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From formic acid to single-cell protein: genome-scale revealing the metabolic network of Paracoccus communis MA5

From formic acid to single-cell protein: genome-scale revealing the metabolic network of... greenhouse gas carbon dioxide which has a wide range Introduction of sources, can also be used for producing formic acid Protein is essential for human survival, and its balance is by electrochemical system (Agarwal et  al. 2011; Kopljar indispensable for human health and well-being. With the et al. 2014; Wang et al. 2014; Lu et al. 2014; Pletcher 2015; development of society, the global demand for protein Taheri and Berben 2016; Liu et al. 2018) and hydrogena- ingredients has surged due to increased usage of protein. tion (Enthaler 2008; Wang et  al. 2015). Therefore, using The global protein ingredient market was valued at USD formic acid as a carbon source to produce protein has the 38 billion in 2019 and is expected to grow at a rate of 9.1% potential to be a sustainable and environmentally friendly from 2020 to 2027 (Pam et  al. 2020), causing enormous technology strategy. pressure on global protein supplies. In addition, accel- To date, some microorganisms that can utilize for- erating environmental pollution and population growth mic acid naturally have been reported (Lidstrom 2006; will lead to insufficient protein supply for traditional agri - Chistoserdova et al. 2009; Schrader et al. 2009; Bar-Even culture and animal husbandry (Myers et al. 2017; Awange et al. 2013), and among them, two main metabolic strate- and Kiema 2019; Lv et al. 2021). Therefore, a new produc - gies have been found (Bar-Even et  al. 2013; Yishai et  al. tion mode is urgently needed to ensure the nutritional 2017). In the first strategy, formic acid is oxidized to car - value, safety and sustainability of protein. bon dioxide, and the electrons derived from the oxidation Single-cell protein (SCP), also known as microbial pro- reaction are used to support autotrophic growth through tein, is produced using agro-industrial byproducts based CO fixation and ATP supply (Bar-Even et  al. 2012). In on the cultivation of protein-producing microorganisms, the other strategy, formic acid is assimilated into central including bacteria, yeasts, fungi, and microalgae (Ji et al. carbon metabolism through the tetrahydrofolate cycle 2021; Spalvins et  al. 2021). SCP consists of 80% protein and participates in biomass synthesis via assimilation and contains all essential amino acids (Spalvins et  al. pathways such as the Calvin cycle, the RuBisCo pathway 2021; Matassa et  al. 2016). Compared to proteins from and the serine cycle (Goldberg et  al. 1976; Attwood and plants and animals, the production of SCP has many Harder 1978; Jansen et al. 1984; Vuilleumier et al. 2009). advantages, such as environmental friendliness, water However, these natural formate-utilizing microorganisms and soil savings, and short production cycles and is not generally grow slowly and metabolize formic acid inef- easily affected by the environment (Spalvins et al. 2021). ficiently (Xu et  al. 2020). Therefore, a detailed and com - Therefore, single-cell proteins are a good alternative for prehensive regulatory network of formic acid metabolism substituting animal- and plant-derived dietary proteins. urgently needs to be explored to provide guidance for the Formic acid, an important organic one-carbon source, optimization and modification of formic acid metabo - is widely used in the industrial manufacturing of leather, lism. In recent years, formic acid has been biologically pesticides, medicine and rubber. It is found in the secre- utilized to produce organic compounds such as ethanol tions of ants and caterpillars in nature (Bennett et  al. and acetic acid (Kim et al. 2019; Xu et al. 2020); however, 1996; Xu et al. 2020). At present, methods for producing single-cell protein synthesis from formic acid has yet to formic acid mainly include selective biomass oxidation be reported. The strain showing high homology with P. (Wolfel et al. 2011; Albert et al. 2012, 2014), oxidation of communis G9-1 was isolated from soil and named P. com- methane and ethylene (Sorokin et al. 2010), hydration of munis MA5 (Zhao et  al. 2021). Previous studies have carbon monoxide and carbonylation of methanol with shown that strain MA5 could produce SCP directly from carbon monoxide (Shukla et  al. 2005). In addition, the T ong et al. Bioresources and Bioprocessing (2022) 9:55 Page 3 of 12 sodium formate. When cultured with different nitrogen microplate reader (BioTek, Neo2), and the protein con- sources or sodium formate concentrations, the growth centration was calculated based on a bovine serum albu- rate and protein content were significantly changed. To min standard curve. The experiments were performed achieve efficient utilization of formic acid and high pro - in triplicate on three biological replicates, and error bars duction of SCP, comprehensive metabolic pattern analy- ( ±) represent the standard deviation of three replicates. ses of strain MA5 were carried out at the genomic and transcriptomic levels. Determination of formic acid content The formic acid content was determined using high-per - Materials and methods formance liquid chromatography as previously described Strain and culture conditions (Morris 1987). Briefly, the sample was diluted with P. communis strain MA5 was isolated by our group 10  mM sulfuric acid to a suitable concentration (within and deposited at the CGMCC with accession number the range of the standard curve). Then, formic acid con - CGMCC No.21106. Strain MA5 was routinely grown in tent was measured by an Aminex HPX-87H column (Bio- LB broth (10 g/L tryptone, 5 g/L yeast extract and 10 g/L Rad, USA) with 5  mM sulfuric acid as the mobile phase NaCl) overnight at 37 ℃ and then spread onto a screen- at a 0.5  mL/min flow rate and a 10  μL injection volume. ing plate containing sodium formate (8  g/L HCOONa, Chromatographic pure formic acid was used to prepare 0.5  g/L NaCl, 0.5  g/L M gCl .6H O, 0.1  g/L CaCl .2H O, the standard curve, and the formic acid content in the 2 2 2 2 0.5  g/L KCl, 0.2  g/L KH PO , 1  g/L yeast powder and sample was calculated (g/L). The experiments were per - 2 4 15  g/L agar) at 37 ℃ to maintain bacterial activity. For formed in triplicate on three biological replicates, and transcriptome sequencing, bacteria were washed from error bars ( ±) represent the standard deviation of three LB plates with sterile saline solution and inoculated replicates. at 1% (v/v) into three different fermentation media at 37 ℃ for 48  h. These treatments were named Group A Genome sequencing and analysis (8  g/L HCOONa, 1  g/L yeast powder), Group B (20  g/L Genomic DNA was isolated from strain MA5 using a HCOONa, 1  g/L yeast powder) and Group C (8  g/L Bacterial Genome Extraction Kit according to the manu- HCOONa, 2 g/L (NH ) SO ), and the other components facturer’s instructions (Tiangen, China). DNA integrity 4 2 4 were consistent with the screening medium above. Each was detected by agarose gel electrophoresis (1% w/v), group was analyzed in three independent biological sam- and the concentration was measured in a NanoDrop ples. The bacteria grown in Group A were also selected 2000 (Thermo, USA). The genome of strain MA5 was for genome sequencing. sequenced by the third-generation sequencer PacBio. The SMRT Bell Template Kit was used to construct a 10K Analysis of cell growth SMRT Bell library, and the size of the inserted fragment Cell growth was indirectly observed by measuring the was detected using an Agilent 2100 BioAnalyzer. Library OD . After centrifugation at 10,000  g for 10  min, the construction and sequencing were performed at Beijing cells cultured at 37 ℃ for 48  h in fermentation media Novogene Bioinformatics Technology Co., Ltd. After were washed in sterile water twice and diluted to a suit- removing low-quality sequences from raw data, the high- able concentration. Then, 200  μL of cell suspensions quality clean data that could be used for analysis were were added to a 96-well plate, and the OD values were assembled using SMRT Link v5.0.1 software. The coding measured by a microplate reader (BioTek, Neo2). The sequences were predicted using GeneMarkS software, experiments were performed in triplicate on three bio- and their functions were annotated through comparisons logical replicates, and error bars ( ±) represent the stand- with the NR, COG and KEGG databases. The genome ard deviation of three replicates. sequenced has been deposited at GenBank under acces- sion number CP087597-CP087599. Determination of protein content Determination of the total protein content was per- Transcriptome sequencing and analysis formed as described (Chen et al. 2016) with some modi- Total RNA was extracted from strain MA5 using the fications. Briefly, the bacteria cultured at 37  ℃ for 48  h RNAprep pure Cell/Bacteria Kit according to the instruc- in fermentation media were washed with PBS 3 times tions (Tiangen, China). RNA integrity and concentrations and then resuspended in 10  mL of distilled water. After were detected by agarose gel electrophoresis (1% w/v) ultrasonication for 10 min, 1 mL of the above mentioned and an Agilent 2100 BioAnalyzer. Transcriptomic librar- suspension was mixed with 5 mL of NaOH (0.1 M), and ies were constructed according to the manufacturer’s the final sample was obtained by boiling for 10 min. The instructions, and 2 × 150-bp paired-end sequencing was OD was measured using the Bradford method by a performed on an Illumina platform at Beijing Novogene 595 Tong et al. Bioresources and Bioprocessing (2022) 9:55 Page 4 of 12 Bioinformatics Technology Co., Ltd. The filtered reads Table 1 Physiological parameter detection of strain MA5 under different culture conditions were mapped to the annotated genome of strain MA5 using Bowtie2 (Langmead and Salzberg 2012). BAM files Sample Formic acid Biomass (OD ) Protein content (%) were then converted from alignment files using SAM - absorption (g/L) tools v1.3.1 (Li and Durbin 2009). Read counts per gene were collected using HTSeq v0.6.1 (Anders et  al. 2015), A 4.61 ± 0.24 0.56 ± 0.07 44 ± 1.55 followed by exploration of the differential expression a b a B 1.51 ± 0.22 0.49 ± 0.09 30 ± 1.79 across pairwise comparisons of the three conditions b a a C 4.40 ± 0.37 0.21 ± 0.05 53 ± 1.31 using DESeq v1.26.0 (Anders and Huber 2010). KEGG Culture condition A: 8 g/L HCOONa, 1 g/L yeast powder, 0.5 g/L NaCl, 0.5 g/L enrichment analysis of differentially expressed genes (p MgCl .6H O, 0.1 g/L CaCl .2H O, 0.5 g/L KCl, 0.2 g/L KH PO . Culture condition 2 2 2 2 2 4 B: 20 g/L HCOONa, 1 g/L yeast powder, 0.5 g/L NaCl, 0.5 g/L MgCl .6H O, 0.1 g/L value < 0.05) was performed in ClusterProfiler software 2 2 CaCl .2H O, 0.5 g/L KCl, 0.2 g/L KH PO . Culture condition C: 8 g/L HCOONa, 2 2 2 4 (Morin et  al.  2018), and a heatmap was drawn using 2 g/L (NH4) SO , 0.5 g/L NaCl, 0.5 g/L MgCl .6H O, 0.1 g/L CaCl .2H O, 0.5 g/L 2 4 2 2 2 2 TBtools (Chen et al. 2020). KCl, 0.2 g/L KH PO . Error bars ( ±) represent the standard deviation of three 2 4 replicates. Letters indicate statistically significant differences, as determined by a b Student’s t test ( indicates p < 0.01; indicates no significance) Gene expression level detection by real‑time polymerase chain reaction (PCR) Total RNA was isolated as described above and converted Table 2 Genome statistics to cDNA using a FastKing RT Kit (Tiangen, China). The Attribute Value % of total* expression of target genes was analyzed by real-time PCR with dnaN as the reference gene, and the PCR pro- Genome size (bp) 4,473,736 100 cedure was set according to the instructions of the Pow- Chr1 (bp) 1,412,597 31.58 erUp SYBR Green Master Mix Kit (Thermo, USA). The Chr2 (bp) 2,389,614 53.41 primers used in this study are listed in Additional file  1: Plas1 (bp) 671,525 15.01 Table S1. GC content (bp) 3,067,193 68.56 Coding region (bp) 4,013,268 89.71 Results Coding genes (No.) 4361 Physiological parameter detection of P. communis MA5 Protein-coding genes (No.) 4301 98.62 To investigate the characteristics of formic acid metab- RNA genes (No.) 60 1.38 olism and protein synthesis of strain MA5 in sodium Prophages (No.) 5 formate stress or different nitrogen sources (organic or CIRSPR region (No.) 1 inorganic nitrogen), formic acid consumption, biomass The total is based on either the size of the genome in base pairs or the total and protein content were monitored under selected cul- number of protein-coding genes in the annotated genome ture conditions. Compared to the standard culture con- dition (A: normal sodium formate concentration and RIN > 8). Moreover, the concentrations of RNA (> 300 ng/ organic nitrogen), formic acid consumption and protein μL) and genomic DNA (84.2  ng/μL) both reached the content were decreased significantly by 67% and 31.8% in requirements of library construction (Additional file  1: the sodium formate stress treatment (B: sodium formate Table  S2 and S3). These results suggest that the above stress and organic nitrogen), while a 62.5% decrease in samples can be used for whole-genome and transcrip- growth rate and a 20.4% increase in protein content were tome sequencing. observed under the culture conditions with ammonium sulfate instead of yeast extract (C: normal sodium for- Genome assembly, gene prediction and functional mate concentration and inorganic nitrogen) (Table 1). annotation The draft genome of strain MA5 consists of 3 contigs, Extraction and quality analysis of genomic DNA and total including two annular chromosomes and an annu- RNA from P. communis MA5 lar plasmid (Additional file  1: Figure S2). The genome For omics analysis, the genomic DNA and total RNA of size is approximately 4,473,736  bp with a GC content of P. communis MA5 were first extracted and their qual - 68.56%, and the lengths of the 3 contigs are 1,412,597 bp ity were evaluated. The electrophoresis results revealed (Chr1), 2,389,614  bp (Chr2) and 671,525  bp (Plas1). A that the main bands of genomic DNA and RNA showed total of 4361 genes comprising 4,013,268  bp were pre- almost no degradation (Additional file  1: Figure S1), and dicted, 4301 of which were protein-coding genes, and 60 RNA integrity was further confirmed by RNA integ - were RNA genes. In addition, five prophages with sizes rity number (RIN) detection (Additional file  1: Table S2, T ong et al. Bioresources and Bioprocessing (2022) 9:55 Page 5 of 12 from 7164 bp to 44,730 bp and a CIRSPR region together formaldehyde dehydrogenase system, tetrahydrofolate with cas3 upstream of this region were identified in the cycle, serine cycle, glycolytic pathway, TCA cycle and genome of MA5 (Table 2). nitrogen metabolism (Fig. 2). In strain MA5, formic acid According to the KO assignment and KEGG pathway is transformed to methylene-tetrahydrofolate through mapping, 2355 (54.75%) protein-coding genes could be tetrahydrofolate ligase, methylene-tetrahydrofolate dehy- assigned to 222 metabolic pathways (one gene may cor- drogenase and methylene-tetrahydrofolate hydrolase. respond to many pathways). These pathways are classi - The ligase is encoded by the fhs gene, and the dehydro- fied into 6 categories: cellular processes, environmental genase and hydrolase are encoded by the same gene, information processing, genetic information processing, named folD. Thereafter, methylene-tetrahydrofolate human diseases, metabolism, and organismal systems. reacts with glycine via glycine hydroxymethyltransferase The vast majority of genes (2230) were concentrated in (glyA) to produce l-serine, entering biomass synthesis the category of metabolism, and among them, 13% of metabolism through the serine cycle. The serine cycle is genes (289/2230) were focused on amino acid metabo- the only way to assimilate formic acid into central carbon lism (the 2nd most in metabolism) (Fig.  1A). Similarly, metabolism and consists of two parts. One is the serine using COG function assignment, 3477 (80.84%) protein- regeneration cycle, and the other is the glyoxylic acid coding genes could be classified into 24 COG catego - regeneration cycle. The common branch of both is com - ries, and the most abundant category of genes (465) was posed of serine glyoxylate aminotransferase encoded by related to amino acid transport and metabolism (Fig.  1B the sga gene, hydroxypyruvate reductase encoded by the and Additional file  1: Table  S4). Based on the results hpr gene, and glycerate kinase encoded by the gck gene. above, we suspect that active amino acid metabolic path- Moreover, a phosphoenolpyruvate carboxylase was found ways in strain MA5 can explain the efficient synthesis of in MA5. The enzyme can catalyze the carboxylation of protein. carbon dioxide with phosphoenolpyruvate to produce oxaloacetic acid (an important intermediate in the TCA Genome‑scale reconstruction of the central metabolic cycle), which is able to neutralize carbon dioxide (Puri networks in strain MA5 et  al. 2020). The common glycolytic pathway and TCA The central metabolic networks of strain MA5 were cycle were also activated in MA5. Through these path - proposed according to the genome analysis results ways, formic acid is drastically introduced into the cen- and included an integrated glutathione-dependent tral metabolism. Fig. 1 KEGG pathway mapping (A) and COG functional assignment (B) of genes in the genome of strain MA5. The numerical value above the column represents the number of genes in the corresponding pathway or classification. The genes were mainly concentrated in the category of metabolism in KEGG pathway mapping and on amino acid transport and metabolism in COG function assignment Tong et al. Bioresources and Bioprocessing (2022) 9:55 Page 6 of 12 Fig. 2 Proposed central metabolism pathways of strain MA5. maxF methanol dehydrogenase, gfa S-(hydroxymethyl)glutathione synthase, frmA S-(hydroxymethyl)glutathione dehydrogenase, frmB S-formylglutathione hydrolase, fdh formate dehydrogenase, fhs formate-tetrahydrofolate ligase, purU formyltetrahydrofolate deformylase, folD methylene-tetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase, glyA glycine hydroxymethyltransferase, sga serine glyoxylate transaminase, hprA glycerate dehydrogenase, gckA glycerate 2-kinase, gpmI 2,3-bisphosphoglycera te-independent phosphoglycerate mutase, serA D-3-phosphoglycerate dehydrogenase, serC phosphoserine aminotransferase’, serB phosphoserine phosphatase, eno enolase, ppc phosphoenolpyruvate carboxylase, pckA phosphoenolpyruvate carboxykinase, mdh malate dehydrogenase, mtkB malate-CoA ligase, mcl malyl-CoA/(S)-citramalyl-CoA lyase, agxt2 alanine-glyoxylate transaminase, tpiA triosephosphate isomerase, fbaA fructose-bisphosphate aldolase, pfkA 6-phosphofructokinase, fbp fructose-1,6-bisphosphatase, pgi glucose-6-phosphate isomerase, gck glucokinase, pyk pyruvate kinase, gltA citrate synthase, acnA aconitate hydratase, icd isocitrate dehydrogenase, sucA 2-oxoglutarate dehydrogenase E1 component, sucB 2-oxoglutarate dehydrogenase E2 component, sucD succinyl-CoA synthetase alpha subunit, aarC succinyl-CoA:acetate CoA-transferase, frdA fumarate reductase flavoprotein subunit, fumAB fumarate hydratase, narGHI nitrate reductase, nirBS nitrite reductase, norB nitric oxide reductase, nosZ nitrous oxide reductase, gdhA glutamate dehydrogenase. Dotted arrows indicate multistep reactions For nitrogen metabolism, there are two branches in via RNA-seq analyses as detailed in the Materials and MA5. One is the dissimilative nitrate reduction path- methods section. A total of 1.55, 1.51, and 1.53  million way catalyzed by nitrate reductase and nitrite reductase. reads were generated, and 99.3%, 99.4%, and 98.9% were In this pathway, nitrate is first reduced to ammonia, and mapped to the strain MA5 genome in the A-C sample, then ammonia is transformed to glutamate via gluta- respectively. A comparison of differentially expressed mate dehydrogenase and subsequently enters glutamate genes (DEGs) between B (or C) and A was performed. metabolism. The other branch is the denitrification path - The analysis results revealed a total of 123 transcripts way catalyzed by nitrate reductase, nitrite reductase, and upregulated and 119 downregulated in the B vs. A group nitric oxide reductase, in which nitrate is finally reduced (Fig.  3A), and 935 transcripts were upregulated and to nitrogen and discharged from the body. With the help 350 were downregulated in the C vs. A group (Fig.  3B). of the entire central metabolic network, protein synthe- Cross-comparison between the B vs. A group and the C sis based on formic acid as a carbon source proceeds vs. A group showed 149 DEGs shared within both groups smoothly. (Fig. 3C). Among them, more than half of the genes were downregulated compared with the control treatment RNA‑seq and differential expression analysis (Fig.  3D), and the specific DEG annotations are listed in Based on the experimental results, gene expression pro- Additional file 1: Table S5. filing of bacteria grown in different media was performed T ong et al. Bioresources and Bioprocessing (2022) 9:55 Page 7 of 12 Fig. 3 Analysis of differentially expressed genes (DEGs) under three conditions. A Volcano plot of DEGs in the B vs. A group. B Volcano plot of DEGs in the C vs. A group. Red dots represent upregulated DEGs, green dots represent downregulated DEGs, and blue dots represent unchanged genes. The DEGs were identified with a p value < 0.05 and |log2FoldChang|> 0. C Venn diagram between the B vs. A group and the C vs. A group. D Heatmap of shared genes involved in both groups. The heatmap was drawn using TBtools software, and the rows were normalized and clustered Sodium formate stress results in elevated expression is the main component of the bacterial cell wall, offer - of peptidoglycan synthesis‑related genes but a weakening ing the function of increasing resistance to environmental in the sulfur metabolic pathway stress. Therefore, the upregulated expression of peptidogly - KEGG pathway enrichment network analyses revealed that can synthesis-related genes under sodium formate stress the upregulated DEGs of the B vs. A group showed signifi - is reasonable. Similarly, sharply grouped genes mapped to cant enrichment in the peptidoglycan synthesis pathway microbial metabolism in diverse environments were upreg- containing ligase, N-acetylglucosamine transferase, and ulated in response to stress (Fig.  4A). Of note, the down- peptidoglycan glycosyltransferase (Fig.  4A). Peptidoglycan regulated DEGs were significantly enriched in the sulfur Tong et al. Bioresources and Bioprocessing (2022) 9:55 Page 8 of 12 Fig. 4 KEGG pathway enrichment of DEGs in the B vs. A group. A KEGG pathway enrichment of the upregulated DEGs. B KEGG pathway enrichment of the downregulated DEGs. The size of the circle represents the number of DEGs in the pathway, and the color of the circle represents the padj value (p value adjusted to false discovery rate), reflecting the enrichment degree. C The DEGs involved in the denitrification pathway. The genes encoding enzymes in the rectangular box with a red border were significantly upregulated, and the FPKM of each gene is shown in the rectangle inside the ellipse metabolic pathway (Fig.  4B). Sulfur is an essential compo- Ammonium sulfate (nitrogen source) stimulates nent of sulfur-containing amino acids, such as cysteine and the expression of a suite of metabolic pathways associated methionine, which play a crucial role in protein synthesis. with protein synthesis and inhibits the tetrahydrofolate The significant downregulation of sulfite reductase and sul - cycle associated with formic acid assimilation fate/thiosulfate transporters under sodium formate stress The experimental results above showed an increase in treatment indicated that high concentrations of sodium protein content and a decrease in growth rate when formate could inhibit amino acid synthesis, resulting in a ammonium sulfate (replacing yeast extract) as a nitro- decrease in protein content. Furthermore, the key genes gen source. To explain this phenomenon, the influence of the denitrification pathway in nitrogen metabolism, in of ammonium sulfate on the global gene expression was which nitrate is ultimately reduced to nitrogen, were sig- analyzed via transcriptome sequencing. KEGG pathway nificantly upregulated (Fig.  4C). This was also one of the enrichment of C vs. A group DEGs was represented in factors causing the decrease in protein content. Intrigu- ribosome, oxidative phosphorylation, aminoacyl-tRNA ingly, moderately weakened expression was observed in the biosynthesis, citrate cycle (TCA cycle), biosynthesis of key genes of the tetrahydrofolate cycle associated with for- amino acids, etc. (Fig.  5A). Based on the transcriptom- mic acid utilization, although strain MA5 was cultured in ics data, these pathways were significantly promoted by high sodium formate medium. A complete list of the DEGs ammonium sulfate to provide more venues, vehicles, mentioned above is provided in Additional file  1: Table S6. precursor substances and energy for protein synthesis. The expression levels of some genes in the B vs. A group Besides, genes participating in protein export and bac- were further confirmed by real-time PCR (Additional file 1 : terial secretion system pathways were also activated, Figure S3). indicating the extracellular protein content might also T ong et al. Bioresources and Bioprocessing (2022) 9:55 Page 9 of 12 Fig. 5 KEGG pathway enrichment (A) and gene information display (B) of DEGs in the C vs. A group. The size of the circle represents the number of DEGs in the pathway, and the color of the circle represents padj values (p value adjusted to false discovery rate), reflecting the enrichment degree be increased (Fig.  5A). The DEG-related information with formic acid fixation and assimilation was found is listed in Additional file  1: Table  S7. In contrast, some (Fig.  2). In this cycle, the key enzymes dehydrogenase amino acid transporters, such as general l-amino acid and hydrolase are encoded by the same gene in MA5, transporters, branched-chain amino acid transport- named folD, while the different coding genes correspond - ers and d-methionine transporters, were significantly ing to two enzymes existed in methanotrophs Methy- downregulated because ammonium sulfate is a rapidly locystis SB2 (Vorobev et al. 2014) and Methylobacterium available nitrogen source and can be directly absorbed extorquens AM1 (Crowther et  al. 2008). The catalytic (Fig.  5B and Additional file  1: Figure S4). The tetrahy - reaction from formic acid and tetrahydrofolate to meth- drofolate cycle is a key pathway to formic acid metabo- yltetrahydrofolate by tetrahydrofolate ligase is an irre- lism. Under culture conditions with ammonium sulfate, versible reaction, and the reverse reaction is mediated by formate-tetrahydrofolate ligase and methylene-tetrahy- formyltetrahydrofolate deformylase. However, in Methy- drofolate dehydrogenase participating in the formic acid lobacterium extorquens AM1, this catalytic reaction is assimilation pathway were significantly downregulated, reversible (Marx et al. 2003). The coding gene of metha - while formate dehydrogenase catalyzing formic acid to nol dehydrogenase catalyzing methanol to formaldehyde carbon dioxide was significantly upregulated (Fig.  5B). and the genes for the glutathione-dependent formalde- Interestingly, the quorum sensing pathway related to cell hyde dehydrogenase system leading to the production of density regulation was repressed, which might be a signal formic acid from formaldehyde were annotated in strain of reduced growth rate (Additional file  1: Figure S4). The MA5 (Fig.  2). This methanol–formaldehyde–formic acid expression levels of some genes in the C vs. A group were pathway was also reported in Paracoccus AK26 (Puri further confirmed by real-time PCR (Additional file  1: et al. 2020). However, the key genes encoding 3-hexulose- Figure S5). 6-phosphate synthase and hexulose phosphate isomer- ase in the formaldehyde assimilation pathway were not Discussion found. This result suggests that formic acid rather than Strain MA5 can grow in a medium containing formic formaldehyde enters biomass synthesis metabolism when acid as a single carbon source. Based on the genome strain MA5 grows in a medium with methanol as a single sequences, a complete tetrahydrofolate cycle associated carbon source. Tong et al. Bioresources and Bioprocessing (2022) 9:55 Page 10 of 12 Studies have reported that three assimilation pathways to produce l-glutamic acid (Schure et  al. 1995), was sig- of formic acid, namely, the Calvin cycle, the RuBisCo nificantly upregulated. We believe that the increased pathway and the serine cycle, were identified in different expression of these genes is the main reason for the organisms (Chistoserdova 2011; Puri et al. 2020). Except increased protein content in the C sample (Table  1). for the key genes in the serine cycle, the genes encoding Notably, the significant downregulation of formic acid enzymes for the Calvin cycle and the RuBisCo pathway assimilation-relevant genes and the significant upregu - were not found in the genome of strain MA5. Therefore, lation of formate dehydrogenase-encoding genes (Li the serine cycle is the only pathway for the assimilation et  al. 2019) resulted in carbon escape from formic acid of formic acid in strain MA5, which is consistent with (Fig. 5B). We speculate that it is the “murderer” of reduc- what has been reported in Paracoccus AK26 (Puri et  al. tion in biomass when strain MA5 grows under culture 2020). Furthermore, the complete serine pathway was conditions with ammonium sulfate (Table 1). The expres - also found in Methylocystis SB2 (Vorobev et  al. 2014), sion levels of some representative genes detected by and both the serine cycle and the Calvin cycle are present real-time PCR were consistent with the transcriptome in Paracoccus sp. N5 (Puri et  al. 2020). Surprisingly, the profile analysis, which clearly validated the reliability of gene encoding phosphoenolpyruvate carboxylase cata- the DEGs in transcriptome analysis (Additional file  1: lyzing the carboxylation of carbon dioxide and phospho- Figure S3, S5). Overall, our results provide a potential enolpyruvate into oxaloacetic acid was also annotated central metabolic network for understanding formic acid (Fig. 2). This discovery raises the prospect of strain MA5 metabolism and protein synthesis in P. communis MA5. and indicates that it has the ability to neutralize carbon However, some specific metabolic and regulatory mech - dioxide. anisms still need to be explored to provide theoretical In this study, comparative transcriptomic results indi- ground for metabolic engineering designs of this strain in cated that a high concentration of sodium formate can the future. stimulate the expression of genes responding to envi- ronmental stress, including gene enrichment in the pep- Conclusions tidoglycan synthesis pathway and microbial metabolism In conclusion, in order to detect the excellent ability to in diverse environments (Fig.  4A). Studies have reported synthesize SCP directly from sodium formate, genome- that Lactobacillus and Streptococcus can increase the pro- scale analyses of P. communis MA5 were performed. portion of monounsaturated fatty acids in the cell mem- Our results indicated that the complete tetrahydrofolate brane when faced with environmental stress (Quivey cycle, serine cycle, glycolytic pathway, TCA cycle and et  al. 2000; Montanari et  al. 2010). In strain MA5, the nitrogen metabolism playing key roles in the conver- expression of genes encoding acyl carrier protein syn- sion of formic acid into proteins were annotated in the thetase and 3-oxygen acetyl-CoA carboxylase involved genome. Transcriptional analysis showed sodium for- in monounsaturated fatty acid synthesis metabolism was mate stress could increase resistance of MA5 to environ- also increased dramatically. Here, we focused on the key mental stress by stimulating the peptidoglycan synthesis nodes of protein synthesis and formic acid metabolism. pathway and microbial metabolism in diverse environ- Based on omics analysis, an enhanced denitrification ments, but inhibit amino acid synthesis by weakening the pathway and attenuated sulfur metabolism could account sulfur metabolic pathway, resulting in a 31.8% decrease for the experimental result that the protein content was in protein content. Moreover, ammonium sulfate as a decreased in strain MA5 under sodium formate stress nitrogen source (replacing yeast extract) could increase (Fig.  4B, C and Table  1). Intriguingly, formic acid con- protein content (20.4%) due to stimulation the pathways sumption was decreased significantly along with mod - associated with protein synthesis, such as ribosome, erately decreased expression levels of key genes in the aminoacyl-tRNA biosynthesis, TCA cycle, biosynthesis tetrahydrofolate cycle under sodium formate stress treat- of amino acids, etc.; while decrease growth rate (62.5%) ment (Table 1 and Additional file  1: Table S6), suggesting due to suppression the tetrahydrofolate cycle associated that potential negative feedback regulation of formic acid with formic acid assimilation. Our study provides theo- metabolism in response to sodium formate stress exists. retical guidance for the optimization of fermentation sys- Compared to yeast extract, metabolic pathways asso- tems using formic acid as a carbon source and lays the ciated with protein synthesis, including ribosome, oxi- foundation for further study on the regulatory mecha- dative phosphorylation, aminoacyl-tRNA biosynthesis, nism of formic acid metabolism. In the future, genetic citrate cycle (TCA cycle), and biosynthesis of amino transformation system and gene editing system such as acids, showed higher activity under ammonium sulfate CRISPR–Cas9 should been established, to advance the culture conditions (Fig. 5A). l-Glutamate dehydrogenase, development of P. communis MA5 as a SCP-based cell reported to catalyze ammonium and α-ketoglutaric acid factory through metabolic engineering, and to elucidate T ong et al. Bioresources and Bioprocessing (2022) 9:55 Page 11 of 12 Competing interests the regulatory mechanism of efficient metabolism of for - The authors declare no competing interest. mic acid in this strain. Author details Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Abbreviations Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy SCP: Single-cell protein; TCA: Tricarboxylic acid; PCR: Polymerase chain reac- of Sciences, Tianjin 300308, China. Tianjin Key Laboratory of Brine Chemi- tion; DEGs: Differentially expressed genes. cal Engineering and Resource Eco-Utilization, Tianjin University of Sciences and Technology, Tianjin 300457, China. National Innovation Centre for Syn- thetic Biology, Tianjin 300308, China. Supplementary Information The online version contains supplementary material available at https:// doi. Received: 2 March 2022 Accepted: 29 April 2022 org/ 10. 1186/ s40643- 022- 00544-0. Additional file1: Table S1. Primers used in this study. Table S2. Concen- tration detection and integrity analysis of RNA. Table S3. Concentration References detection and integrity analysis of DNA. Table S4. Number of genes Agarwal AS, Zhai Y, Hill D, Sridhar N (2011) The electrochemical reduction associated with the 24 general COG functional categories. Table S5. Dif- of carbon dioxide to formate/formic acid: engineering and economic ferentially expressed genes (DEGs) in both the B vs. A group and the C vs. feasibility. Chemsuschem 4(12):1705–1705. https:// doi. org/ 10. 1002/ cssc. A group. Table S6. Significant KEGG pathway-relevant DEGs in the B vs. 20110 0752 A group. Table S7. Significant KEGG pathway-relevant DEGs in the C vs. Albert J, Luders D, Bsmann A, Guldi DM, Wasserscheid P (2014) Spectroscopic A group. Fig. S1 Agarose electrophoresis results of genomic DNA (A) and and electrochemical characterization of heteropoly acids for their opti- total RNA (B). Genomic DNA was isolated from strain MA5 grown in Group mized application in selective biomass oxidation to formic acid. Green A. A1-A3: total RNA extraction from Group A; B1-B3: total RNA extraction Chem 16:226–237. https:// doi. org/ 10. 1039/ C3GC4 1320A from Group B; C1-C3: total RNA extraction from Group C. Fig. S2 Circular Albert J, Wolfel R, Bosmann A, Wasserscheid P (2012) Selective oxidation of representation of the genome of strain MA5. The genome of strain MA5 complex, water-insoluble biomass to formic acid using additives as reac- consists of 3 contigs, Chr1 (1.41 MB), Chr2 (2.39 MB) and Plas1 (0.67 MB). tion accelerators. Energ Environ Sci 5(7):7956–7962. https:// doi. org/ 10. Fig. S3 Expression of the selected DEGs in the B vs. A group was examined 1039/ C2EE2 1428H by real-time PCR with dnaN as the reference gene. Error bars represent the Anders S, Huber W (2010) Differential expression analysis for sequence standard deviation of three replicates. odc: ornithine decarboxylase; fadH: count data. Genome Biol 11(10):R106. https:// doi. org/ 10. 1186/ 3-oxoacyl-[acyl-carrier-protein] synthase; cysI: sulfite reductase; cdp: cell gb- 2010- 11- 10- r106 division protein; katG: peroxidase; nir: nitrite reductase. Fig. S4 KEGG path- Anders S, Pyl PT, Huber W (2015) HTSeq-a Python framework to work with way enrichment of the downregulated DEGs in the C vs. A group. The size high-throughput sequencing data. Bioinformatics 31(2):166–169. https:// of the circle represents the number of DEGs in the pathway, and the color doi. org/ 10. 1101/ 002824 of the circle represents the padj value (p value adjusted to false discovery Attwood MM, Harder W (1978) Formate assimilation by Hyphomicrobium X. rate), reflecting the enrichment degree. 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From formic acid to single-cell protein: genome-scale revealing the metabolic network of Paracoccus communis MA5

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Abstract

greenhouse gas carbon dioxide which has a wide range Introduction of sources, can also be used for producing formic acid Protein is essential for human survival, and its balance is by electrochemical system (Agarwal et  al. 2011; Kopljar indispensable for human health and well-being. With the et al. 2014; Wang et al. 2014; Lu et al. 2014; Pletcher 2015; development of society, the global demand for protein Taheri and Berben 2016; Liu et al. 2018) and hydrogena- ingredients has surged due to increased usage of protein. tion (Enthaler 2008; Wang et  al. 2015). Therefore, using The global protein ingredient market was valued at USD formic acid as a carbon source to produce protein has the 38 billion in 2019 and is expected to grow at a rate of 9.1% potential to be a sustainable and environmentally friendly from 2020 to 2027 (Pam et  al. 2020), causing enormous technology strategy. pressure on global protein supplies. In addition, accel- To date, some microorganisms that can utilize for- erating environmental pollution and population growth mic acid naturally have been reported (Lidstrom 2006; will lead to insufficient protein supply for traditional agri - Chistoserdova et al. 2009; Schrader et al. 2009; Bar-Even culture and animal husbandry (Myers et al. 2017; Awange et al. 2013), and among them, two main metabolic strate- and Kiema 2019; Lv et al. 2021). Therefore, a new produc - gies have been found (Bar-Even et  al. 2013; Yishai et  al. tion mode is urgently needed to ensure the nutritional 2017). In the first strategy, formic acid is oxidized to car - value, safety and sustainability of protein. bon dioxide, and the electrons derived from the oxidation Single-cell protein (SCP), also known as microbial pro- reaction are used to support autotrophic growth through tein, is produced using agro-industrial byproducts based CO fixation and ATP supply (Bar-Even et  al. 2012). In on the cultivation of protein-producing microorganisms, the other strategy, formic acid is assimilated into central including bacteria, yeasts, fungi, and microalgae (Ji et al. carbon metabolism through the tetrahydrofolate cycle 2021; Spalvins et  al. 2021). SCP consists of 80% protein and participates in biomass synthesis via assimilation and contains all essential amino acids (Spalvins et  al. pathways such as the Calvin cycle, the RuBisCo pathway 2021; Matassa et  al. 2016). Compared to proteins from and the serine cycle (Goldberg et  al. 1976; Attwood and plants and animals, the production of SCP has many Harder 1978; Jansen et al. 1984; Vuilleumier et al. 2009). advantages, such as environmental friendliness, water However, these natural formate-utilizing microorganisms and soil savings, and short production cycles and is not generally grow slowly and metabolize formic acid inef- easily affected by the environment (Spalvins et al. 2021). ficiently (Xu et  al. 2020). Therefore, a detailed and com - Therefore, single-cell proteins are a good alternative for prehensive regulatory network of formic acid metabolism substituting animal- and plant-derived dietary proteins. urgently needs to be explored to provide guidance for the Formic acid, an important organic one-carbon source, optimization and modification of formic acid metabo - is widely used in the industrial manufacturing of leather, lism. In recent years, formic acid has been biologically pesticides, medicine and rubber. It is found in the secre- utilized to produce organic compounds such as ethanol tions of ants and caterpillars in nature (Bennett et  al. and acetic acid (Kim et al. 2019; Xu et al. 2020); however, 1996; Xu et al. 2020). At present, methods for producing single-cell protein synthesis from formic acid has yet to formic acid mainly include selective biomass oxidation be reported. The strain showing high homology with P. (Wolfel et al. 2011; Albert et al. 2012, 2014), oxidation of communis G9-1 was isolated from soil and named P. com- methane and ethylene (Sorokin et al. 2010), hydration of munis MA5 (Zhao et  al. 2021). Previous studies have carbon monoxide and carbonylation of methanol with shown that strain MA5 could produce SCP directly from carbon monoxide (Shukla et  al. 2005). In addition, the T ong et al. Bioresources and Bioprocessing (2022) 9:55 Page 3 of 12 sodium formate. When cultured with different nitrogen microplate reader (BioTek, Neo2), and the protein con- sources or sodium formate concentrations, the growth centration was calculated based on a bovine serum albu- rate and protein content were significantly changed. To min standard curve. The experiments were performed achieve efficient utilization of formic acid and high pro - in triplicate on three biological replicates, and error bars duction of SCP, comprehensive metabolic pattern analy- ( ±) represent the standard deviation of three replicates. ses of strain MA5 were carried out at the genomic and transcriptomic levels. Determination of formic acid content The formic acid content was determined using high-per - Materials and methods formance liquid chromatography as previously described Strain and culture conditions (Morris 1987). Briefly, the sample was diluted with P. communis strain MA5 was isolated by our group 10  mM sulfuric acid to a suitable concentration (within and deposited at the CGMCC with accession number the range of the standard curve). Then, formic acid con - CGMCC No.21106. Strain MA5 was routinely grown in tent was measured by an Aminex HPX-87H column (Bio- LB broth (10 g/L tryptone, 5 g/L yeast extract and 10 g/L Rad, USA) with 5  mM sulfuric acid as the mobile phase NaCl) overnight at 37 ℃ and then spread onto a screen- at a 0.5  mL/min flow rate and a 10  μL injection volume. ing plate containing sodium formate (8  g/L HCOONa, Chromatographic pure formic acid was used to prepare 0.5  g/L NaCl, 0.5  g/L M gCl .6H O, 0.1  g/L CaCl .2H O, the standard curve, and the formic acid content in the 2 2 2 2 0.5  g/L KCl, 0.2  g/L KH PO , 1  g/L yeast powder and sample was calculated (g/L). The experiments were per - 2 4 15  g/L agar) at 37 ℃ to maintain bacterial activity. For formed in triplicate on three biological replicates, and transcriptome sequencing, bacteria were washed from error bars ( ±) represent the standard deviation of three LB plates with sterile saline solution and inoculated replicates. at 1% (v/v) into three different fermentation media at 37 ℃ for 48  h. These treatments were named Group A Genome sequencing and analysis (8  g/L HCOONa, 1  g/L yeast powder), Group B (20  g/L Genomic DNA was isolated from strain MA5 using a HCOONa, 1  g/L yeast powder) and Group C (8  g/L Bacterial Genome Extraction Kit according to the manu- HCOONa, 2 g/L (NH ) SO ), and the other components facturer’s instructions (Tiangen, China). DNA integrity 4 2 4 were consistent with the screening medium above. Each was detected by agarose gel electrophoresis (1% w/v), group was analyzed in three independent biological sam- and the concentration was measured in a NanoDrop ples. The bacteria grown in Group A were also selected 2000 (Thermo, USA). The genome of strain MA5 was for genome sequencing. sequenced by the third-generation sequencer PacBio. The SMRT Bell Template Kit was used to construct a 10K Analysis of cell growth SMRT Bell library, and the size of the inserted fragment Cell growth was indirectly observed by measuring the was detected using an Agilent 2100 BioAnalyzer. Library OD . After centrifugation at 10,000  g for 10  min, the construction and sequencing were performed at Beijing cells cultured at 37 ℃ for 48  h in fermentation media Novogene Bioinformatics Technology Co., Ltd. After were washed in sterile water twice and diluted to a suit- removing low-quality sequences from raw data, the high- able concentration. Then, 200  μL of cell suspensions quality clean data that could be used for analysis were were added to a 96-well plate, and the OD values were assembled using SMRT Link v5.0.1 software. The coding measured by a microplate reader (BioTek, Neo2). The sequences were predicted using GeneMarkS software, experiments were performed in triplicate on three bio- and their functions were annotated through comparisons logical replicates, and error bars ( ±) represent the stand- with the NR, COG and KEGG databases. The genome ard deviation of three replicates. sequenced has been deposited at GenBank under acces- sion number CP087597-CP087599. Determination of protein content Determination of the total protein content was per- Transcriptome sequencing and analysis formed as described (Chen et al. 2016) with some modi- Total RNA was extracted from strain MA5 using the fications. Briefly, the bacteria cultured at 37  ℃ for 48  h RNAprep pure Cell/Bacteria Kit according to the instruc- in fermentation media were washed with PBS 3 times tions (Tiangen, China). RNA integrity and concentrations and then resuspended in 10  mL of distilled water. After were detected by agarose gel electrophoresis (1% w/v) ultrasonication for 10 min, 1 mL of the above mentioned and an Agilent 2100 BioAnalyzer. Transcriptomic librar- suspension was mixed with 5 mL of NaOH (0.1 M), and ies were constructed according to the manufacturer’s the final sample was obtained by boiling for 10 min. The instructions, and 2 × 150-bp paired-end sequencing was OD was measured using the Bradford method by a performed on an Illumina platform at Beijing Novogene 595 Tong et al. Bioresources and Bioprocessing (2022) 9:55 Page 4 of 12 Bioinformatics Technology Co., Ltd. The filtered reads Table 1 Physiological parameter detection of strain MA5 under different culture conditions were mapped to the annotated genome of strain MA5 using Bowtie2 (Langmead and Salzberg 2012). BAM files Sample Formic acid Biomass (OD ) Protein content (%) were then converted from alignment files using SAM - absorption (g/L) tools v1.3.1 (Li and Durbin 2009). Read counts per gene were collected using HTSeq v0.6.1 (Anders et  al. 2015), A 4.61 ± 0.24 0.56 ± 0.07 44 ± 1.55 followed by exploration of the differential expression a b a B 1.51 ± 0.22 0.49 ± 0.09 30 ± 1.79 across pairwise comparisons of the three conditions b a a C 4.40 ± 0.37 0.21 ± 0.05 53 ± 1.31 using DESeq v1.26.0 (Anders and Huber 2010). KEGG Culture condition A: 8 g/L HCOONa, 1 g/L yeast powder, 0.5 g/L NaCl, 0.5 g/L enrichment analysis of differentially expressed genes (p MgCl .6H O, 0.1 g/L CaCl .2H O, 0.5 g/L KCl, 0.2 g/L KH PO . Culture condition 2 2 2 2 2 4 B: 20 g/L HCOONa, 1 g/L yeast powder, 0.5 g/L NaCl, 0.5 g/L MgCl .6H O, 0.1 g/L value < 0.05) was performed in ClusterProfiler software 2 2 CaCl .2H O, 0.5 g/L KCl, 0.2 g/L KH PO . Culture condition C: 8 g/L HCOONa, 2 2 2 4 (Morin et  al.  2018), and a heatmap was drawn using 2 g/L (NH4) SO , 0.5 g/L NaCl, 0.5 g/L MgCl .6H O, 0.1 g/L CaCl .2H O, 0.5 g/L 2 4 2 2 2 2 TBtools (Chen et al. 2020). KCl, 0.2 g/L KH PO . Error bars ( ±) represent the standard deviation of three 2 4 replicates. Letters indicate statistically significant differences, as determined by a b Student’s t test ( indicates p < 0.01; indicates no significance) Gene expression level detection by real‑time polymerase chain reaction (PCR) Total RNA was isolated as described above and converted Table 2 Genome statistics to cDNA using a FastKing RT Kit (Tiangen, China). The Attribute Value % of total* expression of target genes was analyzed by real-time PCR with dnaN as the reference gene, and the PCR pro- Genome size (bp) 4,473,736 100 cedure was set according to the instructions of the Pow- Chr1 (bp) 1,412,597 31.58 erUp SYBR Green Master Mix Kit (Thermo, USA). The Chr2 (bp) 2,389,614 53.41 primers used in this study are listed in Additional file  1: Plas1 (bp) 671,525 15.01 Table S1. GC content (bp) 3,067,193 68.56 Coding region (bp) 4,013,268 89.71 Results Coding genes (No.) 4361 Physiological parameter detection of P. communis MA5 Protein-coding genes (No.) 4301 98.62 To investigate the characteristics of formic acid metab- RNA genes (No.) 60 1.38 olism and protein synthesis of strain MA5 in sodium Prophages (No.) 5 formate stress or different nitrogen sources (organic or CIRSPR region (No.) 1 inorganic nitrogen), formic acid consumption, biomass The total is based on either the size of the genome in base pairs or the total and protein content were monitored under selected cul- number of protein-coding genes in the annotated genome ture conditions. Compared to the standard culture con- dition (A: normal sodium formate concentration and RIN > 8). Moreover, the concentrations of RNA (> 300 ng/ organic nitrogen), formic acid consumption and protein μL) and genomic DNA (84.2  ng/μL) both reached the content were decreased significantly by 67% and 31.8% in requirements of library construction (Additional file  1: the sodium formate stress treatment (B: sodium formate Table  S2 and S3). These results suggest that the above stress and organic nitrogen), while a 62.5% decrease in samples can be used for whole-genome and transcrip- growth rate and a 20.4% increase in protein content were tome sequencing. observed under the culture conditions with ammonium sulfate instead of yeast extract (C: normal sodium for- Genome assembly, gene prediction and functional mate concentration and inorganic nitrogen) (Table 1). annotation The draft genome of strain MA5 consists of 3 contigs, Extraction and quality analysis of genomic DNA and total including two annular chromosomes and an annu- RNA from P. communis MA5 lar plasmid (Additional file  1: Figure S2). The genome For omics analysis, the genomic DNA and total RNA of size is approximately 4,473,736  bp with a GC content of P. communis MA5 were first extracted and their qual - 68.56%, and the lengths of the 3 contigs are 1,412,597 bp ity were evaluated. The electrophoresis results revealed (Chr1), 2,389,614  bp (Chr2) and 671,525  bp (Plas1). A that the main bands of genomic DNA and RNA showed total of 4361 genes comprising 4,013,268  bp were pre- almost no degradation (Additional file  1: Figure S1), and dicted, 4301 of which were protein-coding genes, and 60 RNA integrity was further confirmed by RNA integ - were RNA genes. In addition, five prophages with sizes rity number (RIN) detection (Additional file  1: Table S2, T ong et al. Bioresources and Bioprocessing (2022) 9:55 Page 5 of 12 from 7164 bp to 44,730 bp and a CIRSPR region together formaldehyde dehydrogenase system, tetrahydrofolate with cas3 upstream of this region were identified in the cycle, serine cycle, glycolytic pathway, TCA cycle and genome of MA5 (Table 2). nitrogen metabolism (Fig. 2). In strain MA5, formic acid According to the KO assignment and KEGG pathway is transformed to methylene-tetrahydrofolate through mapping, 2355 (54.75%) protein-coding genes could be tetrahydrofolate ligase, methylene-tetrahydrofolate dehy- assigned to 222 metabolic pathways (one gene may cor- drogenase and methylene-tetrahydrofolate hydrolase. respond to many pathways). These pathways are classi - The ligase is encoded by the fhs gene, and the dehydro- fied into 6 categories: cellular processes, environmental genase and hydrolase are encoded by the same gene, information processing, genetic information processing, named folD. Thereafter, methylene-tetrahydrofolate human diseases, metabolism, and organismal systems. reacts with glycine via glycine hydroxymethyltransferase The vast majority of genes (2230) were concentrated in (glyA) to produce l-serine, entering biomass synthesis the category of metabolism, and among them, 13% of metabolism through the serine cycle. The serine cycle is genes (289/2230) were focused on amino acid metabo- the only way to assimilate formic acid into central carbon lism (the 2nd most in metabolism) (Fig.  1A). Similarly, metabolism and consists of two parts. One is the serine using COG function assignment, 3477 (80.84%) protein- regeneration cycle, and the other is the glyoxylic acid coding genes could be classified into 24 COG catego - regeneration cycle. The common branch of both is com - ries, and the most abundant category of genes (465) was posed of serine glyoxylate aminotransferase encoded by related to amino acid transport and metabolism (Fig.  1B the sga gene, hydroxypyruvate reductase encoded by the and Additional file  1: Table  S4). Based on the results hpr gene, and glycerate kinase encoded by the gck gene. above, we suspect that active amino acid metabolic path- Moreover, a phosphoenolpyruvate carboxylase was found ways in strain MA5 can explain the efficient synthesis of in MA5. The enzyme can catalyze the carboxylation of protein. carbon dioxide with phosphoenolpyruvate to produce oxaloacetic acid (an important intermediate in the TCA Genome‑scale reconstruction of the central metabolic cycle), which is able to neutralize carbon dioxide (Puri networks in strain MA5 et  al. 2020). The common glycolytic pathway and TCA The central metabolic networks of strain MA5 were cycle were also activated in MA5. Through these path - proposed according to the genome analysis results ways, formic acid is drastically introduced into the cen- and included an integrated glutathione-dependent tral metabolism. Fig. 1 KEGG pathway mapping (A) and COG functional assignment (B) of genes in the genome of strain MA5. The numerical value above the column represents the number of genes in the corresponding pathway or classification. The genes were mainly concentrated in the category of metabolism in KEGG pathway mapping and on amino acid transport and metabolism in COG function assignment Tong et al. Bioresources and Bioprocessing (2022) 9:55 Page 6 of 12 Fig. 2 Proposed central metabolism pathways of strain MA5. maxF methanol dehydrogenase, gfa S-(hydroxymethyl)glutathione synthase, frmA S-(hydroxymethyl)glutathione dehydrogenase, frmB S-formylglutathione hydrolase, fdh formate dehydrogenase, fhs formate-tetrahydrofolate ligase, purU formyltetrahydrofolate deformylase, folD methylene-tetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase, glyA glycine hydroxymethyltransferase, sga serine glyoxylate transaminase, hprA glycerate dehydrogenase, gckA glycerate 2-kinase, gpmI 2,3-bisphosphoglycera te-independent phosphoglycerate mutase, serA D-3-phosphoglycerate dehydrogenase, serC phosphoserine aminotransferase’, serB phosphoserine phosphatase, eno enolase, ppc phosphoenolpyruvate carboxylase, pckA phosphoenolpyruvate carboxykinase, mdh malate dehydrogenase, mtkB malate-CoA ligase, mcl malyl-CoA/(S)-citramalyl-CoA lyase, agxt2 alanine-glyoxylate transaminase, tpiA triosephosphate isomerase, fbaA fructose-bisphosphate aldolase, pfkA 6-phosphofructokinase, fbp fructose-1,6-bisphosphatase, pgi glucose-6-phosphate isomerase, gck glucokinase, pyk pyruvate kinase, gltA citrate synthase, acnA aconitate hydratase, icd isocitrate dehydrogenase, sucA 2-oxoglutarate dehydrogenase E1 component, sucB 2-oxoglutarate dehydrogenase E2 component, sucD succinyl-CoA synthetase alpha subunit, aarC succinyl-CoA:acetate CoA-transferase, frdA fumarate reductase flavoprotein subunit, fumAB fumarate hydratase, narGHI nitrate reductase, nirBS nitrite reductase, norB nitric oxide reductase, nosZ nitrous oxide reductase, gdhA glutamate dehydrogenase. Dotted arrows indicate multistep reactions For nitrogen metabolism, there are two branches in via RNA-seq analyses as detailed in the Materials and MA5. One is the dissimilative nitrate reduction path- methods section. A total of 1.55, 1.51, and 1.53  million way catalyzed by nitrate reductase and nitrite reductase. reads were generated, and 99.3%, 99.4%, and 98.9% were In this pathway, nitrate is first reduced to ammonia, and mapped to the strain MA5 genome in the A-C sample, then ammonia is transformed to glutamate via gluta- respectively. A comparison of differentially expressed mate dehydrogenase and subsequently enters glutamate genes (DEGs) between B (or C) and A was performed. metabolism. The other branch is the denitrification path - The analysis results revealed a total of 123 transcripts way catalyzed by nitrate reductase, nitrite reductase, and upregulated and 119 downregulated in the B vs. A group nitric oxide reductase, in which nitrate is finally reduced (Fig.  3A), and 935 transcripts were upregulated and to nitrogen and discharged from the body. With the help 350 were downregulated in the C vs. A group (Fig.  3B). of the entire central metabolic network, protein synthe- Cross-comparison between the B vs. A group and the C sis based on formic acid as a carbon source proceeds vs. A group showed 149 DEGs shared within both groups smoothly. (Fig. 3C). Among them, more than half of the genes were downregulated compared with the control treatment RNA‑seq and differential expression analysis (Fig.  3D), and the specific DEG annotations are listed in Based on the experimental results, gene expression pro- Additional file 1: Table S5. filing of bacteria grown in different media was performed T ong et al. Bioresources and Bioprocessing (2022) 9:55 Page 7 of 12 Fig. 3 Analysis of differentially expressed genes (DEGs) under three conditions. A Volcano plot of DEGs in the B vs. A group. B Volcano plot of DEGs in the C vs. A group. Red dots represent upregulated DEGs, green dots represent downregulated DEGs, and blue dots represent unchanged genes. The DEGs were identified with a p value < 0.05 and |log2FoldChang|> 0. C Venn diagram between the B vs. A group and the C vs. A group. D Heatmap of shared genes involved in both groups. The heatmap was drawn using TBtools software, and the rows were normalized and clustered Sodium formate stress results in elevated expression is the main component of the bacterial cell wall, offer - of peptidoglycan synthesis‑related genes but a weakening ing the function of increasing resistance to environmental in the sulfur metabolic pathway stress. Therefore, the upregulated expression of peptidogly - KEGG pathway enrichment network analyses revealed that can synthesis-related genes under sodium formate stress the upregulated DEGs of the B vs. A group showed signifi - is reasonable. Similarly, sharply grouped genes mapped to cant enrichment in the peptidoglycan synthesis pathway microbial metabolism in diverse environments were upreg- containing ligase, N-acetylglucosamine transferase, and ulated in response to stress (Fig.  4A). Of note, the down- peptidoglycan glycosyltransferase (Fig.  4A). Peptidoglycan regulated DEGs were significantly enriched in the sulfur Tong et al. Bioresources and Bioprocessing (2022) 9:55 Page 8 of 12 Fig. 4 KEGG pathway enrichment of DEGs in the B vs. A group. A KEGG pathway enrichment of the upregulated DEGs. B KEGG pathway enrichment of the downregulated DEGs. The size of the circle represents the number of DEGs in the pathway, and the color of the circle represents the padj value (p value adjusted to false discovery rate), reflecting the enrichment degree. C The DEGs involved in the denitrification pathway. The genes encoding enzymes in the rectangular box with a red border were significantly upregulated, and the FPKM of each gene is shown in the rectangle inside the ellipse metabolic pathway (Fig.  4B). Sulfur is an essential compo- Ammonium sulfate (nitrogen source) stimulates nent of sulfur-containing amino acids, such as cysteine and the expression of a suite of metabolic pathways associated methionine, which play a crucial role in protein synthesis. with protein synthesis and inhibits the tetrahydrofolate The significant downregulation of sulfite reductase and sul - cycle associated with formic acid assimilation fate/thiosulfate transporters under sodium formate stress The experimental results above showed an increase in treatment indicated that high concentrations of sodium protein content and a decrease in growth rate when formate could inhibit amino acid synthesis, resulting in a ammonium sulfate (replacing yeast extract) as a nitro- decrease in protein content. Furthermore, the key genes gen source. To explain this phenomenon, the influence of the denitrification pathway in nitrogen metabolism, in of ammonium sulfate on the global gene expression was which nitrate is ultimately reduced to nitrogen, were sig- analyzed via transcriptome sequencing. KEGG pathway nificantly upregulated (Fig.  4C). This was also one of the enrichment of C vs. A group DEGs was represented in factors causing the decrease in protein content. Intrigu- ribosome, oxidative phosphorylation, aminoacyl-tRNA ingly, moderately weakened expression was observed in the biosynthesis, citrate cycle (TCA cycle), biosynthesis of key genes of the tetrahydrofolate cycle associated with for- amino acids, etc. (Fig.  5A). Based on the transcriptom- mic acid utilization, although strain MA5 was cultured in ics data, these pathways were significantly promoted by high sodium formate medium. A complete list of the DEGs ammonium sulfate to provide more venues, vehicles, mentioned above is provided in Additional file  1: Table S6. precursor substances and energy for protein synthesis. The expression levels of some genes in the B vs. A group Besides, genes participating in protein export and bac- were further confirmed by real-time PCR (Additional file 1 : terial secretion system pathways were also activated, Figure S3). indicating the extracellular protein content might also T ong et al. Bioresources and Bioprocessing (2022) 9:55 Page 9 of 12 Fig. 5 KEGG pathway enrichment (A) and gene information display (B) of DEGs in the C vs. A group. The size of the circle represents the number of DEGs in the pathway, and the color of the circle represents padj values (p value adjusted to false discovery rate), reflecting the enrichment degree be increased (Fig.  5A). The DEG-related information with formic acid fixation and assimilation was found is listed in Additional file  1: Table  S7. In contrast, some (Fig.  2). In this cycle, the key enzymes dehydrogenase amino acid transporters, such as general l-amino acid and hydrolase are encoded by the same gene in MA5, transporters, branched-chain amino acid transport- named folD, while the different coding genes correspond - ers and d-methionine transporters, were significantly ing to two enzymes existed in methanotrophs Methy- downregulated because ammonium sulfate is a rapidly locystis SB2 (Vorobev et al. 2014) and Methylobacterium available nitrogen source and can be directly absorbed extorquens AM1 (Crowther et  al. 2008). The catalytic (Fig.  5B and Additional file  1: Figure S4). The tetrahy - reaction from formic acid and tetrahydrofolate to meth- drofolate cycle is a key pathway to formic acid metabo- yltetrahydrofolate by tetrahydrofolate ligase is an irre- lism. Under culture conditions with ammonium sulfate, versible reaction, and the reverse reaction is mediated by formate-tetrahydrofolate ligase and methylene-tetrahy- formyltetrahydrofolate deformylase. However, in Methy- drofolate dehydrogenase participating in the formic acid lobacterium extorquens AM1, this catalytic reaction is assimilation pathway were significantly downregulated, reversible (Marx et al. 2003). The coding gene of metha - while formate dehydrogenase catalyzing formic acid to nol dehydrogenase catalyzing methanol to formaldehyde carbon dioxide was significantly upregulated (Fig.  5B). and the genes for the glutathione-dependent formalde- Interestingly, the quorum sensing pathway related to cell hyde dehydrogenase system leading to the production of density regulation was repressed, which might be a signal formic acid from formaldehyde were annotated in strain of reduced growth rate (Additional file  1: Figure S4). The MA5 (Fig.  2). This methanol–formaldehyde–formic acid expression levels of some genes in the C vs. A group were pathway was also reported in Paracoccus AK26 (Puri further confirmed by real-time PCR (Additional file  1: et al. 2020). However, the key genes encoding 3-hexulose- Figure S5). 6-phosphate synthase and hexulose phosphate isomer- ase in the formaldehyde assimilation pathway were not Discussion found. This result suggests that formic acid rather than Strain MA5 can grow in a medium containing formic formaldehyde enters biomass synthesis metabolism when acid as a single carbon source. Based on the genome strain MA5 grows in a medium with methanol as a single sequences, a complete tetrahydrofolate cycle associated carbon source. Tong et al. Bioresources and Bioprocessing (2022) 9:55 Page 10 of 12 Studies have reported that three assimilation pathways to produce l-glutamic acid (Schure et  al. 1995), was sig- of formic acid, namely, the Calvin cycle, the RuBisCo nificantly upregulated. We believe that the increased pathway and the serine cycle, were identified in different expression of these genes is the main reason for the organisms (Chistoserdova 2011; Puri et al. 2020). Except increased protein content in the C sample (Table  1). for the key genes in the serine cycle, the genes encoding Notably, the significant downregulation of formic acid enzymes for the Calvin cycle and the RuBisCo pathway assimilation-relevant genes and the significant upregu - were not found in the genome of strain MA5. Therefore, lation of formate dehydrogenase-encoding genes (Li the serine cycle is the only pathway for the assimilation et  al. 2019) resulted in carbon escape from formic acid of formic acid in strain MA5, which is consistent with (Fig. 5B). We speculate that it is the “murderer” of reduc- what has been reported in Paracoccus AK26 (Puri et  al. tion in biomass when strain MA5 grows under culture 2020). Furthermore, the complete serine pathway was conditions with ammonium sulfate (Table 1). The expres - also found in Methylocystis SB2 (Vorobev et  al. 2014), sion levels of some representative genes detected by and both the serine cycle and the Calvin cycle are present real-time PCR were consistent with the transcriptome in Paracoccus sp. N5 (Puri et  al. 2020). Surprisingly, the profile analysis, which clearly validated the reliability of gene encoding phosphoenolpyruvate carboxylase cata- the DEGs in transcriptome analysis (Additional file  1: lyzing the carboxylation of carbon dioxide and phospho- Figure S3, S5). Overall, our results provide a potential enolpyruvate into oxaloacetic acid was also annotated central metabolic network for understanding formic acid (Fig. 2). This discovery raises the prospect of strain MA5 metabolism and protein synthesis in P. communis MA5. and indicates that it has the ability to neutralize carbon However, some specific metabolic and regulatory mech - dioxide. anisms still need to be explored to provide theoretical In this study, comparative transcriptomic results indi- ground for metabolic engineering designs of this strain in cated that a high concentration of sodium formate can the future. stimulate the expression of genes responding to envi- ronmental stress, including gene enrichment in the pep- Conclusions tidoglycan synthesis pathway and microbial metabolism In conclusion, in order to detect the excellent ability to in diverse environments (Fig.  4A). Studies have reported synthesize SCP directly from sodium formate, genome- that Lactobacillus and Streptococcus can increase the pro- scale analyses of P. communis MA5 were performed. portion of monounsaturated fatty acids in the cell mem- Our results indicated that the complete tetrahydrofolate brane when faced with environmental stress (Quivey cycle, serine cycle, glycolytic pathway, TCA cycle and et  al. 2000; Montanari et  al. 2010). In strain MA5, the nitrogen metabolism playing key roles in the conver- expression of genes encoding acyl carrier protein syn- sion of formic acid into proteins were annotated in the thetase and 3-oxygen acetyl-CoA carboxylase involved genome. Transcriptional analysis showed sodium for- in monounsaturated fatty acid synthesis metabolism was mate stress could increase resistance of MA5 to environ- also increased dramatically. Here, we focused on the key mental stress by stimulating the peptidoglycan synthesis nodes of protein synthesis and formic acid metabolism. pathway and microbial metabolism in diverse environ- Based on omics analysis, an enhanced denitrification ments, but inhibit amino acid synthesis by weakening the pathway and attenuated sulfur metabolism could account sulfur metabolic pathway, resulting in a 31.8% decrease for the experimental result that the protein content was in protein content. Moreover, ammonium sulfate as a decreased in strain MA5 under sodium formate stress nitrogen source (replacing yeast extract) could increase (Fig.  4B, C and Table  1). Intriguingly, formic acid con- protein content (20.4%) due to stimulation the pathways sumption was decreased significantly along with mod - associated with protein synthesis, such as ribosome, erately decreased expression levels of key genes in the aminoacyl-tRNA biosynthesis, TCA cycle, biosynthesis tetrahydrofolate cycle under sodium formate stress treat- of amino acids, etc.; while decrease growth rate (62.5%) ment (Table 1 and Additional file  1: Table S6), suggesting due to suppression the tetrahydrofolate cycle associated that potential negative feedback regulation of formic acid with formic acid assimilation. Our study provides theo- metabolism in response to sodium formate stress exists. retical guidance for the optimization of fermentation sys- Compared to yeast extract, metabolic pathways asso- tems using formic acid as a carbon source and lays the ciated with protein synthesis, including ribosome, oxi- foundation for further study on the regulatory mecha- dative phosphorylation, aminoacyl-tRNA biosynthesis, nism of formic acid metabolism. In the future, genetic citrate cycle (TCA cycle), and biosynthesis of amino transformation system and gene editing system such as acids, showed higher activity under ammonium sulfate CRISPR–Cas9 should been established, to advance the culture conditions (Fig. 5A). l-Glutamate dehydrogenase, development of P. communis MA5 as a SCP-based cell reported to catalyze ammonium and α-ketoglutaric acid factory through metabolic engineering, and to elucidate T ong et al. Bioresources and Bioprocessing (2022) 9:55 Page 11 of 12 Competing interests the regulatory mechanism of efficient metabolism of for - The authors declare no competing interest. mic acid in this strain. Author details Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Abbreviations Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy SCP: Single-cell protein; TCA: Tricarboxylic acid; PCR: Polymerase chain reac- of Sciences, Tianjin 300308, China. Tianjin Key Laboratory of Brine Chemi- tion; DEGs: Differentially expressed genes. cal Engineering and Resource Eco-Utilization, Tianjin University of Sciences and Technology, Tianjin 300457, China. National Innovation Centre for Syn- thetic Biology, Tianjin 300308, China. Supplementary Information The online version contains supplementary material available at https:// doi. Received: 2 March 2022 Accepted: 29 April 2022 org/ 10. 1186/ s40643- 022- 00544-0. Additional file1: Table S1. Primers used in this study. Table S2. Concen- tration detection and integrity analysis of RNA. Table S3. Concentration References detection and integrity analysis of DNA. Table S4. Number of genes Agarwal AS, Zhai Y, Hill D, Sridhar N (2011) The electrochemical reduction associated with the 24 general COG functional categories. Table S5. Dif- of carbon dioxide to formate/formic acid: engineering and economic ferentially expressed genes (DEGs) in both the B vs. A group and the C vs. feasibility. Chemsuschem 4(12):1705–1705. https:// doi. org/ 10. 1002/ cssc. A group. Table S6. Significant KEGG pathway-relevant DEGs in the B vs. 20110 0752 A group. Table S7. Significant KEGG pathway-relevant DEGs in the C vs. Albert J, Luders D, Bsmann A, Guldi DM, Wasserscheid P (2014) Spectroscopic A group. Fig. S1 Agarose electrophoresis results of genomic DNA (A) and and electrochemical characterization of heteropoly acids for their opti- total RNA (B). Genomic DNA was isolated from strain MA5 grown in Group mized application in selective biomass oxidation to formic acid. Green A. A1-A3: total RNA extraction from Group A; B1-B3: total RNA extraction Chem 16:226–237. https:// doi. org/ 10. 1039/ C3GC4 1320A from Group B; C1-C3: total RNA extraction from Group C. Fig. S2 Circular Albert J, Wolfel R, Bosmann A, Wasserscheid P (2012) Selective oxidation of representation of the genome of strain MA5. The genome of strain MA5 complex, water-insoluble biomass to formic acid using additives as reac- consists of 3 contigs, Chr1 (1.41 MB), Chr2 (2.39 MB) and Plas1 (0.67 MB). tion accelerators. Energ Environ Sci 5(7):7956–7962. https:// doi. org/ 10. Fig. S3 Expression of the selected DEGs in the B vs. A group was examined 1039/ C2EE2 1428H by real-time PCR with dnaN as the reference gene. Error bars represent the Anders S, Huber W (2010) Differential expression analysis for sequence standard deviation of three replicates. odc: ornithine decarboxylase; fadH: count data. Genome Biol 11(10):R106. https:// doi. org/ 10. 1186/ 3-oxoacyl-[acyl-carrier-protein] synthase; cysI: sulfite reductase; cdp: cell gb- 2010- 11- 10- r106 division protein; katG: peroxidase; nir: nitrite reductase. Fig. S4 KEGG path- Anders S, Pyl PT, Huber W (2015) HTSeq-a Python framework to work with way enrichment of the downregulated DEGs in the C vs. A group. The size high-throughput sequencing data. Bioinformatics 31(2):166–169. https:// of the circle represents the number of DEGs in the pathway, and the color doi. org/ 10. 1101/ 002824 of the circle represents the padj value (p value adjusted to false discovery Attwood MM, Harder W (1978) Formate assimilation by Hyphomicrobium X. rate), reflecting the enrichment degree. Fig. S5 Expression of the selected FEMS Microbiol Lett 3(2):111–114. https:// doi. org/ 10. 1111/j. 1574- 6968. DEGs in the C vs. A group was examined by real-time PCR with dnaN as 1978. tb018 95.x the reference gene. Error bars represent the standard deviation of three Awange J, Kiema J (2019) Environmental pollution extreme hydro-climatic and replicates. agxT: serine transaminase; aspB: aspartate transaminase; gdh: food security challenges: exploiting the Big Data. 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Journal

Bioresources and BioprocessingSpringer Journals

Published: May 18, 2022

Keywords: Formic acid; Single-cell protein; Metabolic network; Genome; Transcriptome; Paracoccus communis

References