Effects of corn steep liquor on β-poly(l-malic acid) production in Aureobasidium melanogenum

β-poly(l-malic acid) (PMLA) is a water-soluble biopolymer used in medicine, food, and other industries. However, the low level of PMLA biosynthesis in microorganisms limits its further application in the biotechnological industry. In this study, corn steep liquor (CSL), which processes high nutritional value and low-cost characteristics, was selected as a growth factor to increase the PMLA production in strain, Aureobasidium melanogenum, and its metabolomics change under the CSL addition was investigated. The results indicated that, with 3 g/L CSL, PMLA production, cell growth, and yield (Yp/x) were increased by 32.76%, 41.82%, and 47.43%, respectively. The intracellular metabolites of A. melanogenum, such as amino acids, organic acids, and key intermediates in the TCA cycle, increased after the addition of CSL, and the enrichment analysis showed that tyrosine may play a major role in the PMLA biosynthesis. The results presented in this study demonstrated that the addition of CSL would be an efficient approach to improve PMLA production.


Introduction
Polymalic acid (PMLA) is a polyester of L-malic acid with a wide range of applications in the medical, food, and environmental industries due to its excellent biochemical properties, including biocompatibility, biodegradability, and chemical modi ability (Zeng et al. 2019). Several chemical synthesis routes are available in the production of PMLA (Kajiyama et al. 2004;Portilla-Arias et al. 2008;Vert 1998), but these methods are costly, polluted, and di cult to scale up for commercial applications. Microorganisms, such as Aureobasidium melanogenum, can also produce PMLA from sugar during fermentation at high production rates, optical purity, and high molecular weights suitable in many applications (Zou et al. 2019).
Regardless of the microorganism used in PMLA production, L-malic acid is the only precursor in PMLA biosynthesis (Zeng et al. 2019). The three major metabolic pathways in PMLA biosynthesis are the Tricarboxylic acid cycle(TCA), Reductive TCA (rTCA), and Glyoxylate pathway (Chi et al. 2016). In recent years, Several factors were tested to increase PMLA production, including the screening of mutant strains, optimizing fermentation conditions, selecting suitable carbon sources, and adding growth factors (Cao et al. 2019a; Cao et al. 2019b). Moreover, the researcher speculated that the metabolic pathway of PMLA may vary in different strains. The research found that PMLA production is considerably associated with the glyoxylate pathway in Physarum polycephalum with the addition of intermediates and inhibitors (Lee et al. 1999 ) , and it can also be catalyzed by nonbiotin-dependent carboxylation, which is related to the rTCA pathway in A. pullulan (Cao et al. 2014). Due to the varied biosynthesis pathways for PMLA production, it is challenging to construct the genetically engineered strains. As a result, selecting a suitable growth factor becomes an easy way to improve PMLA production.
Corn steep liquor (CSL) is a by-product of the corn wet-milling industry that contains nutrients essential for microbial growth. It has been proposed as a potentially effective substrate for many target metabolites produced by microorganisms (Amado et al. 2017). Researchers found that CSL increases the production of citric acid and calcium malate in Yarrowia lipolytica (Cavallo et al. 2020) and Penicillium viticola (Khan et al. 2014), respectively. However, in recent years research of PMLA biosynthesis, little attention has been paid to investigate the mechanism from a metabolomics aspect.
Gas chromatography-mass spectrometry (GC-MS) is a widely used analytical technique with high separation e ciency and sensitivity detection in resolving complex biological mixtures (He et al. 2018).
Moreover, the intracellular metabolites produced by microorganisms in uenced by the growth factor during fermentation could be detected via GC-MS. The researcher investigated the various effects of deregulating enzymes on the metabolites between engineered l-lysine-producing Corynebacterium glutamicum and the wild-type strain was investigated through intracellular metabolite pro les(da Luz et al. 2017). Some other researchers combined intracellular metabolites with proteomics to identify the difference between marine sediment (Beale et al. 2017) and trace pollution (Beale et al. 2018). This approach could easily detect the main difference between microorganisms under different conditions or growth factors.
In this study, we aimed to explore the effect of CSL on the enhancement of cell growth and PMLA production in A. melanogenum. Metabolomics technology was used to gain insight into the working mechanism and to analyze the change in the key intracellular metabolites of A. melanogenum after CSL addition. The results will help to determine an e cient approach to improve PMLA production.

Microorganism and Medium
Aureobasium. melanogenum CGMCC18996 was isolated in our laboratory and then preserved in the China General Microbiological Culture Collection Center (Beijing, China No. CGMCC18996). The strain was stored in potato dextrose agar (PDA) slants at 4 °C and subcultured every 2 weeks. The seed medium contained 60 g/L sucrose, 3 g/L yeast extract, 2 g/L succinic acid, 1 g/L ammonium sulfate, 0.4 g/L

Fermentation conditions
The primary seed culture of Aureobasidium. melanogenum CGMCC18996 was prepared by inoculating cells grown on solid medium into 500 mL Erlenmeyer asks containing 100 mL seed culture medium and then cultured at 25 °C for approximately 40 h in a rotary shaker (IS-RDS3, Crystal Technology and Industries, Inc., USA). CSL at 1, 3, 5, 7, and 9 g/L was placed into 500 mL Erlenmeyer asks containing 100 mL fermentation medium with primary seed culture (10%, v/v), and fermentation cultivation was conducted at 25 °C for 144 h in a rotary shaker at 200 rpm. Fed-batch fermentation kinetics was investigated in a 5 L stirred tank fermenter (GRJB-5D, Zhenjiang Gree Co., Ltd., China) containing 3 L fermentation medium inoculated with 300 mL seed culture, and the fermentation medium was operated at 25 °C for 156 h with an agitation speed and aeration rate of 500 rpm and 1.3 vvm, respectively.
Assay of PMLA production Fermentation broth (10 mL) was collected at different time points and centrifuged at 15,000 r/min. The resulting supernatant (5 mL) was mixed with 5 mL 2 M H 2 SO 4 and then incubated at 110 °C for 11 h.
After neutralization, the sample was analyzed with HPLC (L-2000, Hitachi Ltd., Japan) by using a PrevailC18 organic acid column at 25 °C eluted with 25 mM KH 2 PO 4 at a rate of 1.0 mL/min. The PMLA concentration was determined by comparing the difference in L-malic acid concentrations before and after hydrolysis.

Assay of fermentation parameters
Cell density was determined via the method of dry cell weight (DCW) in three steps. Prior to measurement, HCl (3 M) was added to 10 mL of fermentation broth to eliminate the excess concentration of CaCO 3 . The fermentation broth (10 mL) was centrifuged at 5,000 rpm for 10 min, and the resulting precipitate was washed twice with phosphate buffer saline (PBS) buffer. After recentrifugation, the precipitates were dried overnight at 80 °C and then weighed.
Speci c growth rate, PMLA productivity, and PMLA yield (Yp/x) calculation The speci c growth rate was measured using the increased biomass versus interval time (Liu et al. 2005), and Y p/x was measured by determining the ratio of increased PMLA to the increased cell biomass concentration over the interval time (Yin et al. 2019).

Extraction of intracellular metabolites and metabolomics analysis
In the extraction of intracellular metabolites, three independent fermentation broth samples (50 mL) at the fermentation times of 72, 96, 120, and 144 h were collected from the 5 L stirred tank fermenter. The excess concentration of CaCO 3 that was not consumed by A. melanogenum was removed by centrifuging the samples at 5,000 rpm for 30 s. The resulting supernatant was centrifuged at 15,000 rpm for 5 min.
The precipitate was collected, washed twice with normal saline at -4 °C, and ground in liquid nitrogen for 25 min. The cell fragment (200 mg) of liquid nitrogen grind was collected and mixed with 1 mL of precooling methanol (60%). The resulting mixture was then centrifuged at 10,000 r/min for 5 min. After the derivatization process, the resulted mixture was subjected to refrigerated centrifugation at 10,000 rpm for 5 min. GC-MS pre-processing data were wrote as .csv les and imported to the MetaboAnalyst(Chong et al. 2019) for data normalization. The peak intensity was represented by the relative concentration, and the downstream analysis was performed by R studio using package BiocManager version 1.30.10.

Results
Effect of CSL addition on PMLA production of A. melanogenum In this study, the effects of different CSL concentrations on PMLA production of A. melanogenum cultured in a rotary shaker were evaluated. The accumulation of biomass increased with increasing CSL concentration after 144 h of fermentation. However, after the addition of 3 g/L CSL, PMLA production reached the maximum of 69.8 g/L, which was 36.8% higher than the control (Fig. 1). The effect of the addition of 3 g/L CSL on the PMLA production of A. melanogenum was further veri ed by culturing A. melanogenum in a 5 L fermenter (Fig. 2). The addition of 3 g/L CSL facilitated the growth and PMLA production of A. melanogenum. After 156 h of fermentation, PMLA production reached 73.72 g/L, as shown in Fig. 2[a], which was 32.76% higher than that of the control group. The biomass reached 62.83 g/L, as shown in Fig. 2[b], which was 41.82% higher than that of the control group. Meanwhile, after 24 h of fermentation, the highest speci c growth rate reached 0.19 h − 1 , as shown in Fig. 2[c], which was 37.72% higher than that of the control group. However, a rapid decline in the speci c growth rate was detected from 0.19 h − 1 to 0.021 h − 1 after 24 h when no distinct difference existed between the control and CSL groups. The PMLA yield (Y p/x ) in CSL showed a signi cant increase in the late stage of fermentation after the addition of 3 g/L CSL, as shown in Fig. 2[d]. After 120 h, the highest Y p/x reached 1.82 g/g, which was 47.43% higher than that of the control group. Moreover, the rate of residual sugar (Fig. 2[e]) was consumed rapidly after the addition of 3 g/L CSL, and the end time was 12 h earlier than that of the control.
Metabolomics analysis of A. melanogenum cultivated with the addition of 3 g/L CSL The PLS-DA scores plot (Fig. 4) showed a clear variation in the metabolite pro les under both groups, and the metabolomics data revealed a total of thirty-six metabolites of A. melanogenum that were detected via GC-MS at 72, 96, 120, and 144 h time points (Fig. 3). Among them, most of the metabolite concentrations in the CSL group were increased compared to the control, especially at 120 h (Fig. 3). The enrichment analysis (Fig S1) indicated that the concentrations of metabolite related to PMLA biosynthesis increased obviously, which were mainly deoxyinosine, homogentisate, fumarate acid, and 5aminolevulinic. These metabolites were involved in the Purine metabolism(P < 0.05), Tyrosine metabolism(P < 0.05), TCA cycle(P < 0.05), and Glycine and serine metabolism.

Metabolomics analysis of PMLA metabolic pathway
The metabolic pathway related to PMLA biosynthesis and the relative concentration changes were depicted (Fig. 5). The results showed that, compared to the control, nearly all of the metabolite concentrations increased and peaked at 120 h. Among them, the concentrations of six metabolites related to the Tryptophan metabolism were improved, which were serotonin, 5-Methoxytryptamine, indole-3-acetonitrile, 2-aminomuconate semialde hyde, L-Kynurenine, and Kynurenate. In addition, the CSL increased the concentration of 5-aminolevulinate involved in the Glycine and serine metabolism. The concentrations of homogentisate and R-reticuline, which related to the Tyrosine metabolism, and Dlactaldehyde, which would ow to the pyruvate, were increased respectively, and that of fumarate and oxalosuccinate, which involved in the TCA cycle, also showed an increasing effect. Meanwhile, the results indicated that a decreasing concentration was observed on the metabolites, L-valine and hydroxypruvate.

Assay of the amino acids on PMLA production
The nutritional substances of CSL were tested. Seventeen kinds of amino acids and three types of vitamins were detected. Table 1 showed the ratio of each amino acid quality to the CSL quality. Among them, four amino acids (tyrosine, serine, glycine, and tryptophan) were selected based on the PMLA pathway of the metabolomics data (Fig. 5) and added to the fermentation broth(without peptone) separately to evaluate the PMLA production. The result demonstrated that the PMLA production increased by 29.5% after tyrosine adding, 21.9% after serine adding, 9.3% after tryptophan adding, 7.6% after glycine adding, and 39.47% after the CSL adding (Fig. 6). Moreover, signi cant changes were observed between tyrosine and the control (P < 0.01) and the CSL and the control (P < 0.01).

Discussion
The use of the growth factor is an economical way to improve PMLA production (Cao et al. 2019b). However, less study on the metabolomics changes under the different growth factor has limited the better understanding of the mechanism for PMLA biosynthesis from the metabolic aspect.
In this study, the addition of 3 g/L CSL facilitates cell growth and PMLA production in A. melanogenum. The maximal PMLA production and biomass increased by 32.76% and 41.82%, respectively. Furthermore, the speci c growth rate revealed a rapid increase during the early phase of fermentation, and Y p/x in the CSL group was higher than that of the control in the late stage. Moreover, CSL contained various amino acids and vitamins ( Table 1) that could function as an effective stimulatory agent of cell growth and PMLA biosynthesis. It was found that the rich nutrients of CSL became a source of nitrogen, essential minerals, and cofactors required for Pichia pastoris cell growth (Zheng et al. 2012). Moreover, the addition of CSL increased the biomass of Trametes versicolor in the initial 12 h of fermentation, and an accelerated growth rate was observed ). The addition of 3 g/L CSL provided various amino acids and vitamins (Table 1), indicating that the CSL could be an effective stimulatory agent of cell growth in A. melanogenum. The high cell growth of A. melanogenum led to a high level of PMLA production. Therefore, we speculated that various nutritional substances provided by CSL bene ted cell growth, which in turn bene ted PMLA production.
With the help of metabolomics technology, the working mechanism underlying the effect of the addition of 3 g/L CSL on the metabolism of A. melanogenum was analyzed. The metabolomics data showed that the relative concentrations of metabolites involved in amino and organic acid metabolism were changed during fermentation after the addition of 3 g/L CSL (Fig. 5). And the PLS-DA showed a clear separation between 2 groups. In addition, the Tyrosine metabolism, Glycine and serine metabolism, and TCA cycle were up-regulated after the 3 g/L CSL addition. Among them, for the tyrosine metabolism, tyrosine is rst converted to 4-hydroxyphenylpyruvate then to homogentisate that is further converted to fumarlacetoacetate, and fumarlacetoacetate is nally transferred to fumarate and enter the TCA cycle (Wang et al. 2019). The metabolomics data showed that the relative concentration of homogentisate increased by 1.44-fold at 144 h compared to the control, and that of fumarate increased by 1.52-fold. Moreover, the concentration of 5-aminolevulinate, which related to the Glycine and serine metabolism, increased by 1.2-fold under the CSL. And a 1.2-fold change on 2-aminomuconate semialde hyde concentration resulted in the Tryptophan up-regulated metabolism that leads tryptophan into the glutaric acid pathway and then the TCA cycle (Fukuwatari et al. 2001). The result also proved that deoxyinosine, involved in purine metabolism, increased by 1.2-fold, and this metabolism is related to the cell's nitrogen absorption, which can provide molecules that are essential to DNA and RNA biosynthesis, energy metabolism and signal transduction(Jessica and James 2017). Combined with the fermentation data( Fig. 2), the result showed that PMLA production sustained and PMLA yield decreased after 120 h. it was consistent with the metabolomics data that the metabolites related to the PMLA biosynthesis reached the highest at 120 h then it began to decrease.
Metabolomics data can express the metabolic changes under different conditions. However, after the peak alignment and the data normalization, some of the target-metabolites may lose in the data. Therefore, we speculated that the concentrations of tyrosine, glycine, serine, and tryptophan, which related to the up-regulated Tyrosine pathway, Glycine and serine pathway, and Tryptophan pathway, were improved in the CSL group. These pathways may further cause the PMLA production increased. Consequently, we tested the nutritional substances of CSL and found seventeen kinds of amino acids and three types of vitamins (Table 1). Among them, serine is 1.28%, tyrosine is 0.62%, tryptophan is 0.17%, and glycine is 2.05% of the total CSL weight.
In order to gure out which amino acid most bene ting PMLA production in the CSL, different amino acids from the up-regulated pathway (Fig. 5) were added to the fermentation broth based on the ratio of their quality to the total CSL quality to evaluate the effect on PMLA production. The result demonstrated that all these four amino acids increased PMLA production by 29.5%, 21.9%, 9.3%, and 7.6%, respectively. Apart from that, a signi cant change between tyrosine and the control(P < 0.01) was observed, and this result is consistent with the enrichment analysis, indicating that the CSL signi cantly in uenced the Tyrosine metabolism(P < 0.05). The data obtained from the PMLA-related pathway (Fig. 5) suggested that tyrosine ows to the TCA cycle by converted to fumarate, which is further converted to malic acid and increases the production of PMLA. Meanwhile, it also illustrated that all of the four amino acids, tyrosine, glycine, serine, and tryptophan, eventually ows to the TCA cycle (Fig. 5). However, due to the relative concentration changes of the metabolites (pyruvate and fumarate), we can speculate that the tyrosine probably plays a crucial role in increasing PMLA production under the CSL.
The results, as mentioned above, showed that the TCA cycle was up-regulated after the addition of 3 g/L CSL and led to an increase in malic acid production, which would further increase PMLA production. Therefore, we can conclude that the up-regulated TCA cycle is the key metabolic pathway under the 3 g/L CSL for the increase of PMLA production among three speculated metabolic pathways which are the glyoxylate acid pathway, the reductive TCA pathway, and the TCA pathway. (Zou et al. 2019). Meanwhile, the energy provided by the up-regulated purine metabolism may accelerate this process.
The improvement in amino acid and organic acid metabolism in A. melanogenum from the addition of 3 g/L CSL generated various amino acids and organic acids to improve cell growth. The conversions of the metabolites related to the TCA cycle were enhanced after the addition of 3 g/L CSL. Therefore, we speculated that CSL is an effective stimulatory agent for cell growth and PMLA biosynthesis in A. melanogenum. Meanwhile, CSL could be used as an economic nitrogen source due to its high nutrition and low cost. As a potential replacement of peptone and yeast extract in PMLA production, CSL has satisfactory use prospects in the PMLA industry.

Declarations
Ethics approval and consent to participate not applicable Consent for publication not applicable Availability of data and material All data generated or analysed during this study are included in this paper.