Constructing yeast chimeric pathways to boost lipophilic terpene

Subscriber access provided by Macquarie University

Article

Constructing yeast chimeric pathways to boost lipophilic terpene synthesis Duo Liu, Hong Liu, Hao Qi, Xue-Jiao Guo, Bin Jia, Jin-lai Zhang, and Ying-Jin Yuan ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00360 • Publication Date (Web): 19 Feb 2019 Downloaded from http://pubs.acs.org on February 20, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

Constructing yeast chimeric pathways to boost lipophilic terpene synthesis Duo Liu1,2, Hong Liu1,2, Hao Qi1,2, Xue-Jiao Guo1,2, Bin Jia1,2, Jin-Lai Zhang1,2, YingJin Yuan1,2*

1: Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, P. R. China 2: SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, 300072, P. R. China *: Corresponding author: Ying-Jin Yuan, E-mail: [email protected]

Abstract Synthetic chimeric biological system offers opportunities to illuminate principles of designing life and a primary step is constructing synthetic chimeric pathways. Here, we constructed yeast chimeric pathways by transferring the genes from Saccharomyces cerevisiae pathways into another budding yeast Yarrowia lipolytica for in vivo assembly. We efficiently diversified gene option, combination, localization order and copy number as expected. Convergence of two yeast pathways, especially mevalonic acid (MVA) pathways, remarkably enhanced synthesis of a lipophilic terpene, lycopene. In the selected champion strain with 50-fold of enhanced lycopene production, the chimeric MVA pathway gathered three S. cerevisiae genes with particular copies and locations. Amazingly, therein we discovered distinct transcriptional up-regulation of three significant pathways correlated with acetyl-CoA supply and dramatical tuning of cellular lipid amounts and composition. Modulating these pathways further improved lycopene production to 150-fold, a final 259 mg/L (approximately 80 mg/g DCW). We primarily showed the design of boosting the synthesis of lipophilic products with yeast chimeric pathways. Key words: synthetic biology, DNA assembly, transcriptional unit, terpene, 1

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Saccharomyces cerevisiae, Yarrowia lipolytica In nature, even organisms belonging to the same genus often evolve into distinguished species, resulting in significant differences in genome composition, gene coding, proteome function, cell metabolism and reproductive isolation. As a typical example, although both are popularly used budding yeasts, Saccharomyces cerevisiae evolve to be adept at ethanol production, while Yarrowia lipolytica evolve to gain a significant accumulation of lipid droplets1. The two yeasts with lowest homology in Saccharomyces genus own quite differentiate genome sizes (12.1 Mb vs 20.5 Mb), chromosome numbers (16 vs 6), GC contents (38.3% vs 49.0%) and lowest mean amino-acid homology and conservation of gene adjacency, but common conserved genes and pathways2-5. Although both yeasts have been respectively engineered to synthesize value-added biochemicals with high productivity6-13, we are confused whether we can design high-efficient yeast chimeric pathways by recruiting both yeast genes. A promising way is directly constructing a synthetic chimeric biological system containing respective genetic materials, but still faces several essential difficulties. A primary work is to test whether a gene transferred from one specie to another does work to present particular function, especially given the very low gene homology or widely diverged natural sources. A recent remarkable work researched on systematic humanization of yeast genes and proved that hundreds of yeast essential genes could be replaced by their human orthologs in modular manner14. This build-and-test work expanded the feasibility of recruiting alternatives based on the comparative analysis of gene-expression patterns, chemogenomic profiling and genetic interaction maps15-17. The species with extremely close genetic backgrounds can be hybridized to get a combined genome, offering a platform to study on genetic evolution. Quite recently, Shen and Wu developed a heterozygous and interspecies SCRaMbLE system based on mating of two genetically close yeasts, S. cerevisias and S. paradoxus18. The rearrangement of inclusive synthetic chromosomes (synV and/or synX) led to discovery of new gene potentially attributable to caffeine tolerance. The first synthetic bacteria Syn 1.0 constructed by JCVI is also a proof of feasibility of interchanging close genomes19. The de novo synthesized genome of Mycoplasma mycoides was transferred 2


Page 2 of 29

Page 3 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


into the cell of Mycoplasma capri and gradually determined the new cell’s phenotype along with keeping the cell alive. The few examples illustrated a still common challenge to overcome genetic segregation. Here we focus on the pathway-oriented gene level between gene level and whole genome level by constructing synthetic chimeric pathways. Natural evolved pathway is always not optimal, offering chance to be modified with better-performing reactions. We decide to transfer genes from the illustrated pathways of S. cerevisiae into Y. lipolytica to test their potential benefits on boosting lipophilic product synthesis (Figure 1). Here the transcription of S. cerevisiae genes is still to be controlled by Y. lipolytica’s promoters because the transcription segregation restricts direct physical pathway transplantation between two yeasts. A typical lipophilic terpene, lycopene is chosen as target product. Our aim is to investigate whether the intrinsic terpene and lipid metabolism can be tuned significantly by genes of heterologous yeast pathways genes. The experience of previous works is also referenced. The strategy of overexpression of the genes in intrinsic upstream pathway worked well both in S. cerevisiae and Yarrowia lipolytica, gaining outstanding production of terpenes such as artemisinic acid and carotenoids20-24. Combining host pathway engineering and geneknockout, Chen et al. improved lycopene production to 1.65 g/L (55.56 mg/g DCW) from fermentation of S. cerevisiae21; Matthäus et al. got 16 mg/g DCW lycopene and Schwartz et al. obtained 21.1 mg/g DCW lycopene both in Y. lipolytica22, 23; Gao et al. improved fermentative production of beta-carotene in Y. lipolytica to 4 g/L (49 mg/g DCW)24. Larroude et al. reported that the strain accumulating higher level of lipids (as 42.6 g/L) also produced higher level of carotenoids as fermentative 6.5 g/L (90 mg/g DCW)25. This work suggested the existing but not fully elucidated metabolic correlation between the biosynthesis of two categories of lipophilic products. In order to realize large-scale gene-transfer from S. cerevisiae to Y. lipolytica, we designed a “Y. lipolytica transcriptional-unit (Yl-TU) assembly” toolkit for multi-gene assembly (Figure 2a, Supporting Figure S1, S2). We chose 10 pathways in S. cerevisiae and the repertoire of total 115 genes for construction of corresponding YlTUs and their assembly. The in vivo assembled Yl-TUs were integrated into particular 3



chromosomal sites, resulting in effective selection of diversified gene options, combinations, transcription strengths, localization orders and copy numbers. Through this approach, we constructed different versions of these chimeric pathways and investigated their effects on lycopene synthesis. The introduction of different heterologous reactions might contribute to distinguished degrees of boosting lycopene synthesis and the underlying metabolic correlations were explored.

Results 1.

Testing feasibility of Yl-TU assembly for modulating heterologous product

synthesis To testify the feasibility of Yl-TU assembly, we assembled heterologous pathways in Y. lipolytica for synthesis of prodeoxyviolacein (PDV), lycopene and beta-carotene. Assembly of three-gene pathways of vioABE and crtEBI and integration at chromosomal rDNA sites showed higher than 30% correct rate and four-gene pathway crtEBIY got 25.8%. In contrast, the correct rates for chromosomal GUT2 site were 7.3%~11.7% (Supporting Note 1). Although the DNA assembly and integration efficiency at GUT2 were lower, this single-copy site offered us a chance to exactly investigate effects of different gene combinations, and the crtEBI pathway was integrated in rDNA site as 1-2 copies (Supporting Figure S1). With the primarily assembled lycopene pathways both at the rDNA sites and the GUT2 site, we only got limited amount of lycopene as respective 1.71 mg/L and 0.42 mg/L (Supporting Note 1, Supporting Figure S1). Higher flux towards terpene pathway was still required in Y. lipolytica. Based on the superior starting performer, we moved forward to construct chimeric pathways by incorporating Yl-TUs of heterologous S. cerevisiae genes at Y. lipolytica’s GUT2 site (Figure 2, 3). 100 genes in 10 pathways from “B” to “I” were directly cloned from S. cerevisiae genome and recruited, major of which owned counterparts in Y. lipolytica (Figure 2b, Supporting Table S1). An extra pathway “X” containing different resources of crt genes was also included for later consideration, together with pathway “A” (mevalonate (MVA) 4


Page 4 of 29

Page 5 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pathway). Genes of each pathway were ligated into Yl-TUs Yl-LD04 to Yl-LD08 and then co-transformed into yeast cells along with Yl-LDL02 and one appropriate YlLDR-G (Yl-LDR-G04 to Yl-LDR-G08) (Figure 2a, Table 1). For every assembly of one to five Yl-TUs of each pathway, one deeply-colored yeast colony was picked, and its lycopene production was measured (Figure 2b). Additionally, the genotypes of the assembled Yl-TUs were sequenced (Supporting Table S2, S3). The incorporation of different combinations of genes from a common heterologous pathway, together with native genes, generated different versions of chimeric pathway. The assembly process and possible assembled gene combination and their verification method were illustrated in Figure S3 for pathway “B” as an example. The varied impacts of versions of indirect pathways “B” to “I” on lycopene production were analyzed (Figure 2c, Supporting Table S3). The highest value of lycopene amount was 15.8 mg/L in strain D2, and the lowest value in strain K3 was even less than half of the initial value. Most strains reached 2~8 mg/L lycopene, approximately 0~3-fold higher than the initial value. In most chimeric pathways, higher production was attributed to combinations of less than three genes. In strain B1 (7.5 mg/L lycopene), ERG25 governed the first key nonessential enzyme for synthesis of the basic sterol structure. In strain C2 (15.8 mg/L lycopene), only the first gene was verified, namely, PDA1, which might act in the mitochondria or cytoplasm to catalyze the direct oxidative decarboxylation of pyruvate to synthesize acetyl-CoA. Strain E5 was an example that the lycopene production was slightly tuned, although up to four genes of TRP1, GCN4, ARO7 and ARO3 were combined. In strain H1 (7.2 mg/L lycopene), YMR226C expressed an NADP(+)-dependent serine dehydrogenase. In strain I2 (7.5 mg/L lycopene), both ADH1 and LOT6 governed the reactions supplying NAD+ to glycolysis, and LOT6 also occurred in strain I3 (12.6 mg/L lycopene). A recent work engineered on the conversion of glycolytic NADH into NADPH or acetyl-CoA, which is required for lipid biosynthesis in Y. lipolytica, and the strain gained a 25% improvement of high-level lipid yield26. Our pathways “H” and “I” were set for similar purpose. These results offered potential targets for engineering terpene synthesis. 5



2.

Boosting lycopene production with chimeric MVA pathways containing

different multigene combinations and copy numbers The improved lycopene production was still not satisfactory and we speculated that the direct upstream crt pathway (pathway “A”) and MVA pathway (pathway “B”) would play more significant roles. We constructed all five Yl-TUs for pathway-B genes and mixed them with pathway-A genes only inserted in Yl-LD08. The only last extra pathway-A gene was set for alleviating potential crtEBI bottlenecks. The cotransformation of these Yl-TUs into Y. lipolytica cells generated a library of A4AX1 (First four Yl-TUs from pathway “B” and one last from pathway “B” or “A”). Amazingly, we got highly diversified phenotypes of colonies with different depths of red color. We picked up 25 out of total 50 deep-colored colonies from the plate containing a sum of 300~500 colonies and measured their genotypes and capacities of producing lycopene (Figure 3, Supporting Figure S4). The lycopene concentrations were ranged from 7.8 mg/L to 89.7 mg/L, 4.6-fold to 52.5-fold higher than the initial production. The diversification of versions of chimeric pathways were testified by occurrence of every gene including ERG8, ERG10, ERG13, ERG12, ERG20, tHMGR, IDI1, MVD1, BTS1 and some crt genes in different locations. The adequate selection of these genes suggested that they were all beneficial to lycopene production, similar with their roles in S. cerevisiae8,20. The markedly increased lycopene amounts proved that these genes did work in Y. lipolytica and performed significant roles. Among different chimeric pathways, IDI1 was the highest-frequently selected gene, demonstrating its indispensable role; however, only appropriate gene combinations largely enhanced lycopene production. The strains with much higher lycopene synthesis carried stoichiometric expression control of special genes. The Yl-TUs of gene IDI1, ERG20 and ERG8 accounted for 69% of all pathway-X gene’s TUs identified in all selected strains, suggesting their high frequency to be selected and crucial roles of boosting lycopene biosynthesis in Y. lipolytica. All the three genes were only gathered in one champion strain A4AX1-25 producing 89.7 mg/L lycopene. The unique genotype in the champion strain was (TU1-IDI1, TU2-ERG20, TU3-ERG20, TU4-ERG8, TU56


Page 6 of 29

Page 7 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


IDI1), offering the best performed chimeric MVA pathway. The impacts of each one of the genes on lycopene production were tested (Supporting Information Figure S5).

3.

Exploring significantly regulated intrinsic pathways relevant with boosting of

lycopene synthesis We were interested in the phenomenon that particular version of chimeric pathway in champion strain led to outstanding production of lycopene. We did comparative transcriptome analysis for three diverged strains, A4AX1-1, A4AX1-24 and A4AX125 (Figure 4a, Supporting Figure S6-S9, Supporting Table S4), to investigated the underlying causes. There were 329 differentially expressed genes between A4AX1-1 and A4AX1-24, including 163 up-regulated genes and 166 down-regulated genes. 490 genes were differentially expressed between A4AX1-1 and A4AX1-25, covering 265 up-regulated genes and 225 down-regulated genes. Of these genes, 217 common targets were shared by both pairs compared. KEGG pathway enrichment of the up-regulated and down-regulated genes in each comparison provided distinct results. The comparison of A4AX1-1 and A4AX1-24 showed no obvious up-regulated or downregulated pathways with relatively high enrichment factors (RF>0.50) except for an upregulated alpha-linolenic acid metabolism pathway (RF=0.6) (Supporting Figure S7). In contrast, in the comparison of A4AX1-1 and A4AX1-25, three up-regulated pathways exhibited remarkably high enrichment factors, namely, the synthesis and degradation of ketone bodies (RF=1.00), valine, leucine and isoleucine degradation (RF=0.68), and alpha-linolenic acid metabolism (RF=0.60) (Supporting Figure S8). All three pathways synthesized acetyl-CoA as the end product or intermediate and included several up-regulated genes which were summarized in Figure 4b~e and Supporting Table S5-S7. The pathway of valine, leucine and isoleucine degradation was also explored by another recent work27. Seven key targets were selected for further overexpression in A4AX1-25 to test their effects on lycopene production. Their single TU on the vector pLD-EcYl had been constructed in our previous work, where we had proved their benefits to slight improvement of basic beta-carotene synthesis27. Here we showed that the targets of 7



Page 8 of 29

YALI0E11099g(ERG10, expressing acetoacetyl-CoA thiolase), YALI0F30481g(ERG13, expressing

3-hydroxy-3-methylglutaryl-CoA

expressing

succinyl-CoA:

3-ketoacid-CoA

synthase),

YALI0F26587g(OXCT,

transferase),

YALI0F10010g(TGL4,

expressing multifunctional lipase/hydrolase/phospholipase), YALI0F10857g(POX2, expressing peroxisomal acyl-CoA oxidase) and YALI0E18568g(POT1, expressing peroxisomal 3-oxoacyl-CoA thiolase) were shown to enhance lycopene synthesis to varying degrees (Figure 4f, Supporting Figure S9). Only one target, YALI0B22550g(HMGL, expressing 3-hydroxy-3-methylglutaryl-CoA lyase) had no effect, as the associated reaction was the synthesis of acetoacetate from HMG-CoA. As previously mentioned, the alpha-linolenic acid degradation pathway was enriched by KEGG but actually did not exist in Y. lipolytica28. The real role of coupled YALI0F10857g(POX2) and YALI0E18568(POT1) should be performing repeated betaoxidation of acyl-CoA from C18 to final acetyl-CoA (Figure 4d). Another gene YALI0F10010g(TGL4) actually should be responsible for the degradation of multiple compounds in recycling free fatty acids (Figure 4d). The highest lycopene production was obtained in the strain with YALI0E18568(POT1) overexpression, reaching 259.5± 7.4 mg/L (approximately 80 mg/g DCW), which was 2.9-fold of that in A4AX1-25 and 152.6-fold of initial. We discovered that special S. cerevisiae gene combinations and consequent chimeric pathway triggered dramatic tuning of native genes and pathways highly relevant with lipid metabolism in Y. lipolytica. Here we also checked the genotypes of typical strains as A4AX1-1, -24, -25 and former C2, C5, D1, F5 and I3 by genome sequencing. We found that the already checked integration was also one copy at the exact GUT2 site (YALI0B13970g) in most strains except that when the gene could not be verified by PCR. In C2, only the first TU of PDA1 was integrated correctly at GUT2 and the second TU was not lost. In D1, the unique TU of POT1 was integrated correctly at GUT2. In I3, only the first TU of LOT6 was inserted to the 3,223,423 of chromosome E but other two TU were not integrated. The TUs in C5, F5, A4AX1-1, A4AX1-24, A4AX1-25 were all exactly integrated in GUT2 site as expected except for the not inserted TUs that were also not verified by PCR. We believed that our special design of 200 bp high-AT-content homologous arms 8


Page 9 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


contributed to this exact TU assembly and integration

(Supporting Information

Table S9).

4.

Modulating cell’s lipid amounts and composition along with enhancing

synthesis of lipophilic terpene According to the previous works on tuning of lipid metabolism, we speculated that some of qualitative changes of lipid metabolism had been triggered in the champion strain A4AX1-25. As a proof, we measured and compared cell’s lipid amounts and compositions in several strains (Figure 5). Although the strains were cultivated in different media according to their nutrition deficiency markers (-Leu, -Ura) and resistance markers (HygR), they showed similar growth abilities, all slightly weaker than the blank strain (Figure 5a). All strains with lycopene biosynthesis ability exhibited enhanced amounts of lipids detected as total fatty acids (Figure 5b). In the initial strain with only the lycopene pathway integrated at the rDNA site, the lipid titer was 64.2 mg/g DCW, approximately a 20% increase over the titer in the blank strain (53.4 mg/g DCW). In other strains containing A4AX1 gene incorporation, the titers of total fatty acids were from 67.0 mg/g DCW to 77.5 mg/g DCW, approximately 25% to 45% increases from the value in the blank strain. Although not quantitatively, the differences of lipid droplets could also be seen in microphotographs of the cells (Figure 5c). We proved that the chimeric MVA pathways boosted enhancement of both lycopene and lipid synthesis as well. In particular, we observed a distinct fatty acid composition in strain A4AX1-25 compared with other strains (Figure 5b). The 9-octadecenoic acid constituted 56.7% of the total fatty acids in A4AX1-25 but at most 1/3 in other strains. This change was derived from the unique genotype and transcriptome changes obtained. The 9octadecenoic acid almost occupied both proportions of upstream metabolite octadecanoic acid and downstream metabolite 9,12-octadecenoic acid. The delta-9 fatty acid desaturase might play a role but the transcription of its related gene (YALI0C05951g) was not obviously changed in A4AX1-25 compared with that in A4AX1-1. The changed composition might stem instead from broad metabolic 9



modulation.

Furthermore,

when

the

intrinsic

Page 10 of 29

YALI0F10857g(POX2)

and

YALI0E18568(POT1) was respectively overexpressed, fatty acid degradation was enhanced and 9-octadecenoic acid was transformed, recovering normal lipid composition (Figure 5d). These results proved again the capability of different yeast chimeric pathways for boosting total synthesis of lipophilic products. In this last part, we tried to improve the performance of our method in two aspects. In the first aspect, another recent work used Δku70 strain for the integration of large DNA fragments and obtained a clear increase in frequency24. In our test, the correct assembly rates in the Δku70 strain could be improved at both sites (90.0% in rDNA and 22.0% in GUT2), increasing the probability of obtaining multiple TU combinations (Figure S10). Higher-efficient genome integration site still required to be further explored. In the second aspect, we directly introduced the best players in Chen’s work as TmCrtE (from Taxus x media), PaCrtB (from Pantoea agglomerans), BtCrtI (from Blakeslea trispora) into Y. lipolytica21(Figure S11). The gene expression cassettes were constructed and assembled with genome integration as the exactly same way as our ENcrtEBI pathway. We also overexpressed each one gene based on our selected champion strain A4AX1-25. The results suggested it was promising to successively improve the performance of the terpene-producing strain.

Conclusion Here, we designed a strategy of constructing chimeric pathways of two budding yeasts, S. cerevisiae and Y. lipolytica, to boost lipophilic terpene synthesis. To our knowledge, this was the first time to transfer genes directly cloned from S. cerevisiae genome into Y. lipolytica at this large scale without adjusting codon usage. The options of heterologous yeast genes and their localization and copy numbers were effectively diversified to tune related enzyme expression. Previous works developed several library-screening methods for mainly testing single gene overexpression or knockout and carotenoid synthesis pathway itself25,29,30. Only a recent work of in vitro SCRaMbLE screened multi-gene combinations which were assembled by Cre 10


Page 11 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


recombinase31. In our work, the diversified chimeric pathways especially different versions of chimeric MVA pathway dramatically affected lycopene synthesis. The collaboration of special chimeric MVA pathway and modified intrinsic pathway attributed to about 150-fold increment of lycopene production from 1.7 mg/L to 259.3 mg/L. The incorporation of critical heterologous genes also affected the contents of intracellular lipids. An unusual increase of 9-octadecenoic acid in the selected lycopene-producing champion strain indicated a qualitive change of lipid metabolism. In our previous work, a distinguished strategy of global transcriptional engineering was used to screen strains with enhanced beta-carotene synthesis28. Two common pathways of ketone bodies metabolism and fatty acids metabolism were explored as distinct regulated in both works. We established a high-efficient metabolic manner for boosting lipophilic product synthesis and supplied some clues of how to design the synthetic biological system. Synthesis of other value-added products could also be optimized, considering many highthroughput in vivo library screening strategies had been set up in eukaryote systems32-34. The genome editing tools could also be utilized to facilitate efficient chromosomal integration of gene assemblies35. Our strategy also attributed to the establishment of chimeric genome consisting of genes from different resources, a potentially valuable extension of the de novo chemically synthesized genome and genome rearrangement36-39.

Materials and Methods Strain and media. All the Y. lipolytica strains in this work were constructed based on the starting strain ATCC 201249 (MATA ura3-302 leu2-270 lys8-11 PEX17-HA). The S. cerevisiae strain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was used for genome extraction and cloning of its genes as heterologous gene groups. E. coli strain DH5α (Biomed company) was used for DNA transformation. YPD medium containing dextrose (20 g/L), yeast extract (10 g/L) and peptone (20 g/L) was used to cultivate starting yeast strains. Synthetic complete (SC) medium (0.67 11



% yeast nitrogen base without amino acids, 2 % glucose, and appropriate amino acid drop-out mix), lacking uracil (SC-Ura) and/or leucine (SC-Leu), was used to select and cultivate Y. lipolytica transformants. LB medium containing NaCl (10 g/L), yeast extract (5 g/L), peptone (10 g/L) and ampicillin (100 μg/mL) was used to select E. coli strains. The Y. lipolytica strains synthesizing prodeoxyviolacein, lycopene or betacarotene were firstly inoculated in 4 mL of SC medium in glass tubes for overnight growth, and then were cultivated in 50 ml of SC medium in flask by three-parallel at 28 ℃ and 250 rpm for 96 h until the intracellular products were extracted and detected. Some selected strains were quick-frozen by liquid nitrogen at 72 h for transcriptome analysis.

Design of standardized Yl-TU for gene expression. The gene expression was performed under the control of a series of standardized Yl-TUs (Supporting Figure S1, S2; Table 1). First, a single Yl-TU on a plasmid was constructed combining the digestion of an open reading frame (ORF) and “promoterterminator” couples with a type IIS restriction enzyme and ligation with T4 ligase, similar to previous methods of yeast Golden Gate (yGG) in S. cerevisiae and in Y. lipolytica 40,41. Considering the GC content of Y. lipolytica genome is nearly 49%, we have to carefully coupled certain pairs of promoters and terminators not containing a common IIS enzyme site3. With some exceptions, most sequences of promoters and terminators were chosen according to Blazeck’s work (Supporting Table S8)42. Two of the same enzyme site (BsaI or BsmBI) were set in back-to-back mode between promoter-terminator couples. One of six type IIS enzyme sites (BsaI, BbsI, BfuAI, BsmBI, BsmFI and BtgZI) was added flanking the ORF. After digestion by corresponding enzyme, the left stick end 5’-AATG-3’ of ORF complemented the downstream site 3’-TTAC-5’ of promoter, and the right stick end 3’-ATTT-5’ complemented the site of 5’-TAAA-3’ before terminator. Multiple Yl-TUs were assembled through yeast in vivo recombination, similar to the description in previous reports43, 44. Two homologous ends (Hx) were localized outside the promoter (Px) and terminator (Tx) (Supporting Figure S1). Hx sequences 12


Page 12 of 29

Page 13 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with a length of 200 bp were directly cloned from the S. cerevisiae genome and had clearly lower GC contents than the Y. lipolytica genome, making them highly distinct with the promise of specific and correct recombination (Supporting Table S9). Our Yl-TU was also compatible with other gene part libraries such as BioBrick45, BglBrick46, VEGAS44 and MoClo47 (Supporting Figure S2). The sticky ends could be rapidly switched by the pop-in and pop-out of an RFP cassette (an integrated RFP expression cassette in E. coli, Supporting Table S9). A left cassette of “Yl-LDL” series and a right cassette of “Yl-LDR” series were employed to assist integration of the in vivo assembled TUs into the chromosome (Table 1, Supporting Figure S1).

DNA manipulation for construction of Yl-TU. For gene part (promoter, terminator, ORF, homologous end) cloning, a standard PCR mixture was used containing 10 ng/μL templates of genomic DNA, 5× FastPfu buffer, 0.02 U/μL FastPfu DNA polymerase (TRANSGENE), 0.2 mM each dNTP (TIANGEN Biotech), 0.2 μM each primer, and ddH2O. A common PCR condition was used. Step 1: initial denaturation at 95 °C (FastPfu) for 10 min; step 2 (30 cycles): denaturation at 95 °C for 30 s, annealing at 50~55 °C for 30 s, and elongation at 72 °C, 1 kb/30 s; step 3: a final elongation at 72 °C for 10 min. PCR products were verified on a 1% agarose gel and purified using the TIANgel Midi Purification Kit (TIANGEN Biotech, DP209). The primers and sequences were summarized in Supporting Table S9-S11. All blank Yl-TUs including the “Yl-LD”, “Yl-LDL” and “Yl-LDR” series were constructed by overlap-extension PCR (OE-PCR) from basic part. The primers were omitted here. These PCR products were digested with NotI and ligated into plasmid pLD-Ec, This plasmid was newly constructed from pEASY-Blunt (TransGen Biotech Company) where the ORF of KanR was eliminated and all the reorganization sites of BsaI, BbsI, BfuAI, BsmBI, BsmFI and BtgZI were mutated. The ORFs of heterologous genes were digested with alternative appropriate IIS enzyme and ligated into the blank Yl-TUs (Supporting Figure S2). When the mixed Yl-TUs containing gene libraries were constructed, the constructed plasmid DNA were 13



directly extracted from water-washed plates of E. coli colonies. The mixed Yl-TUs were directly recovered after NotI digestion without purified on gel electrophoresis. Then all the DNA segments were co-transformed into yeast for assembly of chimeric pathways. This design allowed simultaneous and rapid recovery of mixed Yl-TUs libraries for their assembly. The desirable colonies were picked and their genotypes were verified by PCR with couples of forward and reverse primers (Supporting Table S2). For testing the impacts of overexpression of single-gene on lycopene production, a series of plasmids containing single TU was transformed into strain A4AX1-25. Each gene was ligated in a blank Yl-TU “TEFp-(back-to-back BsmBI sites)-LIP2t” localized on the vector pLD-EcYl constructed from pMCSCen141 by replacing the previous URA marker with a hygromycin resistance marker (HygR).

E. coli and yeast transformation. E. coli transformation was performed according to the instructions of BioMed’s product (BC102-02). We used classical LiAc/SS carrier DNA/PEG method for chemical transformation of Y. lipolytica cells without large modification. Specially in our work, the 74 μL DNA mixture included 5-20 μL or 600 ng of each NotI-digested TU product and 1000 ng of each NotI-digested “Yl-LDL” and “Yl-LDR” product. The transformed Y. lipolytica colonies were cultivated on Sc-Ura&/-Leu agar plates at 28 ℃ for 3-5 days. The transformation efficiency of Y. lipolytica’s was calculated as described in DiCarlo’s work48. In our work, the product colors facilitated detection and calculation of the correct assembly rate which was shown as the ratios of the numbers of colored colonies to the number of total colonies.

Extraction and HPLC detection of heterologous products. After 96 h of cultivation, the Y. lipolytica cells were collected for the extraction and detection of different products. The prodeoxyviolacein producing strains were treated using our previous protocol developed for S. cerevisiae cells49. Finally, the prodeoxyviolacein samples were dissolved in methanol and detected on HPLC by using 14


Page 14 of 29

Page 15 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the same method. The alternative lycopene and beta-carotene producing strains were treated according to Xie’s method previously used for treating S. cerevisiae cells50. Briefly, 1 mL of 3 M hydrochloric acid was added to the cell pellets after centrifugation, and the cells were shocked for blending. The centrifuge tube was placed in boiling water for 5 min, then immediately put in ice bath for 5 min. The supernatant was discarded after centrifugation and the pellets were washed with sterile water. 1 mL of acetone containing 1 mg BHT (butylated hydroxytoluene) was added to the cells and the tubes were shaken vigorously on a vortex mixer for 10 min. After centrifugation, the supernatant was filtered with 0.2 μm hydrophobic membranes for collecting products. A 20 μL lycopene or beta-carotene sample diluted in acetone was measured on a Waters 2695-2489 UV detector with a SUPELCOSIL LC18 (33 mm × 4.6 mm, 3 μm) column and monitored at 470 nm (lycopene) and 450 nm (beta-carotene). The mobile phase was acetonitrile: methanol: dichloromethane (9: 40: 1). The column temperature was 25 ℃ and the flow rate was 0.5 mL/min.

Extraction and GC detection of total fatty acids. Cells were harvested by centrifugation. For this purpose, 1 mL of methanol containing 3 M HCl and 10 mg/L heptadecanoic acid and 100 μL chloroform was added to the cell pellets, which were shocked for blending. The tubes were incubated at 70 °C for 3 h and reversed several times per 40 min, then cooled down to room temperature. Next, sodium chloride was added to the point of saturation, and the tubes were shaken for 1 min. Then 500 μL of hexyl hydride was added and the tube was shaken for 1 min. The supernatant was filtered through 0.2 μm hydrophobic membranes to collect fatty ester products. For product detection, 10 μL of sample was injected into a 6890N GC (Agilent Corp., USA) coupled with a GCT PremierTM MICROMASS mass spectrometer (Waters Corp., USA), on a DB-5 MS column with a split injection of 5:1. The GC conditions were as follows: carrier gas (helium) at 91 kPa per min in constant pressure mode, programmed oven temperature from 70 °C (3 min) to 280 °C at a rate of 3 °C per min, source temperature at 250 °C and interface temperature at 250 °C. Electron impact (EI) spectra were obtained at -70 eV. Raw GC-MS data were analyzed using the software package Masslynx4.1(Waters Corp., USA), avoiding detector 15



Page 16 of 29

overload and isotope fractionation as described.

Transcriptome sequencing and analysis. Transcriptome sequencing was performed using the following main steps. First, the purity of the RNA was checked, and its concentration was measured. RNA integrity was assessed by the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Then, for each sample, 3 μg of RNA was used for library preparation. Sequencing libraries were generated using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s instructions. Finally, PCR products were purified by the AMPure XP system, and the library quality was assessed by the Agilent Bioanalyzer 2100 system. Sample clustering was performed, the prepared libraries were sequenced on an Illumina HiSeq 4000 platform and 150 bp paired-end reads were generated. After these steps, the data were analyzed by the Novogene Company. The utilized detailed methods for data analysis were not listed here. The raw transcriptome data and the manipulation processes performed in this study were uploaded to GEO, and the accession number is GSE93919.

Table 1. The standardized Yl-TU toolkit used in this work. ID

Content

Abbreviation

Length (bp) (without RFP)

Yl-LD01

H0-PEXP1-(RFP)-TXPR2-H1

H0-P1-T1-H1

1670

Yl-LD02

H1-PTEF-(RFP)-TLIP2-H2

H1-P2-T2-H2

1393

Yl-LD03

H2-PGPD-(RFP)-TOCT-H3

H2-P3-T3-H3

1684

Yl-LD04

H3-PGPAT-(RFP)-TPEX16-H4

H3-P4-T4-H4

1783

Yl-LD05

H4-PYAT1-(RFP)-TLIP1-H5

H4-P5-T5-H5

1679

Yl-LD06

H5-PXPR2-(RFP)-TPEX20-H6

H5-P6-T6-H6

1689

Yl-LD07

H6-PFBA-(RFP)-TCYC1-H7

H6-P7-T7-H7

1662

Yl-LD08

H7-PLEU-(RFP)-TACO-H8

H7-P8-T8-H8

1672

Yl-LDL01

rDNAL-Ura3-H0

rDNAL-Ura3-H0

2823

16


Page 17 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Yl-LDL02

GUT2L-Leu2-H3

GUT2L-Leu2-H3

3006

Yl-LDR-r01

H1-rDNAR

H1-rDNAR

800

Yl-LDR-r02

H2-rDNAR

H2-rDNAR

800

Yl-LDR-r03

H3-rDNAR

H3-rDNAR

800

Yl-LDR-r04

H4-rDNAR

H4-rDNAR

800

Yl-LDR-

H4-GUT2R

H4-GUT2R

1200

H5-GUT2R

H5-GUT2R

1200

H6-GUT2R

H6-GUT2R

1200

H7-GUT2R

H7-GUT2R

1200

H8-GUT2R

H8-GUT2R

1200

G04 Yl-LDRG05 Yl-LDRG06 Yl-LDRG07 Yl-LDRG08

Acknowledgments We thank Prof. Sheng Yang in Shanghai Institute of Life Sciences, Chinese Academy of Sciences for offering us the Y. lipolytica strain ATCC 201249. This work was funded by the Ministry of Science and Technology of China (“973” Program: 2014CB745100), and the National Natural Science Foundation of China (Major Program: 21750001, 21621004).

Author contributions D. L. designed the work and planed the major experiments; D. L., H. Q., J-L. Z. designed all the primers and did all the DNA manipulations. H. L. and X-J. G. did all the fermentations and detected the heterologous products synthesized by cells. D. L. and B. J. optimized library construction and screening methods. All of the work was 17



guided by Y-J. Y.

Competing interests The authors declare no competing financial interests.

Supporting information Supporting Figures S1-S11 and Supporting Tables S1-S11 are available online.

Reference 1.

Löbs, A. K., Schwartz, C. & Wheeldon, I. (2017) Genome and metabolic engineering in non-conventional yeasts: Current advances and applications. Synth Syst Biotechnol. 2, 198-207.

2.

Dujon, B. et al. (2004) Genome evolution in yeasts. Nature 430, 35-44.

3.

Duina, A. A., Miller, M. E. & Keeney, J. B. (2014) Budding yeast for budding geneticists: a primer on the Saccharomyces cerevisiae model system. Genetics 197, 33-48.

4.

Dujon B. A., Louis E. J. (2017) Genome diversity and evolution in the budding yeasts (Saccharomycotina). Genetics. 206, 717-750.

5.

Patterson, K. et al. (2018) Functional genomics for the oleaginous yeast Yarrowia lipolytica. Metab Eng. 48, 184-196.

6.

Luo, Y. et al. (2015) Engineered biosynthesis of natural products in heterologous hosts. Chem. Soc. Rev. 44, 5265–5290.

7.

Zhou, Y. J. et al. (2016) Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories. Nat. Commun. 7, 11709.

8.

Paddon, C. J. et al. (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496, 528-32.

9.

Xue, Z. et al. (2013) Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica. Nat. Biotechnol. 31, 734-740.

10. Qiao, K. et al. (2015) Engineering lipid overproduction in the oleaginous yeast 18


Page 18 of 29

Page 19 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Yarrowia lipolytica. Metab. Eng. 29, 56-65. 11. Zhu, Q. & Jackson, E. N. (2015) Metabolic engineering of Yarrowia lipolytica for industrial applications. Curr. Opin. Biotechnol. 36, 65-72. 12. Madzak, C. (2018) Engineering Yarrowia lipolytica for use in biotechnological applications: a review of major achievements and recent innovations. Mol Biotechnol. doi: 10.1007/s12033-018-0093-4. 13. Blazeck, J. et al. (2014) Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production. Nat. Commun. 5, 3131. 14. Kachroo, A. H. et al. (2015) Systematic humanization of yeast genes reveals conserved functions and genetic modularity. Science. 348, 921-925. 15. Frost, A. et al. (2012) Functional repurposing revealed by comparing S. pombe and S. cerevisiae genetic interactions. Cell 149, 1339-52. 16. Kapitzky, L. et al. (2010) Cross-species chemogenomic profiling reveals evolutionarily conserved drug mode of action. Mol Syst Biol. 6, 451. 17. Ryan, C. J. et al. (2012) Hierarchical modularity and the evolution of genetic interactomes across species. Mol Cell. 46, 691-704. 18. Shen, M. J. et al. (2018) Heterozygous diploid and interspecies SCRaMbLEing. Nat Commun. 9, 1934. 19. Gibson, D. G. et al. (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52-56. 20. Westfall, P. J. et al. (2012) Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Proc Natl Acad Sci U S A. 109, E111-118. 21. Chen, Y. et al. (2016) Lycopene overproduction in Saccharomyces cerevisiae through combining pathway engineering with host engineering. Microb Cell Fact. 15, 113. 22. Matthäus F., Ketelhot M., Gatter M., Barth G. (2014) Production of lycopene in the non-carotenoid-producing yeast Yarrowia lipolytica. Appl Environ Microbiol. 80, 1660-9. 23. Schwartz C., Frogue K., Misa J., Wheeldon I. (2017) Host and pathway 19



engineering for enhanced lycopene biosynthesis in Yarrowia lipolytica. Front Microbiol. 8, 2233. 24. Gao, S. et al. (2017) Iterative integration of multiple-copy pathway genes in Yarrowia lipolytica for heterologous β-carotene production. Metab. Eng. 41, 192201. 25. Larroude M. et al. (2018) A synthetic biology approach to transform Yarrowia lipolytica into a competitive biotechnological producer of β-carotene. Biotechnol Bioeng. 115, 464–72. 26. Qiao, K., Wasylenko, T. M., Zhou, K., Xu, P. & Stephanopoulos, G. (2017) Lipid production in Yarrowia lipolytica is maximized by engineering cytosolic redox metabolism. Nat. Biotechnol. 35, 173-177. 27. Zhao, C. et al. (2017) Metabolomic changes and metabolic responses to expression of heterologous biosynthetic genes for lycopene production in Yarrowia lipolytica. J Biotechnol. 251, 174-185. 28. Wang, M. et al. (2018) Engineering global transcription to tune lipophilic properties in Yarrowia lipolytica. Biotechnol Biofuels. 11, 115. 29. Jin, Y. S. & Stephanopoulos, G. (2007) Multi-dimensional gene target search for improving lycopene biosynthesis in Escherichia coli. Metab. Eng. 9, 337−347. 30. Özaydın, B., Burd, H., Lee, T. S. & Keasling, J. D. (2013) Carotenoid-based phenotypic screen of the yeast deletion collection reveals new genes with roles in isoprenoid production. Metab. Eng. 15, 174-183. 31. Wu, Y. et al. (2018) In vitro DNA SCRaMbLE. Nat Commun. 9, 1935. 32. Rogers, J. K., Taylor, N. D. & Church, G. M. (2016) Biosensor-based engineering of biosynthetic pathways. Curr. Opin. Biotech. 42, 84-91. 33. Li, S., Si, T., Wang, M. & Zhao, H. (2015) Development of a synthetic malonylCoA sensor in Saccharomyces cerevisiae for intracellular metabolite monitoring and genetic screening. ACS Synth. Biol. 4, 1308-1315. 34. Feng, J. et al. (2015) A general strategy to construct small molecule biosensors in eukaryotes. eLIFE. 4, e10606. 35. Schwartz, C. M., Hussain, M. S., Blenner, M. & Wheeldon, I. (2016) Synthetic 20


Page 20 of 29

Page 21 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RNA polymerase III promoters facilitate high-efficiency CRISPR-Cas9-mediated genome editing in Yarrowia lipolytica. ACS Synth. Biol. 5, 356-359. 36. Xie, Z. X. et al. (2017) "Perfect" designer chromosome V and behavior of a ring derivative. Science 355, 6329. pii: eaaf4704. 37. Wu, Y. et al. (2017) Bug mapping and fitness testing of chemically synthesized chromosome X. Science 355, 6329. pii: eaaf4706. 38. Xie, Z. X. et al. (2017) Design and chemical synthesis of eukaryotic chromosomes. Chem. Soc. Rev. 46, 7191-7207. 39. Jia, B. et al. (2017) Precise control of SCRaMbLE in synthetic haploid and diploid yeast. Nat Commun. 9, 1933. 40. Agmon, N. et al. (2015) Yeast Golden Gate (yGG) for the efficient assembly of S. cerevisiae transcription units. ACS Synth. Biol. 4, 853-859. 41. Celińska, E. et al. (2017) Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica. Microb Biotechnol. 10, 450-455. 42. Blazeck, J., Liu, L., Redden, H. & Alper, H. (2011) Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach. Appl. Environ. Microbiol. 77, 7905. 43. Shao, Z., Zhao, H. & Zhao, H. (2009) DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res. 37, e16. 44. Gao, S. et al. (2014) One-step integration of multiple genes into the oleaginous yeast Yarrowia lipolytica. Biotechnol. Lett. 36, 2523-2528. 45. Shetty, R. P., Endy, D. & Knight, T. F. Jr. (2008) Engineering BioBrick vectors from BioBrick parts. J. Biol. Eng. 2, 5. 46. Lee, T. S. et al. (2011) BglBrick vectors and datasheets: A synthetic biology platform for gene expression. J. Biol. Eng. 5, 12. 47. Weber, E., Engler, C., Gruetzner, R., Werner, S. & Marillonnet, S. (2011) A modular cloning system for standardized assembly of multigene constructs. PLoS One. 6, e16765. 48. DiCarlo, J. E. et al. (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 4336-4343. 21



49. Liu, D. et al. (2016) Multigene pathway engineering with regulatory linkers (MPERL). ACS Synth. Biol. 5, 1535-1545. 50. Xie, W. et al. (2014) Construction of a controllable beta-carotene biosynthetic pathway by decentralized assembly strategy in Saccharomyces cerevisiae. Biotechnol. Bioeng. 111, 125-133.

22


Page 22 of 29

Page 23 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure Captions

Figure 1. Concept illustration of constructing yeast chimeric pathways. The yeast chimeric pathways were constructed by incorporating genes from S. cerevisiae into Y. lipolytica. The goal was to boost heterologous lipophilic terpene synthesis and the superior strains were selected from yeast libraries containing different gene combinations.

23



Figure 2. Testing the feasibility of Yl-TU assembly toolkit. (a) The way of utilizing Yl-TU assembly toolkit to incorporate groups of S. cerevisiae genes into Y. lipolytica. (b) A series of Yl-TU libraries were constructed for pathways of A to K. The details of pathways were given in Table S1. (c) A single colony with deeper color in each library was picked for lycopene detection. All error bars indicate ±SD, n=3.

24


Page 24 of 29

Page 25 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. The chimeric MVA pathway library drastically affected lycopene production. Genotypes of the gene combinations in the selected strains were sequenced. The “NL” indicates that no gene was ligated, and the “ND/NA” indicates “not detected or not assembled”. The most frequently selected genes were IDI1, ERG20 and ERG8.

25



Figure 4. Comparative transcriptome analysis of the selected strains. (a) Compared with those in strain A4AX1-1, the expression levels of several genes in strains A4AX124 and A4AX1-25 were up-regulated or down-regulated. (b) Three pathways of P1, P2 and P3 were clearly up-regulated only in A4AX1-25 and got the highest rich factors in KEGG pathway analysis. Seven genes were considered playing key roles (labelled in green circle and in accordance with those labelled in figure 4c, 4d, 4e and 4f). (c) P1 26


Page 26 of 29

Page 27 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pathway: synthesis and degradation of ketone bodies. HMG-CoA: (S)-3-hydroxy-3methyl-glutaryl-CoA. (d) P2 pathway: alpha-linolenic acid metabolism. PTC: phosphatidylcholine. JA-CoA: (+)-7-Isojasmonic acid CoA. (e) P3 pathway: valine, leucine, and isoleucine degradation. In all figures, red solid lines represented upregulated genes; gray solid lines meant unchanged genes; and gray dotted lines were reactions that had not been verified in Y. lipolytica according to KEGG. (f) An extra Yl-TU of the seven key genes was transferred to strain A4AX1-25 and further affected lycopene production. All error bars indicate ±SD, n=3.

27



Figure 5. Modulating cell’s lipid contents along with enhancing lycopene synthesis. (a) Growth curves of typical strains. (b) Amounts of intrinsic lipids were also enhanced concomitantly with improved lycopene production. The composition of fatty acids was drastically altered in A4AX1-25, where 9-octadecenoic acid was the major component. (c) Microscopic images of the strains. (d) Extra overexpression of key enzymes in the fatty acid degradation pathway participated in the tuning of fatty acid composition.

28


Page 28 of 29

Page 29 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Constructing yeast chimeric pathways to boost lipophilic terpene synthesis. Duo Liu, Hong Liu, Hao Qi, XueJiao Guo, Bin Jia, Jin-Lai Zhang, Ying-Jin Yuan 309x90mm (150 x 150 DPI)


Constructing yeast chimeric pathways to boost lipophilic terpene

Recommend Documents