Jinping Lin

Background: Changes in the tumor microenvironment (TME) are closely involved in hepatocellular carcinoma (HCC) development, and lipid metabolic disorders may take part in this process. STARD3NL is known to be related to cholesterol trafficking and lipid homeostasis. However, little is known about whether STARD3NL is linked to immune features of the HCC microenvironment. Methods: We first explored STARD3NL expression and its prognostic relevance in HCC using The Cancer Genome Atlas (TCGA). To verify the bioinformatic findings, HCC tissue microarrays (TMAs) were analyzed by immunohistochemistry (IHC) for STARD3NL localization and survival evaluation. Multiplex immunohistochemistry (mIHC), combined with digital image analysis, was further used to characterize immune cell infiltration. Clinicopathological differences between tumors with high and low STARD3NL expression were also examined. In addition, spatial distance analysis was performed to assess how immune cells were arranged in STARD3NL-high regions. Results: TCGA-based analysis and mIHC validation both showed higher STARD3NL expression in HCC tissues. Patients with increased STARD3NL expression had poorer survival (p = 0.029), and multivariable analysis supported STARD3NL as an independent risk factor. STARD3NL-high tumors showed increased proportions of CD68+ macrophages and FOXP3+ T cells (p < 0.005). Spatial analysis further indicated that Tregs were more frequently located near macrophages (p = 0.001), with a shorter mean macrophage-Treg distance (p = 0.027). This macrophage-Treg proximity was associated with worse prognosis (p = 0.030). Conclusion: High STARD3NL expression is significantly associated with immune-suppressive features and unfavorable outcomes in HCC. These findings indicate that STARD3NL may have value as a prognostic biomarker associated with an immunosuppressive tumor microenvironment.

(R)-Citronellal is a key chiral precursor of high-value chemicals, such as the best-selling flavor compound (-)-menthol; however, the conventional synthesis suffers from low yield and unsatisfactory enantioselectivity. In this study, we developed a highly atom-efficient hydrogen-borrowing cascade for the synthesis of (R)-citronellal from geraniol using alcohol dehydrogenase from Escherichia coli K12 (AdhP) and ene-reductase from Saccharomyces cerevisiae YJM1341 (OYE2p). The key rate-limiting enzyme, AdhP, was subjected to structure-guided semirational engineering, and the triple mutant AdhP^{260T/284A/268P} (M3) was obtained that demonstrated a 1.28-fold improvement in catalytic efficiency (k_cat/K_m) toward geraniol. After optimization of the reaction conditions, the hydrogen-borrowing cascade system achieved the conversion of 23.14 g/L geraniol into (R)-citronellal at a conversion rate of 98.23% with 96.7% ee. This work represents an alternative approach for the biosynthesis of (R)-citronellal without sacrificing a cosubstrate or additional enzymes.

Asymmetric reduction of electronically activated alkenes by ene reductases (ERs) is an attractive approach for the production of enantiopure chiral products. Herein, a novel FMN-binding ene reductase (PaER) from Pichia angusta was heterologously expressed in Escherichia coli BL21(DE3), and the recombinant enzyme was characterized for its biocatalytic properties. PaER displayed optimal activity at 40 °C and pH 7.5, respectively. The purified enzyme was quite stable below 30 °C over a broad pH range of 5.0–10.0. PaER was identified to have a good ability to reduce the CC bond of various α,β-unsaturated compounds in the presence of NADPH. In addition, PaER exhibited a high reduction rate (k_cat = 3.57 s⁻¹) and an excellent stereoselectivity (>99%) for ketoisophorone. Engineered E. coli cells harboring PaER and glucose dehydrogenase (for cofactor regeneration) were employed as biocatalysts for the asymmetric reduction of ketoisophorone. As a result, up to 1000 mM ketoisophorone was completely and enantioselectively converted to (R)-levodione with a >99% ee value in a space–time yield of 460.7 g L⁻¹ d⁻¹. This study provides a great potential biocatalyst for practical synthesis of (R)-levodione.

Background: d-Xylonic acid is a versatile platform chemical with broad potential applications as a water reducer and disperser for cement and as a precursor for 1,4-butanediol and 1,2,4-tributantriol. Microbial production of d-xylonic acid with bacteria such as Gluconobacter oxydans from inexpensive lignocellulosic feedstock is generally regarded as one of the most promising and cost-effective methods for industrial production. However, high substrate concentrations and hydrolysate inhibitors reduce xylonic acid productivity. Results: The d-xylonic acid productivity of G. oxydans DSM2003 was improved by overexpressing the mGDH gene, which encodes membrane-bound glucose dehydrogenase. Using the mutated plasmids based on pBBR1MCS-5 in our previous work, the recombinant strain G. oxydans/pBBR-R3510-mGDH was obtained with a significant improvement in d-xylonic acid production and a strengthened tolerance to hydrolysate inhibitors. The fed-batch biotransformation of d-xylose by this recombinant strain reached a high titer (588.7 g/L), yield (99.4%), and volumetric productivity (8.66 g/L/h). Moreover, up to 246.4 g/L d-xylonic acid was produced directly from corn stover hydrolysate without detoxification at a yield of 98.9% and volumetric productivity of 11.2 g/L/h. In addition, G. oxydans/pBBR-R3510-mGDH exhibited a strong tolerance to typical inhibitors, i.e., formic acid, furfural, and 5-hydroxymethylfurfural. Conclusion: Through overexpressing mgdh in G. oxydans, we obtained the recombinant strain G. oxydans/pBBR-R3510-mGDH, and it was capable of efficiently producing xylonic acid from corn stover hydrolysate under high inhibitor concentrations. The high d-xylonic acid productivity of G. oxydans/pBBR-R3510-mGDH made it an attractive choice for biotechnological production.

Summary: Bioproduction of optical pure ( R )‐citronellal from ( E /Z)‐citral at high substrate loading remains challenging. Low catalytic efficiency of ( R )‐stereoselective ene reductases towards crude citral mixture is one of the major bottlenecks. Herein, a structure‐based engineering strategy was adopted to enhance the catalytic efficiency and stereoselectivity of an ene reductase (OYE2p) from Saccharomyces cerevisiae YJM1341 towards ( E / Z )‐citral. On basis of homologous modelling, molecular docking analysis and alanine scanning at the binding pocket of OYE2p, a mutant Y84A was obtained with simultaneous increase in catalytic efficiency and stereoselectivity. Furthermore, site‐saturation mutagenesis of Y84 yielded seven mutants with improved activity and stereoselectivity in the ( E / Z )‐citral reduction. Among them, the variant Y84V exhibited an 18.3% and 71.3% rise in catalytic efficiency ( k _cat / K _m ) for ( Z )‐citral and ( E )‐citral respectively. Meanwhile, the stereoselectivity of Y84V was improved from 89.2% to 98.0% in the reduction in ( E / Z )‐citral. The docking analysis and molecular dynamics simulation of OYE2p and its variants revealed that the substitution Y84V enabled ( E )‐citral and ( Z )‐citral to bind with a smaller distance to the key hydrogen donors at a modified ( R )‐selective binding mode. The variant Y84V was then co‐expressed with glucose dehydrogenase from Bacillus megaterium in E . coli D4, in which competing prim ‐alcohol dehydrogenase genes were deleted to prevent the undesired reduction in the aldehyde moiety of citral and citronellal. Employing this biocatalyst, 106 g l ⁻¹ ( E / Z )‐citral was completely converted into ( R )‐citronellal with 95.4% ee value and a high space‐time yield of 121.6 g l ⁻¹ day ⁻¹ . The work highlights the synthetic potential of Y84V, which enabled the highest productivity of ( R )‐citronellal from ( E / Z )‐citral in high enantiopurity so far.

Objectives 3,4-Dihydroxybutyric acid (3,4-DHBA) is a multifunctional C4 platform compound widely used for the synthesis of various materials, including pharmaceuticals. Although, a biosynthetic pathway for 3,4-DHBA production has been developed, its low yield still precludes large-scale use. Here, a heterologous four-step biosynthetic pathway was established in recombinant Escherichia coli (E. coli) using a combinatorial strategy. Results Several aldehyde dehydrogenases (ALDHs) were screened, using in vitro enzyme assays, to identify suitable catalysts for the dehydrogenation of 3,4-dihydroxybutanal (3,4-DHB) to 3,4-DHBA. A pathway containing glucose dehydrogenase (BsGDH) from Bacillus subtilis, d-xylonate dehydratase (YagF) from E. coli, benzoylformate decarboxylase (PpMdlC) from Pseudomonas putida and ALDH was introduced into E. coli, generating 3.04 g/L 3,4-DHBA from d-xylose (0.190 g 3,4-DHBA/g d-xylose). Disruption of competing pathways by deleting xylA, ghrA, ghrB and adhP contributed to an 87% increase in 3,4-DHBA accumulation. Expression of a fusion construct containing PpMdlC and YagF enhanced the 3,4-DHBA titer, producing the highest titer and yield reported thus far (7.71 g/L; 0.482 g 3,4-DHBA/g d-xylose). Conclusions These results showed that deleting genes from competing pathways and constructing fusion proteins significantly improved the titer and yield of 3,4-DHBA in engineered E. coli.

Background: D-xylonic acid is a versatile platform chemical with wide potential applications as the water reducer and disperser for cement and the precursor for 1,4-butanediol and 1,2,4-tributantriol. Microbial production of D-xylonic acid with bacteria like Gluconobacter oxydans from cheap lignocellulosic feedstock is generally regarded as one of the most promising and cost-effective methods to industrial production, but xylonic acid productivity is reduced by high substrate inhibition and hydrolysate inhibitors. Results: D-xylonic acid productivity of G. oxydans DSM2003 was improved by overexpressing the mGDH gene, which encoded the membrane-bound glucose dehydrogenase. Using the mutated plasmids based on pBBR1MCS-5 in our previous work, the recombinant strain G. oxydans/pBBR-R3510-mGDH with the significant improvement in D-xylonic acid production and the strengthened tolerance to hydrolysate inhibitors was obtained. The fed-batch biotransformation of D-xylose by this recombinant strain reached the record high titer (588.7 g/L), yield (99.4%), and space-time yield (8.72 g/L/h). Moreover, up to 246.4 g/L D-xylonic acid was produced directly from corn stover hydroxylates without detoxification at a yield of 98.9% and a space-time yield of 11.2 g/L/h. In addition, G. oxydans/pBBR-R3510-mGDH performed strong tolerance to typical inhibitors, i.e., formic acid, furfural, and 5-hydroxymethylfurfural. Conclusion: Through overexpression of mgdh in G. oxydans, we obtained a recombinant strain G. oxydans/pBBR-R3510-mGDH. It was capable to efficiently produce xylonic acid from the corn stover hydrolysate under high concentrations of inhibitors. The high D-xylonic acid productivity made G. oxydans/pBBR-R3510-mGDH an attractive choice for biotechnical production.