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Identification of a novel metabolic engineering target for carotenoid production in Saccharomyces cerevisiae via ethanol-induced adaptive laboratory evolution

Identification of a novel metabolic engineering target for carotenoid production in Saccharomyces... Carotenoids are a large family of health‑beneficial compounds that have been widely used in the food and nutra‑ ceutical industries. There have been extensive studies to engineer Saccharomyces cerevisiae for the production of carotenoids, which already gained high level. However, it was difficult to discover new targets that were relevant to the accumulation of carotenoids. Herein, a new, ethanol‑induced adaptive laboratory evolution was applied to boost carotenoid accumulation in a carotenoid producer BL03‑D ‑4, subsequently, an evolved strain M3 was obtained with a 5.1‑fold increase in carotenoid yield. Through whole ‑ genome resequencing and reverse engineering, loss‑ of‑function mutation of phosphofructokinase 1 (PFK1) was revealed as the major cause of increased carotenoid yield. Transcrip‑ tome analysis was conducted to reveal the potential mechanisms for improved yield, and strengthening of gluconeo‑ genesis and downregulation of cell wall‑related genes were observed in M3. This study provided a classic case where the appropriate selective pressure could be employed to improve carotenoid yield using adaptive evolution and elucidated the causal mutation of evolved strain. Keywords: Saccharomyces cerevisiae, Adaptive laboratory evolution, Carotenoid, PFK1, Reverse engineering, Cell wall Introduction carotenoid yield in S. cerevisiae (Wang et  al. 2019). Carotenoids are a large kind of isoprenoid pigment Many strategies have been applied and high levels that can be used in many fields such as colorants, of yields were obtained (Chen et  al. 2016; Hong et  al. antioxidants, and nutrients (Saini and Keum 2019). 2019; Shi et al. 2019; Xie et al. 2015), while the highest Currently, carotenoids are produced by chemical syn- yield of 73 mg/g CDW was achieved through lipid engi- thesis or extraction from natural sources, which are neering (Ma et  al. 2019). However, it was difficult to mostly restricted by complicated structures and short- discover new targets that were relevant to the heterolo- age of raw materials (Mussagy et  al. 2019). Wild-type gous production of carotenoids and, furthermore pro- Saccharomyces cerevisiae does not normally accu- mote the production of carotenoids in recombinant S. mulate carotenoids but engineered strains, obtained cerevisiae based on previous studies. Fortunately, ran- through rational engineering, have achieved enhanced dom disturbance in the genes at a whole-genome scale (through mutagenesis or evolution) provided a choice for acquiring a carotenoid hyper-producer (Zhu et  al. *Correspondence: dengmr@gdim.cn; zhuhh@gdim.cn 2018). For instance, using a single atmospheric and Guangdong Microbial Culture Collection Center (GDMCC), Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, room temperature plasma (ARTP) treatment, astaxan- State Key Laboratory of Applied Microbiology Southern China, Institute thin yield was improved by 0.83-fold over the parental of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, strain and three potential targets related to astaxanthin People’s Republic of China © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://crea‑ tivecommons.org/licenses/by/4.0/. Su et al. Bioresour. Bioprocess. (2021) 8:47 Page 2 of 10 biosynthesis in yeast were revealed (Jin et  al. 2018). cell wall-related genes were identified as the likely regula - Furthermore, a combined strategy composing physical tion that resulted in increased carotenoid yield. mutagenesis by ARTP and adaptive laboratory evolu- tion (ALE), driven by hydrogen peroxide, was executed Material and methods and the titer of astaxanthin was increased nearly four- Microorganisms and growth conditions fold ( Jiang et al. 2020). S. cerevisiae strain BL03-D-4, derived from BY4742 ALE is a widely used technology to achieve insights (MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0), was chosen into the mechanisms of adaptive mutations that accu- as the parental strain. All S. cerevisiae strains used in mulate under designed culture conditions for long peri- this study are listed in Additional file  1: Table  S1. Prim- ods of screening (Dragosits and Mattanovich 2013). ers are provided in Additional file  1: Table  S2. For culti- ALE has been proven to be a potent tool in metabolic vation, a single colony was picked up from a fresh YPD engineering, both for the revelation of new design prin- plate and transferred to a 5 mL YPD medium. After being ciples and the engineering of excellent strains (Zhu cultured at 200  rpm and 30  °C, 1  mL seed culture was et  al. 2018). Many superior phenotypes have been inoculated into a 250  mL shake-flask with 50  mL YPD achieved through ALE, such as improvement of the medium, or modified YPD medium (YPM) to an opti - growth rate and production, the adaption of strains to cal density (OD ) of about 0.05 and then incubated at utilize non-native substrates, and enhancement of the 30  °C for 24 to 96  h. After incubation, the cultures were resistance towards environmental stresses (Sandberg analyzed for biomass and carotenoid content. The YPD −1 −1 et  al. 2019). Furthermore, the relationship between medium contained 20  g L tryptone, 20  g L glucose, −1 genomic changes and the excellent phenotype could be and 10  g L yeast extract (Oxoid, LOTs of 2,665,431– −1 uncovered by whole-genome resequencing combined 02). YPM medium contained 20  g L tryptone, 20  g −1 −1 with systems biology (Yang et  al. 2019). For example, L glucose, 10 g L yeast extract (Angel, FM802, LOT: −1 −1 ALE has been applied to adapting hydrogen perox- 2018082210C9), salt (10 g L KH PO , 2.5 g L MgSO , 2 4 4 −1 −1 ide-tolerant yeast to improve carotenoids production 3.5 g L K SO , 0.25 g L Na SO ) and 1 mL trace metal 2 4 2 4 (Reyes et  al. 2014), and the beneficial mutations led to solution (TMS) described in previous literature (Su et al. increased yield have been revealed (Godara et al. 2019). 2020b). Compared to random mutagenesis approaches such as ARTP and ultraviolet light, ALE with sequential pas- Adaptive laboratory evolution experiments sages is a reasonably easy technique for confirming Sequential passages and batch cultures were performed crucial mutations related to the beneficial phenotype using the parental strain (BL03-D-4) and YPD media due to its low mutation probability. Furthermore, the with different concentrations of ethanol under the cul - production of the target product could be improved by ture conditions illustrated in Fig. 1. Cells were inoculated introducing the identified mutations into an engineered into 50 mL YPD medium at OD of 0.05 to maintain the producer (Lee and Kim 2020). growth phase. After each cultivation, cells with appropri- ALE used suitable stress as a driving force for screening ate concentrations were plated on YPD plates for single mutants with superior phenotypes. Because carotenoid colony isolation, based on cell color. The single colonies yield was facilitated by engineering microbial membranes obtained were inoculated in the YPD medium for further which could also increase ethanol tolerance (Guo et  al. screening. 2020; Hong et  al. 2019), we hypothesized that ethanol stress might be used as a driving force for the adaptation Whole‑genome resequencing and transcriptome analysis of yeast for increased carotenoids accumulation. As a The strains chosen for whole-genome resequencing were proof of concept, ethanol-induced ALE was applied to a cultivated in 50 mL YPD medium at 30 °C in a shaker at recently constructed S. cerevisiae strain, BL03-D-04, har- 200  rpm for 24  h. Genomic DNA was extracted accord- boring the carotenoid synthetic pathway (Su et al. 2020b). ing to the manufacturer’s protocol using the HiPure Yeast Improvement of carotenoid yield was obtained through DNA Kit (Magen, Guangzhou, China). At least 5  μg of this novel ALE strategy to screen for higher carotenoid each genomic DNA sample was provided to Shanghai producers. The underlying cause related to the promo - Majorbio Bio-pharm Technology Co. Ltd, for sequenc- tion of carotenoids accumulation in the evolved strain ing using the Illumina HiSeq 2000 platform. Paired-end were assessed using whole-genome resequencing and reads of ~ 250 bp were generated. The average sequencing transcriptome analysis. The inactivation of phosphofruc - depths of the samples were 70 to 90. Fastq DNA-seq raw tokinase 1 (PFK1) was determined as the causal mutation data were deposited in the Genome Sequence Archive of the evolved strain with improved carotenoid yield, and (GSA) server at the BIG Data Center (http:// bigd. big. ac. strengthening of gluconeogenesis and downregulation of cn, GSA accession No. CRA003704). Su  et al. Bioresour. Bioprocess. (2021) 8:47 Page 3 of 10 Fig. 1 Schematic diagram of the ethanol‑induced adaptive laboratory evolution. Gradually increasing (from 2 to 12% or from 4 to 12%) and specific concentrations (2%, 4%, 8%, and 10%) of ethanol were selected as the driving forces for the ALE. After screening and analytical certification, the excellent strain will be turned into another round of evolution. The mutations of excellent strain were uncovered through whole‑ genome resequencing and then parental strain was reverse engineered to gain a rationally designed strain Total RNA from yeast cells was extracted according diagnostic PCR using paired primers PFK1-F-2/PFK1- to our previous work (Su et  al. 2020a). The concentra - CHECK-R and PFK2-F-2/PFK2-CHECK-R, respectively. tions and quality of the RNA samples were examined by Biospec-nano (Shimadzu, Kyoto, Japan). 1  μg total RNA Cell dry weight, specific growth rate and carotenoid sample was used for mRNA library preparation and RNA quantification sequencing (Illumina HiSeq), performed by Shanghai Cell dry weight (CDW) was determined according to the Majorbio Bio-pharm Technology Co. Ltd. Fastq RNA- OD value and correlated to CDW by the CDW/OD 600 600 seq raw data were deposited into NCBI (GEO accession standard curve [y = 0.184x + 0.891 (x is OD , y is CDW, number GSE164470). Data processing was accomplished R = 0.992]. The specific growth rate (μ) was calculated by by the online Majorbio Cloud Platform (www. major bio. the equation, μ = (ln X2 − ln X1)/(t2 − t1), where X1 and com). X2 are the biomasses at time t1 and t2, respectively. For carotenoid quantification, strain culture after fer - Gene deletions mentation was transferred to a 1.5-mL sample tube and Auxotroph marker HIS3 including a promoter and termi- the cells were collected by centrifugation at 12,000  rpm nator, was amplified from the genomic DNA of S. cerevi- for 5  min. The sedimentary cells were disrupted using −1 siae S288C. The homologous arm (~ 50 bp) was designed 3 mol L HCl and boiling for 4 min. Then, cell debris was in primers. For deletions of target genes, one-step inte- washed thrice by sterile water, resuspended in acetone for gration of PCR-amplified deletion cassettes, including extraction, and centrifuged at 12,000 rpm for 2 min. The HIS3, was adopted (Chen et  al. 2016). Primers PFK1-F supernatant was transferred to a new tube for quantifica - and PFK1-R were used for amplification of HIS3 from tion of total carotenoids using a UV/Vis spectrometer at genomic DNA of S. cerevisiae S288C. Primers PFK1-F-2 470 nm. The extinction coefficient was adopted using an and PFK1-R-2 were used for the addition of homologous A 1% 1 cm of 3450 (Su et al. 2020b). arms targeted to PFK1. Primers PFK2-F and PFK2-R were used for amplification of HIS3 from genomic DNA Morphological observation of S. cerevisiae S288C. Primers PFK2-F-2 and PFK2-R-2 The morphology of the strains was observed by a light were used for the addition of homologous arms targeted microscope (DM6/MC190, Leica). In brief, the strains to PFK2. PFK1 and PFK2 deletions were verified by were cultivated in 50 mL YPD medium at 30 °C for 48 h, Su et al. Bioresour. Bioprocess. (2021) 8:47 Page 4 of 10 followed by centrifuging and washing with PBS three genetic mutations and systematic transcriptional regula- times. They were then observed through the microscope. tion, which needs to be further investigated. Results Whole‑genome resequencing of the evolved strain M3 Adaptive laboratory evolution of BL03‑D‑4 under ethanol To identify the genetic basis of the improved carot- stress enoids yield, whole-genome resequencing of strain Because the heterologous accumulation of carotenoids BL03-D-4 and M3 was performed using the Illumina in S. cerevisiae poses a metabolic burden on host cells, a HiSeq platform through paired-end sequencing. Cov- suitable pressure was needed to endow the higher pro- erage depth was approximately 70 to 90 reads. Overall, ducers a striking feature (Reyes et al. 2014). Based on the 14 single nucleotide polymorphisms (SNPs) and 100 already known mechanisms of ethanol tolerance (Ma and insertion and deletions (INDEL) were detected in M3, Liu 2010), we hypothesized that yeast strain would pre- compared to BL03-D-4 after the evolution process (full sent different levels of carotenoids yield with the fluctu - mutation lists are available in Additional file  2). The ant composition of the membrane under ethanol stress, resequencing results suggested that M3 had acquired a and there might be some status fitted to the carotenoids loss-of-function mutation of PFK1 since M3 generated accumulation. Thus, in this study, an ethanol-induced a stop codon in the front part of the PFK1 gene, which ALE strategy was developed for improving carotenoid encoded 6-phosphofructokinase 1. It has been reported yield. For the ALE experiment, sequential and batch cul- that PFK1 was involved in glycolysis and gluconeogen- tures were performed in YPD medium supplemented esis which was related to NADPH generation (Kwak et al. with different concentrations of ethanol (v/v) (Fig.  1). 2020). Furthermore, the E. coli central metabolic network When the cell growth reached a plateau, the ALE process was rewired after deletion of pfkA which indicated that was terminated and the evolved cells were plated on YPD reduced glycolysis by weakening 6-phosphofructokinase plates without ethanol to screen darkened colonies. Sub- had a profound effect on metabolism (Hollinshead et  al. sequently, an evolved strain M3 (deposited at Guangdong 2016). Thus, combined with the genome resequencing Microbial Culture Collection Center with GDMCC No. results, we speculated that the loss-of-function mutation 61336) was obtained in the sequential cultures, through a of PFK1 in M3 was related to the promoted carotenoid range of adaptive experiments with gradually rising con- yield. centrations of ethanol (the first horizontal panel of Fig.  1 at 10% after three subcultures) based on darkened cell Determining causal mutation color (Additional file  1: Figure S1). Furthermore, M3 was To determine whether the loss-of-function mutation also turned into another round of evolution. However, no of PFK1 confers the improvement of carotenoids yield, better strain was obtained using the above ALE strategy. the inactivation of PFK1 was reverse-engineered into Shake-flask fermentation showed that the carotenoid BL03-D-4. The reverse-engineered strain BE1 showed yield of M3 increased about 5.1- and 2.4-fold compared remarkably increased carotenoid yield relative to BL03- with BL03-D-4 in YPD and YPM media, respectively D-4, which was not significantly different compared to (Fig. 2a). After 96 h of incubation, the carotenoid yield of M3 (Fig.  3) without ethanol or under different concen - M3 reached 42.4 mg/g CDW in the YPM medium (YPM trations of ethanol, fully recovering the evolved carot- medium facilitated carotenoid accumulation (Su et  al. enoids phenotype (Additional file  1: Figure S3). BE1 2021)). The specific growth rate of strain M3 increased represented a similar colonial morphology compared markedly, compared to that of the parental strain BL03- with M3, but with a different morphology to parental D-4 in the concentration from 2 to 8% ethanol (Fig.  2b). strain BL03-D-4 (Additional file  1: Figure S1 and S4). Both strains grew poor in the presence of 10% etha- Deletion of PFK1 had little effect on the growth of nol (Additional file  1: Figure S2). We also observed that strain BE1 without ethanol or under different concen - the additional supplement of ethanol had a remarkably tration of ethanol (Fig.  3), these results suggested that repressive effect on the carotenoid yield of M3 especially mutations other than PFK1 mutation contributed to under a high concentration of ethanol (Fig.  2c). How- the normal growth of M3 and counteracted the nega- ever, there was no significant difference in cell growth tive effect of the loss-of-function mutation of PFK1 . between BL03-D-4 and M3 during the cultivation with- Since there was an isoenzyme (PFK2) of PFK1 in S. cer- out ethanol (Fig. 2d), indicating that the ALE process had evisiae, BL03-D-4 was engineered with the deletion of no significant influence on the biomass of M3. Therefore, PFK2 to generate BE2 to test whether PFK2 mutation the improvement of carotenoids yield in M3 was not due has any similar effect on the pentose phosphate path - to facilitating the strain growth, but was probably con- way (PPP). As shown in Fig.  3b, PFK2 deletion exerted cerned with the change of metabolic pathway involved in a severe suppression on the growth of BE2 and no Su  et al. Bioresour. Bioprocess. (2021) 8:47 Page 5 of 10 Fig. 2 Characterization of parental (BL03‑D ‑4) and evolved (M3) strains. Carotenoid yields and biomasses of BL03‑D ‑4 and M3 in YPD and YPM media after 96 h of incubation (a). Specific growth rates of BL03‑D ‑4 and M3 under different concentrations of ethanol (b). Eec ff t of different concentrations of ethanol on carotenoid yields of BL03‑D ‑4 and M3 (c). Growth curves of BL03‑D ‑4 and M3 in YPM media without ethanol stress (d). Error bars represent standard deviations of three replicates increase of carotenoids was detected in this strain after (FBP) in S. cerevisiae which was different compared to 96 h fermentation. However, BE2 represented a similar E. coli (Hollinshead et  al. 2016). Morphology observa- colonial morphology compared with M3 and BE1 after tion of BL03-D-4, M3, BE1, and BE2 was carried out long cultivation (about 8  days) (Additional file  1: Fig- through a microscope at 1000 × magnification. These ure S4). These results suggested that PFK2 might be the pictures showed that evolved (M3) and knockout key enzyme responsible for the conversion of glucose- strains (BE1 and BE2) presented a larger size of mor- 6-phosphate (G6D) to fructose 1,6-bisphosphatase phology compared to the parental strain (BL03-D-4) Su et al. Bioresour. Bioprocess. (2021) 8:47 Page 6 of 10 Fig. 3 Carotenoid yields (a) of evolved (M3), and knockout strains (BE1 and BE2), and growth patterns (b) of M3, BE1, and BE2. Eec ff t of different concentrations of ethanol on carotenoid yields of M3 and BE1 (c). Specific growth rates of M3 and BE1 under different concentrations of ethanol (d). These strains were, respectively, grown in the YPM media at 30 °C for 96 h. Error bars represent standard deviations of three replicates. Two‑tailed Student’s t test was carried out, ns (not significant) (Additional file  1: Figure S5). This change of morphol - carotenoids yield, we performed a transcriptome analy- ogy might be responsible for the promoted carotenoid sis of BL03-D-4 and M3. For the transcriptome analy- accumulation. sis, strains were cultured in YPM media without the addition of ethanol, and cells were collected after 24  h. Transcriptome analysis of evolved strain M3 We defined the gene sets that showed greater than two - To gain insights into the phenotypic changes that fold differences in expression levels between BL03-D-4 were generated during the ALE process to improved and M3. There were 37 up-regulated genes and 162 Su  et al. Bioresour. Bioprocess. (2021) 8:47 Page 7 of 10 Fig. 4 Differentially expressed genes generated by transcriptome analysis in YPM media after 24 h of incubation. a Volcano plot of the differentially expressed genes in M3. The horizontal coordinate represented the fold change, and the vertical coordinate represented a significant difference Padjust (logarithmic transformation at the base of 10). Red dots represented up‑regulated genes, and green dots represented down‑regulated genes. b, c Histogram of GO enrichment and KEGG pathway enrichment analysis. The vertical coordinate shows the enriched GO terms and the pathway names, and the horizontal coordinate represents the number of genes and Padjust of differentially expressed genes in the term and the pathway, respectively. d Overview of significantly changed genes in metabolic pathways. Pentose phosphate pathway (oxidative phase, yellow; non‑ oxidative phase, blue): GND, glucose 6‑phosphate dehydrogenase; 6PGL, 6‑phosphogluconolactonase; 6PGD, 6‑phosphogluconate dehydrogenase; TKL, transketolase; TAL, transaldolase. Glycolysis and gluconeogenesis (green): HXK, hexokinase; PGI, phosphoglucose isomerase; INO1, inositol‑3‑phosphate synthase; PFK, phosphofructokinase; FBP1, fructose 1,6‑bisphosphatase; FBPA, fructose ‑bisphosphate aldolase; GAPD: glyceraldehyde phosphate dehydrogenase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; ENO, enolase down-regulated genes in M3 compared with BL03-D-4 were down-regulated in this study (Fig. 4b). These results through differential expression analysis (Fig.  4a, full lists indicated that there might be a relationship between are available in Additional file  3). We also screened gene PFK1 mutation-improved carotenoid accumulation and functions using GO classification and KEGG enrich - the cell wall systems. ment and found enrichment in both up-regulated and down-regulated gene sets in M3 (Fig. 4). KEGG pathway Discussion enrichment analysis was executed to further explore the Currently, there are hundreds of genes involved in etha- cause of PFK1 mutation promoting carotenoids accumu- nol stress, including glycolysis, ethanol metabolism, lation (Fig.  4c). The differentially expressed genes were plasma membrane composition and cell wall biogenesis primarily enriched in the glycolysis/gluconeogenesis in S. cerevisiae (Ma and Liu 2010). Ethanol resistance pathway and the majority were down-regulated indicat- was a complex phenotype regulated by multiple genes, ing that the loss-of-function mutation of PFK1 might in addition to the molecular genetics for enhancing S. change G6P biosynthesis in S. cerevisiae and regulate the cerevisiae ethanol tolerance such as global transcription cell wall systems involved in carotenoid accumulation. machinery engineering (Alper et  al. 2006), transposon Furthermore, the GO enrichment analysis demonstrated mutation (Kim et al. 2011), genome shuffling (Snoek et al. that the differentially expressed genes were mainly 2015), and metabolic engineering (Lam et  al. 2014). The involved in cell wall organization, and almost all genes ALE was also used as a conventional approach to improve Su et al. Bioresour. Bioprocess. (2021) 8:47 Page 8 of 10 the ethanol tolerance (Voordeckers et al. 2015). Ma et al. pressure, the variation of membrane fluidity was the also found that many genes relating to cell wall composi- major way in S. cerevisiae (Wang et  al. 2018; Yang et  al. tion were vital for cell wall organization and most of them 2019). In this study, it might be the cell wall remodeling were down-regulated under ethanol pressure. They pro - that mainly stands up to ethanol stress. For cell wall bio- posed that cell wall structures might undergo significant genesis, many genes involved in cell wall organization remodeling processes in response to ethanol stress (Ma were down-regulated without ethanol pressure in M3 and Liu 2010). Furthermore, a crucial factor for the ratio (Additional file  1: Table  S3). It is worth mentioning that of glucan and mannan in the walls could be the direction thickened cell walls and larger yeast were observed in the of the glucose 6-phosphate/mannose 6-phosphate inter- micafungin resistant yeast (Li et  al. 2014). Furthermore, conversion (Kratky et al. 1975). secretory pathways transported cell wall proteins onto As described above, the loss-of-function mutation of the plasma membrane, as well as transferring lipids, via PFK1 was identified in M3. Previous researches showed vesicles, to repair membrane destruction under ethanol that PFK1 was involved in glycolysis and gluconeogen- stress and they might contribute to ethanol tolerance esis (Tripodi et al. 2015). In E. coli, two isoenzymes (pfkA in Kluyveromyces marxianus (Mo et  al. 2019). Changes and pfkB) referred to the phosphofructokinase and pfkA of the cell wall might be responsible for the change of was considered to be the key enzyme accounting for membranes, which further affected the storage ability of the conversion of G6P to FBP. The deletion of pfkA pro - those fat-soluble carotenoids. The strategy based on this longed lag phase, impaired both cell growth and acetate probable mechanism could supplement the previously overflow, accumulated G6P, relieved glucose catabolite reported approaches about improving carotenoid yield in repression, and alleviated the Embden–Meyerhof path- S. cerevisiae. way (EMP) repression on gluconeogenesis. The glycolytic flux redistribution resulted in metabolic burdens, cofac - Conclusions tor imbalances, and decreasing carbon yield (Hollinshead In conclusion, a new ethanol-induced ALE was success- et  al. 2016). Similarly, it was reported that the EMP was fully applied to improve carotenoid yield in engineered replaced (deletion of PGI) with the Entner–Doudoroff S. cerevisiae and a hyper-producer was isolated from pathway (EDP) and oxidative PPP to boost isoprenoid evolution with a 5.1-fold increase in carotenoid yield. biosynthesis, along with overexpression of zwf and pgl The loss-of-function mutation of PFK1 was revealed as genes, leading to a 104% squalene increase in E. coli (Xu being the cause of increased carotenoid yield through et  al. 2019). However, there is little reference to PFK1 whole-genome resequencing and reverse engineering. mutation in S. cerevisiae. Transcriptomic analysis revealed the strengthening of To clarify the effect of the inactivation of PFK1 on gluconeogenesis and downregulation of cell wall-related cell metabolism, we screened the genes whose expres- genes, as a potential perturbation for the improvement of sion levels were dramatically changed in M3. As shown carotenoid yield. This study provided a classic case where in Fig.  4d and Additional file  1: Table  S3, M3 generally the appropriate selective pressure could be employed showed lower expression levels in the PPP and cell wall- to improve carotenoid yield using ALE and identified a related genes than BL03-D-4. NADPH generation was novel metabolic engineering target PFK1 for carotenoid highly reliant on the oxidative PP pathway. The meta - production in S. cerevisiae. bolic flux toward the oxidative PPP was always limited due to the rigid glycolysis flux in S. cerevisiae (Minard and McAlister-Henn 2005; Zampar et  al. 2013). There - Abbreviations ARTP: Atmospheric and room temperature plasma; ALE: Adaptive laboratory fore, the increase of G6P fluxes toward the oxidative PPP evolution; PFK1: Phosphofructokinase 1; YPM: Modified YPD medium; TMS: through glucose-6-phosphate dehydrogenase (G6PD), Trace metal solution; GSA: Genome Sequence Archive; CDW: Cell dry weight; instead of glycolysis, was necessary for efficient NADPH SNPs: Single nucleotide polymorphisms; INDEL: Insertion and deletions; PPP: Pentose phosphate pathway; G6P: Glucose‑6‑phosphate; FBP: Fructose production and enhanced production of isoprenoids 1,6‑bisphosphatase; EMP: Embden–Meyerhof pathway; EDP: Entner–Doudor ‑ (Kwak et al. 2020). Similarly, efficient carotenoid biosyn - off pathway; G6PD: Glucose ‑6‑phosphate dehydrogenase. thesis needed NADPH providing reducing power (Hong et  al. 2019; Zhao et  al. 2015). Surprisingly, and in con- Supplementary Information trast, the first and second steps of the oxidative PPP were The online version contains supplementary material available at https:// doi. significantly down-regulated in M3, which presented a org/ 10. 1186/ s40643‑ 021‑ 00402‑5. remarkable improvement of carotenoid yield in our study (Fig.  4d). This result suggested that the loss-of-function Additional file 1: Table S1. Strains used in this study. Table S2. Primers used in this study. Table S3. Differential expression of cell wall‐related mutation of PFK1 might contribute to a new mechanism genes. Figure S1. BL03‑D ‑4 and M3 on YPD plates. Figure S2. Shake ‑flask for improving carotenoid yield. For resisting the ethanol Su  et al. Bioresour. Bioprocess. (2021) 8:47 Page 9 of 10 Guo L, Pang Z, Gao C, Chen X, Liu L (2020) Engineering microbial cell morphol‑ fermentations of BL03‑D ‑4 and M3 in YPM medium with different concen‑ ogy and membrane homeostasis toward industrial applications. Curr tration of ethanol. Figure S3. Shake‑flask fermentations of BL03‑D ‑4, M3 Opin Biotechnol 66:18–26. https:// doi. org/ 10. 1016/j. copbio. 2020. 05. 004 and BE1 in YPM medium and YPD medium. Figure S4. Colony morphol‑ Hollinshead WD, Rodriguez S, Martin HG, Wang G, Baidoo EE, Sale KL, Keasling ogy of BL03‑D ‑4, M3, BE1 and BE2. Figure S5 Morphology observation of JD, Mukhopadhyay A, Tang YJ (2016) Examining Escherichia coli glycolytic BL03‑D ‑4, M3, BE1 and BE2. pathways, catabolite repression, and metabolite channeling using Delta pfk mutants. Biotechnol Biofuels 9:212. https:// doi. org/ 10. 1186/ Additional file 2: Full mutation lists of SNPs and INDEL. s13068‑ 016‑ 0630‑y Additional file 3: Differential expression analysis. Hong J, Park SH, Kim S, Kim SW, Hahn JS (2019) Efficient production of lyco ‑ pene in Saccharomyces cerevisiae by enzyme engineering and increasing membrane flexibility and NAPDH production. Appl Microbiol Biotechnol Acknowledgements 103(1):211–223. https:// doi. org/ 10. 1007/ s00253‑ 018‑ 9449‑8 Not applicable. Jiang G, Yang Z, Wang Y, Yao M, Chen Y, Xiao W, Yuan Y (2020) Enhanced asta‑ xanthin production in yeast via combined mutagenesis and evolution. Authors’ contributions Biochem Eng J 156:107519. https:// doi. org/ 10. 1016/j. bej. 2020. 107519 BS conceived and designed the research. BS conducted the experiments. BS Jin J, Wang Y, Yao M, Gu X, Li B, Liu H, Ding M, Xiao W, Yuan Y (2018) Astax‑ wrote the manuscript. AL revised the manuscript. M‑RD and HZ supervised anthin overproduction in yeast by strain engineering and new gene the study. All authors have read and approved the manuscript. target uncovering. Biotechnol Biofuels 11:230. https:// doi. org/ 10. 1186/ s13068‑ 018‑ 1227‑4 Funding Kim HS, Kim NR, Yang J, Choi W (2011) Identification of novel genes responsi‑ This work is financially supported by the Key‑Area Research and Development ble for ethanol and/or thermotolerance by transposon mutagenesis in Program of Guangdong Province (Grant No. 2018B020206001) and the Sci‑ Saccharomyces cerevisiae. Appl Microbiol Biotechnol 91(4):1159–1172. ence and Technology Plan Project of Guangdong Province (2016A010105013, https:// doi. org/ 10. 1007/ s00253‑ 011‑ 3298‑z 2019B030316017). Kratky Z, Biely P, Bauer S (1975) Mechanism of 2‑ deoxy‑D ‑ glucose inhibition of cell‑ wall polysaccharide and glycoprotein biosyntheses in Saccharomyces Availability of data and materials cerevisiae. Eur J Biochem 54(2):459–467. https:// doi. org/ 10. 1111/j. 1432‑ Fastq DNA‑seq raw data were deposited in the Genome Sequence Archive 1033. 1975. tb041 57.x (GSA) server at the BIG Data Center in Beijing Institute of Genomics (http:// Kwak S, Yun EJ, Lane S, Oh EJ, Kim KH, Jin YS (2020) Redirection of the glyco‑ bigd. big. ac. cn, GSA accession No. CRA003704), RNA‑seq raw data were lytic flux enhances isoprenoid production in Saccharomyces cerevisiae. deposited into NCBI (GEO accession number GSE164470). The dataset gener‑ Biotechnol J 15(2):e1900173. https:// doi. org/ 10. 1002/ biot. 20190 0173 ated during and/or analyzed during the current study are available from the Lam FH, Ghaderi A, Fink GR, Stephanopoulos G (2014) Biofuels. Engineering corresponding author on reasonable request. Strain M3 was deposited at alcohol tolerance in yeast. Science 346(6205):71–75. https:// doi. org/ 10. Guangdong Microbial Culture Collection Center (GDMCC No. 61336). The 1126/ scien ce. 12578 59 materials that support the findings of this study are available from the cor ‑ Lee S, Kim P (2020) Current status and applications of adaptive laboratory evo‑ responding author on request. lution in industrial microorganisms. J Microbiol Biotechnol 30(6):793–803. https:// doi. org/ 10. 4014/ jmb. 2003. 03072 Li X‑E, Wang J‑ J, Phornsanthia S, Yin X, Li Q (2014) Strengthening of cell wall Declarations structure enhances stress resistance and fermentation performance in lager yeast. J Am Soc Brew Chem 72(2):88–94 Ethical approval and consent to participate Ma M, Liu ZL (2010) Mechanisms of ethanol tolerance in Saccharomyces Not applicable. cerevisiae. Appl Microbiol Biotechnol 87(3):829–845. https:// doi. org/ 10. 1007/ s00253‑ 010‑ 2594‑3 Consent for publication Ma T, Shi B, Ye Z, Li X, Liu M, Chen Y, Xia J, Nielsen J, Deng Z, Liu T (2019) Lipid Not applicable. engineering combined with systematic metabolic engineering of Sac- charomyces cerevisiae for high‑ yield production of lycopene. Metab Eng Competing interests 52:134–142. https:// doi. org/ 10. 1016/j. ymben. 2018. 11. 009 The authors declare that they have no competing interests. Minard KI, McAlister‑Henn L (2005) Sources of NADPH in yeast vary with car ‑ bon source. J Biol Chem 280(48):39890–39896. https:// doi. org/ 10. 1074/ Received: 27 March 2021 Accepted: 3 June 2021 jbc. M5094 61200 Mo W, Wang M, Zhan R, Yu Y, He Y, Lu H (2019) Kluyveromyces marxianus developing ethanol tolerance during adaptive evolution with significant improvements of multiple pathways. 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Identification of a novel metabolic engineering target for carotenoid production in Saccharomyces cerevisiae via ethanol-induced adaptive laboratory evolution

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Abstract

Carotenoids are a large family of health‑beneficial compounds that have been widely used in the food and nutra‑ ceutical industries. There have been extensive studies to engineer Saccharomyces cerevisiae for the production of carotenoids, which already gained high level. However, it was difficult to discover new targets that were relevant to the accumulation of carotenoids. Herein, a new, ethanol‑induced adaptive laboratory evolution was applied to boost carotenoid accumulation in a carotenoid producer BL03‑D ‑4, subsequently, an evolved strain M3 was obtained with a 5.1‑fold increase in carotenoid yield. Through whole ‑ genome resequencing and reverse engineering, loss‑ of‑function mutation of phosphofructokinase 1 (PFK1) was revealed as the major cause of increased carotenoid yield. Transcrip‑ tome analysis was conducted to reveal the potential mechanisms for improved yield, and strengthening of gluconeo‑ genesis and downregulation of cell wall‑related genes were observed in M3. This study provided a classic case where the appropriate selective pressure could be employed to improve carotenoid yield using adaptive evolution and elucidated the causal mutation of evolved strain. Keywords: Saccharomyces cerevisiae, Adaptive laboratory evolution, Carotenoid, PFK1, Reverse engineering, Cell wall Introduction carotenoid yield in S. cerevisiae (Wang et  al. 2019). Carotenoids are a large kind of isoprenoid pigment Many strategies have been applied and high levels that can be used in many fields such as colorants, of yields were obtained (Chen et  al. 2016; Hong et  al. antioxidants, and nutrients (Saini and Keum 2019). 2019; Shi et al. 2019; Xie et al. 2015), while the highest Currently, carotenoids are produced by chemical syn- yield of 73 mg/g CDW was achieved through lipid engi- thesis or extraction from natural sources, which are neering (Ma et  al. 2019). However, it was difficult to mostly restricted by complicated structures and short- discover new targets that were relevant to the heterolo- age of raw materials (Mussagy et  al. 2019). Wild-type gous production of carotenoids and, furthermore pro- Saccharomyces cerevisiae does not normally accu- mote the production of carotenoids in recombinant S. mulate carotenoids but engineered strains, obtained cerevisiae based on previous studies. Fortunately, ran- through rational engineering, have achieved enhanced dom disturbance in the genes at a whole-genome scale (through mutagenesis or evolution) provided a choice for acquiring a carotenoid hyper-producer (Zhu et  al. *Correspondence: dengmr@gdim.cn; zhuhh@gdim.cn 2018). For instance, using a single atmospheric and Guangdong Microbial Culture Collection Center (GDMCC), Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, room temperature plasma (ARTP) treatment, astaxan- State Key Laboratory of Applied Microbiology Southern China, Institute thin yield was improved by 0.83-fold over the parental of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, strain and three potential targets related to astaxanthin People’s Republic of China © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://crea‑ tivecommons.org/licenses/by/4.0/. Su et al. Bioresour. Bioprocess. (2021) 8:47 Page 2 of 10 biosynthesis in yeast were revealed (Jin et  al. 2018). cell wall-related genes were identified as the likely regula - Furthermore, a combined strategy composing physical tion that resulted in increased carotenoid yield. mutagenesis by ARTP and adaptive laboratory evolu- tion (ALE), driven by hydrogen peroxide, was executed Material and methods and the titer of astaxanthin was increased nearly four- Microorganisms and growth conditions fold ( Jiang et al. 2020). S. cerevisiae strain BL03-D-4, derived from BY4742 ALE is a widely used technology to achieve insights (MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0), was chosen into the mechanisms of adaptive mutations that accu- as the parental strain. All S. cerevisiae strains used in mulate under designed culture conditions for long peri- this study are listed in Additional file  1: Table  S1. Prim- ods of screening (Dragosits and Mattanovich 2013). ers are provided in Additional file  1: Table  S2. For culti- ALE has been proven to be a potent tool in metabolic vation, a single colony was picked up from a fresh YPD engineering, both for the revelation of new design prin- plate and transferred to a 5 mL YPD medium. After being ciples and the engineering of excellent strains (Zhu cultured at 200  rpm and 30  °C, 1  mL seed culture was et  al. 2018). Many superior phenotypes have been inoculated into a 250  mL shake-flask with 50  mL YPD achieved through ALE, such as improvement of the medium, or modified YPD medium (YPM) to an opti - growth rate and production, the adaption of strains to cal density (OD ) of about 0.05 and then incubated at utilize non-native substrates, and enhancement of the 30  °C for 24 to 96  h. After incubation, the cultures were resistance towards environmental stresses (Sandberg analyzed for biomass and carotenoid content. The YPD −1 −1 et  al. 2019). Furthermore, the relationship between medium contained 20  g L tryptone, 20  g L glucose, −1 genomic changes and the excellent phenotype could be and 10  g L yeast extract (Oxoid, LOTs of 2,665,431– −1 uncovered by whole-genome resequencing combined 02). YPM medium contained 20  g L tryptone, 20  g −1 −1 with systems biology (Yang et  al. 2019). For example, L glucose, 10 g L yeast extract (Angel, FM802, LOT: −1 −1 ALE has been applied to adapting hydrogen perox- 2018082210C9), salt (10 g L KH PO , 2.5 g L MgSO , 2 4 4 −1 −1 ide-tolerant yeast to improve carotenoids production 3.5 g L K SO , 0.25 g L Na SO ) and 1 mL trace metal 2 4 2 4 (Reyes et  al. 2014), and the beneficial mutations led to solution (TMS) described in previous literature (Su et al. increased yield have been revealed (Godara et al. 2019). 2020b). Compared to random mutagenesis approaches such as ARTP and ultraviolet light, ALE with sequential pas- Adaptive laboratory evolution experiments sages is a reasonably easy technique for confirming Sequential passages and batch cultures were performed crucial mutations related to the beneficial phenotype using the parental strain (BL03-D-4) and YPD media due to its low mutation probability. Furthermore, the with different concentrations of ethanol under the cul - production of the target product could be improved by ture conditions illustrated in Fig. 1. Cells were inoculated introducing the identified mutations into an engineered into 50 mL YPD medium at OD of 0.05 to maintain the producer (Lee and Kim 2020). growth phase. After each cultivation, cells with appropri- ALE used suitable stress as a driving force for screening ate concentrations were plated on YPD plates for single mutants with superior phenotypes. Because carotenoid colony isolation, based on cell color. The single colonies yield was facilitated by engineering microbial membranes obtained were inoculated in the YPD medium for further which could also increase ethanol tolerance (Guo et  al. screening. 2020; Hong et  al. 2019), we hypothesized that ethanol stress might be used as a driving force for the adaptation Whole‑genome resequencing and transcriptome analysis of yeast for increased carotenoids accumulation. As a The strains chosen for whole-genome resequencing were proof of concept, ethanol-induced ALE was applied to a cultivated in 50 mL YPD medium at 30 °C in a shaker at recently constructed S. cerevisiae strain, BL03-D-04, har- 200  rpm for 24  h. Genomic DNA was extracted accord- boring the carotenoid synthetic pathway (Su et al. 2020b). ing to the manufacturer’s protocol using the HiPure Yeast Improvement of carotenoid yield was obtained through DNA Kit (Magen, Guangzhou, China). At least 5  μg of this novel ALE strategy to screen for higher carotenoid each genomic DNA sample was provided to Shanghai producers. The underlying cause related to the promo - Majorbio Bio-pharm Technology Co. Ltd, for sequenc- tion of carotenoids accumulation in the evolved strain ing using the Illumina HiSeq 2000 platform. Paired-end were assessed using whole-genome resequencing and reads of ~ 250 bp were generated. The average sequencing transcriptome analysis. The inactivation of phosphofruc - depths of the samples were 70 to 90. Fastq DNA-seq raw tokinase 1 (PFK1) was determined as the causal mutation data were deposited in the Genome Sequence Archive of the evolved strain with improved carotenoid yield, and (GSA) server at the BIG Data Center (http:// bigd. big. ac. strengthening of gluconeogenesis and downregulation of cn, GSA accession No. CRA003704). Su  et al. Bioresour. Bioprocess. (2021) 8:47 Page 3 of 10 Fig. 1 Schematic diagram of the ethanol‑induced adaptive laboratory evolution. Gradually increasing (from 2 to 12% or from 4 to 12%) and specific concentrations (2%, 4%, 8%, and 10%) of ethanol were selected as the driving forces for the ALE. After screening and analytical certification, the excellent strain will be turned into another round of evolution. The mutations of excellent strain were uncovered through whole‑ genome resequencing and then parental strain was reverse engineered to gain a rationally designed strain Total RNA from yeast cells was extracted according diagnostic PCR using paired primers PFK1-F-2/PFK1- to our previous work (Su et  al. 2020a). The concentra - CHECK-R and PFK2-F-2/PFK2-CHECK-R, respectively. tions and quality of the RNA samples were examined by Biospec-nano (Shimadzu, Kyoto, Japan). 1  μg total RNA Cell dry weight, specific growth rate and carotenoid sample was used for mRNA library preparation and RNA quantification sequencing (Illumina HiSeq), performed by Shanghai Cell dry weight (CDW) was determined according to the Majorbio Bio-pharm Technology Co. Ltd. Fastq RNA- OD value and correlated to CDW by the CDW/OD 600 600 seq raw data were deposited into NCBI (GEO accession standard curve [y = 0.184x + 0.891 (x is OD , y is CDW, number GSE164470). Data processing was accomplished R = 0.992]. The specific growth rate (μ) was calculated by by the online Majorbio Cloud Platform (www. major bio. the equation, μ = (ln X2 − ln X1)/(t2 − t1), where X1 and com). X2 are the biomasses at time t1 and t2, respectively. For carotenoid quantification, strain culture after fer - Gene deletions mentation was transferred to a 1.5-mL sample tube and Auxotroph marker HIS3 including a promoter and termi- the cells were collected by centrifugation at 12,000  rpm nator, was amplified from the genomic DNA of S. cerevi- for 5  min. The sedimentary cells were disrupted using −1 siae S288C. The homologous arm (~ 50 bp) was designed 3 mol L HCl and boiling for 4 min. Then, cell debris was in primers. For deletions of target genes, one-step inte- washed thrice by sterile water, resuspended in acetone for gration of PCR-amplified deletion cassettes, including extraction, and centrifuged at 12,000 rpm for 2 min. The HIS3, was adopted (Chen et  al. 2016). Primers PFK1-F supernatant was transferred to a new tube for quantifica - and PFK1-R were used for amplification of HIS3 from tion of total carotenoids using a UV/Vis spectrometer at genomic DNA of S. cerevisiae S288C. Primers PFK1-F-2 470 nm. The extinction coefficient was adopted using an and PFK1-R-2 were used for the addition of homologous A 1% 1 cm of 3450 (Su et al. 2020b). arms targeted to PFK1. Primers PFK2-F and PFK2-R were used for amplification of HIS3 from genomic DNA Morphological observation of S. cerevisiae S288C. Primers PFK2-F-2 and PFK2-R-2 The morphology of the strains was observed by a light were used for the addition of homologous arms targeted microscope (DM6/MC190, Leica). In brief, the strains to PFK2. PFK1 and PFK2 deletions were verified by were cultivated in 50 mL YPD medium at 30 °C for 48 h, Su et al. Bioresour. Bioprocess. (2021) 8:47 Page 4 of 10 followed by centrifuging and washing with PBS three genetic mutations and systematic transcriptional regula- times. They were then observed through the microscope. tion, which needs to be further investigated. Results Whole‑genome resequencing of the evolved strain M3 Adaptive laboratory evolution of BL03‑D‑4 under ethanol To identify the genetic basis of the improved carot- stress enoids yield, whole-genome resequencing of strain Because the heterologous accumulation of carotenoids BL03-D-4 and M3 was performed using the Illumina in S. cerevisiae poses a metabolic burden on host cells, a HiSeq platform through paired-end sequencing. Cov- suitable pressure was needed to endow the higher pro- erage depth was approximately 70 to 90 reads. Overall, ducers a striking feature (Reyes et al. 2014). Based on the 14 single nucleotide polymorphisms (SNPs) and 100 already known mechanisms of ethanol tolerance (Ma and insertion and deletions (INDEL) were detected in M3, Liu 2010), we hypothesized that yeast strain would pre- compared to BL03-D-4 after the evolution process (full sent different levels of carotenoids yield with the fluctu - mutation lists are available in Additional file  2). The ant composition of the membrane under ethanol stress, resequencing results suggested that M3 had acquired a and there might be some status fitted to the carotenoids loss-of-function mutation of PFK1 since M3 generated accumulation. Thus, in this study, an ethanol-induced a stop codon in the front part of the PFK1 gene, which ALE strategy was developed for improving carotenoid encoded 6-phosphofructokinase 1. It has been reported yield. For the ALE experiment, sequential and batch cul- that PFK1 was involved in glycolysis and gluconeogen- tures were performed in YPD medium supplemented esis which was related to NADPH generation (Kwak et al. with different concentrations of ethanol (v/v) (Fig.  1). 2020). Furthermore, the E. coli central metabolic network When the cell growth reached a plateau, the ALE process was rewired after deletion of pfkA which indicated that was terminated and the evolved cells were plated on YPD reduced glycolysis by weakening 6-phosphofructokinase plates without ethanol to screen darkened colonies. Sub- had a profound effect on metabolism (Hollinshead et  al. sequently, an evolved strain M3 (deposited at Guangdong 2016). Thus, combined with the genome resequencing Microbial Culture Collection Center with GDMCC No. results, we speculated that the loss-of-function mutation 61336) was obtained in the sequential cultures, through a of PFK1 in M3 was related to the promoted carotenoid range of adaptive experiments with gradually rising con- yield. centrations of ethanol (the first horizontal panel of Fig.  1 at 10% after three subcultures) based on darkened cell Determining causal mutation color (Additional file  1: Figure S1). Furthermore, M3 was To determine whether the loss-of-function mutation also turned into another round of evolution. However, no of PFK1 confers the improvement of carotenoids yield, better strain was obtained using the above ALE strategy. the inactivation of PFK1 was reverse-engineered into Shake-flask fermentation showed that the carotenoid BL03-D-4. The reverse-engineered strain BE1 showed yield of M3 increased about 5.1- and 2.4-fold compared remarkably increased carotenoid yield relative to BL03- with BL03-D-4 in YPD and YPM media, respectively D-4, which was not significantly different compared to (Fig. 2a). After 96 h of incubation, the carotenoid yield of M3 (Fig.  3) without ethanol or under different concen - M3 reached 42.4 mg/g CDW in the YPM medium (YPM trations of ethanol, fully recovering the evolved carot- medium facilitated carotenoid accumulation (Su et  al. enoids phenotype (Additional file  1: Figure S3). BE1 2021)). The specific growth rate of strain M3 increased represented a similar colonial morphology compared markedly, compared to that of the parental strain BL03- with M3, but with a different morphology to parental D-4 in the concentration from 2 to 8% ethanol (Fig.  2b). strain BL03-D-4 (Additional file  1: Figure S1 and S4). Both strains grew poor in the presence of 10% etha- Deletion of PFK1 had little effect on the growth of nol (Additional file  1: Figure S2). We also observed that strain BE1 without ethanol or under different concen - the additional supplement of ethanol had a remarkably tration of ethanol (Fig.  3), these results suggested that repressive effect on the carotenoid yield of M3 especially mutations other than PFK1 mutation contributed to under a high concentration of ethanol (Fig.  2c). How- the normal growth of M3 and counteracted the nega- ever, there was no significant difference in cell growth tive effect of the loss-of-function mutation of PFK1 . between BL03-D-4 and M3 during the cultivation with- Since there was an isoenzyme (PFK2) of PFK1 in S. cer- out ethanol (Fig. 2d), indicating that the ALE process had evisiae, BL03-D-4 was engineered with the deletion of no significant influence on the biomass of M3. Therefore, PFK2 to generate BE2 to test whether PFK2 mutation the improvement of carotenoids yield in M3 was not due has any similar effect on the pentose phosphate path - to facilitating the strain growth, but was probably con- way (PPP). As shown in Fig.  3b, PFK2 deletion exerted cerned with the change of metabolic pathway involved in a severe suppression on the growth of BE2 and no Su  et al. Bioresour. Bioprocess. (2021) 8:47 Page 5 of 10 Fig. 2 Characterization of parental (BL03‑D ‑4) and evolved (M3) strains. Carotenoid yields and biomasses of BL03‑D ‑4 and M3 in YPD and YPM media after 96 h of incubation (a). Specific growth rates of BL03‑D ‑4 and M3 under different concentrations of ethanol (b). Eec ff t of different concentrations of ethanol on carotenoid yields of BL03‑D ‑4 and M3 (c). Growth curves of BL03‑D ‑4 and M3 in YPM media without ethanol stress (d). Error bars represent standard deviations of three replicates increase of carotenoids was detected in this strain after (FBP) in S. cerevisiae which was different compared to 96 h fermentation. However, BE2 represented a similar E. coli (Hollinshead et  al. 2016). Morphology observa- colonial morphology compared with M3 and BE1 after tion of BL03-D-4, M3, BE1, and BE2 was carried out long cultivation (about 8  days) (Additional file  1: Fig- through a microscope at 1000 × magnification. These ure S4). These results suggested that PFK2 might be the pictures showed that evolved (M3) and knockout key enzyme responsible for the conversion of glucose- strains (BE1 and BE2) presented a larger size of mor- 6-phosphate (G6D) to fructose 1,6-bisphosphatase phology compared to the parental strain (BL03-D-4) Su et al. Bioresour. Bioprocess. (2021) 8:47 Page 6 of 10 Fig. 3 Carotenoid yields (a) of evolved (M3), and knockout strains (BE1 and BE2), and growth patterns (b) of M3, BE1, and BE2. Eec ff t of different concentrations of ethanol on carotenoid yields of M3 and BE1 (c). Specific growth rates of M3 and BE1 under different concentrations of ethanol (d). These strains were, respectively, grown in the YPM media at 30 °C for 96 h. Error bars represent standard deviations of three replicates. Two‑tailed Student’s t test was carried out, ns (not significant) (Additional file  1: Figure S5). This change of morphol - carotenoids yield, we performed a transcriptome analy- ogy might be responsible for the promoted carotenoid sis of BL03-D-4 and M3. For the transcriptome analy- accumulation. sis, strains were cultured in YPM media without the addition of ethanol, and cells were collected after 24  h. Transcriptome analysis of evolved strain M3 We defined the gene sets that showed greater than two - To gain insights into the phenotypic changes that fold differences in expression levels between BL03-D-4 were generated during the ALE process to improved and M3. There were 37 up-regulated genes and 162 Su  et al. Bioresour. Bioprocess. (2021) 8:47 Page 7 of 10 Fig. 4 Differentially expressed genes generated by transcriptome analysis in YPM media after 24 h of incubation. a Volcano plot of the differentially expressed genes in M3. The horizontal coordinate represented the fold change, and the vertical coordinate represented a significant difference Padjust (logarithmic transformation at the base of 10). Red dots represented up‑regulated genes, and green dots represented down‑regulated genes. b, c Histogram of GO enrichment and KEGG pathway enrichment analysis. The vertical coordinate shows the enriched GO terms and the pathway names, and the horizontal coordinate represents the number of genes and Padjust of differentially expressed genes in the term and the pathway, respectively. d Overview of significantly changed genes in metabolic pathways. Pentose phosphate pathway (oxidative phase, yellow; non‑ oxidative phase, blue): GND, glucose 6‑phosphate dehydrogenase; 6PGL, 6‑phosphogluconolactonase; 6PGD, 6‑phosphogluconate dehydrogenase; TKL, transketolase; TAL, transaldolase. Glycolysis and gluconeogenesis (green): HXK, hexokinase; PGI, phosphoglucose isomerase; INO1, inositol‑3‑phosphate synthase; PFK, phosphofructokinase; FBP1, fructose 1,6‑bisphosphatase; FBPA, fructose ‑bisphosphate aldolase; GAPD: glyceraldehyde phosphate dehydrogenase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; ENO, enolase down-regulated genes in M3 compared with BL03-D-4 were down-regulated in this study (Fig. 4b). These results through differential expression analysis (Fig.  4a, full lists indicated that there might be a relationship between are available in Additional file  3). We also screened gene PFK1 mutation-improved carotenoid accumulation and functions using GO classification and KEGG enrich - the cell wall systems. ment and found enrichment in both up-regulated and down-regulated gene sets in M3 (Fig. 4). KEGG pathway Discussion enrichment analysis was executed to further explore the Currently, there are hundreds of genes involved in etha- cause of PFK1 mutation promoting carotenoids accumu- nol stress, including glycolysis, ethanol metabolism, lation (Fig.  4c). The differentially expressed genes were plasma membrane composition and cell wall biogenesis primarily enriched in the glycolysis/gluconeogenesis in S. cerevisiae (Ma and Liu 2010). Ethanol resistance pathway and the majority were down-regulated indicat- was a complex phenotype regulated by multiple genes, ing that the loss-of-function mutation of PFK1 might in addition to the molecular genetics for enhancing S. change G6P biosynthesis in S. cerevisiae and regulate the cerevisiae ethanol tolerance such as global transcription cell wall systems involved in carotenoid accumulation. machinery engineering (Alper et  al. 2006), transposon Furthermore, the GO enrichment analysis demonstrated mutation (Kim et al. 2011), genome shuffling (Snoek et al. that the differentially expressed genes were mainly 2015), and metabolic engineering (Lam et  al. 2014). The involved in cell wall organization, and almost all genes ALE was also used as a conventional approach to improve Su et al. Bioresour. Bioprocess. (2021) 8:47 Page 8 of 10 the ethanol tolerance (Voordeckers et al. 2015). Ma et al. pressure, the variation of membrane fluidity was the also found that many genes relating to cell wall composi- major way in S. cerevisiae (Wang et  al. 2018; Yang et  al. tion were vital for cell wall organization and most of them 2019). In this study, it might be the cell wall remodeling were down-regulated under ethanol pressure. They pro - that mainly stands up to ethanol stress. For cell wall bio- posed that cell wall structures might undergo significant genesis, many genes involved in cell wall organization remodeling processes in response to ethanol stress (Ma were down-regulated without ethanol pressure in M3 and Liu 2010). Furthermore, a crucial factor for the ratio (Additional file  1: Table  S3). It is worth mentioning that of glucan and mannan in the walls could be the direction thickened cell walls and larger yeast were observed in the of the glucose 6-phosphate/mannose 6-phosphate inter- micafungin resistant yeast (Li et  al. 2014). Furthermore, conversion (Kratky et al. 1975). secretory pathways transported cell wall proteins onto As described above, the loss-of-function mutation of the plasma membrane, as well as transferring lipids, via PFK1 was identified in M3. Previous researches showed vesicles, to repair membrane destruction under ethanol that PFK1 was involved in glycolysis and gluconeogen- stress and they might contribute to ethanol tolerance esis (Tripodi et al. 2015). In E. coli, two isoenzymes (pfkA in Kluyveromyces marxianus (Mo et  al. 2019). Changes and pfkB) referred to the phosphofructokinase and pfkA of the cell wall might be responsible for the change of was considered to be the key enzyme accounting for membranes, which further affected the storage ability of the conversion of G6P to FBP. The deletion of pfkA pro - those fat-soluble carotenoids. The strategy based on this longed lag phase, impaired both cell growth and acetate probable mechanism could supplement the previously overflow, accumulated G6P, relieved glucose catabolite reported approaches about improving carotenoid yield in repression, and alleviated the Embden–Meyerhof path- S. cerevisiae. way (EMP) repression on gluconeogenesis. The glycolytic flux redistribution resulted in metabolic burdens, cofac - Conclusions tor imbalances, and decreasing carbon yield (Hollinshead In conclusion, a new ethanol-induced ALE was success- et  al. 2016). Similarly, it was reported that the EMP was fully applied to improve carotenoid yield in engineered replaced (deletion of PGI) with the Entner–Doudoroff S. cerevisiae and a hyper-producer was isolated from pathway (EDP) and oxidative PPP to boost isoprenoid evolution with a 5.1-fold increase in carotenoid yield. biosynthesis, along with overexpression of zwf and pgl The loss-of-function mutation of PFK1 was revealed as genes, leading to a 104% squalene increase in E. coli (Xu being the cause of increased carotenoid yield through et  al. 2019). However, there is little reference to PFK1 whole-genome resequencing and reverse engineering. mutation in S. cerevisiae. Transcriptomic analysis revealed the strengthening of To clarify the effect of the inactivation of PFK1 on gluconeogenesis and downregulation of cell wall-related cell metabolism, we screened the genes whose expres- genes, as a potential perturbation for the improvement of sion levels were dramatically changed in M3. As shown carotenoid yield. This study provided a classic case where in Fig.  4d and Additional file  1: Table  S3, M3 generally the appropriate selective pressure could be employed showed lower expression levels in the PPP and cell wall- to improve carotenoid yield using ALE and identified a related genes than BL03-D-4. NADPH generation was novel metabolic engineering target PFK1 for carotenoid highly reliant on the oxidative PP pathway. The meta - production in S. cerevisiae. bolic flux toward the oxidative PPP was always limited due to the rigid glycolysis flux in S. cerevisiae (Minard and McAlister-Henn 2005; Zampar et  al. 2013). There - Abbreviations ARTP: Atmospheric and room temperature plasma; ALE: Adaptive laboratory fore, the increase of G6P fluxes toward the oxidative PPP evolution; PFK1: Phosphofructokinase 1; YPM: Modified YPD medium; TMS: through glucose-6-phosphate dehydrogenase (G6PD), Trace metal solution; GSA: Genome Sequence Archive; CDW: Cell dry weight; instead of glycolysis, was necessary for efficient NADPH SNPs: Single nucleotide polymorphisms; INDEL: Insertion and deletions; PPP: Pentose phosphate pathway; G6P: Glucose‑6‑phosphate; FBP: Fructose production and enhanced production of isoprenoids 1,6‑bisphosphatase; EMP: Embden–Meyerhof pathway; EDP: Entner–Doudor ‑ (Kwak et al. 2020). Similarly, efficient carotenoid biosyn - off pathway; G6PD: Glucose ‑6‑phosphate dehydrogenase. thesis needed NADPH providing reducing power (Hong et  al. 2019; Zhao et  al. 2015). Surprisingly, and in con- Supplementary Information trast, the first and second steps of the oxidative PPP were The online version contains supplementary material available at https:// doi. significantly down-regulated in M3, which presented a org/ 10. 1186/ s40643‑ 021‑ 00402‑5. remarkable improvement of carotenoid yield in our study (Fig.  4d). This result suggested that the loss-of-function Additional file 1: Table S1. Strains used in this study. Table S2. Primers used in this study. Table S3. Differential expression of cell wall‐related mutation of PFK1 might contribute to a new mechanism genes. Figure S1. BL03‑D ‑4 and M3 on YPD plates. Figure S2. Shake ‑flask for improving carotenoid yield. For resisting the ethanol Su  et al. Bioresour. Bioprocess. (2021) 8:47 Page 9 of 10 Guo L, Pang Z, Gao C, Chen X, Liu L (2020) Engineering microbial cell morphol‑ fermentations of BL03‑D ‑4 and M3 in YPM medium with different concen‑ ogy and membrane homeostasis toward industrial applications. Curr tration of ethanol. Figure S3. Shake‑flask fermentations of BL03‑D ‑4, M3 Opin Biotechnol 66:18–26. https:// doi. org/ 10. 1016/j. copbio. 2020. 05. 004 and BE1 in YPM medium and YPD medium. Figure S4. Colony morphol‑ Hollinshead WD, Rodriguez S, Martin HG, Wang G, Baidoo EE, Sale KL, Keasling ogy of BL03‑D ‑4, M3, BE1 and BE2. Figure S5 Morphology observation of JD, Mukhopadhyay A, Tang YJ (2016) Examining Escherichia coli glycolytic BL03‑D ‑4, M3, BE1 and BE2. pathways, catabolite repression, and metabolite channeling using Delta pfk mutants. Biotechnol Biofuels 9:212. https:// doi. org/ 10. 1186/ Additional file 2: Full mutation lists of SNPs and INDEL. s13068‑ 016‑ 0630‑y Additional file 3: Differential expression analysis. Hong J, Park SH, Kim S, Kim SW, Hahn JS (2019) Efficient production of lyco ‑ pene in Saccharomyces cerevisiae by enzyme engineering and increasing membrane flexibility and NAPDH production. Appl Microbiol Biotechnol Acknowledgements 103(1):211–223. https:// doi. org/ 10. 1007/ s00253‑ 018‑ 9449‑8 Not applicable. Jiang G, Yang Z, Wang Y, Yao M, Chen Y, Xiao W, Yuan Y (2020) Enhanced asta‑ xanthin production in yeast via combined mutagenesis and evolution. Authors’ contributions Biochem Eng J 156:107519. https:// doi. org/ 10. 1016/j. bej. 2020. 107519 BS conceived and designed the research. BS conducted the experiments. BS Jin J, Wang Y, Yao M, Gu X, Li B, Liu H, Ding M, Xiao W, Yuan Y (2018) Astax‑ wrote the manuscript. AL revised the manuscript. M‑RD and HZ supervised anthin overproduction in yeast by strain engineering and new gene the study. All authors have read and approved the manuscript. target uncovering. Biotechnol Biofuels 11:230. https:// doi. org/ 10. 1186/ s13068‑ 018‑ 1227‑4 Funding Kim HS, Kim NR, Yang J, Choi W (2011) Identification of novel genes responsi‑ This work is financially supported by the Key‑Area Research and Development ble for ethanol and/or thermotolerance by transposon mutagenesis in Program of Guangdong Province (Grant No. 2018B020206001) and the Sci‑ Saccharomyces cerevisiae. Appl Microbiol Biotechnol 91(4):1159–1172. ence and Technology Plan Project of Guangdong Province (2016A010105013, https:// doi. org/ 10. 1007/ s00253‑ 011‑ 3298‑z 2019B030316017). Kratky Z, Biely P, Bauer S (1975) Mechanism of 2‑ deoxy‑D ‑ glucose inhibition of cell‑ wall polysaccharide and glycoprotein biosyntheses in Saccharomyces Availability of data and materials cerevisiae. Eur J Biochem 54(2):459–467. https:// doi. org/ 10. 1111/j. 1432‑ Fastq DNA‑seq raw data were deposited in the Genome Sequence Archive 1033. 1975. tb041 57.x (GSA) server at the BIG Data Center in Beijing Institute of Genomics (http:// Kwak S, Yun EJ, Lane S, Oh EJ, Kim KH, Jin YS (2020) Redirection of the glyco‑ bigd. big. ac. cn, GSA accession No. CRA003704), RNA‑seq raw data were lytic flux enhances isoprenoid production in Saccharomyces cerevisiae. deposited into NCBI (GEO accession number GSE164470). The dataset gener‑ Biotechnol J 15(2):e1900173. https:// doi. org/ 10. 1002/ biot. 20190 0173 ated during and/or analyzed during the current study are available from the Lam FH, Ghaderi A, Fink GR, Stephanopoulos G (2014) Biofuels. Engineering corresponding author on reasonable request. Strain M3 was deposited at alcohol tolerance in yeast. Science 346(6205):71–75. https:// doi. org/ 10. Guangdong Microbial Culture Collection Center (GDMCC No. 61336). The 1126/ scien ce. 12578 59 materials that support the findings of this study are available from the cor ‑ Lee S, Kim P (2020) Current status and applications of adaptive laboratory evo‑ responding author on request. lution in industrial microorganisms. J Microbiol Biotechnol 30(6):793–803. https:// doi. org/ 10. 4014/ jmb. 2003. 03072 Li X‑E, Wang J‑ J, Phornsanthia S, Yin X, Li Q (2014) Strengthening of cell wall Declarations structure enhances stress resistance and fermentation performance in lager yeast. J Am Soc Brew Chem 72(2):88–94 Ethical approval and consent to participate Ma M, Liu ZL (2010) Mechanisms of ethanol tolerance in Saccharomyces Not applicable. cerevisiae. Appl Microbiol Biotechnol 87(3):829–845. https:// doi. org/ 10. 1007/ s00253‑ 010‑ 2594‑3 Consent for publication Ma T, Shi B, Ye Z, Li X, Liu M, Chen Y, Xia J, Nielsen J, Deng Z, Liu T (2019) Lipid Not applicable. engineering combined with systematic metabolic engineering of Sac- charomyces cerevisiae for high‑ yield production of lycopene. Metab Eng Competing interests 52:134–142. https:// doi. org/ 10. 1016/j. ymben. 2018. 11. 009 The authors declare that they have no competing interests. Minard KI, McAlister‑Henn L (2005) Sources of NADPH in yeast vary with car ‑ bon source. J Biol Chem 280(48):39890–39896. https:// doi. org/ 10. 1074/ Received: 27 March 2021 Accepted: 3 June 2021 jbc. M5094 61200 Mo W, Wang M, Zhan R, Yu Y, He Y, Lu H (2019) Kluyveromyces marxianus developing ethanol tolerance during adaptive evolution with significant improvements of multiple pathways. 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