Highly Ecient Biosynthesis of Indole-3-acetic acid by Enterobacter xiangfangensis BHW6

Background: Indole-3-acetic acid (IAA) plays an important role in the growth and development of plants. Various bacteria in the rhizosphere are capable to produce IAA that acts as a signaling molecule for the communication between plants and microbes to promote the plant growth. Due to the low IAA content and various interfering analogs, it is dicult to detect and isolate IAA from microbial secondary metabolites. Results: A predominant strain with a remarkable capability to secrete IAA was identied as Enterobacter xiangfangensis BHW6 based on 16S rRNA gene sequence, the determination of average nucleotide identity (ANI) and digital DDH (dDDH). The maximum IAA content (134-1129 μg/mL) was found with the addition of 0.2-15 g/L of L-tryptophan at pH 5 for 6 days, which was 4-40 fold higher than that in the absence of L-tryptophan. The highest yield of IAA was obtained at the stationary phase of bacterial growth. An acidic culture medium was preferred for the IAA biosynthesis of the strain. The strain was tolerant and stable to produce IAA in the presence 2.5%-5% (w/v) of NaCl. IAA was then isolated through column chromatography with a mobile phase of hexane/ethyl acetate (1/2, v/v) and characterized by 1 H Nuclear Magnetic Resonance ( 1 H NMR). Conclusions: A remarkable IAA production was obtained from E. xiangfangensis BHW6 that was tryptophan–dependent. According to genomic analysis, the ipdC gene coding for the key enzyme (indole-3-pyruvate decarboxylase) was identied indicating that IAA biosynthesis was mainly through the indole-3-pyruvia acid (IPyA) pathway, which was further conrmed by intermediate assay. E. xiangfangensis BHW6 with an important economic value has great prospect in agricultural and industrial application.


Introduction
Phytohormones including abscisic acid (ABA), gibberellins (GAs), ethylene, indole-3-acetic acid (IAA), and cytokinins (CKs), and brassinosteroids (BRs) participate in many physiological and biochemical processes in plants (Fahad et al. 2015). Indole-3-acetic acid (IAA) as one of the main phytohormones plays a key role in the regulation of various physiological processes, such as cell division and elongation, vascular differentiation, gravitropism and phototropism. It inhibits or delays abscission of leaves and induces owering and fruiting (Boopathi et al. 2013).
IAA can be synthesized in plants and also in microorganisms. A large number of bacteria such as Bacillus, Pseudomonas, Enterobacter, Azospirillum, Agrobacterium and Rhizobium has been reported to generate the capability to synthesize IAA (Oberhänsli, et al. 1991;Lim & Kim, 2009). These microbes act as a communication system with the host plants. Exogenous application of IAA can improve the endogenous levels of plants and thus alter the plant ontogeny (Lin et al. 2018).
The biosynthesis of IAA in microorganisms is accomplished by a variety of pathways. Tryptophan is an e cient precursor for the biosynthesis of IAA. In general, the followings are the main pathways for the conversion of tryptophan to IAA (Patten and Glick, 1996). The IPyA pathway (tryptophan→IPyA→IAAld→IAA) has been studied in detail in Enterobacter cloacae (Koga et al. 1991) and Bacillius amloliquefaciens FZB42 (Idris et al. 2007). The IAM pathway (tryptophan→IAM→IAA) has been discovered in Streptomyces spp (Manulis et al. 1998). The TAM pathway (tryptophan→TAM→IAAld→IAA) has been reported in Bacillus cereus (Perley and Stowe, 1966). The TSO pathway (tryptophan→IAAld→IAA) has been demonstrated in Pseudomonas uorescens CHA0 (Oberhänsli et al. 1991). The IAN pathway (tryptophan→IAN→IAA) has been con rmed in several strains of Agrobacterium and Rhizobium (Kobayashi et al. 1995).
Due to the low IAA content and various interfering analogs, it is di cult to detect and isolate IAA from microbial secondary metabolites. Cultivation surrounding is crucial to improve the capability of the microorganisms to synthesize IAA. L-tryptophan supplementation, pH, and NaCl concentration have an important effect on bacterial growth and IAA yield. Ultraviolet spectrometry is widely used to determine the IAA content. High performance liquid chromatography is a well-recognized method to enable the sensitive and accurate measurement of the substances in crude extracts (Li et al. 2018). Common puri cation procedures including liquid-liquid extraction and column chromatography are employed for the isolation and puri cation of IAA products (Lim and Kim, 2009).
In this study, highly e cient biosynthesis of IAA was found in a strain identi ed as Enterobacter xiangfangensis BHW6, which was tryptophan-dependent. Cultivation parameters including L-tryptophan supplementation, fermentation time, initial pH, NaCl concentration were determined in order to improve the IAA production. Genome analysis was conducted to investigate the IAA biosynthesis of the strain.
Immediate assay was also employed to identify the IAA synthetic pathways. IAA was further isolated and puri ed using column chromatography, which was characterized by 1 H NMR for its chemical structure.

Sample collection
Soil samples were collected from the corn farm in the Jining of the Shandong province, P. R. China, which was stored in sealed and sterile bags at 4°C. All chemicals were purchased from Aladdin Reagent Co., Isolation of the strain and determination of IAA content 10 g of the soil sample was mixed with 90 mL of distilled sterile water, which was stirred at 30 °C and 150 rpm for 30 min. The resultant soil solution was diluted and plated on Luria-Bertan (LB) agar medium including NaCl (10 g), yeast extract (5 g), tryptone (10 g), agar (18 g). IAA content of the strains was determined according to the method of Gordon and Weber (1951) with some modi cations. The strain was incubated in LB liquid medium supplemented with 0.2 mg/mL of L-tryptophan at 30 °C for 6 days.
Uninoculated LB liquid medium was used as a control. Each experiment was conducted in triplicate. After that, the fermentation broth was centrifuged at 10000 rpm for 10 min. 2 mL of supernatant was combined with 4 mL of Salkowski reagent (50 mL of 35% HClO 4 & 1 mL of 0.5 M FeCl 3 ), which was incubated in darkness for 30 min at 40 ℃. The absorbance of Indole-3-acetic acid (IAA) was measured at 530 nm using a UV-VIS spectrophotometer (UV-6100, Metash, China).

Molecular identi cation
The DNA of the enriched bacteria was extracted using a DNA kit according to the manufacturer's instructions (OMEGA, USA). 16S rRNA gene sequence was ampli ed by PCR with 27F (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492R (5'-GGT TAC CTT GTT ACG ACT T-3'). The polymerase chain reaction (PCR) was performed in a 50 μL system (template DNA 2 μL, forward primer 2 μL, reverse primer 2 μL, 2×mastermix 25μL, and DdH 2 O 19 μL) (Tiangen, China). PCR products were sequenced at the Huada gene company (Beijing, China). The sequences were deposited into Genebank and the accession numbers were obtained as MN696244.
Cultivation factors for IAA production The reactions were performed in LB medium with 1% inoculation and 150 rpm. L-tryptophan as a precursor was examined at the concentrations of 0, 0.2, 0.5, 1, 2, 5, 10 and 15 (mg/mL) at pH 7.0 and 30 °C for 6 days. Incubation time of 1-10 days was investigated at pH 7.0 and 30 °C. Initial pH value was detected in the range of 3.0-11.0 at 30 °C for 6 days. NaCl concentration was determined at 0%, 2.5%, 5.0%, 7.5% and 10.0% (w/v) at 30°C and initial pH 5 for 6 days.
Genomic sequencing analysis Genome analysis was performed by Personalbio Company (Beijing, China). The DNA was prepared using a genomic DNA extraction kit (Tiangen, China). The whole genome shotgun (WGS) and next generation sequencing (NGS) were employed. Illumina NovaSeq Sequencing platform was used to sequence the paired-end (PE). GeneMarkS software was used to predict the protein coding genes in the bacterial genome. Protein coding gene was annotated by NR, KEGG, Swiss-Prot, eggNOG, and Go and Pfam.
Intermediate assay for IAA biosynthesis Intermediate assay for IAA synthetic pathway was based on monitoring the utilization of an intermediate and the determination of the resulting product after enzyme reactions as described by Bunsangiam et al. (2019) with some modi cations. The precultured strains were resuspended in LB medium were supplemented with 0.2% (w/v) of one of IAA precursors such as L-tryptophan, IPyA, IAM, TAM and IAN, which were incubated at 30 °C for 24 h. The metabolites were quanti ed by HPLC analysis (Waters 2695, America).

Isolation and puri cation of IAA
The strain was incubated at 30 °C and pH 5 with 1% inoculation and 150 rpm for 6 days in LB liquid medium supplemented with 10 mg/mL of L-tryptophan. After that, the solution was centrifuged at 5000 rpm for 15 min. The supernatant was acidi ed to pH 2.5 with 1 mol/L HCl, which was then extracted twice with double volume of ethyl acetate. The ethyl acetate was evaporated at 40-50 ℃ by rotary evaporation (EYELAN-1100, Japan) to give the crude product. The crude product was then subjected to column chromatography (1.5×30 cm), which was eluted using hexane/ethyl acetate (1/2, v/v). The solvent was evaporated at 40°C-50 and the resulting product was dried in a vacuum for 2 h. 1 H nuclear magnetic resonance ( 1 H NMR) of IAA was conducted using a spectrometer (Bruker AVI 400 MHz, Germany).

Results
Determination of IAA production of the strain Han et al. isolated 55 strains with the capability of producing IAA in a range of 1-10 μg/mL with Ltryptophan as a precursor from rice root endophytes. Xu et al. (2018) screened 12 strains with the capability of producing IAA in 0.2 g/L of L-tryptophan-supplemented medium from watermelon rhizosphere soil with a high yield of IAA (115.3 μg/mL) obtained from streptomycete CL05. In current studies, IAA productions of 14 strains were obtained in a range of 4.62 μg/mL to 109.72 μg/mL with a supplement of L-tryptophan (0.2 g/L) (Fig.1a).
Particularly, 28.36 μg/mL of IAA was obtained in the absence of L-tryptophan, whereas 115.72 μg/mL was achieved in the presence of 0.2 g/L of L-tryptophan. An increase of 4.1-fold was observed with the addition of L-tryptophan, thus the biosynthesis IAA in strain BHW6 was tryptophan-dependent. Similar results were found in the previous study. IAA production in a plant-bene cial bacterium Arthrobacter pascens ZZ21 was increased 4.5-fold with the addition of 0.2 mg/L of L-tryptophan, indicating that IAA biosynthesis in ZZ21 was induced by L-tryptophan (Li et al. 2018). The low quantity of IAA was also detected in Enterobactersp. I-3 in the culture medium without L-tryptophan and the high concentration of IAA (approximately 220 μg/mL) was observed in 2 g and 3 g of L-tryptophan-supplemented medium (Park et al. 2015).

Molecular identi cation of the strain
A number of microorganisms such as Agrobacterium, Pseudomonas, Bacillus, Rhizobium and Azospirillum are known to be capable to produce IAA (Mohite, 2013). Microbes such as Bacillus Tp. 1B-7B and Penicillium Tp. 1F-5F could produce IAA, especially when the growth media are supplemented with Ltryptophan as a precursor (Hassan, 2017). In this study, molecular identi cation of the strain was based on 16S rRNA gene sequence and the phylogenetic tree was shown in Fig. 2 Average nucleotide identity (ANI) and digital DDH (dDDH) have been most widely used to identify the microbes. Draft genomes were sequenced and assembled in this study. ANI value was calculated according to the orthoANIu algorithm (Lee et al. 2016). ANI value between two genome sequences and the DNA G+C content of a single genome sequence was calculated based on an online ANI calculator (www.ezbiocloud.net/tools/ani). As shown in Table 1, the ANI value between Enterobacter sp. BHW6 and the type strain of phylogenetically related species were more than 97.12%. Moreover, dDDH value was calculated according to Genome-to-Genome Distance Calculator of dDDH Web service (http://ggdc.dsmz.de/ggdc.php#). The dDDH value between Enterobacter sp. BHW6 and the type strain of phylogenetically related species was more than 77.6%. Therefore, the strain was identi ed as Enterobacter xiangfangensis BHW6.
L-tryptophan concentration for IAA production and bacterial growth L-tryptophan is considered as a precursor of IAA. IAA production was improved with an increase of Ltryptophan concentration. Klebsiella pneumoniae Psn8 was found to have the maximum IAA production (277 μg/mL) at 1.6% (w/v) of L-tryptophan concentration. IAA production (1.5 μg/mL) of R.benzoatilyticus JA2 was obtained in the absence of L-tryptophan, which was enhanced to 12-14 μg/mL with 0.5-2 μg/mL of L-tryptophan (Mujahid et al. 2011).
Different concentrations of L-tryptophan were studied for E. xiangfangensis BHW6. An increase of IAA production was found with the increase of L-tryptophan concentration (Fig. 3). Signi cantly, the maximum IAA content (1078 μg/mL) was found with the addition of 15 g/L of L-tryptophan at pH 7 for 6 days, which was 38-fold higher than the IAA content (28.33 μg/mL) in the absence of L-tryptophan. Therefore, IAA biosynthesis in E. xiangfangensis BHW6 was mainly dependent on the exogenous Ltryptophan concentration.
Meanwhile, the strains grew well at 10 g/L of L-tryptophan and produced good yield of IAA. Although the bacterial growth was reduced remarkably at 15 g/L, IAA content was still increase to the maximum amount. Therefore, the highest IAA production was obtained at the stationary phase of bacterial growth. With the further increase of L-tryptophan concentration to 20 g/L, bacterial growth was inhibited with a signi cant reduced OD 600 value, which was also accompanied by the decreased IAA production.
Incubation time for IAA production and bacterial growth Swain et al. (2007) described that IAA production of B. subtilis was increased linearly from 2 days to 8 days with the maximum IAA content at 8th day of incubation, which was accompanied with the growth of the strain. Bharucha et al. (2013) found that IAA production was improved from 12 h to 96 h and then was reduced from 96 h to 144 h for Pseudomonas putida UB1, which was consistent with that for bacterial growth.
As shown in Fig. 4a, IAA production of E. xiangfangensis BHW6 was increased notably from 1 day to 6 days with a high IAA yield at 6th in the presence of 0.2 g/L of L-tryptophan. After 6 days, the ability of the strain to produce IAA was decreased slightly. However, the growth of strain was increased remarkably within 2 days of cultivation.
Similar results were obtained in the presence of 15 g/L of L-tryptophan (Fig. 4b). The maximum amount of IAA was observed at 6-8 days of cultivation. Meanwhile, the growth of strain was reduced after 2 days, which indicated that IAA was accumulated mainly during stationary phase of the growth of E. xiangfangensis BHW6.
Initial pH for IAA production and bacterial growth Initial pH has a crucial effect on the growth of the strains and their metabolic activity. Acuña et al. (2011) found that the highest IAA production was obtained at pH 6.0 for Bacillus spp. MQH-19. However, 62% of reduction was observed at pH 5.0. In this study, the strain has good growth over a broad range of pH 5.0-10.0, with a strong ability to secrete IAA at pH 5.0-8.0.
As reported, the highest IAA yield for B. pyrrocina JK-SH007 was achieved at pH 7.0 and the fastest growth of this strain was observed at pH 4.0 (Liu et al. 2019). In our study, the highest yield of IAA was found at pH 5.0 and the optimal growth of the strain was found at pH 5.0-7.0 (Fig. 5). An acidic culture medium was preferred for the IAA biosynthesis indicating that pH have a reasonably effect on the metabolic activity of E. xiangfangensis BHW6. When the pH value was below 5.0, the strains hardly survived without the capacity to produce IAA. Therefore, the maximum amount of IAA (134 μg/mL and 1129 μg/mL) was obtained at pH 5 with the addition of L-tryptophan at 0.2 g/L and 15 g/L, respectively (Fig. 5b).
NaCl tolerance of the IAA producing strain As reported, IAA production under salt stress was observed to be 250 μg/ml, 220μg/ml and 200 μg/ml for Rheinheimera sp. Rhizobium sp. and Bacillus subtilis respectively (Rupal K, 2020). The salt tolerance was determined by selecting NaCl concentrations of 2.5%, 5.0%, 7.5% and 10.0% (w/v). E. xiangfangensis BHW6 can grow well and generate good capability to produce IAA in the range of 0%-5% (w/v) NaCl concentration. When the medium was supplemented with 2.5%-5% (w/v) of NaCl, IAA content was stable at 117-115 μg/mL (Fig. 6a) and 1260-1053 μg/mL (Fig. 6b) in the presence of 0.2 g/L and 15 g/L of Ltryptophan, respectively. However, the bacterial growth was inhibited with addition of 7.5%-10.0% (w/v) of NaCl, which caused the poor yield of IAA and the bacterial growth.

Genomic analysis of IAA biosynthesis pathways
There are ve main pathways for the conversion of tryptophan to IAA including IPyA pathway, IAM pathway, TAM pathway, TSO pathway, and IAN pathway. The indole-3-pyruvate acid (IPyA) pathway is the most important pathway for IAA biosynthesis, which has been described in various bacteria such as Enterobacter cloacae, Azospirillum brasilense, Bacillius amloliquefaciens (Zakharova et al. 1999). According to genomic analysis, E. xiangfangensis BHW6 contains the gene (ipdC) encoding the key enzyme (IPDC) that can catalyze the conversion of IPyA to IAAld in the IpyA pathway (Fig.7a). The length of this gene is 1659 bp, encoding 559 amino acid sequences (Fig.7b). The similarity of the gene sequence is 99.82% with the IPDC enzyme of Enterobacter cloacae (KGY930.98.1). The ipdC gene coding for the IPDC enzyme has been isolated and identi ed from Azospirillum brasilense, Pseudomonas putida and Pa. agglomerans (Zakharova et al.1999;Patten & Glick, 2002b;Costacurta et al. 1994).
Intermediates assay for IAA biosynthesis IAA biosynthetic pathway was investigated by intermediates assay using different IAA precursors. The accumulation of IAA was found when the medium was supplemented with IpyA indicating that the strain was tryptophan-dependent IpyA pathway. IAAld and IEt were also detected in the medium containing IpyA. In IPyA pathway, tryptophan is converted into IPyA by an aminotransferase. IPyA is then decarboxylated to indole-3-acetaldehyde (IAAld) by indole-3-pyruvate decarboxylase (IPDC), which is the rate-limiting step. After that, IAAld is oxidized in IAA (Fig. 8). The intermediates assay con rmed that IPyA pathway was the main IAA biosynthetic pathway.

Discussion
IAA plays a central role in modulating plant growth and development, which can be synthesized by both plants and microbes. Various bacteria in the rhizosphere are capable to produce IAA that acts as a signaling molecule for the communication between plants and microbes to promote the plant growth. Generally, IAA production was obtained in a range of 10-200 µg/mL with the addition of 0.2-2 µg/mL of L-tryptophan (Mujahid et al. 2011;Park et al. 2015;Xu et al. 2018). In this study, a predominant strain was identi ed as Enterobacter xiangfangensis BHW6 that was capable of producing 134-1129 µg/mL of IAA in the presence of 0.2-15 g/L of L-tryptophan indicating that the strain was highly tryptophan-dependent.
A good IAA production of E. xiangfangensis BHW6 was bene cial for agricultural application. In the presence of L-tryptophan, a remarkable IAA yield of E. xiangfangensis BHW6 as a cell factory has great prospect in industrial application.
Tryptophan is the main precursor for IAA biosynthesis. Tryptophan could be obtained from rot exudates or released from degrading roots and microbial cells, which are the natural sources of tryptophan for the rhizosphere microbes that may enhance auxin biosynthesis in the rthizosphere (Marten & Frankenberger, 1994). In plants, four tryptophan-dependent pathways have been identi ed including indole-3-acetamide (IAM), indole-3-pyruvic acid (IPyA), tryptamine (TAM), and indole-3-acetaldoxime (IAOX) pathway (Mano & Nemoto, 2012). Actually, IAA can be synthesized via tryptophan-dependent pathway and tryptophanindependent pathway in microbes. However, tryptophan-independent pathway was still unclear. In bacterial, ve pathways including IAM pathway, IPyA pathway, TAM pathway, IAN (indole-3-acetonitrile) pathway and tryptophan side-chain oxidase pathway were identi ed using tryptophan as a precursor. A combination of genetic analysis and chemical analysis was employed in this study. A candidate gene (ipdC) was identi ed for E. xiangfangensis BHW6 based on genomic analysis. This gene is similar with the IPDC enzyme of Enterobacter cloacae. The key IPDC enzyme is capable to convert IPyA into IAAld in the IpyA pathway. The following intermediate assay con rmed the IpyA pathway for E. xiangfangensis BHW6, as the accumulation of desired products was observed in the medium with the addition of precursors. Therefore, IpyA pathway is the major IAA biosynthetic pathway for E. xiangfangensis BHW6.
Environmental factors have an important effect on the IAA synthesis of microbes. An acidic pH is typical for the rhizosphere environment due to proton extrusion through membranes of root cells. Thus IAA biosynthesis of E. xiangfangensis BHW6 is probably tuned to encounter environmental stresses associated with the soil and plant environment (Spaepen et al. 2007). An alternative sigma factor that is acid-regulated could be responsible in passing on the pH response to ipdC expression (Ona et al. 2003). With the increase of culture time, the nutrient in the medium was gradually depleted and the proportion of each nutrient was maladjusted, which resulted in the reduction of multiplication of the strain. Meanwhile, a number of harmful metabolite was possibly accumulated, which caused the cell death. Herein, the physiological metabolic activity of the strain gradually slows down and IAA production tends to stagnate. Moreover, bacteria such as rhizosphere growth-promoting bacteria, nitrogen-xing bacteria, phosphorus bacteria, potassium bacteria and rhizobia are very limited in their ability to resist salinity and alkali, leading to their seriously restricted application in agricultural production. A high NaCl tolerance and a broad pH value of E. xiangfangensis BHW6 are promising in agricultural application. Furthermore, as the low IAA content and various interfering analogs, it is di cult to isolate IAA from microbial secondary metabolites. HPLC, HPLC-MS, GC-MS are always applied to measure IAA content and determine its chemical structure. In this study, IAA was successfully isolated and puri ed by column chromatography, which was characterized by NMR. Conclusions E. xiangfangensis BHW6 was identi ed to possess a predominant ability to secrete IAA in the presence of L-tryptophan. The maximum IAA content (134-1129 µg/mL) was found with the addition of 0.2-15 g/L of L-tryptophan at pH 5 for 6 days, which was 4-40 fold higher than the IAA content in the absence of Ltryptophan. IAA was produced at the stationary phase of bacterial growth. The strain was tolerant and stable to 2.5%-5% (w/v) of NaCl. Genomic sequencing analysis indicated that IAA biosynthesis of the strain was mainly through the IPyA pathway, which was con rmed by intermediates assay as the accumulation of desired products was found in the media containing IAA precursors of IpyA pathway. IAA was isolated with column chromatography and characterized with NMR. E. xiangfangensis BHW6 with a crucial economic value has great prospect for the effective and inexpensive production of IAA.

Figure 8
Intermediates assay for IAA biosynthesis Figure 8 Intermediates assay for IAA biosynthesis Figure 9 1H NMR spectrum of IAA from Enterobacter sp. BHW6