Valorization of xylose mother liquor into salidroside by engineered Kluyveromyces marxianus

TL;DR

Scientists engineered a yeast to convert industrial waste into a valuable pharmaceutical compound, salidroside, offering a sustainable production method.

Contribution

The first gram-scale production of salidroside in Kluyveromyces marxianus directly from xylose mother liquor using metabolic engineering.

Findings

Engineered K. marxianus produced 10.17 g/L salidroside in shake-flask cultivation.

Fermentation using xylose mother liquor achieved 15.49 g/L salidroside in a bioreactor.

Strain YSAL07 reached a productivity of 84.75 mg/(L·h), surpassing previous reports.

Abstract

Large amounts of xylose mother liquor (XML) are produced as a byproduct of the xylose industry and are often treated as waste. Salidroside is a high-value glycoside with pharmaceutical and nutraceutical applications, but conventional extraction from plants cannot meet market demand and threatens the sustainability of Rhodiola resources. Here, XML was valorized for de novo salidroside production using engineered Kluyveromyces marxianus. The KmPDC1 disruption promoted pyruvate accumulation, which in turn enhanced the accumulation of a shikimate pathway precursor (phosphoenolpyruvate). Introduction of the salidroside biosynthesis pathway enabled salidroside production in K. marxianus. Additionally, salidroside production was improved through three strategies: expressing feedback-resistant 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase in the shikimate pathway, expressing phosphoketolase to boost erythrose-4-phosphate (another precursor of the shikimate pathway), and expressing chorismate mutase/prephenate dehydrogenase to facilitate tyrosine synthesis. The control of pH during fermentation further enhanced salidroside production. The final strain YSAL07 produced 10.17 ± 0.12 g/L salidroside in shake-flask cultivation, with a productivity of 84.75 mg/(L·h), which is higher than previously reported values. In the bioreactor, YSAL07 produced 13.18 ± 0.18 g/L salidroside within 96 h using glucose as the substrate, whereas fermentation with XML achieved a salidroside titer of 15.49 ± 0.69 g/L within 120 h. This study demonstrates the first gram-scale (≥10 g/L) salidroside production in K. marxianus directly from XML, providing a sustainable and economically viable route for the manufacture of high-value natural products.

Image 1

Figures

Construction process of the engineered strains. Strains were cultivated at 42 °C in YPD medium containing 4% (w/v) glucose. (A) Platform strain construction: deletion of *KmPDC1* blocked ethanol formation and enhanced pyruvate accumulation. (B) Overexpression of *KmMTH1-ΔT* partially rescued the growth defect caused by *KmPDC1* deletion. (C) Genetic modifications and their effects on salidroside and tyrosol production in the engineered strains.Fig. 1

Optimization of fermentation conditions for salidroside production. (A) Effects of temperature, (B) initial sugar concentration, and (C) pH on salidroside production by YSAL06. (D) Growth curves and salidroside production of YSAL06 and YSAL07. (E) Effects of carbon source composition (glucose-to-xylose ratios in mixed sugars) on salidroside production by YSAL07. (F) Shake-flask fermentation of YSAL07 using 20% (v/v) xylose mother liquor as the carbon source.Fig. 2

Fed-batch fermentation of YSAL07 using glucose or xylose mother liquor in a 2-L bioreactor. (A) Glucose as the carbon source. (B) Xylose mother liquor as the carbon source.Fig. 3

Full text

Introduction

1

With rising demand for low-calorie sweeteners, the industrial xylitol production has increased, resulting in large quantities of xylose mother liquor (XML) as a byproduct [1,2]. XML is a viscous, brown liquid residue generated during xylose extraction from corncob acid hydrolysate. It is composed primarily of​ xylose (35–40%, w/w), l-arabinose (10–15%, w/w), glucose (8–10%, w/w), and galactose (8–10%, w/w) [2], as well as​ the major inhibitory compounds 5-hydroxymethylfurfural (HMF) and furfural [3]. Due to its complex composition and high viscosity, XML is primarily limited to low-value products such as caramel coloring [4]. However, given its abundance and low cost, XML represents an attractive feedstock for reducing raw material costs in fermentation-based production of high-value natural products.

Salidroside (8-O-β-D-glucoside of tyrosol), the primary active compound in Rhodiola species, exhibits anti-fatigue, antidepressant, anti-aging, and antitumor activities and has shown pharmacological potential in central nervous system and cardiovascular diseases [5]. However, extraction of salidroside from wild Rhodiola has led to increasing depletion of natural resources [6]. Therefore, sustainable production strategies are urgently needed to meet the growing market demand.

Microbial synthesis has emerged as a promising approach for salidroside production. Although engineered Saccharomyces cerevisiae has achieved high salidroside titers [7,8], the reported engineered strains still rely on refined sugars such as glucose and require additional engineering to efficiently utilize xylose-rich feedstocks [9,10]. Kluyveromyces marxianus, a thermotolerant yeast capable of growth up to 52 °C with rapid proliferation (μ ≈ 0.99 h^−1^ at 40 °C), offers advantages for high-temperature fermentation, including reduced cooling costs and lower contamination risk [11,12]. This yeast can naturally utilize xylose and exhibits relative tolerance to lignocellulosic biomass-derived inhibitors, making it particularly suitable for converting agricultural residues and industrial byproducts into high-value products [12].

In this study, a modular metabolic engineering strategy was developed in K. marxianus for de novo salidroside biosynthesis. By optimizing the host strain, introducing a biosynthetic pathway, enhancing shikimate and erythrose-4-phosphate (E4P) precursor supply, and tuning fermentation conditions, high-level salidroside production (15.49 ± 0.69 g/L) was achieved using XML. This approach provides a sustainable and economically viable platform for industrial salidroside production.

Materials and methods

2

Microorganisms and media

2.1

Yeast strains were cultivated in YPD medium (20 g/L peptone, 10 g/L yeast extract, and 20 g/L glucose). Synthetic dropout (SD) medium containing 0.67 g/L yeast nitrogen base (YNB) and 20 g/L glucose, supplemented with appropriate amino acids, was used for transformant screening. Escherichia coli DH5α was used for gene cloning and was cultivated in LB medium (5 g/L yeast extract, 10 g/L tryptone, and 10 g/L NaCl), supplemented with 100 mg/L ampicillin**.** Fed-batch fermentation was conducted in a 2-L bioreactor using an inorganic salt medium containing 20 g/L glucose, 15 g/L (NH_4_)2_SO_4, 8 g/L KH_2_PO_4_, 6.15 g/L MgSO_4_·7H_2_O, supplemented with 12 mL/L vitamin solution and 10 mL/L trace metal solution [13]. The compositions of trace element, vitamin, and feeding solutions are provided in the Supplementary Materials. XML was obtained from Shandong Lujian Biological Technology Co., Ltd. and detoxified by overliming followed by activated carbon treatment (see the Supplementary Materials) [14].

Plasmid and strain construction

2.2

The primers, plasmids, and strains used in this study, together with their construction details, are provided in the Supplementary Materials (Table S1 and S2).

The KmPDC1 gene in parental strain YHJ010 was disrupted by homologous recombination [15] to generate YΔPDC1. After URA3 marker recycling, the linearized plasmid pZB013 carrying KmMTH1-ΔT was integrated into the chromosome to obtain strain YΔPDC1-MTH1. Then, PcAAS was expressed in YΔPDC1-MTH1 to yield strain YSAL01. Subsequent introduction of RrU8GT33 generated YSAL02, thereby enabling heterologous salidroside biosynthesis. To enhance the shikimate pathway and increase E4P supply, ScARO4^K229L^ and ScARO7^G141S^ were introduced to generate strains YSAL03 and YSAL04, respectively. Based on YSAL03, subsequent introduction of BbXFPK yielded YSAL05, and further incorporation of EcTYRA^M53I/A354V^ resulted in YSAL06. The URA3 marker was recycled between transformations as described in Supplementary Materials and previous report [15]. Finally, strain YSAL06 was complemented with KmTRP1 to restore tryptophan prototrophy, resulting in YSAL07. All plasmids, designed for constitutive promoter-driven heterologous gene expression, were linearized prior to chromosomal integration to ensure genetic stability.

Fermentation experiments

2.3

Shake-flask fermentations (50 mL medium in 250-mL flasks, 250 rpm, initial OD_600_ = 0.3) were performed at 30, 37, and 42 °C. For pH maintenance, 3% (w/v) CaCO_3_ was added upon cultivation initiation in shake-flask cultures.

Fed-batch fermentation was conducted in a 2-L bioreactor (Baoxin, Shanghai) with an initial working volume of 1 L at 37 °C. The aeration rate was set to 2 vvm, and pH was controlled at 5.5 using NH_3_·H_2_O. Agitation was maintained between 400 and 800 rpm. The substrates were fed after 12 h of cultivation at 8 mL/h (XML) or 6 mL/h (600 g/L glucose).

Real-time qPCR analysis

2.4

Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to verify the transcription of heterologous genes introduced to enhance precursor availability for salidroside biosynthesis.

The transcript levels of ScARO4^K229L^, BbXFPK, and EcTYRA^M53I/A354V^ were analyzed in strain YSAL07, while the transcript level of ScARO7^G141S^ was examined in strain YSAL04. K. marxianus strains were cultivated overnight in YPD medium at 37 °C and 250 rpm, then inoculated into fresh YPD medium with an initial OD_600_ of 0.3 and grown under the same conditions. Cells were harvested for RNA extraction after 12 h of cultivation following inoculation. Total RNA was extracted using the Spin Column Yeast Total RNA Purification Kit (Sangon Biotech, Shanghai), and first-strand cDNA was synthesized using ToloScript All-in-one RT EasyMix for qPCR (Tolo Biotech, Shanghai) following the manufacturers’ instructions. RT-qPCR was performed with 2 × Q3 SYBR qPCR Master Mix (Tolo Biotech, Shanghai) on a LightCycler 96 Real-Time PCR System (Roche, Switzerland) using gene-specific primers (Table S1). The reference gene ACT1 was used for normalization.

Analytical methods

2.5

The concentrations of d-xylose, d-glucose, and pyruvate were determined using a high-performance liquid chromatography (HPLC) system (Agilent 1260, USA) equipped with an ROA-Organic Acid H^+^ (8%) column (Phenomenex, USA). The mobile phase was 0.005 M H_2_SO_4_, the column temperature was 75 °C, and the flow rate was 0.3 mL/min [15]. Xylitol and glycerol were analyzed using an RCM Ca^2+^ column (Phenomenex, USA) with degassed water as the mobile phase at a flow rate of 0.6 mL/min and a column temperature of 80 °C. Tyrosol and salidroside were determined by HPLC using a C18 column (Phenomenex, USA) with methanol/water (15:85, v/v) as the mobile phase at a flow rate of 0.8 mL/min and detected at 225 nm. Cell growth was monitored by measuring the optical density at 600 nm (OD_600_). All experiments were conducted in triplicate, and results were expressed as mean ± SD.

Results and discussion

3

De novo biosynthesis of salidroside in K. marxianus

3.1

Salidroside biosynthesis proceeds via the shikimate pathway, initiated by phosphoenolpyruvate (PEP) derived from glycolysis and E4P from the pentose phosphate pathway [16] (Fig. S1). Therefore, KmPDC1 was disrupted to enhance the precursor supply for the shikimate pathway by reducing carbon flux toward competing metabolic pathways. After 24 h of fermentation with 4% (w/v) glucose at 42 °C, YΔPDC1 produced 12.18 ± 0.97 g/L pyruvate, whereas no pyruvate was detected in the parental strain YHJ010 (Fig. 1A), indicating that KmPDC1 disruption markedly enhances pyruvate accumulation. In addition, ethanol was detected at 2.10 ± 0.42 g/L in YHJ010 but was not detected in YΔPDC1 (Fig. 1A), suggesting that the conversion of pyruvate to acetaldehyde and ethanol was blocked. This phenotype contrasts with Saccharomyces cerevisiae, in which multiple pyruvate decarboxylase isoenzymes render deletion of a single PDC gene insufficient to block ethanol production [17].Fig. 1. Construction process of the engineered strains. Strains were cultivated at 42 °C in YPD medium containing 4% (w/v) glucose. (A) Platform strain construction: deletion of KmPDC1 blocked ethanol formation and enhanced pyruvate accumulation. (B) Overexpression of KmMTH1-ΔT partially rescued the growth defect caused by KmPDC1 deletion. (C) Genetic modifications and their effects on salidroside and tyrosol production in the engineered strains.Fig. 1

Disruption of KmPDC1 only modestly impaired growth (Fig. 1B) relative to the parental strain YHJ010, in contrast to S. cerevisiae, which cannot grow on glucose in the absence of Pdc activity [18]. Overexpression of KmMTH1-ΔT partially restored growth, thereby alleviating the growth defect associated with KmPDC1 disruption. Accordingly, strain YΔPDC1-MTH1 was selected as the platform strain for salidroside production.

After 72 h fermentation at 42 °C, YΔPDC1-MTH1 accumulated 46.58 ± 2.91 mg/L tyrosol (Fig. 1C), indicating that endogenous metabolic pathways support basal tyrosol formation. As tyrosol is a direct precursor of salidroside, PcAAS was introduced into the strain YΔPDC1-MTH1 to improve tyrosol production, yielding strain YSAL01 (PcAAS). PcAAS converts tyrosine to 4-hydroxyphenylacetaldehyde, which is subsequently reduced to tyrosol. Strain YSAL01 produced 115.59 ± 16.16 mg/L tyrosol, representing a 2.48-fold increase over the strain YΔPDC1-MTH1 (Fig. 1C), indicating that PcAAS effectively enhances tyrosol production.

Salidroside, a glycosylated derivative of tyrosol, is formed from tyrosol and UDP-glucose via UDP-glycosyltransferase-catalyzed reaction [16]. After the introduction of RrU8GT33, strain YSAL02 (PcAAS + RrU8GT33) produced 26.78 ± 3.96 mg/L salidroside and 53.12 ± 9.83 mg/L tyrosol (Fig. 1C). To the best of our knowledge, this represents the first report of de novo salidroside biosynthesis in K. marxianus.

Reinforcement of the shikimate pathway for improved salidroside biosynthesis

3.2

The shikimate pathway is a major regulatory node in aromatic amino acid biosynthesis, and 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase catalyzes the first committed step that controls carbon flux into the pathway [19]. DAHP synthase activity is subject to feedback inhibition by l-tyrosine and l-phenylalanine [20]. In addition, chorismate mutase, which directs chorismate into the l-tyrosine and l-phenylalanine branches, is also subject to feedback regulation by aromatic amino acids [20] (Fig. S1). Feedback-insensitive variants of DAHP synthase (ScAro4^K229L^) and chorismate mutase (ScAro7^G141S^) were expressed, respectively, to relieve feedback inhibition. The strain YSAL03 (PcAAS + RrU8GT33 + ScARO4^K229L^) produced 249.31 ± 18.46 mg/L salidroside, representing a 9.31-fold increase over YSAL02, whereas YSAL04 (PcAAS + RrU8GT33 + ScARO7^G141S^) showed no increase in salidroside production (Fig. 1C), even though ScARO7^G141S^ was transcriptionally active, as confirmed by RT-qPCR (Fig. S2). This result is consistent with previous studies in yeast that identified the Aro4-catalyzed step as controlling the primary flux into the shikimate pathway, and found that overexpression of feedback-insensitive Aro7 alone had limited effects [21,22].

DAHP is formed from the condensation of PEP and E4P [19]. Thus, increasing the availability of these precursors is expected to further enhance salidroside biosynthesis. In the previously constructed platform strain, disruption of KmPDC1 increased PEP availability; however, limited E4P availability may still constrain DAHP synthesis. Overexpression of BbXFPK, which converts fructose-6-phosphate into E4P and acetyl phosphate, in strain YSAL05 (PcAAS + RrU8GT33 + ScARO4^K229L^ + BbXFPK) increased the salidroside titer to 705.60 ± 32.45 mg/L, representing a 2.83-fold increase over YSAL03 (Fig. 1C). BbXfpk provides a direct E4P-generation route from fructose-6-phosphate via the phosphoketolase pathway (Fig. S1), a strategy widely used to alleviate E4P limitation and increase carbon flux into the shikimate pathway after relieving feedback inhibition [22].

To further increase the direct precursor supply for tyrosol, a feedback-resistant chorismate mutase/prephenate dehydrogenase (EcTyrA^M53I/A354V^) was introduced to promote tyrosine biosynthesis. The resulting strain, YSAL06 (PcAAS + RrU8GT33 + ScARO4^K229L^ + BbXFPK + EcTYRA^M53I/A354V^), produced salidroside at the gram-per-liter level (1.03 ± 0.04 g/L) (Fig. 1C), demonstrating that enhancing precursor flux effectively balances upstream and downstream metabolism, thereby facilitating salidroside synthesis in K. marxianus.

Overall, these results show that sequential strengthening of the shikimate pathway by relieving feedback inhibition, increasing DAHP precursor availability, and enhancing tyrosine biosynthesis increased salidroside production in K. marxianus.

Optimization of fermentation conditions for salidroside production

3.3

Although fermentation during strain construction was performed at 42 °C, this temperature may not be optimal for RrU8GT33-mediated glycosylation, as indicated by the substantial accumulation of tyrosol (Fig. 1C). Strain YSAL06 produced 1.29 ± 0.06 g/L salidroside at 30 °C, with a low residual tyrosol level (33.33 ± 5.77 mg/L) (Fig. 2A). At 37 °C, salidroside titer increased to 2.75 ± 0.04 g/L, with moderate tyrosol accumulation (383.33 ± 73.71 mg/L), whereas at 42 °C salidroside production decreased to 1.07 ± 0.04 g/L and was associated with higher tyrosol accumulation (753.33 ± 55.08 mg/L) (Fig. 2A). Biomass was similar at 30 °C and 37 °C but reduced at 42 °C (Fig. 2A). These results indicate that fermentation at 37 °C is optimal among the tested temperatures for salidroside production. The increased tyrosol accumulation at 42 °C may be associated with reduced activity or thermal stability of RrU8GT33. This interpretation is consistent with the phenotype that tyrosol accumulated while salidroside formation decreased at 42 °C, suggesting that glycosylation rather than tyrosol formation becomes the bottleneck at elevated temperature.Fig. 2. Optimization of fermentation conditions for salidroside production. (A) Effects of temperature, (B) initial sugar concentration, and (C) pH on salidroside production by YSAL06. (D) Growth curves and salidroside production of YSAL06 and YSAL07. (E) Effects of carbon source composition (glucose-to-xylose ratios in mixed sugars) on salidroside production by YSAL07. (F) Shake-flask fermentation of YSAL07 using 20% (v/v) xylose mother liquor as the carbon source.Fig. 2

RrU8GT33 is a UDP-dependent glycosyltransferase that catalyzes the glycosylation of tyrosol at the 8-OH group using UDP-glucose to form salidroside [8]. Because UDP-glucose biosynthesis requires ATP and sugar precursors provided by glycolysis, fermentation with higher glucose concentrations was evaluated. Within the range of 4–8% (w/v) glucose, the maximum salidroside titer increased with increasing glucose concentration. Strain YSAL06 produced maximum salidroside with titers of 2.49 ± 0.23 g/L, 2.96 ± 0.25 g/L, and 3.77 ± 0.33 g/L with 4%, 6%, and 8% (w/v) glucose, respectively, and only minor tyrosol accumulation was observed at the time of peak salidroside production (Fig. 2B and Fig. S3A). However, at an initial glucose concentration of 10% (w/v), the salidroside titer decreased to 3.17 ± 0.29 g/L compared with 8% (w/v) glucose (Fig. 2B), and 46.07 ± 3.18 g/L glucose remained at 144 h (Fig. S3B), likely reflecting growth inhibition (Fig. S3C) caused by elevated osmotic stress. Overall, an initial glucose concentration of 8% (w/v) was optimal for salidroside production.

In strain YSAL06, prolonged fermentation resulted in reduced salidroside accumulation (Fig. 2B), which may be associated with acidification of the medium by K. marxianus. To stabilize the fermentation pH, 3% (w/v) CaCO_3_ was added to YPD containing 8% (w/v) glucose. After shake-flask fermentation at 37 °C for 144 h, YSAL06 produced 8.98 ± 0.52 g/L salidroside, representing a 2.38-fold increase compared with the control (without CaCO_3_), with no detectable decline in salidroside titer (Fig. 2C). Tyrosol accumulation was substantially reduced compared with the uncontrolled-pH control, decreasing from 1.90 ± 0.13 g/L at 144 h to 0.23 ± 0.15 g/L upon CaCO_3_ addition, and glucose utilization was accelerated (Fig. S4). Under uncontrolled pH conditions, acidification of the culture medium is likely to impair enzymatic activity and membrane transport processes [23]. The observed reduction in tyrosol accumulation, together with accelerated glucose utilization, suggests that maintaining a more stable pH contributes to improved fermentation performance, thereby promoting salidroside accumulation.

Restoration of tryptophan prototrophy to improve the salidroside production

3.4

During fermentation, auxotrophic strains require additional nutrients and exhibit weaker growth. Therefore, restoring their prototrophy can enhance strain growth and consequently increase salidroside production. The tryptophan biosynthetic capability of YSAL06 was restored, resulting in strain YSAL07. Strain YSAL07 accumulated more biomass than YSAL06 (Fig. 2D), which may contribute to improved salidroside biosynthesis. With 8% (w/v) glucose and CaCO_3_-mediated pH control, strain YSAL07 produced 10.17 ± 0.12 g/L salidroside at 120 h (Fig. 2D), which, to the best of our knowledge, represents the highest titer reported at the shake-flask level. The productivity reached 84.75 mg/(L·h), exceeding the previously reported value of 66.67 mg/(L·h) in yeast under shake-flask conditions [24].

Evaluation of carbon sources for salidroside production

3.5

Since glucose and xylose are the major sugars present in XML, strain YSAL07 was cultured with glucose, xylose, or mixed sugars (glucose-to-xylose ratios of 4:1, 1:1, and 1:4; total sugar concentration of 80 g/L) at 37 °C with 3% (w/v) CaCO_3_ supplementation to evaluate salidroside production. Under single-sugar conditions, glucose was depleted in YPD 24 h earlier than xylose was depleted in YPX (Fig. S5), confirming that glucose is more readily metabolized than xylose. Salidroside reached 10.17 ± 0.12 g/L at 120 h in YPD, as shown above, whereas it reached 7.67 ± 0.19 g/L in YPX (Fig. 2E). The maximum salidroside titers were 9.38 ± 0.80 g/L, 9.22 ± 0.53 g/L, and 8.41 ± 0.50 g/L at glucose-to-xylose ratios of 4:1, 1:1, and 1:4, respectively (Fig. 2E). Among these conditions, a glucose-to-xylose ratio of 1:4 most closely resembled the composition of XML (Fig. S6). Accordingly, 20% (v/v) XML was evaluated as the carbon source, resulting in 9.81 ± 0.51 g/L salidroside after 144 h (Fig. 2F), demonstrating the potential of XML as an effective carbon source for salidroside production. The lower salidroside titer in xylose-containing media likely reflects slower xylose utilization compared with glucose, whereas comparable production in 20% (v/v) XML indicates that strain YSAL07 can effectively utilize XML.

Evaluation of salidroside production in bioreactor using glucose or XML

3.6

Fed-batch fermentations were conducted in a 2-L bioreactor using strain YSAL07 with glucose or XML as the carbon source. With glucose feeding (Fig. 3A), salidroside reached 13.18 ± 0.18 g/L with 0.85 ± 0.03 g/L tyrosol accumulation. During the fermentation, 37.38 ± 0.51 g/L glycerol was produced. The Glycerol accumulation may serve as an alternative NADH sink to maintain redox homeostasis when ethanol formation is constrained by KmPDC1 disruption [25].Fig. 3. Fed-batch fermentation of YSAL07 using glucose or xylose mother liquor in a 2-L bioreactor. (A) Glucose as the carbon source. (B) Xylose mother liquor as the carbon source.Fig. 3

Using XML (Fig. 3B), rapid growth was observed within 48 h, followed by stable biomass accumulation (OD_600_ ≈ 100). Salidroside titer reached 15.49 ± 0.69 g/L at 120 h with minimal tyrosol (0.29 ± 0.04 g/L) accumulation, while xylitol accumulation suggests a redox imbalance associated with XR/XDH-mediated xylose metabolism [26,27]. The superior performance observed with XML compared with pure glucose may be related to the more complex matrix of the mother liquor; however, the specific contributing factors remain to be determined. Together, these results demonstrate that efficient salidroside production can be achieved from both glucose and XML in YSAL07, providing a feasible strategy for valorizing industrial byproducts.

Conclusion

4

This study established a K. marxianus strain for de novo salidroside biosynthesis, achieving efficient production by innovatively using XML as a carbon source. Through platform strain optimization, modular pathway engineering, and optimization of fermentation conditions, salidroside titers were significantly improved. The final strain YSAL07 produced 10.17 ± 0.12 g/L salidroside at 120 h with a productivity of 84.75 mg/(L·h), which, to the best of our knowledge, represents the highest value reported at the shake-flask level. Using XML, YSAL07 produced 9.81 ± 0.51 g/L in shake-flask fermentation and 15.49 ± 0.69 g/L in a 2-L bioreactor. To the best of our knowledge, this is the first report of salidroside produced directly from XML. These findings highlight the potential of K. marxianus and provide a sustainable, cost-effective approach for industrial-scale salidroside production and the valorization of XML.

CRediT authorship contribution statement

Lingya Wang: Writing – review & editing, Writing – original draft, Methodology, Formal analysis, Data curation, Conceptualization. Dongmei Wang: Writing – review & editing, Methodology, Formal analysis. Jiong Hong: Writing – review & editing, Supervision, Methodology, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.