Metabolomic, proteomic and lactylated proteomic analyses indicate lactate plays important roles in maintaining energy and C:N homeostasis in Phaeodactylum tricornutum

Background Phaeodactylum tricornutum accumulates lipids while the growth also increases under high CO2, shedding light on its potential application in the reduction of CO2 emissions and at the same time acquiring biodiesel raw materials. However, the sensing and transducing of high C:N signals and the related response mechanism(s) remained unknown. Results In this study, a multiple omics analysis was performed with P. tricornutum under low nitrogen (LN) and high CO2 (HC) conditions. The results indicated that 2-oxoglutarate was significantly increased under both LN and HC. Meanwhile, proteins involved in carbon concentration mechanism decreased, indicated that 2-oxoglutarate might regulate C:N balance through suppressing carbon fixation. Lactate, which acts in energy metabolism, signal transduction and ‘LactoylLys’ modification on proteins, was the most upregulated metabolite under both LN and HC conditions. Meanwhile, proteins involved in carbon, nitrogen and energy metabolisms were significantly regulated. Western blotting analysis suggested that non-histone L-lactylation modification was enhanced under LN and HC. Moreover, lactylated proteins were enriched in photosynthesis, central carbon metabolism, nitrogen metabolism, fatty acid synthesis and oxidative phosphorylation. Conclusion It is suggested that lactate might play important roles in energy homeostatic maintenance and C:N balance regulation in P. tricornutum through protein lactylation modification. Supplementary Information The online version contains supplementary material available at 10.1186/s13068-022-02152-8.


Background
Diatoms are important phytoplankton which are responsible for approximately 20% of global primary productivity [1,2] and thus play an important role in carbon fixation and the material cycle on earth [3,4]. The great success of diatoms is mainly due to their rapid response to fluctuations in nitrate in the ocean. Diatoms possess a complete urea cycle, which might facilitate the reassimilation of nitrogen in catabolic N-containing components [5], making them response quickly to nitrogen-limited conditions. In contrast to carbon regulation in green algae and higher plants, which increase carbon stores under nitrogen depletion conditions, diatoms accelerate glycolysis and the TCA cycle to provide carbon skeletons for nitrogen redistribution [6]. This response of diatoms under nitrogen-limited conditions is a result

Open Access
Biotechnology for Biofuels and Bioproducts *Correspondence: xiezyscuta@163.com of the superior efficiency of intracellular carbon store breakdown as a source of carbon for the reassimilation of nitrogen than for photosynthesis [6]. Because of the importance of diatoms and their unique characteristics, the response of diatoms to nitrogen depletion has received much attention in the past decade. The pennate diatom Phaeodactylum tricornutum (P. tricornutum) is considered a model organism for the study of physiology, evolution and biochemistry in single-cell organism due to its clear genomic background [7], universal molecular toolbox [8] and stable transgene expression system [9,10]. In the past decade, the physiological response [11], gene expression and metabolic pathway regulation mechanisms of P. tricornutum under nitrogen deficiency have been studied [12][13][14][15]. Under nitrogen deficiency, P. tricornutum accumulates lipids at the expense of depressed growth, photosynthesis, chlorophyll biosynthesis, the Calvin cycle and chrysolaminarin biosynthesis, and increased recycling of N compounds, such as amino acids, proteins, chlorophyll and nucleic acids; high turnover of cell components (e.g. soluble sugar, membrane lipids and phosphatidylcholines) that reallocates cellular nutrients to glycolysis and the TCA cycle, which are accelerated to provide carbon skeletons for redistribution of nitrogen, was all observed [12,16,17]. However, the mechanisms of sensing and transducing nitrogen deficiency signals and the related responses have not been elucidated.
In cyanobacteria, 2-oxoglutarate (2-OG), the metabolic intermediate of TCA cycle, played an important role in the C:N balance regulation by binding to the key regulators PII and NtcA, two receptors in the nitrogen starvation signalling pathways. Our previous study indicated that 2-OG content was upregulated 22-fold in P. tricornutum under nitrogen-deficient conditions [18]. As an important metabolite of central carbon metabolism, 2-OG provides a carbon skeleton for the assimilation of nitrogen through glutamate synthase, connecting intracellular carbon metabolism and nitrogen metabolism [19,20]. Thus, the accumulation of 2-OG indicates a high carbon: nitrogen (C:N) ratio, and accumulated 2-OG emits a nitrogen starvation signal, playing an important role in the regulation of carbon and nitrogen metabolism in cyanobacteria [21]. When the C:N ratio is high, 2-OG suppresses carbon fixation by downregulating genes involved in the Calvin cycle and the carbon concentration mechanism (CCM), which leads to elevated CO 2 concentrations near Rubisco to enhance CO 2 fixation [20,21]. The accumulation of 2-OG in P. tricornutum under nitrogen-deficient conditions, together with changes in carbon metabolism in response to nitrogen starvation in diatoms bears a greater resemblance to the response of cyanobacteria than to that of higher plants or green algae, triggering our interest in investigating the role of 2-OG in P. tricornutum.
If 2-OG indeed plays a role in C:N balance regulation in P. tricornutum, it might also act under other conditions which can result in a high intracellular C:N ratio. Besides nitrogen depletion, high inorganic carbon concentration, i.e. ocean acidification induced by increasing of atmospheric CO 2 , can also result in a high intracellular C:N ratio. In fact, our previous study suggests some similar response of P. tricornutum to high CO 2 concentration as to low nitrogen, including accumulation of lipid, increase in glycolysis and TCA cycle activities and remodelling of cellular components [22,23]. We propose that nitrogen deficiency or high CO 2 concentration essentially disrupts the intracellular C:N balance and energy balance, thus triggering a series of signal transduction and regulation processes to maintain cell homeostasis. In contrast to cyanobacteria, P. tricornutum is an oleaginous microalga in which lipids accumulate under high C:N ratio conditions. This lipid accumulation is accompanied with remodelling of intracellular components and redistributing of metabolites. Hence, carbon and nitrogen level regulation may not be limited to the carbon and nitrogen assimilation; it may also be related to the degradation and redirection of intracellular catabolic products.
Although researchers have investigated the response to nitrogen deficiency in P. tricornutum at various levels [13][14][15][16][17], no studies have been conducted on signal sensing and transduction or their regulatory pathways. In this study, we characterized the proteome, and metabolome of P. tricornutum under normal (NC), low nitrogen (LN) and high CO 2 (HC) conditions. Western blotting was performed to assess lactylation in proteins. Lactylated proteomic analysis for NC was conducted, revealing the probable roles of lactylation in P. tricornutum. The results reveal that lactate might play an important role in C:N balance regulation and energy homeostasis maintaining in P. tricornutum through signal transduction as well as protein lactylation modification.

Differential expression of proteins under HC and LN conditions Overview of the proteomic data
Based on label-free proteome quantification technology, 5958 proteins were identified, among which 5325 proteins were quantified in P. tricornutum under NC, LN and HC conditions. Most peptides consisted of 7-20 amino acids (Additional file 6: Fig. S1a), which conforms to general rules based on enzymatic hydrolysis and mass spectrometry fragmentation. Most proteins corresponded to more than two peptides (Additional file 6: Fig. S1b). The coverage of most proteins was less than 30% (Additional file 6: Fig. S1c). The molecular weights of the identified proteins were evenly distributed (Additional file 6:  Table S1.

Identification and enrichment analysis of differentially expressed proteins (DEPs)
To understand the proteome responses to C:N ratio fluctuation in P. tricornutum, proteomic analysis based on label-free proteome quantification technology was performed to identify the DEPs in P. tricornutum under NC, LN and HC conditions. According to the criteria of a 1.5fold increase (or 0.67-fold decrease) and a P value < 0.05 [24], a total of 1471 DEPs, including 694 differentially upregulated proteins (DUPs) and 777 differentially downregulated proteins (DDPs), were identified under LN conditions (Additional file 3: Table S2), and 327 DEPs, including 215 DUPs and 112 DDPs, were identified under HC conditions (Additional file 4: Table S3). The KEGG pathway-based enrichment analysis revealed significant enrichment of DEPs in pathways involved in carbon metabolism, nitrogen metabolism, lipid metabolism, protein and amino acids metabolism, energy metabolism and signalling, including the enrichment of DUPs under LN conditions in TCA cycle, nitrogen metabolism, phenylpropanoid biosynthesis, phosphatidylinositol signalling system, glycerophospholipid metabolism and oxidative phosphorylation (Fig. 1a); the enrichment of DDPs under LN conditions in photosynthesis, biosynthesis of amino acids, carbon fixation in photosynthetic organisms and nitrogen metabolism (Fig. 1a), the enrichment of DUPs under HC conditions in photosynthesis-antenna proteins, phenylpropanoid biosynthesis and glycolysis / gluconeogenesis (Fig. 1b), and the enrichment of DDPs under HC conditions in biosynthesis of amino acids, pyrimidine metabolism and nitrogen metabolism (Fig. 1b).

Similar expression patterns of DEPs under LN and HC conditions indicated their functions in C:N balance regulation
As showed in Table 2 Table S2 and Additional file 4: Table S3), indicating a frequent exchange of metabolites. In addition, many DEPs are transcription factors or involved in signal transduction (Additional file 3: Table S2 and Additional file 4: Table S3), indicating that in P. tricornutum, the C:N balance might be regulated through signalling pathways. This similar expression pattern of DEPs under LN and HC conditions indicated they might play a role in C:N balance regulation.

DEPs involved in phenylpropanoid biosynthesis and nitrogen metabolism
The most upregulated DEP under both LN (790.8-fold change) and HC (287.8-fold change) was predicted to be peroxiredoxin (Tables S2 & S3), which is involved in phenylpropanoid biosynthesis.

DEPs involved in photosynthesis, carbon concentration mechanism (CCM), central carbon metabolism and chrysolaminarin degradation
Two DUPs and four DDPs in LN were light-harvesting complex-related proteins. A total of 18 DDPs and two DUPs under LN were involved in photosynthesis, indicating a severe downregulated of photosynthesis under LN ( Table 2, Fig. 2). Five light-harvesting complex-related proteins were upregulated under HC condition, indicating enhance of photosynthesis under HC ( Table 2, Fig. 2).

DEPs involved in lactate metabolism and lysine lactylation
D-lactate dehydrogenase (ldhA), which catalyses the dehydration of pyruvate to generate D-lactate, was upregulated under both LN (5.6-fold change) and HC (4.5-fold change) conditions, a finding consistent with lactate accumulation under both LN and HC conditions.     Besides, a l-lactate permease (LCTP), which was reported to expressed under nitrogen-limited conditions [7], and enzymes related to D-lactylation, namely, lactolglutathione lyase (GLXI) and glyoxalase (GLO1 and GLO2) [25], were also detected in the P. tricornutum proteome analysis, although their levels did not vary significantly upon different treatments (Table 2).

Protein validation by PRM and biochemical analysis
We performed PRM to eliminate most of the possible errors though the label-free data in this report had undergone rigorous statistical and bioinformatics analysis. Analysis on FbaC5, ZEP3, bglX, nirA, PPdK, NR, PCCA, LDHA, GLO_2, GLO_1, Formidase, PDH1, PEPCK1, GltX and acnBto was performed to validate the label-free results. The results of target proteins detected by PRM were consistent with the label-free analysis (Fig. 3), which indicated that our proteomics results based on label free were highly reliable and reproducible.

Pan-acetylation and pan-lactyllysine Western blotting analyses
The overall regulation of acetylation and lactylation patterns of all proteins was determined by Western blotting using a pan anti-acetyllysine antibody and a pan-lactyllysine antibody, respectively. As shown in Fig. 4, many proteins were either acetylated or lactylated under NC, LN and HC conditions, but the overall acetylation and were most likely members of the histone family. These findings confirmed that protein lactylation proceeds through temporal dynamics that differ from those of protein acetylation.

Lactylated proteomic analysis under NC in P. tricornutum Global profiling of lactylated proteome
To study the probable function of lysine lactylation in P. tricornutum, we performed global profiling of the lysine lactylome for NC. A total of 463 lactyllysine sites across 212 proteins were identified (Additional file 5: Table S4). To further understand the biological regulations and functions of lactylated proteins in C/N balance in P. tricornutum, we carried out Clusters of Orthologous Groups (COG) annotation and KEGG pathway enrichment analysis. The results of COG functional annotation analysis showed that various lactylated proteins were engaged with crucial biological processes, such as posttranslational modification, protein turnover and chaperones; carbohydrate transport and metabolism; translation, ribosomal structure and biogenesis; energy production and conversion; amino acid transport and metabolism; and lipid transport and metabolism (Fig. 5).

Lactylated proteins are enriched on photosynthesis, central carbon metabolism and fatty acid biosynthesis pathways
Protein domain analysis revealed that lactylated proteins were concentrated in ATP synthase, GADPH, core histone H2A/H2B/H3/H4, phosphoglycerate kinase, fructose-bisphosphate aldolase, carbonic anhydrase, enolase and so on (Fig. 6a). KEGG pathway enrichment analysis illustrated that lactylated proteins were concentrated in photosynthesis, carbon fixation in photosynthetic organisms, photosynthesis-antenna proteins, glycolysis/ gluconeogenesis, oxidative phosphorylation, fatty acid biosynthesis, fructose and mannose metabolism and pentose phosphate pathway (Fig. 6b), indicating that lactylation played an important role in energy balance and carbon metabolism. Lactylated proteins involved in glycolysis/gluconeogenesis, Calvin cycle, pentose phosphate pathway and fatty acid synthesis are shown in Fig. 7. Besides, nitrogen metabolism-related proteins, including ammonium transporter, glutamine synthetase and carbonic anhydrase, were also detected (Fig. 7), indicating that lactylation also played an important role in nitrogen metabolism. Simultaneously, components of oxidative phosphorylation and photosynthetic phosphorylation were also detected, indicating that lactylation participates in energy metabolism.
Interestingly, lactylation mainly occurred in proteins that located in cytoplasm and chloroplast, but rarely in proteins located in mitochondria, implying that protein lactylation modifications are affected by cell compartmentalization. It is probable that there is lactate permease on chloroplast membrane, but not on mitochondria membrane.

Influence of lactate on growth and total lipid content of P. tricornutum
As shown in Fig. 8, lactate at 1 mM, 5 mM and 10 mM could increase (P < 0.05) growth of P. tricornutum, with 5 mM being the most significant. Total lipid content was determined to verify if lactate influenced total lipid content. The results showed that lactate can significantly (P < 0.05) promote the accumulation of lipids in P. tricornutum, and the total lipid content increases with the increase of lactate concentration, indicating that lactate might play an important role in lipid metabolism in P.
tricornutum. Yet as lactate can also function as a carbon source, further studies, such as 13 C labelling, are needed to verify the function of lactate in P. tricornutum.

2-OG might regulate P. tricornutum C:N balance by suppressing CCM
In recent decades, the response of P. tricornutum to nitrogen deficiency has received considerable attention.
Research mainly concerns the physiological response, gene expression and metabolic pathway shift or lipid accumulation process [12][13][14]26]. However, the mechanisms behind P. tricornutum responses, e.g. sensing nitrogen deficiency and transmitting relevant signals to induce the response mechanism, have not been elucidated. To some extent, nitrogen deficiency (LN) and a high CO 2 concentration (HC) indicate a high C:N ratio [23,27], which might trigger the activation of a series of signalling pathways. For example, 2-OG indicates a high C:N ratio and plays an important role in the regulation of carbon and nitrogen metabolism in cyanobacteria [21]. By depressing the expression of genes involved in CCM, 2-OG represses carbon fixation and thus maintains the intracellular C:N balance. In this study, we observed 14-fold and 12-fold upregulation of 2-OG expression under LN and HC conditions (Table 1). In addition, proteins involved in the CCM were significantly downregulated under LN conditions ( Table 2). The accumulation of 2-OG and the downregulation of CCM-related genes under both LN and HC conditions indicated that 2-OG might play an important role in sensing and transducing high C:N ratio signals and act as a ligand that interacts with carbon fixation regulators. This supposition is consistent with the idea that carbon metabolism induced in diatoms in response to nitrogen starvation bears closer resemblance to the response of cyanobacteria than that of higher plants or green algae [6].

Probable role of lactate in P. tricornutum C:N balance regulation
In addition to an increase in 2-OG, we also observed a dramatic increase in lactate content under both LN and HC conditions. Lactate is used to be considered as a metabolism waste by-product of glycolysis. Currently, lactate is considered a crucial regulatory nexus for energy metabolism [28], a signalling molecule participating in a series of metabolic processes [29,30] and a substrate for posttranslational modifications (PTMs) through the 'Lac-toylLys' modification of proteins [25,31]. The increase in lactate under both LN and HC conditions indicated its likely roles in the following processes:

Fig. 5 Clusters of Orthologous Groups (COG) annotation for lactylated proteins
1. Regulation of energy balance. In organisms, the main source of ATP is oxidative phosphorylation, in which electrons in NADH (produced by cell metabolism) are delivered to oxygen via the respiratory chain with the concomitant production of ATP and NAD + . Under hypoxic conditions, oxidative phosphorylation is repressed, NADH accumulates and NAD + regeneration is blocked. To maintain the energy supply under hypoxic conditions, plants switch from respiration to fermentative metabolism by activating lactate dehydrogenase, which catalyses the reduction of pyruvate to generate lactate, accompanied by the consumption of NADH. Thus, lactate dehydrogenase can regulate cellular energy balance. In this study, we observed a significant decrease in AMP under both LN and HC conditions (Table 1), indicating a high ATP:AMP ratio; e.g., cells are in high energy mode. This outcome might be a result of a decrease in the NADH consumption process, i.e. nitrogen reduction. We observed a decrease expression in nitrate reductase (UniProt B7G997) and ferredoxin-nitrite reductase (UnitProt B7G0L4) under both LN and HC conditions ( Table 2, Fig. 2). These results might indicate decreased NADH consumption. Furthermore, processes in which NADH is produced, i.e., the TCA cycle under LN conditions and pyruvate dehydrogenase and photosynthesis under HC conditions, were accelerated. This acceleration might disrupt the energy balance and activate lactate dehydrogenase, resulting in the accumulation of lactate. In this study, we observed a 5.57-fold and 4.46-fold increase in lactate dehydrogenase (ldhA, B7S4E4) under LN and HC conditions (Table2), indicating the upregulation of lactate reductant from pyruvate. Moreover, the shuttling of L-lactate between different organs and cells creates a major circulatory mechanism of carbohydrate sources and NADH/NAD + [32]. In this study, we also detected L-lactate permease (LCTP, Gene ID: 7,195,398) under LN condition (Table 2 and Additional file 2: Table S1), suggesting the important role of lactate in the C:N balance and energy balance regulation.
2. Regulation of lipid metabolism. In contrast to cyanobacteria, P. tricornutum is an oleaginous microalga that accumulates lipids under nitrogen-limited conditions. In this process, P. tricornutum remodels intracellular components and redistributes metabolites [14]. Therefore, its C:N balance regulation may not be limited to the assimilation process of carbon and nitrogen and may also be related to the degradation and redirection of intracellular catabolic products. Lactate itself can act as a signal molecule in the cAMP pathway and regulate a series of cellular processes. In mammalian adipose tissue, for example, lactate can combine with its sensor, an orphan G protein-coupled receptor, activating it and suppressing lipolysis in fat cells [30]. Despite its categorization as a photosynthetic organism, P. tricornutum has an unusual evolutionary history, with secondary endosymbiotic origin and mosaic genome that contains 'animal-like' , 'plant-like' and 'bacteria-like' genes [33][34][35][36][37]. Some of its genes are more similar to those of animals than to their photosynthetic counterparts [38]. Recently, it was reported that in P. tricornutum, phosphatidylinositol 3-kinase (PI3K), which is involved in lipid accumulation, is more closely associated with mammalian homologues than those in higher plants [39]. The accumulation of both lactate and lipids in P. tricornutum under LN and HC conditions indicated that lactate might also suppress lipolysis in P. tricornutum, as it does in mammalian cells, and thus facilitate lipid accumulation in P. tricornutum under LN and HC conditions. 3. 'LactoylLys' modification on proteins (lactylation).
Researchers recently found that lactate can regulate protein functions through posttranslational modifications (PTMs) by conjugating to protein Lys residues and generating a 'LactoylLys' modification on proteins, revealing non-metabolic functions of lactate [31]. Protein lactylation is a new topic currently studied mainly in mammals, as protein lactylation plays an important role in diseases, such as neoplasia, sepsis and autoimmune diseases. Studies related to protein lactylation in algae have not been reported. Protein lactylation is positively correlated with intracellular lactate concentration [25]. In this study, lactate was the most significantly upregulated metabolite under both LN and HC conditions. The WB results (Fig. 4) indicated proteins in P. tricornutum had undergone lactylation, which was particularly evident under LN and HC conditions. Lactylated proteomics under NC condition revealed that lactylated proteins were significantly involved in photosynthesis, oxidative phosphorylation, central carbon metabolism including Calvin cycle, glycolysis and pentose phosphate pathway, fatty acid synthesis and nitrogen metabolism, confirming protein lactylation in P. tricornutum played an important role in C:N balance regulation, energy homeostasis maintenance and fatty acid synthesis.

Probable role of the IP3K signalling pathway under LN conditions
In this study, we found that genes involved in the phosphatidylinositol signalling system were upregulated at the protein level ( Table 2) under LN conditions. It has been reported that in P. tricornutum, the expression of genes involved in MAPK signalling was upregulated, while that in lipid metabolism was decreased under treatment with a phosphatidylinositol 3-kinase inhibitor, demonstrating a role for phosphatidylinositol 3-kinase in the allocation of carbon, including as a factor of lipid reduction in P. tricornutum [39]. In a study of the green alga Chlamydomonas reinhardtii, it was reported that the phosphatidylinositol 3-kinase signalling system is a master regulator of energy and carbon metabolism [40], regulating the homeostasis of membrane lipids, TAGs, starch, free fatty acids (FFAs) and ATP by influencing other components of the signalling network. Here, we also found that the expression of a carboxypeptidase (B5Y4V6), which negatively regulates membrane lipid metabolism, was upregulated under both LN and HC conditions. The enrichment of upregulated proteins in the phosphatidylinositol signalling system and related genes indicated that this pathway might play important regulatory roles (energy and carbon balance regulation and homeostasis maintenance) in the P. tricornutum response to LN conditions. In addition, genes involved in autophagy, plant-pathogen interactions, endocytosis and phagosomes were also enriched, indicating a complex regulatory pattern through signal transduction in P. tricornutum under LN conditions.

Probable role of phenylpropanoids in N recycling under LN and HC conditions
The most upregulated DUP under both LN (790.8-fold change) and HC (287.8-fold change) conditions was PRDX6, which is involved in phenylpropanoid biosynthesis, followed by two predicted transcription factors involved in the regulation of phenylpropanoid biosynthesis ranked second and fifth (LN) and second and fourth (HC) (Tables S2 & S3), indicating an important role for the phenylpropanoid biosynthesis pathway under LN and HC conditions. The phenylpropanoid biosynthesis pathway is involved in the degradation of phenylalanine and tyrosine and thus produces abundant NH 4 + , facilitating the recycling of nitrogen under N-limited conditions, while nitrate inhibits large sectors of phenylpropanoid metabolism [41]. The upregulation of the phenylpropanoid biosynthesis pathway might facilitate nitrogen reallocation under both LN and HC conditions.

Overall response to a high C:N ratio in P. tricornutum under HC and LN conditions
Considering the combined results of the proteome, metabolome and WB analysis and lactylated proteome with respect to P. tricornutum under HC and LN conditions, we propose the following responses of P. tricornutum to C:N imbalance (Fig. 9): a high C:N ratio stimulates 2-OG accumulation. The resulting nitrogen reduction and nitrite reduction were reduced as a result of N limitation, thereby reducing the consumption of NADH. In addition, accelerated glycolysis, photosynthesis (HC conditions) and the TCA cycle (LN conditions) result in increases in NADH and ATP (Fig. 2). A low AMP:ATP ratio (Table 1) depresses the activity of NADH dehydrogenase, and the accumulation of NADH triggers the activity of ldhA (Table 2), resulting in the accumulation of lactate (Table 1). 2-OG and lactate then serve as signalling molecules and regulate a series of pathways. 2-OG senses and transduces signals when the C:N ratio is high, regulates genes involved in the CCM and reduces the extent carbon fixation ( Table 2, Fig. 2). Moreover 2-OG can act on the PHD and E3 ubiquitin ligation complex and induce protein degradation through ubiquitin-mediated proteolysis, providing carbon skeletons and NH 4 + for the reallocation of nitrogen. Lactate can regulate the function of proteins through PTM and regulate cellular components through a series of signalling pathways, i.e. IP3K signalling pathways. Lactylated proteins were involved in central carbon metabolism, photosynthesis, oxidative phosphorylation and nitrogen metabolism, thus can maintain C:N balance and energy homeostasis.

Conclusion
Multiple omics analysis was conducted in P. tricornutum under HC and LN conditions. The results indicated 2-OG and lactate was significantly up-regulated under both LN and HC conditions. Genes involved in CCM were down-regulated, indicating 2-OG regulated C:N balance by suppressing CCM-related genes. Genes involved in lactate redundant and energy metabolism are significant regulated. By WB analysis it was proved that lactylation modification occurred in P. tricornutum. What is more, the non-histone lactylation modification was enhanced under LN and HC. Lactylated proteomic analysis revealing that lactylation might play an important role in energy and carbon balance regulation and homeostasis maintenance in P. tricornutum. Our result reveal C:N regulation mechanism under LN and HC conditions, shedding light on the potential application of P. tricornutum to the reduction of CO 2 emissions from industrial flue gas streams and at the same time acquiring biodiesel raw materials.

Materials and methods
Cell culture and treatments P. tricornutum Bohlin was obtained from the Microalgae Culture Center (MACC) at Ocean University of China. Algal cells were cultured using NaHCO 3 -free sterilized artificial seawater supplemented with f/2 [42] under cool white fluorescent light (~ 100 μmol m -2 s -1 ) at 20 °C and a 12:12 dark:light cycle. A UV/visible spectrophotometer (UV-1800, Shimadzu, Japan) was used to measure the absorbance at 730 nm (A730 nm) to monitor cell growth. For high CO 2 (HC, ~ 2000 μatm) or normal CO 2 (NC, ~ 400 μatm) treatment, the culture was bubbled with ~ 2,000 μatm of CO 2 or ambient air with a flow rate of 500 ml min -1 . For the low nitrogen (LN) treatment, a nitrogen-free f/2 medium was produced with artificial seawater and the cultures were bubbled with ambient air at a flow rate of 500 ml min -1 . For experiments testing the influence of lactate on growth and lipid content, ( ±)-lactate (Xilong Chemical Co., Ltd., Shantou, Guangdong province, China) was added to a final concentration of 1, 5 and 10 mM. Each treatment was administered in triplicate sets. After 8 days, cell pellets were collected and then washed using distilled water at 5,000 g for 4 min. The pellets were frozen in liquid nitrogen and stored at − 80 °C.

Lipid analysis and metabolomic analysis
Total lipids were extracted and weighed as described by previously [43] with minor modifications. Metabolomic analysis was performed as described [18]. Full details are given in Additional file 1: Methods S1.

Proteomic analysis
Algal cells were ground into powder in liquid nitrogen, and proteins were extracted using phenol buffer (containing 10 mM dithiothreitol and a 1% protease inhibitor) and lysed by ultrasound. After adding Tris-balanced phenol and centrifuging (4℃, 5500g, 10 min), the supernatant was maintained overnight to allow precipitation with ammonium acetate/methanol, and then, it was washed with methanol and acetone. The protein precipitate was dissolved with urea, and the concentration was determined with a BCA protein assay kit (Beyotime). Equal amounts of protein were hydrolysed by trypsin and analysed by LC MS/MS as described in Additional file 1: Methods S1. The MS/MS raw data were administered by operating MaxQuant (http:// www. maxqu ant. org/) with an integrated Andromeda search engine (v1.6.15.0). Full detail parameters are given in Additional file 1: Methods S1. Significantly differentially expressed proteins were defined by the criteria of a fold change (FC) ≥ 1.5 or ≤ 0.67 (P < 0.05). Functional annotation of the proteins was performed using non-redundant protein (NR), Swiss Protein (SwissProt), Gene Ontology (GO) and KEGG databases.

Pan anti-acetylation and pan anti-lactyllysine as determined by Western blotting
Proteins used in Western blotting were extracted according to the same operation manual as used in proteomic analysis except that 3 μM TSA (trichostatin A) and 50 mM nicotinamide were added. Protein concentration was determined by BCA reagent. Western blotting samples were prepared as follows: equal amounts of protein (20 µg/sample) were mixed with SDS-PAGE 4 × loading buffer with a ratio of 3:1 (v/v), heating for 10 min at 95 °C. The same volume of 20% protein marker, boiled samples and 4 × loading buffer were loaded into wells. Gel electrophoresis was then performed as follows: at 80 V for 30 min, then at 120 V until loading dye reach the bottom edge of SDS-PAGE gel. Following transfer the protein to nitrocellulose membrane at 100 V for 1 h at 4 °C, the membranes were blocked in 1 × TBST with 5% non-fat milk with gentle shaking for 1 h at RT. By rinsing with 1 × TBST for three times (10 min each), the membrane was incubated in primary antibody (anti-acetyllysine antibody, PTM-101, Lot: 12838533L303, 1:1000 dilution in 1 × TBST with 2.5% BSA; and pan anti-lactyllysine, PTM-1401RM, Lot: L011121, 1:500 dilution in 1 × TBST with 2.5% BSA) at 4 °C with gentle shaking overnight, rinsing with1 × TBST for 10 min three times, incubating for 1 h at RT with secondary antibody (Thermo, Pierce, goat anti-mouse IgG, H + L, peroxidase-conjugated, 31,430 and 31,460, respectively) diluted 1:10,000 in in 1 × TBST with 5% non-fat milk), rinsing with 1 × TBST for 10 min three times, and staining with ECL western blot detection reagent (Beyotime, Beijing, China) for 2 min, followed by quantification using multiple exposure time to acquire the optimal picture.

Lactylated proteomic analysis
Protein was extracted and hydrolysed by trypsin as described in 2.3, following by pan-antibody-based PTM enrichment. Briefly, tryptic peptides dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) were incubated with pre-washed antibody beads (Lot: L011121, PTM Bio) at 4 °C overnight with gentle shaking. After that the beads were washed for four times with NETN buffer and twice with H2O. Then the bound peptides were eluted from the beads with 0.1% trifluoroacetic acid. Finally, the eluted fractions were combined and vacuum-dried and desalted with C18 ZipTips (Millipore) according to the manufacturer's instructions.
LC MS/MS and database search were conducted as described in 2.3. Full detail parameters are given in Methods S1. For motif analysis, an adjacent sequence model of lactyllysine was analysed by motif-x (http:// www. motif-x. med. harva rd. edu), integrating 10 amino acids upstream and downstream of the modification sites (lactyl-21-mers). When the number of a lactyl-21-mer is greater than 20 and P < 0.000001, the lactyl-21-mer is considered to be a motif of modified peptides. Protein sequences for all P. tricornutum proteins were utilized as background databases.

Statistical analysis
The significance of differences between samples was assessed by two-tailed student t-tests and one-way analysis of variance. The statistical significance level was set at P < 0.05.