Metabolic flux analysis of the halophilic archaeon Haladaptatus paucihalophilus

Accepted Manuscript Metabolic flux analysis of the halophilic archaeon Haladaptatus paucihalophilus Guangxiu Liu, Manxiao Zhang, Tianlu Mo, Lian He, Wei Zhang, Yi Yu, Qi Zhang, Wei Ding PII:

S0006-291X(15)30684-7

DOI:

10.1016/j.bbrc.2015.09.174

Reference:

YBBRC 34674

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 14 September 2015 Accepted Date: 30 September 2015

Please cite this article as: G. Liu, M. Zhang, T. Mo, L. He, W. Zhang, Y. Yu, Q. Zhang, W. Ding, Metabolic flux analysis of the halophilic archaeon Haladaptatus paucihalophilus, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.09.174. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Metabolic flux analysis of the halophilic archaeon Haladaptatus

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paucihalophilus

Guangxiu Liu1,2, Manxiao Zhang1,2, Tianlu Mo3, Lian He4, Wei Zhang1,2, Yi Yu4*, Qi Zhang3*,

Key Laboratory of Desert and Desertification, Cold and Arid Regions Environmental and

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1

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Wei Ding1,2,3*

Engineering Research Institute, Chinese Academy of Sciences, Lanzhou 730000, China 2

Key Laboratory of Extreme Environmental Microbial Resources and Engineering, Gansu

Province, Lanzhou 730000, China

Department of Chemistry, Fudan University, Shanghai, 200433, China

4

Key Laboratory of Combinatory Biosynthesis and Drug Discovery (Ministry of Education),

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3

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School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430071, China

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*Corresponding author Email addresses:

YY: [email protected]

QZ: [email protected] WD: [email protected]

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ACCEPTED MANUSCRIPT Abstract This work reports the

13

C-assisted metabolic flux analysis of Haladaptatus paucihalophilus, a

halophilic archaeon possessing an intriguing osmoadaption mechanism. We showed that the

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carbon flow is through the oxidative tricarboxylic acid (TCA) cycle whereas the reductive TCA cycle is not operative in H. paucihalophilus. In addition, both threonine and the citramalate pathways contribute to isoleucine biosynthesis, whereas lysine is synthesized through the

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diaminopimelate pathway and not through the α-aminoadipate pathway. Unexpected, the labeling

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patterns of glycine from the cells grown on [1-13C]pyruvate and [2-13C]pyruvate suggest that, unlike all the organisms investigated so far, in which glycine is produced exclusively from the serine hydroxymethyltransferase (SHMT) pathway, glycine biosynthesis in H. paucihalophilus involves different pathways including SHMT, threonine aldolase (TA) and the reverse reaction of

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glycine cleavage system (GCS), demonstrating for the first time that other pathways instead of SHMT can also make a significant contribution to the cellular glycine pool. Transcriptional analysis confirmed that both TA and GCS genes were transcribed in H. paucihalophilus, and the

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transcriptional level is independent of salt concentrations in the culture media. This study expands

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our understanding of amino acid biosynthesis and provides valuable insights into the metabolism of halophilic archaea.

Keywords 13

C-assisted metabolism analysis | Glycine biosynthesis | Halophilic archaea | Isoleucine

biosynthesis | Lysine biosynthesis

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Abbreviations SHMT, hydroxymethyltransferase; TA, threonine aldolase; GCS, glycine cleavage system; TCA,

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tricarboxylic acid; THF, tetrahydrofolate

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Introduction

Halophilic archaea (order Halobacteriales) are aerobic chemoorganotrophs that thrive in

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hypersaline habitats, where they often comprise the majority of prokaryotic population. To survive the high salinities, these organisms maintain a high intracellular osmotic pressure, by uptake and accumulation of high concentrations of inorganic ions such as K+, or by synthesis and/or uptake of highly soluble organic solutes that do not interfere with intracellular enzymatic activities and

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cellular processes [1]. Although high salt concentration is strictly required for the growth of most halophilic archaea, some members such as Haladaptatus paucihalophilus isolated from Zodletone

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Spring in south-western Oklahoma, USA, grows in a wide range of salt concentrations [2]. Remarkably, H. paucihalophilus remained viable even upon extended incubations in distilled

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water, suggesting its potent osmoadaptation mechanism to cope with the dramatically fluctuating salt conditions [2]. Understanding of the physiology and metabolism of this intriguing archaea is therefore of ecological and biochemical interests and is important for the development of halophiles in biotechnological applications [3,4,5].

Glycine is an essential building block required for the biosynthesis of proteins, nucleic acids, and many other metabolites. Except for some rare cases where glycine is supplemented by a second 3

ACCEPTED MANUSCRIPT pathway catalyzed by threonine aldolases (TAs) [6,7,8], most organisms synthesize glycine from serine by serine hydroxymethyltransferase (SHMT) [9] (Figure 1). Glycine could also be synthesized by glyoxylate aminotransferases and by the reverse reaction of glycine cleavage

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systems (GCSs) [10] (Figure 1), but whether these two glycine biosynthetic pathways play a certain physiological role remains largely unclear. Here we report metabolic flux analysis of the halophilic archaeon Haladaptatus paucihalophilus. We show that, surprisingly, a significant

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proportion of glycine was not synthesized from SHMT pathway but likely from both TA and GCS

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pathways, demonstrating for the first time that other pathways instead of SHMTs can also make a significant contribution to the cellular glycine pool.

Methods

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Materials

Chemicals and biochemicals were obtained from Sigma and Sangon Biotech Co. Ltd. [1-13C]pyruvate and [2-13C]pyruvate were obtained from Cambridge Isotope Laboratories, Inc.

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Primers were synthesized at Invitrogen Biotech Co. Ltd. Enzymes were from Takara

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Biotechnology (Dalian, China) or from Vazyme Biotech (Nanjing, China).

Cell culture

H. paucihalophilus DX253 was from ATCC and was grown in a modified DSMZ1125 media with no yeast extract and with pyruvate as a carbon source (in grams per liter of solution: MgCl2•6H2O, 20; K2SO4, 5.0; CaCl2•2H2O, 0.1; pyruvate, 2.0; NH4Cl, 0.5; KH2PO4, 0.05; NaCl, 90 or 270; HEPES, 6.0). The fresh medium was inoculated with 2% cell culture in the 4

ACCEPTED MANUSCRIPT late exponential growth phase, and the culture was grown at 37oC with shaking at 180 rpm for 2-3 days.

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Isotopomer analysis The isotopomer analysis was performed similarly to that reported previously [11]. Cell pellets were collected by centrifugation at 5000 rpm for 30 min and were subsequently hydrolyzed in 6 M

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HCl at 100°C for 24 hrs. After air-drying overnight, the dried samples containing free amino acids

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were derivatized with N-(tertbutyl-dimethylsilyl)-N-methyl-trifluoroacetamide in tetrahydrofuran at 70°C for 1 hr. Isotopomer measurements were performed on a GC (Hewlett-Packard, model 6890, Agilent Technologies) equipped with a DB5-MS column (J&W Scientific) and a mass spectrometer (MS) (Agilent Technologies). Different groups of charged fragments were detected

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by GC-MS: the [M-57]+ or [M-15]+ group corresponds to the intact amino acids, whereas the [M-159]+ or [M-85]+ group corresponds to the fragments losing the α carboxyl group. For each type of fragments, the labeling patterns were represented by M0, M1, M2, etc, corresponding to

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the unlabeled, singly labeled, and doubly labeled amino acids [11]. The effects of natural isotopes

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on isotopomer labeling patterns were corrected by previously reported algorithms [11].

Transcriptional analysis

The RNA sample was prepared from H. paucihalophilus cells grown for 48 hr at 37oC with TRIzol reagents (Invitrogen) according to manufacturer’s instructions. The DNase I-treated mRNA was reverse transcribed to the first-strand cDNA using the HiScript Q RT SuperMix (Vazyme). For the control of each set of experiment, the cDNA synthesis was carried out in the 5

ACCEPTED MANUSCRIPT absence of reverse transcriptase to verify that genomic DNA did not contaminate the RNA samples. After removal of RNA by incubation with RNase H for 10 min, the resulting cDNAs were used as templates for PCR to analyze the transcription levels of TA and GCS genes, and each

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analysis was performed on three parallel samples. The PCR amplification was performed by using Taq DNA polymerase under the following conditions: pre-denaturation, 2 min at 94°C, and for each cycle (25 cycles in total), 30 s at 94°C followed by 30 s at 65°C and 10 s at 72°C. A 104-bp

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fragment of the 16S rRNA gene was amplified by using a primer pair GAG AGG AGG TGC ATG

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GCC GC and ACC ACA AGG GTG CTG CTG GC; a 98-bp fragment of the TA gene was amplified by using a primer pair GTG GGC GAC GAC GTG TAC GG and GTC GGA ACG TAG AGG GCC GC; a 105-bp fragment of the H-protein gene was amplified by using a primer pair GTA TTC TCC AAC TCG TCG TTG and ACA GGA AGG CAA CCT CGG CG; and a 102-bp

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fragment of the P-protein gene was amplified using a primer pair GGG AGG CCG AGC GCG GCC GC and GTT CTG CCTC GGC ACC GAC C.

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Results and discussion

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C-assisted metabolism

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To investigate the metabolism of H. paucihalophilus, we performed a

analysis by feeding with 13C labeled substrates and analyzed the labeling pattern of proteinogenic amino acids by tert-butyldimethylsilyl (TBDMS) derivatization followed by gas chromatography (GC)/mass spectrometry (MS) analysis. Such a GC/MS-based method has been widely used to study

the

metabolism

of

various

organisms,

including

bacteria,

fungi

and

plants

[11,12,13,14,15,16,17,18,19]. It has been previously shown that the cell growth of H. paucihalophilus was well supported by pyruvate as the sole carbon source, providing a good 6

ACCEPTED MANUSCRIPT system for metabolic flux analysis [2,20]. When H. paucihalophilus was grown on [1-13C]pyruvate, serine was predominantly singly labeled at the C1 position (M1 = 0.80) (Table 1), suggesting that serine was mainly derived from pyruvate. Because in the reductive (reverse)

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tricarboxylic acid (TCA) cycle, oxaloacetate, the precursor of aspartate, is synthesized from pyruvate, a significant proportion of aspartate would be labeled for the cells grown on [1-13C]pyruvate if the carbon flow is through the reductive TCA cycle. However, aspartate was

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mainly not labeled (M0 = 0.80) in this analysis (Table 1), suggesting that although a

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phosphoenolpyruvate carboxykinase gene is present in the genome (ZOD2009_06007), the reductive TCA cycle was not operative in H. paucihalophilus. This is consistent with the chemoorganotrophic nature of halophilic archaea. Glutamate was also barely labeled (M0 = 0.88),

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further confirming that the carbon flow is through the oxidative TCA cycle in H. paucihalophilus.

For the hydrophobic amino acids, valine was predominately singly labeled (M1 = 0.89), whereas leucine and isoleucine are barely labeled by 13C (M0 = 0.86 and 0.88, respectively). Because ～20%

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of threonine is singly labeled at C1, the labeling pattern of isoleucine suggests that both threonine

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and citramalate pathways contributed equally to isoleucine biosynthesis in H. paucihalophilus (threonine pathway: citramalate patway ～ 2: 3) (Figure 2A); similar observation was also reported by Szyperski et al using a 13CNMR-based analysis on the halophilic archaeon Haloarcula hispanica [7].

Lysine can be biosynthesized via two pathways: the α-aminoadipate pathway that involves the condensation of α-ketoglutaric acid and acetyl-CoA, and the diaminopimelate pathway that 7

ACCEPTED MANUSCRIPT involves condensation of pyruvate and oxaloacetate [21] (Figure 2B). Because in this analysis, both glutamate (produced from α-ketoglutaric acid) and aspartate (produced from oxaloacetate) are barely labeled, it is expected that lysine will be predominantly unlabeled if it is produced from

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the α-aminoadipate pathway, and will be half singly labeled if it is from the diaminopimelate pathway (Figure 2B). The result showed that ～ 50% of lysine from the cells grown on [1-13C]pyruvate was singly labeled (M1 = 0.54) (Table 1), suggesting that lysine biosynthesis in H.

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paucihalophilus is via the both the diaminopimelate pathway. This is also consistent with that

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reported by Szyperski et al in the study on Haloarcula hispanica [7], suggesting that halophilc archaea may share a common paradigm for the biosynthesis of isoleucine and lysine.

Unexpectedly, only 55% of glycine from the cells grown on [1-13C]pyruvate is singly labeled 13

C-labeled at the C1 position, the labeling pattern of

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(Table 1). Given that ～80% of serine is

glycine clearly indicated that a significant proportion of glycine in H. paucihalophilus was not synthesized from the SHMT pathway, because otherwise, ～80% of glycine would be expected to 13

C-labeled. This observation is in stark contrast to the metabolic study on Haloarcula

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be

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hispanica, which showed that glycine is synthesized exclusively from SHMT pathway [7]. To the best of our knowledge, such a mixed glycine biosynthetic pathway is unprecedented not only in archaea but also in other organisms.

To further investigate the amino acid biosynthetic pathways in H. paucihalophilus, we grew the cells on [2-13C]pyruvate. In this analysis, valine was predominantly doubly labeled (M2 = 88%), whereas leucine and isoleucine shared a similar labeling pattern and are both predominantly triply 8

ACCEPTED MANUSCRIPT labeled (M3 = 80% and 77% for leucine and isoleucine) (Table 1). Because 33% of threonine is doubly labeled, the labeling pattern of isoleucine further confirmed the proposal that threonine and citramalate pathways contributed equally to isoleucine biosynthesis in H. paucihalophilus. As

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expected, serine is predominantly singly labeled (M1 = 80%) whereas only 50% of glycine is singly labeled, again demonstrating that glycine is produced via a mixed biosynthetic pathway.

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Intriguingly, the labeling ratio of glycine from cells grown on [2-13C]pyruvate (50%) is apparently

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lower than that on [1-13C]pyruvate (55%). Because the labeling ratio of threonine from [2-13C]pyruvate (M1 = 0.57, M2 = 0.33) is significantly higher than that from [1-13C]pyruvate (M1 = 0.22, M2 = 0.01), this observation suggested that glycine in H. paucihalophilus was, at least, not only supplemented by TA (Figure 1B), because otherwise, the labeling ratio of glycine

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from [2-13C]pyruvate is expected to be higher than that from [1-13C]pyruvate. Glycine was also not likely to be mainly supplemented by glyoxylate (Figure 1C), because glyoxylate biosynthesis is closely related to oxaloacetate, the precursor of aspartate, and the labeling ratio of aspartate

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from [2-13C]pyruvate (M1 = 0.62, M2 = 0.32) is significantly higher than that from

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[1-13C]pyruvate (M1 = 0.19, M2 = 0.01). The only explanation for the unusual glycine labeling pattern is that the reverse GCS reaction (Figure 1D) played an important role in glycine biosynthesis, in which some of the CO2 molecules from the C1 of pyruvate are incorporated into glycine.

GCS

consists

of

4

protein

components:

a

glycine

decarboxylase

(P-protein),

an

aminomethyltransferase (T-protein), an H-protein that contains a lipoyl moiety and that serves as a 9

ACCEPTED MANUSCRIPT shuttle to interact with the other protein components, and a commonly-used dihydrolipoyl dehydrogenase (L-protein) [10] (Figure 1D). The H. paucihalophilus genome contains all the required genes of GCS: the T-protein (ZOD2009_05632) and H-protein (ZOD2009_05627) are

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encoded by an operon whereas the P-protein, which consists of two subunits (ZOD2009_05622 and ZOD2009_05617), is encoded by another operon; the L-protein (ZOD2009_17975) is likely from the pyruvate dehydrogenase pathway. To show that the GCS genes were indeed expressed in

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H. paucihalophilus, we performed a semi-quantitative reverse transcription PCR analysis using

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16S rRNA gene as an internal standard. The result showed that GCS genes were indeed transcribed in H. paucihalophilus, and their transcriptional levels are generally higher than that of 16S rRNA. In addition, threonine aldolase was also transcribed with a level similar to those of GCS genes (Figure 3). These results are consistent with the metabolic flux analysis and indicated

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that SHMT, GCS and TA likely all contribute to the glycine biosynthesis in H. paucihalophilus.

Glycine is a precursor of betaine, which is an important solute required for osmoadaptation of

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halophiles [22]. Although H. paucihalophilus possesses of powerful mechanism for betaine uptake,

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it does not have a betaine synthesis gene [20], excluding the possibility that the extra pathways are utilized to increase the glycine pool for betaine biosynthesis. We performed semi-quantitative RT-PCR analysis for the cells grown with very different salt concentration (90 and 270 g/L NaCl), and the results showed that the transcriptional levels of both GCS and TA genes did not change significantly compared with that of 16S rRNA gene (Figure 3), suggesting that these glycine biosynthetic pathways are likely salinity-independent.

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ACCEPTED MANUSCRIPT In summary, this investigation provides the first fluxomic study of H. paucihalophilus, a halophilic archaea with a potent osmoadaptation mechanism. We showed that in H. paucihalophilus the carbon flow is through the oxidative TCA cycle and the reductive TCA cycle

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is not operative. Isoleucine is synthesized by both threonine and citramalate pathway, whereas lysine is synthesized through the diaminopimelate pathway and not through the α-aminoadipate pathway. These observations are consistent with those reported for H. hispanica, suggesting that

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halophilic archaea may generally share common metabolic pathways for amino acid biosynthesis.

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Remarkably, unlike H. hispanica and all other organisms investigated so far, in which glycine biosynthesis is exclusively from SHMT pathway, glycine biosynthesis in H. paucihalophilus is clearly consists of different pathways including SHMT, GCS and TA. Why this archaeal strain utilizes such an unusual mixed pathway for glycine production is unclear, but it seems unrelated to

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the cell osmoadaption mechanism, as no apparent change in the transcriptional levels of GCS and TA genes was observed when cells were grown under different salt concentrations. These understandings could facilitate future efforts in studying the ecology and biochemistry of

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halophilic archaea and in developing these organisms for biotechnological use. It is also remains

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to be seen whether the unusual mixed glycine biosynthetic pathway revealed in this study is present in other organisms.

Acknowledgements This work was supported in part by grants from the International S&T Cooperation Program of 11

ACCEPTED MANUSCRIPT China (No. 2011DFA32520 to M.Z.), from National Natural Science Foundation (2012CB721006 to Y.Y.), from Fudan University (IDH1615002 to Q.Z), and from Natural Science Foundation of Gansu, China (1308RJZA173 to W.D.). W.Z. would also like to thank the West Light Foundation

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of the Chinese Academy of Sciences for support.

References

[1] A. Oren, (Ed.), Life at high salt concentrations. The Prokaryotes—Prokaryotic Communities and

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Ecophysiology (4th edn). Springer: Berlin-Heidelburg, 2013.

[2] K.N. Savage, L.R. Krumholz, A. Oren, M.S. Elshahed, Haladaptatus paucihalophilus gen. nov., sp. Microbiol 57 (2007) 19-24.

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nov., a halophilic archaeon isolated from a low-salt, sulfide-rich spring, Int J Syst Evol [3] W.D. Grant, Life at low water activity, Philosophical Transactions of the Royal Society of London Series B-Biological Sciences 359 (2004) 1249-1266.

[4] S.M.C. Alqueres, R.V. Almeida, M.M. Clementino, R.P. Vieira, W.I. Almeida, A.M. Cardoso, O.B. Martins, Exploring the biotechnologial applications in the archaeal domain, Brazilian Journal of Microbiology 38 (2007) 398-405.

[5] B.S. Zhao, Y.C. Yan, S.L. Chen, How could haloalkaliphilic microorganisms contribute to

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biotechnology?, Canadian Journal of Microbiology 60 (2014) 717-727. [6] J.B. McNeil, E.M. McIntosh, B.V. Taylor, F.R. Zhang, S. Tang, A.L. Bognar, Cloning and molecular characterization of three genes, including two genes encoding serine hydroxymethyltransferases, whose inactivation is required to render yeast auxotrophic for glycine, J Biol Chem 269 (1994) 9155-9165.

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[7] M. Hochuli, H. Patzelt, D. Oesterhelt, K. Wuthrich, T. Szyperski, Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica, J Bacteriol 181 (1999) 3226-3237. [8] G. Liu, M. Zhang, X. Chen, W. Zhang, W. Ding, Q. Zhang, Evolution of threonine aldolases, a diverse

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family involved in the second pathway of glycine biosynthesis, J Mol Evol 80 (2015) 102-107. [9] N. Appaji Rao, M. Ambili, V.R. Jala, H.S. Subramanya, H.S. Savithri, Structure-function relationship in serine hydroxymethyltransferase, Biochim Biophys Acta 1647 (2003) 24-29.

[10] G. Kikuchi, Y. Motokawa, T. Yoshida, K. Hiraga, Glycine cleavage system: reaction mechanism, physiological significance, and hyperglycinemia, Proc Jpn Acad Ser B Phys Biol Sci 84 (2008) 246-263.

[11] S.A. Wahl, M. Dauner, W. Wiechert, New tools for mass isotopomer data evaluation in (13)C flux analysis: mass isotope correction, data consistency checking, and precursor relationships, Biotechnol Bioeng 85 (2004) 259-268. [12] M. Dauner, U. Sauer, GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing, Biotechnol Prog 16 (2000) 642-649. [13] C. Cannizzaro, B. Christensen, J. Nielsen, U. von Stockar, Metabolic network analysis on Phaffia rhodozyma yeast using 13C-labeled glucose and gas chromatography-mass spectrometry, 12

ACCEPTED MANUSCRIPT Metab Eng 6 (2004) 340-351. [14] C. Wittmann, Fluxome analysis using GC-MS, Microbial Cell Factories 6 (2007) 6. [15] X. Feng, K.H. Tang, R.E. Blankenship, Y.J. Tang, Metabolic flux analysis of the mixotrophic metabolisms in the green sulfur bacterium Chlorobaculum tepidum, J Biol Chem 285 (2010) 39544-39550. [16] W. Xiong, L. Liu, C. Wu, C. Yang, Q. Wu, 13C-tracer and gas chromatography-mass spectrometry protothecoides, Plant Physiol 154 (2010) 1001-1011.

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analyses reveal metabolic flux distribution in the oleaginous microalga Chlorella [17] K.H. Tang, X. Feng, W.Q. Zhuang, L. Alvarez-Cohen, R.E. Blankenship, Y.J. Tang, Carbon flow of

heliobacteria is related more to clostridia than to the green sulfur bacteria, J Biol Chem 285 (2010) 35104-35112.

[18] A.M. Varman, L. He, L. You, W. Hollinshead, Y.J.J. Tang, Elucidation of intrinsic biosynthesis yields using C-13-based metabolism analysis, Microbial Cell Factories 13 (2014).

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[19] L. You, B. Zhang, Y.J. Tang, Application of stable isotope-assisted metabolomics for cell metabolism studies, Metabolites 4 (2014) 142-165.

[20] N.H. Youssef, K.N. Savage-Ashlock, A.L. McCully, B. Luedtke, E.I. Shaw, W.D. Hoff, M.S. Elshahed,

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Trehalose/2-sulfotrehalose biosynthesis and glycine-betaine uptake are widely spread mechanisms for osmoadaptation in the Halobacteriales, ISME J 8 (2014) 636-649. [21] F. Fazius, C. Zaehle, M. Brock, Lysine biosynthesis in microbes: relevance as drug target and prospects for beta-lactam antibiotics production, Appl Microbiol Biotechnol 97 (2013) 3763-3772.

[22] J.F. Imhoff, F. Rodriguez-Valera, Betaine is the main compatible solute of halophilic eubacteria, J

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Bacteriol 160 (1984) 478-479.

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ACCEPTED MANUSCRIPT Table 1. Isotopic labeling patterns of the proteinogenic amino acids from H. paucihalophilus grown on [1-13C]pyruvate or or [2-13C] pyruvate. Amino acid

Fragments

[1-13C]pyruvate

Ala

[M-57]+ [M-85]

+

[M-57]

+

[M-85]

+

[M-57]

+

Ser

[M-159] Thr

Val

Ala

Gly

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Ser

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Thr

Val

[M-57]

0.99

0.01

0.19

0.80

0.93 0.88

[M-15]

[M-57]

+

+

+

+

+

0.01

0.01

0.01

0.03

0.04

0.01

0.08

0.02

0.93

0.06

0.01

0.86

0.05

0.02

0.88

0.04

0.01

0.80

0.19

0.01

0.88

0.11

0.01

[M-57]+

0.88

0.11

0.01

[M-159]+

0.95

0.05

[M-57]+

0.05

0.93

0.02

[M-85]+

0.05

0.93

0.02

[M-57]

+

0.48

0.51

0.01

[M-85]

+

0.54

0.46

[M-57]

+

0.15

0.80

0.04

[M-159]

+

M3

0.01

0.89

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[M-15]

0.08 +

+

0.21

0.77

0.01

[M-57]

+

0.10

0.57

0.33

0.01

[M-85]

+

0.24

0.52

0.16

0.07

[M-57]

+

0.03

0.05

0.88

0.03

0.03

0.06

0.87

0.04

0.03

0.03

0.16

0.77

0.03

0.09

0.82

0.05

0.02

0.02

0.12

0.80

0.04

0.06

0.84

0.03

0.06

0.62

0.32

0.88

0.11

0.01

0.03

0.27

0.57

0.12

0.06

0.54

0.38

0.01

[M-15]

+

+

[M-15]

+

[M-57]

[M-57]

+

+

[M-159]

1

+

+

[M-159] Glu

0.55

0.05

[M-159] Asp

0.43

0.95

+

[M-159]+ Leu

0.03

[M-85]+

[M-159] Ile

0.98

0.01

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PRP

0.02

0.20

[M-159]

Glu

0.91

0.98

[M-159]

Asp

0.07

0.78

[M-159]

Leu

M2

[M-57]+

[M-159] Ile

+

M1

SC

Gly

M0

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Carbon source

+

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aminotransferase, and (D) GCS. H in (D) represents the lipoyl-containing H-protein.

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Figure 2. Proposed biosynthetic pathway of isoleucine (A) and lysine (B) in H. paucihalophilus

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cells grown on [1-13C]pyruvate. The 13C atoms are shown by red circles.

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ACCEPTED MANUSCRIPT Figure 3. Transcriptional analysis of the TA and GCS genes by semi-quantitative RT-PCR, and the 16S rRNA genes was used as an internal standard. The analysis was performed for cells grown in media with 90 or 270 g/L sodium chloride. Each analysis was performed for three parallel

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samples.

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ACCEPTED MANUSCRIPT Highlights serine hydroxymethyltransferase, threonine aldolase, and glycine cleavage system all contribute to the glycine biosynthesis in Haladaptatus paucihalophilus

paucihalophilus

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Threonine and the citramalate pathways contribute equally to the isoleucine biosynthesis in H.

through the α-aminoadipate pathway

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Lysine in H. paucihalophilus is synthesized through the diaminopimelate pathway and not

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Glycine biosynthesis is likely unrelated to the cell osmoadaption mechanism

ACCEPTED MANUSCRIPT No conflict of interests in this paper!

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Qi Zhang