Increasing Malonyl-CoA Derived Product through Controlling the

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Increasing Malonyl-CoA derived product through controlling the transcription regulators of phospholipid synthesis in Saccharomyces cerevisiae Xiaoxu Chen, Xiaoyu Yang, Yu Shen, Jin Hou, and Xiaoming Bao ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00346 • Publication Date (Web): 29 Jan 2017 Downloaded from http://pubs.acs.org on January 30, 2017

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ACS Synthetic Biology

Increasing Malonyl-CoA derived product through controlling the transcription regulators of phospholipid synthesis in Saccharomyces cerevisiae

Xiaoxu Chen, Xiaoyu Yang, Yu Shen, Jin Hou*, Xiaoming Bao

State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, 250100, China

* Corresponding author: Dr. Jin Hou, email: [email protected], State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan 250100, China. Tel.: +86 531 8836 5827; Fax: +86 531 8836 5826

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Abstract: Malonyl-CoA is a precursor of a variety of compounds such as polyketides and flavonoids. In Saccharomyces cerevisiae, malonyl-CoA concentration is tightly regulated and therefore maintained at a very low level, limiting the production of malonyl-CoA-derived chemicals. Here we manipulated the phospholipid synthesis transcriptional regulators to control the malonyl-CoA levels and increase the downstream product. Through manipulating different regulators including Ino2p, Ino4p, Opi1p, and a series of synthetic Ino2p variants, combining with studying the inositol and choline effect, the engineered strain achieved a 9-fold increase of the titer of malonyl-CoA-derived product 3-hydroxypropionic acid, which is among the highest improvement relative to previously reported strategies. Our study provides a new strategy to regulate malonyl-CoA availability and will contribute to the production of other highly valued malonyl-CoA-derived chemicals. Key words: malonyl-CoA, INO2, INO4, 3-hydroxypropionic acid, Saccharomyces cerevisiae


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Introduction Malonyl-coenzyme A (malonyl-CoA) is the precursor of a diverse group of valuable compounds, such as flavonoids and polyketides (Figure 1A). A number of flavonoids and polyketides 1 are considered important pharmaceuticals and nutraceuticals that can be used as anticancer, antibacterial, antifungal and veterinary medicines and insecticides

2-6

. These

molecules are usually extracted from plants, and low production largely constrains their application. To circumvent this challenge, the microbial synthesis of these compounds has drawn much attention because of their potential to achieve considerable yields with low cost of feedstock. Additionally, some important platform chemicals, such as 3-hydroxypropionic acid (3-HP) and fatty-acid-derived chemicals, such as biodiesel, alkanes and fatty alcohols, are also generated from malonyl-CoA (Figure 1A) 7.

Saccharomyces cerevisiae is considered a potential microbial cell factory for the production of a range of fuels and chemicals due to its robustness and high tolerance to environmental stresses. It also has advantages in the biosynthesis of many plant secondary metabolites involved in the activity of cytochrome P450 enzymes, which are often difﬁcult to express in bacteria. However, malonyl-CoA concentration is maintained at low levels due to the intrinsic tight regulation of fatty acid biosynthesis in S. cerevisiae. Malonyl-CoA is synthesized by acetyl-CoA carboxylase (Acc1p), and it is the first and rate limiting step for fatty acid synthesis. Acc1p is tightly regulated both transcriptionally and post-translationally, and the phosphorylation of Acc1p by Snf1p protein kinase inactivates its activity. Direct overexpression of native Acc1p increases its activity only slightly 8. As a result, the overexpression of ACC1 has little effect on fatty acid biosynthesis 9, and replacement of the native ACC1 promoter by the TEF1 promoter can only increase polyketide 6-methylsalicylic acid (6-MSA) production by 60% 10. These results revealed that overexpression of the native acetyl-CoA carboxylase did not increase malonyl-CoA-derived products efficiently. A site-direct mutation of Acc1p phosphorylation site ser1157 was performed to increase activity in subsequent study, and the production of both polyketide 6-methylsalicylic acid (6-MSA) and fatty acids were increased approximately 3-fold

11

. Shi et al. introduced two site

mutations, S659A and S1157A, into Acc1p. These mutations resulted in a 3-fold increase of activity and a 3- and 3.5-fold increase of fatty acid ethyl esters and 3-HP production,



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respectively 8. The overexpression of heterologous acetyl-CoA carboxylase genes, such as the

AAE13 gene from Arabidopsis thaliana, can also provide a 1.6- and 2.4-fold increase in lipid and resveratrol accumulation, respectively, in S. cerevisiae 12. Aside from Acc1p overexpression, the deletion of the competing pathways was also performed to direct flux to malonyl-CoA synthesis. For example, the deletion of GSY1/2,

ZWF1, PYC1/2 or YIA6 can improve triacetic acid lactone (TAL) production approximately 2-fold

13

. The combination of multiple deletions that included protease activity (PRB1),

reverse glycolysis (PYC2), cofactor transport (YIA6) and lipid biosynthesis (NTE1) led to 5-fold improvement of TAL production 13. A synthetic malonyl-CoA sensor was also used for high-throughput genetic screening of a genome-wide overexpression library, through which two targets PMP1 and TPI1 were identified. Manipulating these genes could improve intracellular malonyl-CoA concentration

14

. Although these studies have shown positive

impact on increasing malonyl-CoA availability, as a basic precursor of fatty acid synthesis, its concentration is still tightly regulated to control lipid synthesis. The heterodimer Ino2p/Ino4p and Opi1p are transcriptional regulators of phospholipid biosynthetic genes. Transcriptional activators Ino2p and Ino4p play an important role in activating the transcription of structural genes involved in phospholipid biosynthesis, including the fatty acid synthesis genes ACC1, FAS1 and FAS2

15

. It has been reported that

Ino2p and Ino4p form a heteromeric dimer that binds to the inositol-choline-responsive element (ICRE) through a basic helix-loop-helix domain

16

. Negative regulator Opi1p

represses the activation of transcription through interaction with the activation domain of Ino2p 17. Previously,

the

deletion

malonyl-CoA-derived products

of

NTE1

showed

obvious

effect

on

improving

13

. Nte1p participates in the regulation of lipid metabolism,

indicating the importance of regulating lipid metabolism in changing intracellular malonyl-CoA concentration. In this study, we investigated the impact of manipulating the phospholipid synthesis transcriptional regulators on malonyl-CoA levels. The transcriptional activators encoding genes INO2 and INO4 were deleted, and the negative regulator encoding gene OPI1 was overexpressed to inhibit fatty acid generation (Figure 1B). The production of the malonyl-CoA-derived chemical 3-hydroxypropionic acid (3-HP) was used to monitor the


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availability of malonyl-CoA for product synthesis. MCR encodes a bifunctional enzyme, comprising malonyl-CoA reductase and 3-hydroxypropionate dehydrogenase, was introduced to covert malonyl-CoA to 3-HP. We found that INO2 deletion can increase 3-HP production, but INO4 deletion and OPI1 overexpression did not show positive effects. The Ino2p deletion strain and the Ino2p/Ino4p double deletion strain showed 9- and 5-fold increases in 3-HP synthesis, respectively, with inositol and choline addition. Nevertheless, INO2 deletion interfered with cell growth. To solve this problem, we constructed a series of synthetic INO2 variants in the ∆ino2 strain, and one of the INO2 variants can rescue the cell growth defect and increase 3-HP production.

Results The effect of transcriptional activators Ino2p or Ino4p deletion and repressor Opi1p overexpression on 3-HP production Transcriptional activators Ino2p and Ino4p form a heterodimer to regulate the expression of phospholipid biosynthesis through binding ICRE-containing promoters

15

. Negative

transcriptional regulator Opi1p represses genes transcription through interaction with the activation domain of Ino2p 17. To efficiently inhibit fatty acid biosynthesis, we deleted INO2 and INO4 and overexpressed OPI1 as well as Opi1p ER-binding deficiency mutation, which was encoded by OPI1m that constitutively releases Opi1p to the nucleus (details see Figure S1)

18, 19

. As shown in Figure 2A, the ∆ino2 strain achieved a 1.8-fold increase in 3-HP titer

compared with the control strain. However, OPI1 and OPI1m overexpression did not increase 3-HP production. No 3-HP production was detected in the ∆ino4 strain. The 3-HP titer increased 1.5-fold in the ∆ino2/4 strain. We found that the cell growth was rigorously hindered by the deletion of INO2 or INO4 but not by the overexpression of OPI1 and OPI1m (Figure 2B). The final biomass (g/g DCW, DCW: dry cell weight) was approximately 10%, 12% and 17% of the control strain in the ∆ino2, ∆ino4 and ∆ino2/4 strains, respectively (Table 1). Although INO2 deletion largely hindered the cell growth, the specific yield of 3-HP (mg/g DCW) was increased 18-fold. The deletion of both INO2 and INO4 increased the specific yield of 3-HP (mg/g DCW) approximately 8.8-fold. Thus, INO2 deletion showed



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great potential for the synthesis of malonyl-CoA-dependent products.

INO2 and INO4 deletions may cause the repression of phospholipid synthesis-related genes transcription, thereby inhibiting cell growth. It has been reported that the growth deficiency of INO2 and INO4 deletion strains can be relieved by inositol and choline addition 20

. Consistent with previous studies, the growth of ∆ino2, ∆ino4 and ∆ino2/4 strains were

indeed partly recovered when 0.1 mM inositol and 1 mM choline were added in the medium (Figure 2D). The final biomass of the three strains was similar at approximately 55% of the control strain. The ∆ino2 strain improved the 3-HP titer 9-fold, reaching 477 mg/L with inositol and choline addition (Figure 2C), as a result, increased the specific yield of 3-HP (mg/g DCW) 17-fold (Table 1). The 3-HP titer in the ∆ino2/4 strain improved 5-fold with inositol and choline addition compared with the control strain. However, the INO4 deletion did not contribute to the 3-HP titer increase (Figure 2C). In the recombinant strains, we also measured the relative transcription levels of several key genes in the phospholipid biosynthetic pathway, including the genes encoding acetyl-CoA carboxylase (ACC1), fatty acid synthetase (FAS1 and FAS2), inositol-3-phosphate synthase (INO1), and CDP-diglyceride synthetase (CDS1). These genes are crucial for phospholipids synthesis, and they all have at least one ICRE element in the promoter region21. As shown in Figure 3, INO2, INO4, INO2/4 deletions without inositol and choline feeding reduced the transcription of these genes to different levels comparing with the CM. Especially the transcription of INO1 and CDS1, was rigorously repressed. Although OPI1 overexpression also decreased the transcription of ACC1, FAS1, FAS2, it did not increase 3-HP production, probably because the repression caused by OPI1 overexpression did not benefit malonyl-CoA accumulation. Interestingly, we found that with inositol and choline addition, the transcription of INO1 increased in INO2, INO4, INO2/4 deletion strains, and

ACC1 increased in INO2, INO2/4 deletion strains relative to CM strain with same precursors feeding. It has been demonstrated that inositol and choline supplement repressed the transcription of ICRE containing genes through releasing Opi1p from ER to nucleus to prevent Ino2p-Ino4p mediated activation22,

23

.In addition, different constitutes of ICRE

elements also exhibited different regulated activities24. We speculated that the repression of inositol and choline was absent in INO2 or INO4 deletion strains but not in wild-type strain,


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therefore the relative transcription level of the two genes were increased. Meantime, the transcription level of ACC1 in the ∆ino2 and ∆ino2/4 strains are higher than in ∆ino4 strain with inositol and choline addition, which may be one of the reasons for the differences of 3-HP production. To further increase cellular malonyl-CoA availability for 3-HP production, we introduced Acc1p phosphorylation-defected mutation ACC1S659AS1157A to transcriptional activators deletion strains. the mutation of phosphorylated sites Ser659 and Ser1157 has proved to increase the activity of Acc1p8. However, in our study, overexpression of

ACC1S659AS1157A in ∆ino2 or ∆ino4 strain did not further improve 3-HP production, and it only showed positive effect in ∆ino2/4 strain, but the final titer was still lower than in the ∆ino2 strain (Figure S3). The different effect to 3-HP production when reinforcing upstream pathway supply of malonyl-CoA synthesis also indicated that Ino4p and Ino2p may have different regulatory functions.

The effect of inositol or choline concentration on cell growth and 3-HP production in ∆ino2 strain. The deletion of INO2, combined with inositol and choline addition, improved 3-HP production significantly. This result is the highest improvement relative to previously reported single gene manipulation strategies 25, indicating its great potential to enhance other malonyl-CoA-derived products, especially valuable pharmaceuticals and nutraceuticals. Therefore, the minimum concentrations of inositol and choline needed to maintain growth and production were determined in the ∆ino2 strain. We found choline was more important for maintaining cell growth and supporting 3-HP production as well, 0.02 mM of choline was enough to relieve the cell growth defect without greatly affecting 3-HP production (Figure 4A and B). Ino2p variants partly recovered cell growth deficiency of ∆ino2 strain and increased 3-HP production The deletion of INO2 showed great potential in enhancing malonyl-CoA-derived products, especially with choline addition. We further explored other possibilities to recover cell growth and maintain high 3-HP production without choline addition. We first



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overexpressed INO1 (encodes inositol-3-phosphate synthase), which is important for endogenous inositol synthesis. INO1 transcription is repressed by INO2 deletion. We found that INO1 overexpression indeed recovered cell growth to the level of choline addition; however, 3-HP production was even lower than the control strain (Figure S2). A series of synthetic INO2 variants that can maintain the regulatory function of Ino2p at a relatively low level were then constructed and expressed in the ∆ino2 strain. It has been reported that Ino2p has two activation domains (TAD1 and TAD2) and a DNA binding domain (basic helix-loop-helix domain (bHLH)). Mutations in or truncations of activation domains can decrease the activation activity of Ino2p, and DNA binding-domain mutations can decrease its binding affinity for promoters26. We first constructed the activation-domain mutations through specific domain deficiencies or in combination with site-directed mutagenesis (Figure 5A) to maintain the Ino2p activity at different levels. All the Ino2p variants were integrated into the genome and controlled by the PGK1 promoter. Figure 5C shows that the expression of these mutations could all recover the cell growth of the ∆ino2 strain to some extent, and the TAD1, F21L and D20S strains showed the best growth capability. The TAD1 strain not only recovered cell growth capacity but also enabled a 188% increase in 3-HP production compared with the CM strain (Figure 5B and C) (Table 2). The transcription level of the key target genes in the TAD1 strain was also determined, and the transcription levels of

FAS1, FAS2, INO1 and CDS1 were lower than the CM strain, but the transcription of ACC1 was not reduced (Figure 5D). These results imply that a balance was obtained in the TAD1 strain to maintain cell growth and relatively high malonyl-CoA availability. The defects in the bHLH domain of Ino2p resulted in reduced DNA binding activity 27. We therefore intended to reduce the DNA binding activity by altering amino acids of the Ino2p DNA binding domain (bHLH domain). Five mutations in the bHLH domain of Ino2p, R237Q, E257A, P266A, R273Q and D302A, were constructed in the ∆ino2 strain (Figure 6A). Likewise, all INO2 mutations were controlled by the PGK1 promoter. Although these mutations relieved cell growth defect, they did not lead to increased 3-HP levels (Figure 6B and C) (Table 2).

Discussion


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As a basic precursor, malonyl-CoA is used for formation of various valuable chemicals. The amount of cytosolic malonyl-CoA is very low in S. cerevisiae due to the inherent tight regulation. Cytosolic malonyl-CoA is synthesized from acetyl-CoA through the catalysis of acetyl-CoA carboxylase (Acc1p), which is the first step in fatty acid synthesis. Acc1p was engineered to increase malonyl-CoA synthesis

8, 10, 12, 28

. Because the activity of Acc1p is

controlled by phosphorylation and other unknown regulatory mechanisms, direct overexpression is not effective in increasing its activity. The possible phosphorylation sites ser1157 or ser1157 and ser659 were then mutated to avoid Acc1p inactivation. This strategy can increase the downstream products approximately 2- to 3-fold (Table 3) 11. In addition, the competing pathway of acetyl-CoA or malonyl-CoA consumption was also deleted, and the best result, such as with the single deletion of GSY1/2, ZWF1, PYC1/2 or YIA6, can improve TAL production approximately 2-fold. Additionally, the combination of PRB1, PYC2, YIA6 and NTE1 deletions can increase TAL production approximately 5-fold (Table 3)

13

.

Malonyl-CoA is primarily used for fatty acid synthesis. However, fatty acid synthesis cannot be abolished completely because it is essential for cell survival. In this study, we reduced fatty acid synthesis by manipulating the transcription factors that regulate FAS genes. It has been reported that Ino2p and Ino4p co-regulate the transcription of phospholipid synthetic genes,

including

genes

in

fatty

acid,

phosphatidic

acid,

phosphatidylcholine,

phosphatidylinositol and phosphatidylethanolamine synthesis, through binding to ICRE sequence-containing promoters

20

. Opi1p repressed transcription by interacting with the

activation domain of Ino2p 16. In our study, the deletion of INO2 increased the specific yield (mg/g DCW) of 3-HP 18-fold and the titer 1.8-fold. In addition, the increase of the titer was 9-fold with the addition of inositol and choline, which is the best improvement compared with other approaches (Table 3). This result proved the potential of INO2 in controlling the transcription of fatty acids biosynthetic genes and regulating malonyl-CoA availability. In addition, we found that in contrast to INO2 deletion, the deletion of INO4 did not increase 3-HP production, indicating the differences between INO2 and INO4 in regulating their target genes. We did not perform a transcriptome analysis of both strains and can only speculate their differences from our primary data. Ino2p and Ino4p null mutations did reduce the transcription of fatty acid synthesis genes (ACC1, FAS1 and FAS2) without inositol and



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choline addition. However, inositol and choline supplementation induced the transcription of

ACC1 in ∆ino2 and ∆ino2/4 strain, but not in ∆ino4 strain, indicating the different roles of Ino2p and Ino4p in transcriptional regulation. It is reported that although both Ino2p and Ino4p contain basic helix-loop-helix domains required for ICRE binding, the transcription activation mediated exclusively by Ino2p16, 20. The regulation differences may be one of the reasons for the differences in malonyl-CoA accumulation between the ∆ino2 and ∆ino4 strains. Additionally, overexpression of Opi1p and the Opi1p ER-binding deficiency mutation did not contribute to increase 3-HP production, possibly due to the indirect regulation of FAS genes. ACC1 and INO1 acts differently in the presence or absence of inositol and choline. The presence of inositol and choline can release Opi1p from its membrane anchor in ER and subsequent interact with Ino2p to prevent the activation of ICRE containing genes in wild-type strain. Although Ino2p null mutation decreased the transcription of ICRE containing genes directly, it also eliminated the regulation repression effect of inositol and choline, which may be the reason of transcription discrepancy of these two genes. Malonyl-CoA is the precursor of phospholipid synthesis, which is important in maintaining cell membrane stability. Additionally, down-regulating the expression of this pathway by INO2 deletion led to disruption of cell growth, especially without the addition of choline. We therefore intended to express Ino2p at relatively low activity levels to recover the cell growth while increasing 3-HP production. We constructed a series of synthetic INO2 variants. The activation domains and DNA binding domain (bHLH) of Ino2p were mutated to decrease the activation and DNA binding affinity, respectively. We found that expressing Ino2p without the TAD2 activation domain in the ∆ino2 strain could restore cell growth to 55% of the wild strain, similar to the level of inositol and choline addition, and lead to a 188% increase in 3-HP production. However, although the other variants in the activation domains can recover cell growth to some extent, the ability to improve 3-HP production was abolished. As shown in Figure 5, the transcription of the FAS genes was still down-regulated in the TAD1 strain, but transcription of ACC1 was not reduced. This result indicated that the relatively low activity of Ino2p in the TAD1 strain can distribute some malonyl-CoA for product synthesis, but it was enough to maintain cell growth. Higher or lower levels of Ino2p activity may not balance cell growth and product synthesis. The TAD1 strain could efficiently


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improve 3-HP production by reducing fatty acid synthesis to the proper level, which can also benefit the production of other malonyl-CoA-derived chemicals. In this study, we improved the availability of malonyl-CoA through down-regulating lipid synthesis. The best strategy increased the titer of malonyl-CoA-derived product 3-HP 9-fold, which is much higher than the previously reported strategies. Although a small amount of choline was needed to maintain cell growth for a high titer of production, this approach is still attractive for producing highly valued products such as polyketides and flavonoids. Additionally, it is possible to balance fatty acid formation for biomass synthesis and malonyl-CoA-derived product synthesis by expressing an INO2 variant in the ∆ino2 strain. Our strategy shows great potential for malonyl-CoA-derived product biosynthesis and will contribute to the construction of a yeast cell factory for a variety of products.

Materials and Methods Medium and growth conditions

S. cerevisiae strain CEN.PK102-5B (MATa; ura3-52; His3∆1; leu2-3,112) was used as the background strain. Synthetic complete (SC) dropout medium was used for recombinant yeast strain selection. The SC medium contained 1.7 g/L of yeast nitrogen base (BBI Life Science Corporation, China), 5 g/L of ammonium sulfate, 0.77 g/L of complete supplement mixture (CSM, without histidine or tryptophan) (Sunrise Science Products, USA) and 20 g/L of glucose. For routine cloning procedures, Escherichia coli Trans 5α was used and grown in Luria−Bertani (LB) medium with 100 μg/L ampicillin. Plasmid construction All plasmids and strains used in this study are listed in Table S1. All primers used in this study are listed in Table S2. Rhanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China) was used for PCR amplification. A codon-optimized MCRca gene from Chloroflexus aurantiacus was synthesized and ligated into the plasmid pIYC04 under the control of the TEF1 promoter and ADH1 terminator, resulting in plasmid pIYM. The OPI1 gene was amplified from the genomic DNA of CEN.PK102-5B and inserted into pIYM under the control of the PGK1 promoter and

CYC1 terminator to create pJMOPI1. The OPI1m gene, which encodes a OPI1 mutation that



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constitutively releases Opi1p from the ER membrane, was obtained through fusion PCR to create site-direct mutations. The conserved amino acid residues in the ER membrane protein Scs2p binding motif of Opi1p were changed as follows: E200A, F201L, F202L, D203A, and the ER membrane component PA binding motif MSIESKKR (resides from 131 to 138) in Opi1p was substituted with eight alanines (Figure S1)

18, 19

. The OPI1m was then inserted

into pIYM under the control of the PGK1 promoter and CYC1 terminator to create pJMOPI1m. The ACC1S659AS1157A gene was obtained through fusion PCR by designing mutation-containing primers. The ACC1S659AS1157A was inserted into multicopy plasmid pJFE3 under the control of TEF1 promoter and PGK1 terminator by yeast homologous recombination method, resulting in pJFE3-ACC1S659AS1157A.

INO2 variants were obtained through fusion PCR. A TAD1 fragment was amplified by fusing residues 1-35 and 136-304 of INO2. A TAD2 fragment was cloned by removing the N-terminal residues 1-100 of INO2. A TAD1-bHLH fragment was cloned by fusing residues 1-35 and 213-304 of INO2. F21L, D20S, R237Q, E257A, P266A, R273Q and D302A were obtained through fusion PCR to create site-directed mutations. These INO2 variants and

INO1 were inserted into integration plasmid pRS304X under the control of the PGK1 promoter and CYC1 terminator. All INO2 variants were named according to the properties of the mutant fragments. Strain construction CEN.PK102-5B (MATa ura3-52 His3△1 leu2-3,112) was used as the background strain. The disruption cassette loxP-Kanr-loxP, which was amplified from pUG6 plasmid, was used for the deletion of INO2 and INO4. The marker was looped out by transformation of pSH47 plasmid containing CreA. To facilitate INO2 mutation and INO1-containing plasmid integration, the TRP1 gene was disrupted in the ∆ino2 strain to obtain a trp1 auxotrophic strain. The pIYM, pJMOPI1 or pJMOPI1m plasmids were transformed into CEN.PK102-5B to construct OPI1 and OPI1m strains. Similarly, pIYM together with pJFE3-ACC1S659AS1157A plasmid was transformed into CEN.PK102-5B to obtain ACC1S659AS1157A strain. INO1 and INO2 mutation-containing plasmids were digested with Eco81I and transformed into the ∆ino2 strain, and the resulting strains were named INO1, TAD1, TAD2, F21L, D20S,


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TAD1-bHLH, R237Q, E257A, P266A, R273Q and D302A. Real-time qPCR analysis The relative transcription levels of ACC1, FAS1, FAS2, INO1 and CDS1 in the engineered strains were determined by real time qPCR. All strains were inoculated in 5 mL SC drop-out medium overnight for pre-culture. The strains were then cultivated in 40 mL medium at 30 °C and 200 rpm with an initial OD600 of 0.2. CM, OPI1, ∆ino2, ∆ino4 and ∆ino2/4 strains were cultivated either with or without 0.1mM inositol and 1 mM choline addition. Cells were grown until the exponential phase and then frozen with liquid nitrogen. Total RNA was isolated using the UNlQ-10 Column Trizol Total RNA Isolation Kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s recommendations. cDNA was obtained using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa Biotechnology, Dalian, China). One microgram of cDNA was used for SYBR Green I-based real-time PCR analysis (Roche Diagnostic) in a 10-µL reaction mixture. The primers used for qPCR are shown in Table S2. The ACT1 gene was used as an internal control. Transcription levels of target genes were relative to the CM strain. Fermentation and metabolite analysis SC medium was used for cultivation, and different concentrations of inositol and choline were added to the medium as needed. Strains were pre-cultured in tubes with 5 mL of SC medium for 16 h. Seed cultures were inoculated in shake flasks with a working volume of 40 mL at an initial OD600 of 0.2. Cultivation conditions were controlled at 30 °C and 200 rpm. Samples were taken every 12 h during fermentation up to 60 h. 3-HP, glucose, ethanol, acetate and glycerol were detected by an HPLC (Shimadzu Corporation, Japan) equipped with an Aminex HPX-87H column (Bio-Rad, Hercules, USA) at 65 °C using 2.5 mM H2SO4 as the mobile phase at a ﬂow rate of 0.6 mL/min. Biomass determination The dry cell weight was determined as described previously 29. Cell density was detected using an Eppendorf BioPhotometer. The criterion was 1 g/L biomass equals 0.246× (OD600) - 0.0012.



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Competing interests The authors declare that they have no competing interests.

Acknowledgements This work was supported by the National Natural Science Foundation of China (31470163 and 31270151), and the Key R & D Program of Shandong Province (2015GSF121015).

Supporting information: A detailed description of materials used and additional experiments (PDF).

References

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polyketide yield in yeast cells, Appl. Microbiol. Biotechnol. 98, 5435-5447. [13] Cardenas, J., and Silva, N. A. D. (2014) Metabolic engineering of Saccharomyces cerevisiae for the production of triacetic acid lactone, Metabolic engineering 25, 194-203. [14] Li, S., Si, T., Wang, M., and Zhao, H. (2015) Development of a Synthetic Malonyl-CoA Sensor in Saccharomyces cerevisiae for Intracellular Metabolite Monitoring and Genetic Screening, Acs Synthetic Biology. 4, 1308-1315 [15] Schüller, H. J., Hahn, A., Tröster, F., Schütz, A., and Schweizer, E. (1992) Coordinate genetic control of yeast fatty acid synthase genes FAS1 and FAS2 by an upstream activation site common to genes involved in membrane lipid biosynthesis, The EMBO Journal. 11, 107-114. [16] Schwank, S., Ebbert, R., Rautenstrauss, K., Schweizer, E., and Schüller, H. J. (1995) Yeast transcriptional activator INO2 interacts as an Ino2p/Ino4p basic helix-loop-helix heteromeric complex with the inositol/choline, Nucleic Acids Res. 23, 230-237. [17] Wagner, C., Dietz, M., Wittmann, J., Albrecht, A., and Schüller, H. J. (2001) The negative regulator Opi1 of phospholipid biosynthesis in yeast contacts the pleiotropic repressor Sin3 and the transcriptional activator Ino2, Mol. Microbiol. 41, 155–166. [18] Loewen, C. J. R., Roy, A., and Levine, T. P. (2003) A conserved ER targeting motif in three families of lipid binding proteins and in Opi1p binds VAP. The EMBO Journal. 22, 2025-2035. [19] Loewen, C. J. R., Gaspar, M. L., Jesch, S. A., Delon, C., Ktistakis, N. T., Henry, S. A., and Levine, T. P. (2004) Phospholipid Metabolism Regulated by a Transcription Factor Sensing Phosphatidic Acid, Science 304, 1644-1647. [20] Ambroziak, J., and Henry, S. A. (1994) INO2 and INO4 gene products, positive regulators of phospholipid biosynthesis in Saccharomyces cerevisiae, form a complex that binds to the INO1 promoter, J. Biol. Chem. 269, 15344-15349. [21] Henry, S. A., Kohlwein, S. D., and Carman, G. M. (2012) Metabolism and Regulation of Glycerolipids in the Yeast Saccharomyces cerevisiae, Genetics 190, 317-349. [22] Hirsch, J. P., and Henry, S. A. (1986) Expression of the Saccharomyces cerevisiae inositol-1-phosphate synthase (INO1) gene is regulated by factors that affect phospholipid synthesis, Mol. Cell. Biol. 6, 3320-3328. [23] Chen, M., Hancock, L. C., and Lopes, J. M. (2007) Transcriptional regulation of yeast phospholipid biosynthetic genes, Biochim. Biophys. Acta 1771, 310-321. [24] Koipally, J., Ashburner, B. P., Bachhawat, N., Gill, T., Hung, G., Henry, S. A., and Lopes, J. M. (1996) Functional characterization of the repeated UASINO element in the promoters of the INO1 and CHO2 genes of yeast, Yeast 12, 653-665. [25] David, F., Nielsen, J., and Siewers, V. (2016) Flux control at the malonyl-CoA node through hierarchical dynamic pathway regulation in Saccharomyces cerevisiae, Acs Synthetic Biology 9, 785-796. [26] Dietz, M., Heyken, W. T., Hoppen, J., Geburtig, S., and Schüller, H. J. (2003) TFIIB and subunits of the SAGA complex are involved in transcriptional activation of phospholipid biosynthetic genes by the regulatory protein Ino2 in the yeast Saccharomyces cerevisiae, Mol. Microbiol. 48, 1119–1130. [27] Nikoloff, D. M., and Henry, S. A. (1994) Functional characterization of the INO2 gene of Saccharomyces cerevisiae. A positive regulator of phospholipid biosynthesis, J. Biol. Chem. 269, 7402-7411. [28] Kildegaard, K. R., Jensen, N. B., Schneider, K., Czarnotta, E., Özdemir, E., Klein, T., Maury, J., Ebert, B. E., Christensen, H. B., and Yun, C. (2016) Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway, Microbial Cell Factories 15, 1-13.



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[29] Hou, J., Fan, S., Wang, C., Li, X., Yu, S., and Bao, X. (2014) Fine-tuning of NADH oxidase decreases byproduct accumulation in respiration deficient xylose metabolic Saccharomyces cerevisiae, BMC Biotechnol. 14, 1-10. [30] Shi, S., Valle-Rodríguez, J. O., Khoomrung, S., Siewers, V., and Nielsen, J. (2012) Functional expression and characterization of five wax ester synthases in Saccharomyces cerevisiae and their utility for biodiesel production, Biotechnology for Biofuels 5, 7.


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Table 1. The titer and specific yield of 3-HP in INO2, INO4 deletion strains.

strains

Choline and inositola

Titer(mg/L)

Specific yield (mg/g DCW)

Final biomass (g/g DCW)

+

56±2

26±1

2.17±0.02

-

53±0

23±0

2.26±0.00

+

477±1

406±11

1.17±0.03

-

100±3

417±39

0.24±0.01

+

263±3

248±2

1.06±0.02

-

82±1

204±2

0.4±0.01

+

58±1

44±1

1.31±0.01

-

0

0

0.27±0.04

CM

∆ino2

∆ino2/4

∆ino4

a

1 mM choline and 0.1 mM inositol were added to the medium.



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Table 2. The titer and specific yield of 3-HP in ino2p mutation strains.

strains

Titer(mg/L)

Specific yield (mg/g DCW)

Final biomass (g/g DCW)

CM

53±0

23±0

2.26±0.00

TAD1

107±0.5

89±0.3

1.2±0.001

TAD2

40±7.4

55±7.7

0.7±0.03

TAD1-bHLH

14±0.9

36±0.2

0.4±0.02

F21L

54±0.6

44±0.7

1.2±0.008

D20S

37±2.4

31±1.2

1.2±0.03

P266A

46±2.7

16±0.4

2.9±0.09

D302A

62±4.7

22±2.2

2.7±0.06

R237Q

22±2.3

11±1.2

1.9±0.004

E257A

35±1.3

15±0.3

2.2±0.03

R273Q

0

0

1.5±0.01

Note: no choline and inositol were added during cell growth.


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Table 3. Summary of strategies to improve the production of malonyl-CoA-derived chemicals in Saccharomyces cerevisiae.

Strategies

Products

Improvement

Replacing native ACC1 promoter with TEF1 promoter

6-methylsalicylic acid (6-MSA)

160%

Overexpression of native ACC1

Fatty acid

No improvement

Overexpression of a plant malonyl-CoA synthetase gene (AAE13)

Lipid and resveratrol

1.6-fold and 2.4-fold

Overexpression of native ACC1

Wax esters

130%

30

Overexpression of native phosphorylation-defected ACC1 mutation ACC1S1157A

6-methylsalisylic acid (6-MSA)

4-fold

11

Overexpression of native phosphorylation-defected ACC1 mutant ACC1S659A S1157A

fatty acid ethyl esters (FAEE) and 3-HP

3-fold and 3.5-fold

Overexpression of endogenous triose-phosphate isomerase (TPI1)

3-HP

120%

14

Single deletion of GSY1/2, ZWF1, PYC1/2 or YIA6

triacetic acid lactone (TAL)

2-fold

13

Combining of multiple genes deletion including ∆PRB1∆PYC2∆YIA6∆NTE1

triacetic acid lactone (TAL)

5-fold

13


References

10

9

12

8


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Deletion of INO2 with choline and inositol addition

3-HP

9-fold

This study

Expression of the Ino2p activation domain truncation in the ∆ino2 strain

3-HP

1.9-fold

This study


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Figure legends: Figure 1 The metabolic pathway of malonyl-CoA-derived products in S. cerevisiae (A), and the phospholipids synthesis regulation by transcriptional regulators Ino2p, Ino4p and Opi1p in S. cerevisiae (B): Transcriptional activators Ino2p and Ino4p form a heterodimer and activate genes expression through binding to ICRE motifs of promoters. Opi1p repressed gene expression through interaction with the activation domain of Ino2p. Figure 2 3-HP production of the strains with INO2/4 deletion or OPI1 and OPI1m overexpression. A: The titer of 3-HP in the recombinant strains without inositol and choline addition; B: The growth of the recombinant strains without inositol and choline addition; C: The titer of 3-HP in the recombinant strains with 0.1 mM inositol and 1 mM choline addition; D: The growth of the recombinant strains with 0.1 mM inositol and 1 mM choline addition. CM: CEN.PK102-5B transformed with pIYM plasmid. Figure 3 Relative transcription levels of phospholipid synthesis genes ACC1, FAS1,

FAS2, INO1 and CDS1 in the recombinant strains. Strains were cultivated until exponential phase for RNA extraction at initial OD of 0.2. OPI1-, ∆ino2-, ∆ino4-, ∆ino2/4- represent OPI1 overexpression, INO2, INO4, INO2/4 deletion strains without inositol and choline supplement, the transcription level was relative to the CM strain without inositol and choline supplement; OPI1+, ∆ino2+, ∆ino4+, ∆ino2/4+ represent the corresponding recombinant strains with 0.1mM inositol and 1mM choline supplement, the transcription level was relative to the CM strain with inositol and choline supplement. Figure 4 The impact of different amounts of inositol or choline supplementation on 3-HP production and cell growth in ∆ino2 strain. A: The titer of 3-HP in the recombinant strains with different amounts of inositol and choline addition; B: The growth of ∆ino2 strain with different amounts of inositol and choline addition. CM: control strain without inositol or choline addition; ∆ino2: ∆ino2 strain without inositol or choline addition; ino: abbreviation of inositol; cho: abbreviation of choline. Figure 5 3-HP production in Ino2p activation-domain mutation strains. A: The design of

INO2 mutations in the activation domains; B: The titer of 3-HP in the recombinant strains without inositol and choline addition; C: The cell growth of the recombinant strains without inositol and choline addition; D: Relative transcription levels of phospholipids synthesis



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genes ACC1, FAS1, FAS2, INO1 and CDS1 in the recombinant strains. Figure 6 3-HP production in Ino2p DNA binding-domain mutation strains. A: The design of INO2 mutations in DNA binding domain; B: The titer of 3-HP in the recombinant strains without inositol and choline addition; C: The cell growth of the recombinant strains without inositol and choline addition.


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Figure 1 The metabolic pathway of malonyl-CoA-derived products in S. cerevisiae (A), and the phospholipids synthesis regulation by transcriptional regulators Ino2p, Ino4p and Opi1p in S. cerevisiae (B): Transcriptional activators Ino2p and Ino4p form a heterodimer and activate genes expression through binding to ICRE motifs of promoters. Opi1p repressed gene expression through interaction with the activation domain of Ino2p. 152x81mm (300 x 300 DPI)



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Figure 2 3-HP production of the strains with INO2/4 deletion or OPI1 and OPI1m overexpression. A: The titer of 3-HP in the recombinant strains without inositol and choline addition; B: The growth of the recombinant strains without inositol and choline addition; C: The titer of 3-HP in the recombinant strains with 0.1 mM inositol and 1 mM choline addition; D: The growth of the recombinant strains with 0.1 mM inositol and 1 mM choline addition. CM: CEN.PK102-5B transformed with pIYM plasmid. 263x182mm (300 x 300 DPI)


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Figure 3 Relative transcription levels of phospholipid synthesis genes ACC1, FAS1, FAS2, INO1 and CDS1 in the recombinant strains. Strains were cultivated until exponential phase for RNA extraction at initial OD of 0.2. OPI1-, ∆ino2-, ∆ino4-, ∆ino2/4- represent OPI1 overexpression, INO2, INO4, INO2/4 deletion strains without inositol and choline supplement, the transcription level was relative to the CM strain without inositol and choline supplement; OPI1+, ∆ino2+, ∆ino4+, ∆ino2/4+ represent the corresponding recombinant strains with 0.1 mM inositol and 1mM choline supplement, the transcription level was relative to the CM strain with inositol and choline supplement. 229x185mm (300 x 300 DPI)



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Figure 4 The impact of different amounts of inositol or choline supplementation on 3-HP production and cell growth in ∆ino2 strain. A: The titer of 3-HP in the recombinant strains with different amounts of inositol and choline addition; B: The growth of ∆ino2 strain with different amounts of inositol and choline addition. CM: control strain without inositol or choline addition; ∆ino2: ∆ino2 strain without inositol or choline addition; ino: abbreviation of inositol; cho: abbreviation of choline. 304x117mm (300 x 300 DPI)


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Figure 5 3-HP production in Ino2p activation-domain mutation strains. A: The design of INO2 mutations in the activation domains; B: The titer of 3-HP in the recombinant strains without inositol and choline addition; C: The cell growth of the recombinant strains without inositol and choline addition; D: Relative transcription levels of phospholipids synthesis genes ACC1, FAS1, FAS2, INO1 and CDS1 in the recombinant strains. 285x182mm (300 x 300 DPI)



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Figure 6 3-HP production in Ino2p DNA binding-domain mutation strains. A: The design of INO2 mutations in DNA binding domain; B: The titer of 3-HP in the recombinant strains without inositol and choline addition; C: The cell growth of the recombinant strains without inositol and choline addition. 152x75mm (300 x 300 DPI)


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Increasing Malonyl-CoA Derived Product through Controlling the

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