Enhancement of Astaxanthin Biosynthesis in Oleaginous Yeast Yarrowia Lipolytica Via Microalgal Pathway

microorganisms

Article Enhancement of Astaxanthin Biosynthesis in Oleaginous Yeast Yarrowia lipolytica via Microalgal Pathway

Larissa Ribeiro Ramos Tramontin, Kanchana Rueksomtawin Kildegaard, Suresh Sudarsan and Irina Borodina *

The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800 Kgs. Lyngby, Denmark; [email protected] (L.R.R.T.); [email protected] (K.R.K.); [email protected] (S.S.) * Correspondence: [email protected]

Received: 10 October 2019; Accepted: 16 October 2019; Published: 19 October 2019

Abstract: Astaxanthin is a high-value red pigment and antioxidant used by pharmaceutical, cosmetics, and food industries. The astaxanthin produced chemically is costly and is not approved for human consumption due to the presence of by-products. The astaxanthin production by natural microalgae requires large open areas and specialized equipment, the process takes a long time, and results in low titers. Recombinant microbial cell factories can be engineered to produce astaxanthin by fermentation in standard equipment. In this work, an oleaginous yeast Yarrowia lipolytica was engineered to produce astaxanthin at high titers in submerged fermentation. First, a platform strain was created with an optimised pathway towards β-carotene. The platform strain produced 331 66 mg/L of β-carotene ± in small-scale cultivation, with the cellular content of 2.25% of dry cell weight. Next, the genes encoding β-ketolase and β-hydroxylase of bacterial (Paracoccus sp. and Pantoea ananatis) and algal (Haematococcus pluvialis) origins were introduced into the platform strain in diﬀerent copy numbers. The resulting strains were screened for astaxanthin production, and the best strain, containing algal β-ketolase and β-hydroxylase, resulted in astaxanthin titer of 44 1 mg/L. The same strain was ± cultivated in controlled bioreactors, and a titer of 285 19 mg/L of astaxanthin was obtained after ± seven days of fermentation on complex medium with glucose. Our study shows the potential of Y. lipolytica as the cell factory for astaxanthin production.

Keywords: Yarrowia lipolytica; β-carotene; astaxanthin; metabolic engineering; submerged fermentation

1. Introduction Astaxanthin is a keto-carotenoid compound with a red color and strong antioxidant activity. It is mainly used in aquaculture as a feed additive and in human nutrition as a dietary supplement [1]. Astaxanthin and the closely related compound canthaxanthin are also used in the diet of quails and chicken for a more intense color of the flesh and egg yolks [2,3]. Astaxanthin is produced by chemical synthesis (ca. 90%) and by algal fermentation. The chemically synthesized astaxanthin is not allowed for human consumption due to the presence of several chiral forms of astaxanthin as well as some other impurities. Therefore, astaxanthin is mainly used in aquafarming [4,5]. Astaxanthin market price varies from $2500–7000/kg and comprises a significant fraction of the salmon production cost (up to 15%) [6]. The natural astaxanthin is primarily extracted from the freshwater green alga Haematococcus pluvialis, which can accumulate 1.5–3% astaxanthin on a dry cell weight (DCW) basis and is the richest source for natural production of astaxanthin [7,8]. The astaxanthin chemical structure varies between three different stereoisomers, (3S, 30S), (3R, 30S), and (3R, 30R). In chemically synthetized astaxanthin, these

Microorganisms 2019, 7, 472; doi:10.3390/microorganisms7100472 www.mdpi.com/journal/microorganisms Microorganisms 2019, 7, 472 2 of 17

isomers are obtained in the ratio of 1:2:1. The most valuable one is the 3S, 30S stereoisomer, which is predominantly found in H. pluvialis [9]. To address the high demand for astaxanthin, efforts have been made to increase the astaxanthin production in the natural producer organisms, such as H. pluvialis and red yeast Xanthophyllomyces dendrorhous, by metabolic engineering. Studies made by Gassel et al. (2013) used random mutagenesis, overexpression of the bifunctional phytoene synthase/lycopene cyclase (crtYB), and astaxanthin synthase (asy), and selection of an optimum growth medium to reach an astaxanthin content of 9.7 mg/g DCW by X. dendrorhous in fermenters [10]. In another work, Gassel et al. (2014) reported an astaxanthin content of 9 mg/g DCW by X. dendrorhous, which was obtained in shake-flask culture after a combination of classical mutagenesis and simultaneous integration of rate-limiting enzymes encoded by genes from X. dendrorhous (crtYB, asy, geranylgeranyl pyrophosphate synthase (crtE), and 3-hydroxy-3-methylglutaryl-coenzymeA reductase (HMG) in its truncated form lacking the membrane binding region) [11]. The production of astaxanthin by the microalgae H. pluvialis has been improved primarily by classical mutagenesis and selection [9]. In 2006, a transformation protocol for this microalga was reported by Steinbrenner and Sandmann, where they transformed H. pluvialis with a mutated phytoene desaturase (PDS gene) and obtained a transformant with 32% higher astaxanthin content than the wild type. In shake flask cultivation, this strain accumulated 11.4 mg/g DCW astaxanthin [12]. Recently, new approaches such as nuclear transformation vectors [13] and genetic engineering of chloroplasts genome [14] have been developed. However, a feasible natural production of astaxanthin by H. pluvialis, able to compete with chemical synthesis, was not yet achieved. The astaxanthin biosynthesis has also been engineered into noncarotenogenic organisms, such as the bacterium Escherichia coli, and yeasts Saccharomyces cerevisiae and Yarrowia lipolytica. Park et al. (2018) used E. coli as platform for production of astaxanthin by expressing heterologous genes crt (crtE, lycopene cyclase (crtY), phytoene desaturase (crtI), phytoene synthase (crtB), and β-carotene hydroxylase (crtZ)) from Pantoea ananatis and a truncated β-carotene ketolase gene (trCrBKT) from Chlamydomonas reinhardtii. The authors used the signal peptide of OmpF and TrxA to tag the N-terminus and C-terminus of trCrBKT and confer stable expression and to efficiently guide trCrBKT to the E. coli membrane. Further optimization of culture conditions and overexpression of 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (ispD) and 4-diphosphocytidyl-2-C-methyl-D-ery-thritol kinase (ispF) from E. coli lead to an astaxanthin production of 432 mg/l with 7 mg/g DCW in fed-batch fermentation [15]. Another work done in E. coli reported production of 320 mg/L of astaxanthin by simultaneous fermentation and extraction using pathway optimization on transcriptional, translational, and enzyme levels. In this approach, Zhang et al. (2018) expressed 14 genes stepwise (grouped into four major modules) to optimize the production of precursors lycopene and β-carotene, and to reduce the bottlenecks towards the production of astaxanthin [16]. Metabolic engineering done in the yeast S. cerevisiae compared the activity of the β-carotene hydroxylase (CrtZ) from Alcaligenes sp. and CrtZ from Agrobacterium aurantiacum towards the production of astaxanthin. In this study, Jin et al. (2018) integrated heterologous genes (β-carotene ketolase (crtW) from Brevundimonas vesicularis and crtZ from Agrobacterium aurantiacum), and used mutagenesis by atmospheric and room temperature plasma to promote astaxanthin production. After fermentation in 5 L bioreactor, they obtained an astaxanthin content of 13.8 mg/g DCW (217.9 mg/L) [17]. Studies done by Zhou et al. (2017) improved the pathway towards astaxanthin precursors in S. cerevisiae by integrating the gene crtE and the rate-limiting enzymes crtI, crtYB (from X. dendrorhous), and truncated HMG1 (from S. cerevisiae). After expressing OBKTM and OCRTZ (β-carotene ketolase and β-carotene hydroxylase from H. pluvialis, respectively, which were developed by directed evolution), the engineered strain accumulated 8 mg/g DCW (47 mg/l) of (3S, 30S)-astaxanthin in shake-flask cultures [18]. In another work, S. cerevisiae was engineered to produce astaxanthin by expression of crtZ and BKT from H. pluvialis. In this work, a content of 4.7 mg/g DCW of astaxanthin was achieved in the shake-flask cultures [19]. A promising organism for production of a variety of carotenoids, including astaxanthin, is the oleaginous yeast Y. lipolytica. Due to its biosafety record and for the natural production of carotenoid precursors, cytosolic acetyl-CoA, and redox co-factor NADPH, Y. lipolytica has the potential to produce astaxanthin Microorganisms 2019, 7, 472 3 of 17 at high titers [20–22]. Our group reported engineering of Y. lipolytica for astaxanthin production in Kildegaard et al. (2017). After metabolic engineering and integration of several heterologous genes for the production of astaxanthin, we obtained 54.6 mg/L (3.5 mg/g DCW) of astaxanthin. To achieve this titer, we expressed the bifunctional phytoene synthase/lycopene cyclase (crtYB) and the phytoene desaturase (crtI) from X. dendrorhous. We further optimized and expressed HMG1 and compared the activity of the geranylgeranyl diphosphate synthases GGS1 and crtE from Y. lipolytica and X. dendrorhous, respectively. Next, we downregulated the competing squalene synthase SQS1 by truncating the native promoter to 50 bp and introduced the astaxanthin pathway by expressing and optimizing the copy numbers of the β-carotene ketolase (crtW) from Paracoccus sp. and the β-carotene hydroxylase (crtZ) from Pantoea ananatis [23]. A list of the astaxanthin content produced by the above-mentioned organisms and their genotype is presented in Table1. In this present work, we aimed to produce a high astaxanthin producing Y. lipolytica strain. To increase the production of the precursor β-carotene, we expressed and compared two different geranylgeranyl pyrophosphate synthases (CrtE and GGPPs7). The best β-carotene producer strain was used as the platform for integration of heterologous astaxanthin genes (crtW, BKT, and two different crtZ) in different combinations. To optimize the astaxanthin production, different molar ratios of the astaxanthin genes were tested. The best performing astaxanthin producer strain was cultivated in controlled bioreactors.

Table 1. Summary of astaxanthin production by natural producers and engineered non-carotenogenic organisms. Single black arrow ( ) represents downregulation. ↓ Astaxanthin Organism Genotype Titer and Reference Content 9.7 mg/g DCW X. dendrorhous crtYB and asy (native genes) [10] (bioreactor) 9 mg/g DCW X. dendrorhous crtYB, asy, crtE and trHMG (native genes) [11] (shake-flasks) 11.4 mg/g DCW H. pluvialis site-directed mutagenesis of PDS (native gene) [12] (shake-flasks) 432 mg/L, crtE, crtY, crtI, crtB, crtZ (from P. ananatis); trBKT E. coli 7 mg/g DCW [15] (from C. reinhardtii); ispD and ispF (native genes) (bioreactor) Module 1: atoB (native), hmgS (S. cerevisiae), and thmgR (S. cerevisiae); module 2: mevk (S. cerevisiae), pmk 320 mg/L, (S. cerevisiae), pmd (S. cerevisiae), and idi (native); module E. coli 2 mg/g DCW [16] 3: crtEBI (amplified from pAC-LYC plasmid) and ispA (SFE) (native); crtY (P. ananatis), crtZ (from P. ananatis), crtW (Brevundimonas sp.) crtW (from Brevundimonas vesicularis),crtZ (from 217.9 mg/L, S. cerevisiae Agrobacterium aurantiacum), and mutagenesis of CSS1, 13.8 mg/g DCW [17] YBR012W-B and DAN4 (bioreactor) 47 mg/L, 8 mg/g crtE, crtI, crtYB (from X. dendrorhous); trHMG1 S. cerevisiae DCW [18] (native gene); BKT and crtZ (from H. pluvialis) (shake-flasks) 4.7 mg/g DCW S. cerevisiae BKT and crtZ (from H. pluvialis) [19] (shake-flasks) 54.6 mg/L, crtYB, crtI, crtE (from X. dendrorhous); HMG1 3.5 mg/g DCW Y. lipolytica (native gene); SQS1; crtW (from Paracoccus sp.) and crtZ [23] ↓ (microtiter (from P. ananatis) plates) 285 mg/L, GGPPs7 (from Synechococcus sp.), HpBKT, HpcrtZ Y. lipolytica 6 mg/g DCW This study (from H. pluvialis) (bioreactor) Microorganisms 2019, 7, x FOR PEER REVIEW 4 of 17

54.6 mg/L, 3.5 crtYB, crtI, crtE (from X. dendrorhous); HMG1 mg/g DCW Y. lipolytica (native gene); ↓SQS1; crtW (from Paracoccus sp.) [23] (microtiter and crtZ (from P. ananatis) plates) 285 mg/L, 6 Microorganisms 2019, 7, 472GGPPs7 (from Synechococcus sp.), HpBKT, HpcrtZ This 4 of 17 Y. lipolytica mg/g DCW (from H. pluvialis) study (bioreactor) 2. Results 115 2. Results 2.1. Enhancement of Beta-Carotene Production by the Introduction of crtE and GGPPs7 116 2.1. Enhancement of Beta-Carotene Production by the Introduction of crtE and GGPPs7 Astaxanthin is biologically synthesized from β-carotene, which in turn is made from two products 117 of the mevalonateAstaxanthin pathway,is biologic isopentenylally synthesized pyrophosphate from β-carotene, (IPP) which and dimethylallyl in turn is made pyrophosphate from two 118 (DMAPP).products An of e ffithecient mevalonate strategy to enhancepathway, IPP isopen and DMAPPtenyl pyrophosphate production in Y. lipolytica(IPP) andis thedimethylallyl upregulation 119 of thepyrophosphate native mevalonate (DMAPP). pathway An efficient genes strategy and downregulation to enhance IPP of and side DMAPP fluxes, e.g.,production towards in squaleneY. lipolytica [23 ]. 120 Foris biosynthesis the upregulation of β-carotene of the native in Y. mevalonate lipolytica, insertionpathway ofgenes the and heterologous downregulationβ-carotene of side pathway fluxes, e.g., genes 121 towards squalene [23]. For biosynthesis of β-carotene in Y. lipolytica, insertion of the heterologous β- (crtE, crtYB, and crtI) is required. To improve the carbon flux towards β-carotene biosynthesis, we chose 122 carotene pathway genes (crtE, crtYB, and crtI) is required. To improve the carbon flux towards β- to enhance the conversion of FPP into GGPP by inserting two different GGPP synthases, encoded by 123 carotene biosynthesis, we chose to enhance the conversion of FPP into GGPP by inserting two GGPPs7 from Synechococcus sp. and crtE from X. dendrorhous. These two GGPP syntheses showed good 124 different GGPP synthases, encoded by GGPPs7 from Synechococcus sp. and crtE from X. dendrorhous. results increasing the precursor GGPP, which will supply the chain towards the β-carotene formation 125 These two GGPP syntheses showed good results increasing the precursor GGPP, which will supply 126 andthe further chain carotenoidstowards the and,β-carotene therefore, formation were chosen and further to be usedcaroteno in thisids studyand, therefore, [23,24]. Thewere strain chosen ST6899 to 127 wasbe used used as in a this parent study strain [23,24]. to express The strain and ST6899 compare wa thes used activity as a ofparent GGPPs7 strain and to CrtE.express It isand worth compare noting 128 thatthe the activity parent of strain GGPPs7 already and CrtE. bore oneIt is copyworth of notingcrtE fromthat theX. dendrorhousparent strain, onealready additional bore one copy copy of of the 129 nativecrtE HMG1from X., anddendrorhous the genes, onecrtI additionaland crtYB copyfrom of theX.dendrorhous native HMG1. Additionally,, and the genes the crtI squalene and crtYB synthase from 130 SQS1X. dendrorhouswas downregulated. Additionally, by the the shortening squalene ofsynthase the native SQS1 promoter was downregulated to 50 base pairs. by Thethe shortening results showed of 131 thatthe the native strain promoter carrying to the 50 GGPPbase pairs. synthase The result encodeds showed by GGPPs7 that the(ST7434) strain carrying produced the aGGPPβ-carotene synthase titer 132 272%encoded higher by (330 GGPPs766 mg(ST7434)/L) compared produced to a the β-carotene parent strain titer 272% (88 higher11 mg /(330L), whereas ± 66 mg/L) the compared strain ST7433, to ± ± 133 containingthe parent the strain second (88 ± copy11 mg/L), of crtE whereasshowed the anstrain increase ST7433, of containing 48.59% compared the second with copy the of crtE parent showed strain, 134 withan aincreaseβ-carotene of 48.59% production compared of 132 with the11 mgparent/L (Figure strain,1 with). a β-carotene production of 132 ± 11 mg/L ± 135 (Figure 1).

136 137 FigureFigure 1. 1E.ﬀ Effectect of ofSynechococcus Synechococcus GGPPs7 GGPPs7(ST7434) (ST7434) andand thethe 2nd copycopy of of crtEcrtE (ST7433)(ST7433) on on β-caroteneβ-carotene 138 production.production. ST6899 ST6899 is is the the parental parental strain strain to to ST7434 ST7434 andand ST7433.ST7433. (AA. )Yeast Yeast extract extract peptone peptone dextrose dextrose (YPD) plate after 2 days cultivation. (B) Titers measured by HPLC. The error bars represent standard deviations calculated from biological triplicate experiments.

2.2. Expression of Heterologous β-ketolases for the Biosynthesis of Astaxanthin Intermediates The strain ST7434 with the highest titer of β-carotene was further engineered by insertion of β-ketolase coding genes from Paracoccus sp. (PscrtW) or from H. pluvialis (HpBKT), resulting in corresponding strains ST7906 and ST7972. Both strains had an orange-red color due to the formation of echinenone and canthaxanthin, which are β-carotene derivatives with one or two ketone groups, Microorganisms 2019, 7, x FOR PEER REVIEW 5 of 17

139 (YPD) plate after 2 days cultivation. B. Titers measured by HPLC. The error bars represent standard 140 deviations calculated from biological triplicate experiments.

141 2.2. Expression of Heterologous β-ketolases for the Biosynthesis of Astaxanthin Intermediates 142 The strain ST7434 with the highest titer of β-carotene was further engineered by insertion of β- 143 ketolaseMicroorganisms coding2019 ,genes7, 472 from Paracoccus sp. (PscrtW) or from H. pluvialis (HpBKT), resulting5 ofin 17 144 corresponding strains ST7906 and ST7972. Both strains had an orange-red color due to the formation 145 of echinenone and canthaxanthin, which are β-carotene derivatives with one or two ketone groups, respectively. When analyzing the pathway, the keto-carotenoid canthaxanthin is the intermediate 146 respectively. When analyzing the pathway, the keto-carotenoid canthaxanthin is the intermediate closer to astaxanthin and, therefore, a strain that accumulates higher amounts of canthaxanthin would 147 closer to astaxanthin and, therefore, a strain that accumulates higher amounts of canthaxanthin be more relevant since it would have more potential to convert the high amount of canthaxanthin into 148 would be more relevant since it would have more potential to convert the high amount of astaxanthin. The HPLC analysis showed that the strain ST7972, which contained HpBKT, produced a 149 canthaxanthin into astaxanthin. The HPLC analysis showed that the strain ST7972, which contained higher amount of canthaxanthin (30 2 mg/L) when compared to ST7906 (13 0.8 mg/L). The strain 150 HpBKT, produced a higher amount of± canthaxanthin (30 ± 2 mg/L) when compared± to ST7906 (13 ± ST7972 also produced 55 3 mg/L of echinenone, and 82 14 mg/L of β-carotene, while ST7906 151 0.8 mg/L). The strain ST7972± also produced 55 ± 3 mg/L of echinenone,± and 82 ± 14 mg/L of β-carotene, accumulated 109 5 mg/L of echinenone, and 30 1 mg/L of β-carotene (Figure2). The β-ketolases 152 while ST7906 accumulated± 109 ± 5 mg/L of echinenone,± and 30 ± 1 mg/L of β-carotene (Figure 2). The PsCrtW and HpBKT have a bifunctional activity and convert β-carotene into echinenone and echinenone 153 β-ketolases PsCrtW and HpBKT have a bifunctional activity and convert β-carotene into echinenone into canthaxanthin. The higher titer of echinenone produced by ST7906 compared to ST7972 might 154 and echinenone into canthaxanthin. The higher titer of echinenone produced by ST7906 compared to suggest the preference of PsCtrW for β-carotene as substrate compared to echinenone, leading to an 155 ST7972 might suggest the preference of PsCtrW for β-carotene as substrate compared to echinenone, accumulation of this intermediate. 156 leading to an accumulation of this intermediate.

157

158 FigureFigure 2 2.. CarotenoidCarotenoid production production of of strains strains ST7906 ST7906 (expressing (expressing multiple multiple copies copies of of PscrtWPscrtW) )and and ST7972 ST7972 159 (expressing(expressing multiple multiple copies copies of of HpBKTHpBKT).). All All strains strains were were cultivated cultivated in in YP YP ++ 8%8% glucose glucose in in 24-deep-well 24-deep-well 160 platesplates for for 72 72 h. h. The The error error bars bars represent represent standard standard deviations deviations calculated calculated from from triplicate triplicate experiments. experiments.

161 2.3.2.3. Single-Copy Single-Copy Expression Expression of of ββ-Hydroxylase-Hydroxylase for for Production Production of of Astaxanthin Astaxanthin Y. lipolytica 162 ToTo evaluate evaluate the productionproduction ofof astaxanthin astaxanthin in in Y. lipolytica, the, the strains strains ST7906 ST7906 and and ST7972 ST7972 were were used β 163 usedas platforms as platforms for insertion for insertion of -hydroxylases of β-hydroxylases from the from bacteria the bacteriaP. ananatis P.( PaCrtZananatis) or(PaCrtZ the microalgae) or the 164 microalgaeH. pluvialis H.(HpCrtZ pluvialis). Two (HpCrtZ to seven). Two individual to seven clones individual of each clones of the of resulting each of fourthe resulting strains were four screened strains 165 werefor carotenoid screened for production. carotenoid We production. observed We a signiﬁcant observed clonala significant variation, clonal which variation, could possibilitywhich could be β 166 possibilitydue to the be instability due to the of the instability integrated of the-ketolase integrated genes, β-ketolase which were genes, integrated which were into rDNAintegrated regions into of β 167 rDNAthe genome. regions Nevertheless,of the genome. there Nevertheless, was a clear there tendency was a thatclear strain tendency ST7974, that combiningstrain ST7974,-ketolase combining and β 168 β-ketolase-hydroxylase and β genes-hydroxylase from H. genes pluvialis from, had H. the pluvialis highest, had titer the of highest astaxanthin titer (Figureof astaxanthin3; Figure (Figure4), up to3; 20 0.8 mg/L. This strain still produced signiﬁcant amounts of astaxanthin precursors, 40 2 mg/L of ± ± β-carotene, 47 3 mg/L of echinenone, and 3 0.5 mg/L of canthaxanthin. ± ± Microorganisms 2019, 7, x FOR PEER REVIEW 6 of 17 Microorganisms 2019, 7, x FOR PEER REVIEW 6 of 17

169 MicroorganismsFigure 4), up2019 to, 207, 472± 0.8 mg/L. This strain still produced significant amounts of astaxanthin precursors,6 of 17 169 Figure 4), up to 20 ± 0.8 mg/L. This strain still produced significant amounts of astaxanthin precursors, 170 40 ± 2 mg/L of β-carotene, 47 ± 3 mg/L of echinenone, and 3 ± 0.5 mg/L of canthaxanthin. 170 40 ± 2 mg/L of β-carotene, 47 ± 3 mg/L of echinenone, and 3 ± 0.5 mg/L of canthaxanthin.

171 171 172 172 Figure 3. Carotenoid production by strains ST7925 and ST7926. Set of yeast transformants expressing 173 FigureFigure 3. 3.Carotenoid Carotenoid productionproduction by strains ST7925 ST7925 and and ST ST7926.7926. Set Set of ofyeast yeast transformants transformants expressing expressing 173 PscrtW in combination with either PacrtZ or HpcrtZ. Positive control: ST7400; Parent strain: ST7906. 174 PscrtWPscrtWin in combination combination withwith eithereither PacrtZ or HpcrtZHpcrtZ. .Positive Positive control: control: ST7400; ST7400; Parent Parent strain: strain: ST7906. ST7906. 174 All strains were cultivated in YP + 8% glucose in 24-deep-well plates for 72 h. Three dots represent 175 AllAll strains strains were were cultivated cultivated inin YPYP ++ 8% glucose in in 24-deep-well 24-deep-well plates plates for for 72 72 h. h.Three Three dots dots represent represent 175 multiple integrations of genes and one dot represents single integration. The error bars represent 176 multiplemultiple integrations integrations ofof genesgenes andand one dot represen representsts single single integration. integration. The The error error bars bars represent represent 176 standard deviations calculated from triplicate experiments (“iso” after each strain indicates the isolate 177 standardstandard deviations deviations calculated calculated from from triplicate triplicate experi experimentsments (“iso” (“iso” after each after strain each indicates strain indicatesthe isolate the 177 number). isolatenumber). number).

178 178 179 FigureFigure 4. 4.Carotenoid Carotenoid production production by by strains strains ST7973 ST7973 and an ST7974.d ST7974. Set ofSet transformants of transformants expressing expressingHpBKT 179 Figure 4. Carotenoid production by strains ST7973 and ST7974. Set of transformants expressing 180 inHpBKT combination in combination with either withPacrtZ eitheror PacrtZHpcrtZ or. Positive HpcrtZ. control:Positive ST7400.control: ParentST7400. strain: Parent ST7972. strain: ST7972. All strains 180 HpBKT in combination with either PacrtZ or HpcrtZ. Positive control: ST7400. Parent strain: ST7972. 181 wereAll strains cultivated were in cultivated YP + 8% in glucose YP + 8% in glucose 24-deep-well in 24-deep-well plates for plates 72 h. for Three 72 h.dots Three represent dots represent multiple 181 All strains were cultivated in YP + 8% glucose in 24-deep-well plates for 72 h. Three dots represent 182 multiple integrations of genes, and one dot represents single integration. The error bars represent 182 integrationsmultiple integrations of genes, and of genes, one dot and represents one dot repres singleents integration. single integration. The error The bars error represent bars represent standard 183 standard deviations calculated from triplicate experiments (“iso” after each strain indicates the isolate 183 deviationsstandard calculated deviations from calculated triplicate from experiments triplicate experi (“iso”ments after (“iso” each after strain each indicates strain indicates the isolate the number). isolate 184 number). 184 number). 185 2.4. Integration of Multiple Copies of β-ketolase and β-hydroxylase Increases Astaxanthin Production 185 2.4. Integration of Multiple Copies of β-ketolase and β-hydroxylase Increases Astaxanthin Production 186 The high concentrations of astaxanthin precursors (β-carotene, echinenone, and canthaxanthin) 186 The high concentrations of astaxanthin precursors (β-carotene, echinenone, and canthaxanthin) 187 indicated imbalances in the pathway. In attempt to resolve these, we transformed a β-carotene 187 indicated imbalances in the pathway. In attempt to resolve these, we transformed a β-carotene

Microorganisms 2019, 7, 472 7 of 17

2.4. Integration of Multiple Copies of β-ketolase and β-hydroxylase Increases Astaxanthin Production The high concentrations of astaxanthin precursors (β-carotene, echinenone, and canthaxanthin) β indicatedMicroorganisms imbalances 2019, 7, x FOR in the PEER pathway. REVIEW In attempt to resolve these, we transformed a -carotene 7 of 17 producing strain ST7434 with diﬀerent ratios of integration constructs targeting rDNA loci. For each 188 transformation,producing strain two ST7434 DNA constructswith differe werent ratios mixed, of integration one carrying constructs a β-ketolase targeting expression rDNA loci. cassette For each and 189 anothertransformation, carrying a twoβ-hydroxylase DNA constructs expression were mixed, cassette. one As carrying a positive a β-ketolase control, expression we used the cassette astaxanthin and 190 produceranother strain carrying (ST7400) a β-hydroxylase from our expression previousstudy cassette. [23 As]. Ninea positive to seventeen control, we individual used the astaxanthin clones were 191 analysedproducer for strain each of(ST7400) the four from gene our combinations previous study (Figure [23].5; FigureNine to6 ).seventeen The highest individual titers of clones astaxanthin were 192 wereanalysed again obtainedfor each of for the the four combination gene combinations of the genes (Figure from 5; FigureH. pluvialis 6). The; high strainest ST7976 titers of isolate astaxanthin 3 gave 193 astaxanthinwere again titer obtained of 44 for1 mg the/L, combination which was of 2.8-fold the genes higher from than H. pluvialis the previously; strain ST7976 reported isolate strain 3 ST7400gave ± 194 withastaxanthin a titer of titer 15 of0.8 44 ± mg 1 mg/L,/L (Figure which6). was Moreover, 2.8-fold higher ST7976 than (iso the 3) previously accumulated reported 163 strain12 ST7400 mg/L of ± ± 195 β-carotene.with a titer The of carotenoid 15 ± 0.8 mg/L production (Figure details6). Moreover, for all theST7976 strains (iso constructed 3) accumulated in this 163 study ± 12 canmg/L be of found β- 196 in Tablecarotene. S1. The carotenoid production details for all the strains constructed in this study can be found 197 in Table S1.

198

199 FigureFigure 5. Carotenoid5. Carotenoid production production by by strains strains ST7927ST7927 andand ST7928ST7928 transformed with with combinations combinations of of 200 PscrtWPscrtWand andPacrtZ PacrtZ/HpcrtZ/HpcrtZ genes. genes. Molar Molarratios ratios ofof DNADNA constructsconstructs are in br brackets.ackets. Strains Strains used used as as 201 controls:controls: ST7906 ST7906 and and ST7400. ST7400. Three Three dots dots represent represent multiplemultiple integrations of of genes. genes. All All strains strains were were 202 cultivatedcultivated in in YP YP+ 8%+ 8% glucose glucose in in 24-deep-well 24-deep-well platesplates forfor 7272 h.h. The The error error bars bars represent represent standard standard 203 deviationsdeviations calculated calculated from from triplicate triplicate experiments experiments (‘iso’ (‘iso’ afterafter each strain indicates indicates the the isolate isolate number). number).

Microorganisms 2019, 7, 472 8 of 17 Microorganisms 2019, 7, x FOR PEER REVIEW 8 of 17

204 Figure 6. Carotenoid production by strains ST7975 and ST7976 transformed with combinations of 205 Figure 6. Carotenoid production by strains ST7975 and ST7976 transformed with combinations of HpBKT and PacrtZ/HpcrtZ genes. Molar ratios of DNA constructs are in brackets. Strain used as positive 206 HpBKT and PacrtZ/HpcrtZ genes. Molar ratios of DNA constructs are in brackets. Strain used as control: ST7400. Three dots represent multiple integrations of genes. All strains were cultivated in 207 positive control: ST7400. Three dots represent multiple integrations of genes. All strains were YP + 8% glucose in 24-deep-well plates for 72 h. The error bars represent standard deviations calculated 208 cultivated in YP + 8% glucose in 24-deep-well plates for 72 h. The error bars represent standard from triplicate experiments (“iso” after each strain indicates the isolate number). 209 deviations calculated from triplicate experiments (“iso” after each strain indicates the isolate number). 2.5. Fed-batch Fermentation of Astaxanthin Producer Strain 210 2.5. Fed-batch Fermentation of Astaxanthin Producer Strain The production of astaxanthin by strain ST7976 (iso 3) was evaluated in 1 L controlled bioreactors. 211 Fed-batchThe fermentationproduction wasof astaxanthin performed onby rich strain complex ST7976 media (iso with 3) 20was g/ Levaluated yeast extract, in 401 gL/ Lcontrolled peptone, 212 andbioreactors. with glucose. Fed-batch Glucose fermentation was added was at a performed low rate in on order rich to complex maintain me itsdia concentration with 20 g/L yeast below extract, 5 g/L 213 (Figure40 g/L7 ).peptone, After 2 days, and 56with g DCW glucose./L was Glucose accumulated was a anddded the at growth a low stopped.rate in order The carotenoids to maintain were its 214 accumulatedconcentration linearly below from 5 g/L 24 (Figure h of fermentation 7). After 2 untildays, the 56 endg DCW/L at 168 h.was At accumulated the end, the astaxanthin and the growth titer 215 reachedstopped. 285 The19 carotenoids mg/L (6 mg were/g DCW) accumulated with the simultaneouslinearly from production24 h of fermentation of 269 44 until mg/ theL of endβ-carotene, at 168 h. ± ± 216 42At the4 mg end,/L of the echinenone, astaxanthin and titer 7 reached0.8 mg/L 285 of canthaxanthin± 19 mg/L (6 (Figuremg/g DCW)8). At thewith end the of simultaneous the process, ± ± 217 47%production of the total of carotenoids269 ± 44 mg/L was of astaxanthin. β-carotene, HPLC 42 ± proﬁle4 mg/L of of the ec carotenoidshinenone, and produced 7 ± 0.8 by mg/L ST7976 of 218 (isocanthaxanthin 3) at the end (Figure of the 8). fementation At the end process of the (168proces h)s, can 47% be of seen the in total Figure carotenoids S1. The highwas astaxanthin.β-carotene 219 titerHPLC shows profile the of potential the carotenoids for further produced strain optimizationby ST7976 (iso so 3) that at the all endβ-carotene of the fementation can be converted process into (168 220 astaxanthin.h) can be seen The fermentationin Figure S1. results The showhigh theβ-carotene potential titer of Y. shows lipolytica thefor potential production for of further astaxanthin. strain 221 optimization so that all β-carotene can be converted into astaxanthin. The fermentation results show 222 the potential of Y. lipolytica for production of astaxanthin.

Microorganisms 2019, 7, x FOR PEER REVIEW 9 of 17

MicroorganismsMicroorganisms 20192019,, 77,, x 472 FOR PEER REVIEW 9 9 of of 17 17

223 223 224 Figure 7. Concentrations of dry cell weight (DCW), glucose, and carotenoids during the fed-batch 225 Figurecultivations 7. Concentrations of ST7976 (iso of3). dry The cell values weight are averag (DCW),es glucose,from three and independen carotenoidst experiments; during the fed-batch the error 224 Figure 7. Concentrations of dry cell weight (DCW), glucose, and carotenoids during the fed-batch 226 cultivationsbars represent of ST7976show standard (iso 3). Thedeviations. values are averages from three independent experiments; the error 225 cultivations of ST7976 (iso 3). The values are averages from three independent experiments; the error bars represent show standard deviations. 226 bars represent show standard deviations.

227 Figure 8. (A) Carotenoid concentrations during fed-batch cultivations (as in Figure7) of ST7976 (iso 3). 227228 TheFigure values 8. A: are Carotenoid averages concentrations from three independent during fed-batch experiments; cultivations the error (as bars in Figure represent 7) of show ST7976 standard (iso 3). 229 deviations.The values are (B) averages Bioreactor from at the thr endee independent of the fed-batch experiments; fermentation the erro of ST7976r bars represent (iso 3). show standard 228 Figure 8. A: Carotenoid concentrations during fed-batch cultivations (as in Figure 7) of ST7976 (iso 3). 230 deviations. B: Bioreactor at the end of the fed-batch fermentation of ST7976 (iso 3). 229 3. DiscussionThe values are averages from three independent experiments; the error bars represent show standard 230 deviations. B: Bioreactor at the end of the fed-batch fermentation of ST7976 (iso 3). 231 3. DiscussionOwing to its outstanding antioxidant properties, health-related functions and application in the aquaculture and poultry sector, astaxanthin has a crescent market demand that is valued to reach 231232 3. DiscussionOwing to its outstanding antioxidant properties, health-related functions and application in the USD 814 million by 2022 [25]. To meet this demand, astaxanthin production has been investigated in 233 aquaculture and poultry sector, astaxanthin has a crescent market demand that is valued to reach 232 differentOwing microbial to its outstanding hosts. The strategiesantioxidant to improveproperties, the health-related titer of astaxanthin functions varies and from application optimization in the of 234 USD 814 million by 2022 [25]. To meet this demand, astaxanthin production has been investigated in 233 aquaculturethe astaxanthin and biosynthetic poultry sector, pathway astaxanthin in native has producers a crescent such market as X. demand dendrorhous thatand is valuedH. pluvialis to reachand 235 different microbial hosts. The strategies to improve the titer of astaxanthin varies from optimization 234 USDusing 814 random million mutagenesis by 2022 [25]. to To insertion meet this of heterologous demand, astaxanthin genes for production astaxanthin has production been investigated in new hosts in 236 of the astaxanthin biosynthetic pathway in native producers such as X. dendrorhous and H. pluvialis 235 differentsuch as E. microbial coli, S. cerevisiae hosts. ,The and strategiesCandida utilis to improve[7,18,26– the29]. titer In this of astaxanthin study, the oleaginous varies from yeast optimizationY. lipolytica 237 and using random mutagenesis to insertion of heterologous genes for astaxanthin production in new 236 ofwas the engineered astaxanthin for biosynthetic the production pathway of astaxanthin. in native First,producers we evaluated such as theX. dendrorhous effect of two and diff erentH. pluvialis GGPP 238 hosts such as E. coli, S. cerevisiae, and Candida utilis [7,18,26–29]. In this study, the oleaginous yeast Y. 237 and using random mutagenesis toβ insertion of heterologous genes for astaxanthin production in new 239 synthases for the biosynthesis of -carotene. The GGPP syntheses can come from a variety of sources, 238 hostslipolytica such was as E. engineered coli, S. cerevisiae for the, and production Candida utilis of astaxant [7,18,26–29].hin. First, In this we study, evaluated the oleaginous the effect yeast of two Y.

239 lipolytica was engineered for the production of astaxanthin. First, we evaluated the effect of two

Microorganisms 2019, 7, 472 10 of 17 such as bacteria, fungi, or mammals. In this study, we compared the activity of the GGPP synthases CrtE from X. dendrorhous and GGPPs7 from Synechococcus sp. The results obtained in this study show that the expression of GGPPs7 increased, more efficiently, the carbon flux toward the formation of precursors to supply the astaxanthin production. The activity of GGPPs7 has been reported to be high enough to confer toxicity to the cell due to a dramatic increase in GGPP production, which could result in a drain on a downstream pathway such as ergosterol production [24]. Additionally, the expression of GGPPs7 for production of terpenoids has already been described in a patent by Evolva SA [30]. Several studies have successfully reported an increase in the production of isoprenoids by expressing native or heterologous crtE [18,23,31–33], while, to our knowledge, only a few patents have reported the expression of GGPPs7 for production of isoprenoids [24,30]. The results presented in this study show the potential of expressing GGPPs7 in Y. lipolytica to obtain higher titers of β-carotene. Next, we analysed the efficiency of two different β-ketolases. The β-ketolase PsCtrW from Paracoccus sp. produced higher amounts of echinenone, an astaxanthin intermediate, compared to HpBKT from H. pluvialis. These data suggest the preference of this bifunctional enzyme (PsCrtW) for β-carotene as the substrate over echinenone. Then we integrated two different β-hydroxylases from either bacterial or microalgae organisms, into both platforms expressing PsCrtW and HpBKT. The gene expression was optimized by varying the copy number of the integrated genes. The best production of astaxanthin was obtained by the strain expressing the microalgae genes HpBKT and HpcrtZ in a molar ratio of 1:1. Although the transformation was performed in a way to balance the molar ratio between the two genes, the copy number of those need to be further investigated to evaluate if there is any disparity between the copy number of the enzymes. It is worth noting that the β-carotene hydroxylase adds a hydroxyl group to the β-carotene molecule, while the β-carotene ketolase adds a keto group. These two enzymes accept several substrates; thus, the β-carotene hydroxylase is capable of converting β-carotene to zeaxanthin and also canthaxanthin to astaxanthin. On the other hand, the β-ketolase can convert ß-carotene to canthaxanthin and also zeaxanthin to astaxanthin. Therefore, the high asxantathin content obtained in this study is a result of the enzymatic activity of HpcrtZ and HpBKT from H. pluvialis. In this study was used as positive control the astaxanthin producer strain of Y. lipolytica (ST7403) described in the work of Kildegaard et al. [23]. This strain of Y. lipolytica was engineered to produce astaxanthin and showed a production of 54.6 mg/L of astaxanthin. When compared to the positive control, the best astaxanthin producer obtained in this study presented a 145% higher titer in 24-well plates. This shows that the engineering modifications performed to improve the MVA pathway in order to increase the precursor supply plus overexpression of β-ketolase and β-hydrolase with higher activity has successfully increased astaxanthin production. Kildegaard et al. (2017) reported the production of astaxanthin using the bacterial genes PscrtW and PacrtZ, the results showed that the β-hydrolase PaCrtZ was the rate-limiting enzyme. Likewise, the present work has shown that when the bacterial genes are expressed together the best production of astaxanthin is achieved with a molar ratio of 1:3 (PscrtW: PacrtZ), confirming the β-hydrolase to be a rate-limiting step when expressing bacterial genes. Finally, the best astaxanthin producer was cultivated in 1 L bioreactors and achieved a production of 285 19 mg/L (6 mg/g DCW) of astaxanthin after 168 h of fermentation. The strain also produced ± 269 44 mg/L of β-carotene, 42 4 mg/L of echinenone, and 7 0.8 mg/L of canthaxanthin. Previous ± ± ± studies have used the genes BKT and HpCrtZ from H. pluvialis to produce astaxanthin in S. cerevisiae, their results showed that when these enzymes were expressed, the astaxanthin stereoisomer obtained was the optically pure 3S, 30S as the one produced in the microalgae H. pluvialis [18,19]. The chiral analysis for the astaxanthin produced in this present study was not performed, however, as the enzymes, BKT and HpCrtZ from H. pluvialis commonly produce the 3S, 30S configuration, we believe that the same isomer is synthezed in Y. lipolytica. Nonetheless, future analysis to identify the astaxanthin stereoisomer synthetized by ST7976 (iso3) by NMR analysis, for example, is necessary. The high concentration of β-carotene in the strain ST7976 (iso 3) shows that further improvements in the pathway can lead to even higher titers of astaxanthin so that all β-carotene can be converted into astaxanthin. Microorganisms 2019, 7, 472 11 of 17

As demonstrated by Zhou et al. (2017) protein optimization of BKT from H. pluvialis led to higher activity of the enzyme and consequently higher production of astaxanthin [18]. Similarly, strategies to optimize rate-limiting enzymes in the pathways leading to biosynthesis of astaxanthin might be efficient to improve the production of astaxanthin. The fermentation process was carried in glucose limitation regime, since studies show that a high C:N ratio promotes lipid synthesis as well as the synthesis of carbon-based compounds, such as carotenoids [34]. Studies done by Larroude et al. 2017 showed that when glucose was kept at low concentration during the fermentation process, β-carotene production steadily increased [35]. In another study performed by Gao et al. 2017, β-carotene production also increased when glucose was at a low concentration in the medium [36]. Therefore, we selected a limited glucose regime for the fermentation process. Our results show that after nitrogen was exhausted from the medium, the biomass growth stopped, and a continuous increase in carotenoid production was observed. As described by Papanikolaou and Aggelis (2011), lipid production (secondary metabolite) in oleaginous yeasts is only triggered when a growth-required nutrient, in many cases nitrogen, is limited, and the carbon source is still available in the medium [37]. Similarly, our results suggest that a high C:N ratio positively affects astaxanthin production, which is also a secondary metabolite. Additionally, other strategies to improve the fermentation process and increase the astaxanthin biosynthesis could be applied, such as medium supplementation with Fe2+. In the work done by Zhou et al. (2017), the results showed that when Fe2+ was added in the media, the astaxanthin titer increased from 6.95 mg/g DCW to 8.10 mg/g DCW [18]. In another work, an increase in astaxanthin yield of 1.9-fold was reported when iron was supplemented into the media [19]. The positive effect of iron in the activity of the β-ketolase is associated to the histidine motifs present in the protein structure, which are reported to be involved in iron binding. The work done by Ye et al. (2016) reported that mutations in the conserved histidine motifs led to the inefficiency of the enzyme to catalyze the formation of ketocarotenoids [38]. Due to the time limitation, we had to stop the fermentation process at 168 h. Futher improvement of the fermentation process is required to achieve even better titer, rates, and yields. In addition, as the fermentation results showed a constant increase in astaxanthin production throughout the whole process, a longer fermentation might reveal even higher titers. To our knowledge only Kildegaard et al. (2017) reported the production of astaxanthin by engineered Y. lipolytica [23]. The obtained titer was 54.6 mg/L in microtiter plate cultivation, and fermentation was not performed. The results obtained in this work highlight the potential of Y. lipolytica for commercial production of astaxanthin.

4. Materials and Methods

4.1. Strains and Culture Conditions E. coli DH5-α was used for the cloning procedures. The transformed E. coli cells were grown at 37 ◦C and 300 rpm in lysogeny broth (LB) liquid medium and at 37 ◦C on LB solid medium plates supplemented with 20 g/L agar. Ampicillin was supplemented when necessary at a concentration of 100 mg/L. The Y. lipolytica strain ST6899, engineered in previous work of Kildegaard et al. (2017) [23], was used as the parent strain. All strains used in this study are listed in Table S2. Y. lipolytica was grown at 30 ◦C on yeast extract peptone dextrose (YPD), or synthetic complete minus Uracil (SC-Ura) media supplemented with 20 g/L agar. Supplementation with antibiotics was done when required at the following concentrations: hygromycin B at 50 mg/L and nourseothricin at 250 mg/L. The recombinant strains for carotenoids production were cultivated in yeast extract peptone medium containing 80 g/L glucose (YP + 8% glucose). The chemicals were purchased from Sigma-Aldrich, withexception of nourseothricin, which was purchased from Jena Bioscience GmbH (Jena, Germany).

4.2. Plasmid Construction The geranylgeranyl pyrophosphate synthase encoded by GGPPs7 from Synechococcus sp., the β-carotene ketolases encoded by crtW and BKT from Paracoccus sp. and H. pluvialis, respectively, and the β-carotene hydroxylases encoded by crtZ from Pantoea ananatis and H. pluvialis were Microorganisms 2019, 7, 472 12 of 17

Microorganisms 2019, 7, x FOR PEER REVIEW 12 of 17 codon-optimized for Y. lipolytica and synthesized as GeneArt String DNA fragments by Thermo 341 Fisheroptimized Scientific for Y. (Waltham, lipolytica MA,and synthesized USA). The geranylgeranylas GeneArt String diphosphate DNA fragments synthase by CrtEThermo encoded Fisher by 342 crtEScientificfrom X. (Waltham, dendrorhous Massachusetts,was obtained USA). from AddgeneThe geranylgeranyl [31]. The plasmids,diphosphate primers, synthase and CrtE BioBricks encoded used 343 inby this crtE study from can X. dendrorhous be found in was the obtained Tables S3–S5, from Addgene respectively. [31]. TheThe plasmids, BioBricks primers, were amplified and BioBricks by PCR 344 usingused Phusion in this study U polymerase can be found (Thermo in the Fisher Tables Scientific, S3–S5, respectively. Waltham, MA,The BioBricks USA) following were amplified the described by 345 PCR using Phusion U polymerase (Thermo Fisher Scientific, Waltham, Massachusetts, USA) conditions: 98 ◦C for 30 s; 6 cycles of 98 ◦C for 10 s, 51 ◦C for 20 s, and 72 ◦C for 30 s/kb; and 26 cycles 346 following the described conditions: 98 °C for 30 s; 6 cycles of 98 °C for 10 s, 51 °C for 20 s, and 72 °C of 98 ◦C for 10 s, 58 ◦C for 20 s, 72 ◦C for 30 s/kb, and 72 ◦C for 5 min. The BioBricks were purified 347 for 30 s/kb; and 26 cycles of 98 °C for 10 s, 58 °C for 20 s, 72 °C for 30 s/kb, and 72 °C for 5 min. The from 1% agarose gel using the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel, Bethlehem, 348 BioBricks were purified from 1% agarose gel using the NucleoSpin® Gel and PCR Clean-up kit PA, USA). After purification, the BioBricks were assembled into EasyCloneYALI vectors using USER 349 (Macherey-Nagel, Bethlehem, PA, USA). After purification, the BioBricks were assembled into cloning as described in the protocol by Holkenbrink et al. [39]. The USER reactions containing the 350 EasyCloneYALI vectors using USER cloning as described in the protocol by Holkenbrink et al. [39]. 351 desiredThe USER plasmids reactions were containing transformed the intodesired chemically plasmids competent were transformedE. coli DH5. into chemically The correct competent assembly E. was 352 confirmedcoli DH5. by The DNA correct sequencing. assembly Figure was confirmed9 summarizes by DNA the engineered sequencing. pathway Figure for9 summarizes improvement the of 353 precursorengineeredenzymes production for pathway the production and for improvement the of expression astaxanthin. of of precursor heterologous production enzymes and for the the expression production of heterologous of astaxanthin. 354

355 356 FigureFigure 9. 9.Engineered Engineered pathways. pathways. ( AA). ImprovementImprovement of of precursor precursor supply. supply. B (.B Astaxanthin) Astaxanthin biosynthesis biosynthesis 357 pathway.pathway. The The whitewhite boxes indicate indicate enzymes enzymes already already expressed expressed in the in parental the parental strain strainST6899 ST6899 [23]. The [23 ]. 358 Thegreen green boxes boxes indicate indicate enzymes enzymes additionally additionally expresse expressedd in the inparental the parental strain in strain this instudy. this IPP: study. 359 IPP:Isopentenyl Isopentenyl pyrophosphate; pyrophosphate; DMAPP: DMAPP: Dimethylallyl Dimethylallyl pyrophosphate; pyrophosphate; FPP: FPP: Farnesyl Farnesyl pyrophosphate; pyrophosphate; 360 GGPP:GGPP: geranylgeranyl geranylgeranyl pyrophosphate; pyrophosphate; ERG20:ERG20: farnesyl farnesyl pyrophosph pyrophosphateate synthase; synthase; CrtE CrtE and and GGPPs7: GGPPs7: 361 geranylgeranylgeranylgeranyl pyrophosphate pyrophosphate synthase; synthase; CrtYB: CrtYB: phytoene phytoene synthase synthase and lycopeneand lycopene cyclase; cyclase; CrtI: phytoene CrtI: 362 desaturase;phytoene CrtW:desaturase;β-ketolase CrtW: from β-ketolase bacteria; from BKT: bacteria;β-ketolase BKT: from β-ketolase microalgae; from CrtZ:microalgae;β-hydroxylase. CrtZ: β- 363 hydroxylase. 4.3. Construction and Cultivation of Y. lipolytica 364 4.3.Di Constructionfferent previously and Cultivation characterized of Y. lipolytica intergenic loci in Y. lipolytica were used to integrate yeast 365 vectorsDifferent into the previously genome of characterized the parent strain, intergenic as described loci in Y. inlipolytica Holkenbrink were used etal. to [integrate39]. To performyeast 366 DNAvectors transformation into the genome into ofY. the lipolytica parent ,strain, the integrative as described vectors in Holkenbrink were linearized et al. [39]. with To perform FastDigest DNA NotI 367 (Thermotransformation Fisher Scientific, into Y. lipolytica Waltham,, the MA, integrative USA) and vectors transformed were linearized into Y. with lipolytica FastDigestusinga NotI lithium-acetate (Thermo 368 protocolFisher [Scientific,40]. The transformants Waltham, Massachusetts, were selected USA) on YPD and +transformedHygromycin into/Nourseothricin Y. lipolytica using or SC-Ura a lithium- plates. 369 Theacetate yeast protocol transformants [40]. The carrying transformants the correct were integration selected on inYPD the + genome Hygromycin/Nourseothricin were verified by colony or SC- PCR 370 usingUra primersplates. The listed yeast in transformants the Table S4. Thecarrying best theβ-carotenoid correct integration precursor in producerthe genome was were used verified for further by 371 implementationcolony PCR using of theprimers astaxanthin listed in biosyntheticthe Table S4. pathway.The best β-carotenoid For astaxanthin precursor production, producer the was plasmids used 372 werefor further constructed implementation for single of and the multipleastaxanthin integrations biosynthetic in pathway. the Y. lipolyticaFor astaxanthingenome. production, For multiple the 373 integrations,plasmids were the constructed vectors carried for single two homologous and multiple regions integrations targeting in the the Y. ribosomallipolytica genome. DNA (rDNA) For 374 multiple integrations, the vectors carried two homologous regions targeting the ribosomal DNA elements in Y. lipolytica. The transformation for multiple integrations was performed using vectors 375 (rDNA) elements in Y. lipolytica. The transformation for multiple integrations was performed using Microorganisms 2019, 7, 472 13 of 17

Microorganisms 2019, 7, x FOR PEER REVIEW 13 of 17 in three diﬀerent molar ratios, 1:1, 1:2, and 1:3 (PsctrW:PacrtZ, PscrtW:HpcrtZ, HpBKT:PacrtZ, and 376 HpBKT:HpcrtZvectors in three). The different strain molar construction ratios, 1:1, strategy 1:2, and is 1:3 summarized (PsctrW:PacrtZ, in Figure PscrtW:HpcrtZ, 10. After HpBKT:PacrtZ, the screening of 377 transformants,and HpBKT:HpcrtZ 4 to 7). clones The strain of astaxanthin construction producing strategy is strainssummarized were in selected Figure for10. After each transformationthe screening 378 molarof transformants, ratio. The selection 4 to 7 clones of colonies of astaxanthin was based prod inucing color strains screening. were Forselected preculture for each preparation, transformation single 379 molar ratio. The selection of colonies was based in color screening. For preculture preparation, single colonies were inoculated from fresh plates in 3 mL YPD in 24-well plates with an air-penetrable lid 380 colonies were inoculated from fresh plates in 3 mL YPD in 24-well plates with an air-penetrable lid (EnzyScreen, Heemstede, The Netherlands). The strains were grown at 30 C for 18 h with agitation 381 (EnzyScreen, Heemstede, The Netherlands). The strains were grown at 30 °C◦ for 18 h with agitation of 300 rpm at 5 cm orbit cast. The required volume of inoculum was transferred to 3 mL YP + 8% 382 of 300 rpm at 5 cm orbit cast. The required volume of inoculum was transferred to 3 mL YP + 8% glucose into 24-well plates for an initial OD600 of 0.1. The cultivation plates were incubated for 72 h 383 glucose into 24-well plates for an initial OD600 of 0.1. The cultivation plates were incubated for 72 h at 30 C with 300 rpm agitation. After cultivation, 0.5 mL of the cultivation volume was transferred 384 at 30◦ °C with 300 rpm agitation. After cultivation, 0.5 mL of the cultivation volume was transferred β 385 intointo a prelabelleda prelabelled 2 2 mL mL microtube microtube (Sarstedt, (Sarstedt, Numbrecht,Numbrecht, Germany) Germany) for for β-carotenoid-carotenoid extraction extraction and and 386 subsequentlysubsequently quantiﬁcation quantification of of carotenoids carotenoids waswas donedone by HPLC.

387 Figure 10. Flowchart of the strains generated in this study. 388 Figure 10. Flowchart of the strains generated in this study. 4.4. Carotenoid Extraction 389 4.4. Carotenoid Extraction The optical density at 600 nm (OD600) was measured after cultivation, using NanoPhotometer 390 The optical density at 600 nm (OD600) was measured after cultivation, using NanoPhotometer (Implen GmbH, Munchen, Germany). For biomass dry weight measurements, 1 mL of the cultivation 391 (Implen GmbH, Munchen, Germany). For biomass dry weight measurements, 1 mL of the cultivation broth was transferred into a preweighed 2 mL microtube (Sarstedt, Numbrecht, Germany). The tubes 392 broth was transferred into a preweighed 2 mL microtube (Sarstedt, Numbrecht, Germany). The tubes were centrifuged at 10,000 g for 5 min. The supernatant was removed and the samples were washed 393 were centrifuged at 10,000×× g for 5 min. The supernatant was removed and the samples were washed 394 withwith 1 mL1 mL of of sterile sterile water. water. Subsequent Subsequent toto thethe centrifugationcentrifugation and and removal removal of of the the supernatant, supernatant, the the tubes tubes 395 containingcontaining the the biomass biomass pellets pellets werewere placedplaced in the incubator at at 60 60 °C◦C for for 96 96 h. h. After After 96 96 h the h the tubes tubes 396 werewere weighed weighed on on an an analytical analytical scale. scale. ForFor carotenoidscarotenoids extraction, 0.5 0.5 mL mL of of the the cultivation cultivation volume volume was was 397 transferredtransferred into into a 2a 2 mL mL microtube microtube (Sarstedt, (Sarstedt, Numbrecht,Numbrecht, Germany). Germany). Each Each sample sample was was centrifuged centrifuged at at 398 10,00010,000×g forg for 5 min5 min and and the the supernatant supernatant was was removed. removed. Then, Then, 0.5 0.5 mL mL of 0.5–0.75of 0.5–0.75 mm mm acid-washed acid-washed glass × 399 beadsglass were beads added were toadded each to tube each followed tube followed by the by addition the addition of 0.5 of mL 0.5 ofmL ethyl of ethyl acetate acetate supplemented supplemented with 400 0.01%with 3,5-di-tert-4- 0.01% 3,5-di-tert-4- butylhydroxytoluene butylhydroxytoluene (BHT). (B TheHT). BHT The was BHT added was toadded prevent to prevent carotenoid carotenoid oxidation. 401 Theoxidation. cells were The disrupted cells were using disrupted a Precellys using Ra 24Precellys homogenizer R 24 homogenizer (Bertin Corp., (Bertin Montigny-le-Bretonneux, Corp., Montigny-le- 402 France)Bretonneux, in four France) cycles ofin 5500four cycles rpm for of 205500 s. rpm The tubesfor 20 weres. The placed tubes were on ice placed for 1 minon ice in for between 1 min eachin 403 lysisbetween cycle. each After lysis disruption, cycle. After the cellsdisruption, were centrifuged the cells were for centrifuged 5 min at 10,000 for 5 gmin. For at quantiﬁcation10,000× g. For of × 404 β-carotenequantification and individualof β-carotene carotenoids and individual by HPLC, carotenoids 100 µ byL ofHPLC, the solvent100 µL of fraction the solvent was fraction transferred was to 405 HPLCtransferred vials. to HPLC vials.

406 4.5. Carotenoid Quantification by HPLC

Microorganisms 2019, 7, 472 14 of 17

4.5. Carotenoid Quantification by HPLC For HPLC measurements, 100 µL of ethyl acetate extract was evaporated in a rotatory evaporator, and the dry extracts were redissolved in 1 mL 99% ethanol + 0.01% BHT. Then, the extracts were analyzed by HPLC (Thermo Fisher Scientific, Waltham, MA, USA ) equipped with a Discovery HS F5 150 mm 2.1 mm column (particle size 3 mm). For this analysis, the column oven temperature × was set to 30 ◦C. All organic solvents used were HPLC grade (Sigma Aldrich, St. Louis, MO, USA). The flow rate was set to 0.7 mL/min with an initial solvent composition of 10 mM ammonium formate (pH = 3, adjusted with formic acid) (solvent A) and acetonitrile (solvent B) (3:1) until minute 2.0. Solvent composition was then changed at minute 4.0 following a linear gradient until % A = 10.0 and % B = 90.0. The solvent composition was kept until 10.5 min when the solvent was returned to initial conditions and the column was re-equilibrated until 13.5 min. The injection volume was 10 µL. The peaks obtained from the sample analysis were identified by comparison to prepared standards and integration of the peak areas was used to quantify carotenoids from obtained standard curves. The β-carotene and echinenone compounds were detected at retention times of 7.6 min and 6.9 min, respectively, by measuring absorbance at 450 nm, while astaxanthin and canthaxanthin were detected by absorbance at 475 nm and retention times of 5.9 min and 6.4 min, respectively. The results were verified by comparing the samples with the standards. Standards were purchased from Sigma-Aldrich: β-carotene (C4582-5 mg), echinenone (73341-1MG), canthaxanthin (11775-1MG), and astaxanthin (SML0982-50MG).

4.6. Fermentation Procedures For inoculum, the strain glycerol stock was inoculated in 25 mL of media containing 20 g/L of yeast extract, 40 g/L of peptone and 5 g/L of glucose and propagated at 30 ◦C with 250 rpm agitation for 24 h. The OD600 was measured and the volume required to start the fermentation with an initial OD600 of 1.5 was transferred to a 20 mL syringe and used as inoculum. The fermentation was performed as fed-batch cultivation in a 1 L bioreactor (Sartorius Stedim Biotech, Gottingen, Germany). The initial cultivation volume was 0.4 L, the medium contained 20 g/L of yeast extract, 40 g/L of peptone, and 0.5 mL/L Antifoam 204 (Sigma, St. Louis, MO, USA). The 50% glucose stock solution, sterilized by filtration, was used as carbon source. To begin the fermentation, glucose was added to the concentration of 5 g/L. The temperature was kept constant at 28 ◦C, aeration was set to 2 VVM, the agitation was set to 500–1000 rpm, pH was automatically maintained at 5.5 by addition of 5 M KOH and 5 M HCl. The dissolved oxygen was set to a minimum of 20%. The foaming was prevented by automatic addition of Antifoam 204 (Sigma, St. Louis, MO, USA). The feeding of 50% glucose solution was initiated 6 h after inoculation. The glucose concentration was maintained below 5 g/L during the whole fermentation process and the glucose flow rate was adjusted manually according to the cell growth (OD600). The bioreactors were sampled three times a day to measure biomass dry weight, glucose, and carotenoids. For glucose quantification, the sample was immediately centrifuged, and the supernatant was stored at 20 C until HPLC analysis. − ◦ 4.7. Biomass and Glucose Quantification in Bioreactors The OD600 values were detected with UV-1800 Shimadzu UV spectrophotometer. For the dry cell weight (DCW), 3 mL of the fermentation broth was filtered through preweighed cellulose nitrate membranes (0.45 µm pore size, 47 mm circle) using a filtration unit with a vacuum pump. The filters were dried at 60 ◦C for 96 h and weighed on an analytical scale. For glucose quantification, 1 mL of cultivation broth was transferred into a 2 mL microtube (Sarstedt, Numbrecht, Germany). The tubes were centrifuged at 10,000 g for 5 min. The supernatant was removed, filtered, and used × for quantification on HPLC. The analysis on HPLC analyzed 20 µL of the sample for 30 min using an Aminex HPX-87H ion exclusion column with a 5 mM H2SO4 flow of 0.6 mL/min. The column Microorganisms 2019, 7, 472 15 of 17

temperature was set to 30 ◦C, the reﬂective index was set at 45 ◦C, and the glucose was detected using a RI-101 Refractive Index Detector (Dionex, Sunnyvale, CA, USA).

5. Conclusions In this work, β-carotene production in Y. lipolytica was optimized through expression of GGPP synthase from Synechococcus sp. and then turned into astaxanthin producer by integration of heterologous β-ketolase and β-hydroxylase genes. The optimal gene combination was when both genes were from microalgae H. pluvialis. Nearly 0.3 g/L of astaxanthin was produced by the optimized strain in fed-batch cultivation with cellular content of 6 mg/g DCW. These results reinforce the potential of Y. lipolytica for production of carotenoids, in particular, astaxanthin.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/2076-2607/7/10/472/s1. Figure S1. HPLC profile of carotenoids produced after 168 h of fed-batch fermentation of ST7976 (iso 3). Table S1. Production of carotenoids by the strains generated in this study. Supplementary sequences: Sequences of synthetic genes used in the study. Titers were quantified by HPLC analysis. Values are the average of the triplicates. The molar ratio (β-ketolase: β-hydroxylase) is indicated between brackets. The isolate number is indicated after the abbreviation ‘iso’Table S2. List of Yarrowia lipolytica engineered strains. Table S3. List of plasmids used in this study. Table S4. List of primers used in this study. Table S5. List of BioBricks used in this study. Author Contributions: L.R.R.T., K.R.K., and I.B. conceived the study and designed the experiments. S.S. helped to design and perform fermentation experiments. I.B. secured the funding for the research. L.R.R.T. performed the experiments and analyzed the data. L.R.R.T. and I.B. wrote the manuscript. All the authors have checked and approved the manuscript. Funding: Research was funded by the Novo Nordisk Foundation (Grant agreement NNF15OC0016592 and NNF10CC1016517). IB and KRK acknowledge the financial support from the European Union’s Horizon 2020 research and innovation programmes (European Research Council, YEAST-TRANS project No 757384 and OLEFINE project No 760798). Acknowledgments: The authors would like to thank Volker Zickermann from Goethe University Medical School, Institute of Biochemistry II, Germany, who kindly provided the Y. lipolytica strain GB20 which was used as platform for the parent strain ST3683 used in this study. They also thank the researches from the Analytical Core Facility at DTU Biosustain for the support given during HPLC analysis, and to Jacqueline Medina for technical assistance during the fermentation process. Conflicts of Interest: I.B. and K.R.K. have financial interest in BioPhero ApS.

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