SPOCK1 silencing decreases 5-FU resistance through PRRX1 in colorectal cancer.

SPOCK1 is an extracellular proteoglycan and involved in tumor growth and metastasis in various cancers. 5-fluorouracil (5-FU) is commonly used for the treatment of colorectal cancer (CRC) in patients who receive concurrent chemoradiotherapy. However, the relationship between development of resistance to 5-FU and SPOCK1 remain unclear. In this study, we established two 5-fluorouracil (5-FU)-resistant CRC cell lines, HCT116/FU and LOVO/FU, and found that SPOCK1 is upregulated in 5-FU-resistance CRC cells compared with its parental cell line. knockdown of SPOCK1 in 5-FU-resistant CRC cells increases their sensitivity to 5-FU. In contrast, transient transfection of SPOCK1 enhanced HCT116 and LOVO cell resistance to 5-FU and reduced cell apoptosis. Mechanistically, SPOCK1 promoted 5-FU resistance by regulating PRRX1 expression and the downstream apoptosis signaling pathway. Taken together, our results revealed for the first time that SPOCK1 plays a crucial role in the resistance of CRC cells to 5-FU and indicated that targeting SPOCK1 may be a promising therapeutic strategy to overcome 5-FU resistance in CRC.

Elevated ß-Carotene Production Using Codon-Adapted CarRA&B and Metabolic Balance in Engineered Yarrowia lipolytica.

ß-carotene is a precursor of vitamin A and has multiple physiological functions. Producing ß-carotene by microbial fermentation has attracted much attention to consumers' preference for natural products. This study focused on improving ß-carotene production by constructing codon-adapted genes and minimizing intermediate accumulation. The codon-adapted CarRA and CarB genes from the industrial strain of Blakeslea trispora were integrated into the genome of the Yarrowia lipolytica to construct YL-C0, the baseline strain for producing ß-carotene. Thereafter, the ß-carotene biosynthetic pathway's metabolic balance was accurately regulated to reduce the intermediates' accumulation. Notably, the ß-carotene content increased by 21 times to reach 12.5 dry cell weight (DCW) mg/g when minimizing HMG-CoA and FPP accumulation. Further, we improved the expression levels of the CarRA and CarB genes to minimize the accumulation of phytoene and lycopene. Total production of ß-carotene of 1.7 g/L and 21.6 mg/g DCW was achieved. These results reveal that the rate-limiting enzymes CarRA and CarB of B. trispora exhibited higher catalytic activity than the same enzymes from other microorganisms. Promoting metabolic balance by minimizing the accumulation of intermediates is a very effective strategy for increasing ß-carotene. The ß-carotene-producing strain constructed in this study has established the foundation for its potential use in industrial production. These successful engineering strategies also provide a foundation for large-scale production of other terpenoids.

Promoting the Synthesis of Precursor Substances by Overexpressing Hexokinase (Hxk) and Hydroxymethylglutaryl-CoA Synthase (Erg13) to Elevate ß-Carotene Production in Engineered Yarrowia lipolytica.

As a valuable carotenoid, ß-carotene is commercially used in food, cosmetics, animal feeds, and other industries. Metabolic engineering of microorganisms has been widely explored to improve the production of ß-carotene. Compared with the traditional genetic modifications mainly focused on the pathways of mevalonate (MVA) and ß-carotene biosynthesis, this study aims to increase the ß-carotene production through promoting the synthesis of precursor substances by overexpressing hexokinase and hydroxymethylglutaryl-CoA synthase in an engineered Yarrowia lipolytica. In this study, we investigated the effect of the unique hexokinase gene (Hxk) overexpression on ß-carotene accumulation and glucose consumption. The Hxk gene was introduced into a ß-carotene producing strain Y.L-1 to generate strain Y.L-2, and this increased the ß-carotene content by 98%. Overexpression of the Hxk gene led to increasing in hexokinase activity (329% higher), glucose-6-phosphate content (92% higher), and improvement of the transcriptional level of Hxk (315% higher) compared to the control Y.L-1 strain. Moreover, Hxk overexpression accelerated the utilization rate of glucose. The gene erg13 encoding hydroxymethylglutaryl-CoA synthase was also overexpressed to increase the precursor supply for ß-carotene biosynthesis. Recombinant Y.L-4 harboring two copies of erg13 produced 8.41 mg/g dry cell weight (DCW) of ß-carotene, which was 259% higher than Y.L-1. The ß-carotene content of 9.56 mg/g DCW was achieved in strain Y.L-6 by integrating erg13 into the chromosome and Hxk overexpression. The 3-Hydroxy-3-Methylglutaryl-CoA content in the cells was increased by overexpressing two copies of the erg13 gene. Finally, the titer of ß-carotene reached 2.4 g/L using a 50 L bioreactor by the engineered strain, and the fermentation cycle was shortened from 144 to 120 h. Overall, overexpression of Hxk and erg13 could improve ß-carotene production and successfully overcoming the bottleneck of precursor generation to support a more efficient pathway for the production of the target product. Our results revealed a novel strategy to engineer the pathway of ß-carotene synthesis.