Patent ID: 12252724

EXAMPLES

Example 1: General Methods, Strains, and Plasmids

All basic molecular biology and DNA manipulation procedures described herein are generally performed according to Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York (1989) or Ausubel et al. (eds). Current Protocols in Molecular Biology. Wiley: New York (1998).

Shake plate assay. Typically, 800 μl of 0.075% Yeast extract, 0.25% peptone (0.25×YP) is inoculated with 10 μl of freshly grownYarrowiaand overlaid with 800 μl of mineral oil (Drakeol 5, Penreco Personal Care Products, Karns City, PA, USA) carbon source 5% corn oil in mineral oil and/or 5% in glucose in aqueous phase. Transformants were grown in 24 well plates (Microplate Devices 24 Deep is Well Plates Whatman 7701-5102), covered with mat seal (Analytical Sales and Services Inc. Plate Mats 24010CM), sterile sealed with Qiagen Airpore Tape Sheets (19571) and shaken in Infors multi plate shaker (Multitron), 30° C., 800 RPM in YPD media with for 4 days. The mineral oil fraction was removed from the shake plate wells and analyzed by HPLC on a normal phase column, with a photo-diode array detector. This method is used in Examples 2, 3, 4.

DNA transformation. Strains are transformed by overnight growth on YPD plate media 50 μl of cells is scraped from a plate and transformed by incubation in 500 μl with 1 μg transforming DNA, typically linear DNA for integrative transformation, 40% PEG 3550MW, 100 mM lithium acetate, 50 mM Dithiothreitol, 5 mM Tris-Cl pH 8.0, 0.5 mM EDTA for 60 minutes at 40° C. and plated directly to selective media or in the case of dominant antibiotic marker selection the cells are out grown on YPD liquid media for 4 hours at 30° C. before plating on the selective media.

DNA molecular biology. Genes were synthesized with NheI and MluI ends in pUC57 vector (GenScript, Piscataway, NJ). Typically, the genes were subcloned to the MB5082 ‘URA3’, MB6157 HygR, and MB8327 NatR vectors for marker selection inYarrowia lipolyticatransformations, as in WO2016172282. For clean gene insertion by random nonhomologous end joining of the gene and marker HindIII/XbaI (MB5082) or PvuII (MB6157 and MB8327), respectively purified by gel electrophoresis and Qiagen gel purification column. MB5082 ‘URA3’ marker could be reused due to gratuitous repeated flanking sequences that enable selection of circular excisants of the URA3 cassette on FOA. The NatR and HygR markers can be removed by transient expression of Cre recombinase that results in excisants due to the flanking Lox sites.

Plasmid list. Plasmid, strains, nucleotide and amino acid sequences to be used are listed in Table 1, 2 and the sequence listing. Nucleotide sequence ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 35, 37, and 39 are codon optimized for expression inYarrowia.

TABLE 1list of plasmids used for construction of the strains carryingthe heterologous BCO, RDH and ATF1-genes. The sequence IDNOs refer to the inserts. For more details, see text.SEQ ID NO:MB plasmidBackbone MBInsert(aa/nt)84575082UmCCO11/284565082FfCarX3/467035082CsZCO5/667025082DmNinaB7/890685082DrBCO9/1092795082DrBCO-TPI11/1291235082IpBCO13/1491215082ElBCO15/1691265082LcBCO17/1882005082FfRDH1219/2080645082SbATF121/2285096157FaATF23/2485106157EcCAT25/2685116157EaCAcT27/2885126157MdATF29/3085136157PhATF31/3288495082LmATF133/3586105082LfATF136/3788065082LffATF138/39

TABLE 2list ofYarrowiastrains used for productionof retinoids carrying the heterologous BCO,RDH and ATF1-genes. For more details, see text.ML strainDescriptionFirst described in7788Carotene strainWO201617228215710ML7788 transformed withWO2016172282MB7311 -Mucor CarG17544ML15710 cured of URA3 byhereFOA and HygR by Cre/lox17767ML17544 transformed withhereMB6072 DmBCO-URA3and MB6732 SbATF1-HygRand cured of markers17968ML17544 transformed withhereMB8457 UmCCO1-URA3 and cured of markers17978ML17968 transformed withhereMB8200 FfRDH-URA3and cured of markers

Normal phase retinol method. A Waters 1525 binary pump attached to a Waters 717 auto sampler were used to inject samples. A Phenomenex Luna 3p Silica (2), 150×4.6 mm with a security silica guard column kit was used to resolve retinoids. The mobile phase consists of either, 1000 mL hexane, 30 mL isopropanol, and 0.1 mL acetic acid for astaxanthin related compounds, or 1000 mL hexane, 60 mL isopropanol, and 0.1 mL acetic acid for zeaxanthin related compounds. The flow rate for each is 0.6 mL per minute. Column temperature is ambient. The injection volume is 20 μL. The detector is a photodiode array detector collecting from 210 to 600 nm. Analytes were detected according to Table 3.

TABLE 3list of analytes using normal phase retinol method.The addition of all added intermediates gives the amountof total retinoids. For more details, see text.RetentionLambdatimemaxIntermediates[min][nm]11-cis-dihydro-retinol7.129311-cis-retinal436411-cis-retinol8.631813-cis-retinal4.1364dihydro-retinol9.2292retinyl-acetate3.5326retinyl-ester3325trans-retinal4.7376trans-retinol10.5325

Sample preparation. Samples were prepared by various methods depending on the conditions. For whole broth or washed broth samples the broth was placed in a Precellys® tube weighed and mobile phase was added, the samples were processed in a Precellys® homogenizer (Bertin Corp, Rockville, MD, USA) on the highest setting 3× according to the manufactures directions. In the washed broth the samples were spun in a 1.7 ml tube in a microfuge at 10000 rpm for 1 minute, the broth decanted, 1 ml water added mixed pelleted and decanted and brought up to the original volume the mixture was pelleted again and brought up in appropriate amount of mobile phase and processed by Precellys® bead beating. For analysis of mineral oil fraction, the sample was spun at 4000 RPM for 10 minutes and the oil was decanted off the top by positive displacement pipet (Eppendorf, Hauppauge, NY, USA) and diluted into mobile phase mixed by vortexing and measured for retinoid concentration by HPLC analysis.

Fermentation conditions. Fermentations were identical to the previously described conditions using preferably a silicone oil or a mineral oil overlay and stirred tank that was preferably glucose or corn oil fed in a bench top reactor with 0.5 L to 5 L total volume (see WO2016172282). Generally, the same results were observed with a fed batch stirred tank reactor with an increased productivity demonstrating the utility of the system for the production of retinoids. Preferably, fermentations were batched with 5% glucose and 20% silicone oil was added after dissolved oxygen plummeted and feed was resumed to achieve 20% dissolved oxygen throughout the feeding program. Alternatively, corn oil was used as a feed and mineral oil was used as a second phase to collect the aliphatic retinoids.

Example 2: Conversion of Beta-Carotene to Retinal inYarrowia lipolytica

For expression of heterologous BCOs, a beta carotene strain ML17544 was transformed with purified linear DNA fragment by HindII and XbaI mediated restriction endonucleotide cleavage and gel purification of beta carotene oxidase (BCO) containing codon optimized fragments linked to a URA3 nutritional marker. Transforming DNA were derived from MB6702DrosophilaNinaB BCO gene, MB6703CrocusBCO gene, MB8456FusariumBCO gene, and MB8457UstilagoBCO gene and MB6098 Dario BCO gene, whereby the codon-optimized sequences (SEQ ID NOs:2, 4, 6, 8, 10, 12) had been used. The genes were then grown screening 6-8 isolates in a shake plate analysis, and isolates that is performed well were run in a fed batch stirred tank reaction for 8-10 days. Detection of cis- and trans-retinal was made by HPLC using standard parameters as described in WO2014096992, but calibrated with purified standards for the retinoid analytes. The amount of trans-retinal in the retinal mix could be increased to 90% (using theCrocusBCO), 95% (using theFusariumBCO), 98% (using theUstilagoBCO) and 98% (using Dario BCO), respectively. A comparison with the BCO fromDrosophila melanogaster(SEQ ID NO:7) resulted in 61% of trans-retinal based on the total amount of retinal (see Table 4).

TABLE 4Retinal production inYarrowiaas enhanced by action ofheterologous BCOs. “% trans” means percentage oftrans-retinal in the mix of retinoids. For more details, see text.% retinoids/MLMBOrganismBCO gene% trans-DCWstrainplasmidDrosophilaDmNinB6114175446702UstilagoUmCCO1988175448457FusariumFfCarX955175448456CrocusZsZCO900.01175446703DarioDrBCO986175449068DarioDrBCO-TPI986175449279IctalurusIpBCO985175449123EsoxElBCO983175449121LatimeriaLcBCO982175449126

Example 3: Conversion of Retinal to Retinol inYarrowia lipolytica

For expression of heterologous RDHs, the beta carotene strain ML17767 was transformed with purified HinDIII/XbaI fragments derived from plasmids containing retinol dehydrogenase (RDH) gene fragments linker to a URA3 promoter. Six to eight isolates were screened for a decrease in the retinol:retinal ratio in a shake plate assay and successful isolates were run in a fed batch stirred tank reactor for eight days which showed an order of magnitude increase in the productivity of the process which indicates a utility in large scale production. The best results were obtained with theFusariumRDH12 homolog with only 2% or residual retinal maintained after 8 days of shake-flask incubation as described above. The isolate derived from theFusariumsequence demonstrated an increased reduction of retinol.

Example 4: Conversion of Retinol to Retinyl Acetate inYarrowia lipolytica

For expression of heterologous ATF1, the trans retinol producing strain ML17968 was transformed with purified PvuII gene fragments containing acetyltransferase gene fragments linked to a Hygromycin resistance marker (HygR) for selection rich media (YPD) containing 100 ug/ml hygromycin. Prior to plating the cultures were outgrown in YPD for four hours to synthesize the antibiotic resistance genes. Isolates were screened for acylation in shake plate assays and successful isolates were screened in fed batch stirred tank reactor which showed an order of magnitude increased productivity indicating utility in the production of retinoids. The data from the analysis are shown in Table 5).

TABLE 5Trans retinoid production inYarrowiaas enhancedby action of heterologous ATF1 enzymes. “% acetylation”means percentage of trans-retinyl acetate in the mixof retinoids. For more details, see text.MLMBOrganismATF1 gene% acetylation-strainplasmidS. bayanusSbATF110.3179686832P. hybridaPhATF2.1179688513E. alatusEaCAcT0.45179688511E. coliEcCAT0.35179688510L. fermentataLfATF19.6185238610L. fermentataLffATF111.7185238806L. mirantinaLmATF140.4185238849

Example 5: ATF1 Activity Assay

For expression of heterologous ATF1, the trans retinol producing strain ML17968 was transformed with purified PvuII gene fragments containing acetyltransferase gene fragments linked to a Hygromycin resistance marker (HygR) for selection rich media (YPD) containing 100 ug/ml hygromycin. Prior to plating the cultures were outgrown in YPD for four hours to synthesize the antibiotic resistance genes. Isolates were screened for acylation in shake plate assays, specifically using 10% glucose as a carbon source in 0.25×YP with silicone oil as an overlay and successful isolates were further screened in fed batch stirred tank reactor with glucose feed and silicone oil overlay, which showed an order of magnitude increased productivity indicating utility in the production of retinoids. The data from the analysis are shown in Table 5.

Example 6: Conversion of Beta-Carotene to Retinyl Acetate inSaccharomyces cerevisiae

Typically, a beta carotene strain is transformed with heterologous genes encoding for enzymes such as geranylgeranyl synthase, phytoene synthase, lycopene synthase, lycopene cyclase constructed that is producing beta carotene according to standard methods as known in the art (such as e.g. as described in US20160130628 or WO2009126890). Further, when transformed with beta carotene oxidase genes retinal can be produced. Further, when transformed with retinol dehydrogenase, then retinol can be produced. The retinol can be acetylated by transformation with genes encoding alcohol acetyl transferases. Optionally, the endogenous retinol acylating genes can be deleted. Further, the enzymes can be selected to produce and acylate the trans form of retinol to yield all trans retinyl acetate, and long chain esters of trans retinol, respectively. With this approach, similar results regarding specificity for trans-isoform or productivity towards retinyl acetate are obtained.