l-arabinose, a valuable C5 pentose sugar in xylose mother liquor (a low-cost cellulose hydrolysis byproduct), remains underutilized due to costly separation requirements, resulting in significant waste of fermentable carbon resources. In this study, Escherichia coli W3110 was systematically engineered to efficiently convert l-arabinose into xylitol, a low-calorie sweetener with significant commercial value. Firstly, the l-arabinose metabolic network was reconstructed and glucose catabolic repression was alleviated through coordinated pathway modifications, enabling simultaneous utilization of both l-arabinose and glucose. Subsequently, a xylitol synthesis module consisting of l-arabinose isomerase (AraA), l-xylulose reductase (LXR), and d-psicose-3-epimerase (DPE) was systematically optimized via gene arrangement, promoter and RBS engineering. The optimized pathway was integrated into the E. coli W3110 genome at the IS5 locus using MUCICAT technology, generating a plasmid-free production strain and reducing plasmid-segregation concerns. Fed-batch fermentation in a 3 L bioreactor yielded 64.07 g/L xylitol at a productivity of 1.46 g/L/h with 90.77% l-arabinose conversion in 44 h. This achievement overcomes two critical metabolic bottlenecks: (1) glucose catabolite repression, which normally prevents pentose utilization in the presence of glucose, and (2) the successful stoichiometric balancing of three enzymatic steps (AraA, LXR, DPE). The engineered strain achieves simultaneous glucose-arabinose co-metabolism, and glucose co-utilization supports xylitol formation in a manner consistent with an endogenous reducing-power contribution, thereby eliminating the requirement for exogenous glycerol supplementation in the optimized process. This work establishes a defined-substrate engineering platform for l-arabinose-to-xylitol conversion and provides a strategic basis for future evaluation using complex industrial carbohydrate streams.
Isodon rubescens
f. lushanensis, a typical form of
I. rubescens
, is characterized by its natural deficiency in oridonin and the presence of a unique ent-kaurane diterpenoid, lushanrubescensin.
To elucidate the molecular basis underlying the distinct chemical profiles between
I. rubescens
f. lushanensis and
I. rubescens
(Hemsl.) Hara, we conducted a comprehensive analysis utilizing high-quality genomic and transcriptomic data of
I. rubescens
f. lushanensis.
Through a genome-wide survey, our study identified 83 terpene synthase (
TPS
) genes, including 13
TPS
-C and 9
TPS-e
/f subfamily genes, which are implicated in diterpenoid biosynthesis. By integrating conserved motif analysis, differential expression profiling (FPKM), and RT-qPCR validation, we successfully screened key candidate genes. Subsequent heterologous expression in
Saccharomyces cerevisiae
enabled the functional characterization of five diterpene synthase genes, specifically CPS and KSL enzymes, involved in the central modules of diterpenoid synthesis.
These findings not only expand the repertoire of known biosynthetic genes in
I. rubescens
but also offer valuable insights into the divergent biosynthetic pathways of oridonin and lushanrubescensin, paving the way for future metabolic engineering and synthetic biology studies.
Hao Yang, Jin-Lu Liu, Conglong Lian et al.· Frontiers in Plant Science· 0 citations
Xylitol is a highly functional sweetener with extensive applications. Sustainable biosynthesis from glucose is desirable yet metabolically challenging. Here, we engineered Yarrowia lipolytica as a cell factory by constructing a core biosynthetic route via combinatorial screening and multicopy integration of d-arabitol dehydrogenases (ArDH) and an NADPH-dependent xylitol dehydrogenase (XDH) in the robust chassis NBRC1631. To further drive the metabolic flux and alleviate bottlenecks, we employed a synergistic push-and-pull strategy: overexpressing glucose transporters (YH3 and YH4), while upregulating pentose phosphate pathway enzymes (ZWF1 and GND1) to enhance NADPH regeneration, matching the redox demand of the synthetic cascade. Following two-stage pH-controlled fed-batch fermentation in a 3 L bioreactor, the final engineered strain achieved a record-high xylitol titer of 39.0 g/L with a yield of 0.09 g/g glucose. This study establishes a productive platform for microbial de novo xylitol biosynthesis from glucose, offering a green and economically viable route for industrial production.
Bingbing Liu, Xi Yao, Jianping Lin et al.· Journal of Agricultural and...· 0 citations
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