransformations.

(R)-mandelic acid is an industrially important chemical, especially used for producing antibiotics. Its chemical synthesis often uses highly toxic cyanide to produce its racemic form, followed by kinetic resolution with 50% maximum yield. Here we report a green and sustainable biocatalytic method for producing (R)-mandelic acid from easily available styrene, biobased L-phenylalanine, and renewable feedstocks such as glycerol and glucose, respectively. An epoxidation-hydrolysis-double oxidation artificial enzyme cascade was developed to produce (R)-mandelic acid at 1.52 g/L from styrene with > 99% ee. Incorporation of deamination and decarboxylation into the above cascade enables direct conversion of L-phenylalanine to (R)-mandelic acid at 913 mg/L and > 99% ee. Expressing the five-enzyme cascade in an L-phenylalanine-overproducing E. coli NST74 strain led to the direct synthesis of (R)-mandelic acid from glycerol or glucose, affording 228 or 152 mg/L product via fermentation. Moreover, coupling of E. coli cells expressing L-phenylalanine biosynthesis pathway with E. coli cells expressing the artificial enzyme cascade enabled the production of 760 or 455 mg/L (R)-mandelic acid from glycerol or glucose. These simple, safe, and green methods show great potential in producing (R)-mandelic acid from renewable feedstocks.

De Novo Biosynthesis of (S)- and (R)-Phenylethanediol in Yeast via Artificial Enzyme Cascades.

Due to oil depletion and global climate change, sustainable manufacturing of fine chemicals from renewable feedstocks has gained increasing attention in the scientific community. In the present study, we attempted to engineer Saccharomyces cerevisiae toward de novo synthesis of (S)- or (R)-phenylethanediol, an important pharmaceutical intermediate. More specifically, the biocatalytic cascades contain the following: l-phenylalanine undergoes deamination/decarboxylation to styrene by using phenylalanine ammonia lyase (PAL) and ferulic acid decarboxylase (FDC), followed by S-selective epoxidation of styrene to give (S)-styrene oxide with styrene monooxygenase (SMO); regioselective hydrolysis of (S)-styrene oxide with epoxide hydrolase from Sphingomonas HXN-200 (SpEH) or from potato (StEH) gives rise to (S)- or (R)-phenylethanediol. In this work, we found that the artificial enzyme cascades could be functionally expressed in the heterologous host of S. cerevisiae. Small-scale shake flask studies revealed that the engineered yeast cell factories produced approximately 100-120 mg/L of (S)- or (R)-phenylethanediol after 96 h cultivation. To the best of our knowledge, this is the first attempt to explore an artificial route with styrene as an intermediate for producing phenylethanediol in S. cerevisiae. We envision that our engineering strategy will open a new research field for synthesizing other vicinal diol derived chemicals in yeast.