代谢改造酿酒酵母合成萜类化合物的研究进展

萜类化合物是一类种类繁多、功能多样的化合物,部分具有抗癌、增强免疫力等作用,具有良好的生物活性,在食品、保健品以及医疗等领域应用广泛。近年来,随着对萜类化合物生物合成途径研究的深入,研究人员采用代谢工程手段构建了多种萜类产物的高产酿酒酵母工程菌株,部分已经达到或者接近工业化生产水平。因此,采用合成生物学相关技术手段合成萜类化合物,有望取代化学合成或者传统的提取模式,成为天然萜类产物的新型生产方法。文中以常见的几种萜类产物为例,介绍并探讨萜类产物的生物合成策略以及合成生物学方面的研究进展。

A novel Trmt5-deficient zebrafish model with spontaneous inflammatory bowel disease-like phenotype

Dear Editor,Inflammatory bowel disease(IBD),a complex syndrome characterized by chronic inflammation of the gastrointestinal tract,is considered a global health problem,especially prevalent in western developed countries and with accelerating incidence in the developing world over the last decade.1 To date,the primary etiology of IBD remains elusive.

Combinatorial metabolic engineering of Saccharomyces cerevisiae for improved production of 7-dehydrocholesterol

7-Dehydrocholesterol(7-DHC),a key pharmaceutical intermediate in the production of vitamin D3,has a wide range of applications.To explore fermentative synthesis of 7-DHC,a 7-DHC-producing Saccharomyces cerevisiae strain was constructed by blocking the competitive pathway,eliminating rate-limiting steps,altering global reg-ulation,and pathway compartmentalization.After blocking the competitive pathway by disrupting ERG5 and ERG6 and introducing DHCR24 from Gallus gallus,S.cerevisiae produced 139.72 mg/L(17.04 mg/g dry cell weight,hereafter abbreviated as DCW)7-DHC.Subsequent alteration of global regulation by deleting ROX1 and overexpressing UPC2-1 increased 7-DHC production to 217.68 mg/L(37.56 mg/g DCW).To remove the accu-mulated squalene,the post-squalene pathway was strengthened by co-overexpression of PGAL1-driven ERG11 and PGAL10-driven ERG1,which improved 7-DHC titer and yield to 281.73 mg/L and 46.78 mg/g DCW,respectively,and reduced squalene content by 90.12%.We surmised that the sterol precursors in the plasma membrane and peroxisomes may not be accessible to the pathway enzymes,thus we re-localized DHCR24p and Erg2p-GGGGS-Erg3p to the plasma membrane and peroxisomes,boosting 7-DHC production to 357.53 mg/L(63.12 mg/g DCW).Iron supplementation further increased 7-DHC production to 370.68 mg/L in shake flasks and 1.56 g/L in fed-batch fermentation.This study demonstrates the power of global regulation and subcellular relocalization of key enzymes to improve 7-DHC synthesis in yeast.

Assembly of biosynthetic pathways in Saccharomyces cerevisiae using a marker recyclable integrative plasmid toolbox

A robust and versatile tool for multigene pathway assembly is a key to the biosynthesis of high- value chemicals. Here we report the rapid construction of biosynthetic pathways in Saccharomyces cerevisiae using a marker recyclable integrative toolbox （pUMRI） devel- oped in our research group, which has features of ready-to- use, convenient marker recycling, arbitrary element replacement, shuttle plasmid, auxotrophic marker inde- pendence, GAL regulation, and decentralized assembly. Functional isoprenoid biosynthesis pathways containing 4-11 genes with lengths ranging from -10 to -22 kb were assembled using this toolbox within 1-5 rounds of reiterative recombination. In combination with GAL- regulated metabolic engineering, high production of isoprenoids （e.g., 16.3 mg.g-1 dcw carotenoids）was achieved. These results demonstrate the wide range of application and the efficiency of the pUMRI toolbox in multigene pathway construction of S. cerevisiae.

Development of a dual temperature control system for isoprene biosynthesis in Saccharomyces cerevisiae

Conflict between cell growth and product accumulation is frequently encountered in the biosynthesis of secondary metabolites. To address the growth-production conflict in yeast strains harboring the isoprene synthetic pathway in the mitochondria, the dynamic control of isoprene biosynthesis was explored. A dual temperature regulation system was developed through engineering and expression regulation of the transcriptional activator Gal4p. A cold-sensitive mutant, Gal4ep19, was created by directed evolution of Gal4p based on an internally developed growth-based high-throughput screening method and expressed under the heat-shock promoter PSSA4 to control the expression of PGAL-driven pathway genes in the mitochondria. Compared to the control strain with constitutively expressed wild-type Gal4p, the dual temperature regulation strategy led to 34.5% and 72% improvements in cell growth and isoprene production, respectively. This study reports the creation of the first cold-sensitive variants of Gal4p by directed evolution and provides a dual temperature control system for yeast engineering that may also be conducive to the biosynthesis of other high-value natural products.

Redesign of a short-chain dehydrogenase/reductase for asymmetric synthesis of ethyl (R)-2-hydroxy-4-phenylbutanoate based on per-residue free energy decomposition and sequence conservatism analysis

As an important building block for the synthesis of angiotensin-converting enzyme inhibitors,ethyl(R)-2-hy-droxyl-4-phenylbutanoate[(R)-HPBE]has attracted increasing attention.The key to industrial biosynthesis of(R)-HPBE is a biocatalyst that efficiently reduces ethyl 2-oxo-4-phenylbutanoate(OPBE)with high R-enantiose-lectivity.This paper proposed a strategy for identifying key residues involved in enantioselectivity control based on per-residue free energy decomposition and sequence conservatism analysis.Using this strategy,4 noncon-servative sites with high energy contribution to binding of OPBE were chosen as engineering targets,generating variant Mu27 with 99%conversion and 98%(R)ee value at substrate loading of up to 500 mmol/L.MD simu-lations suggested the higher stability and formation probability of Mu27-OPBEproR prereaction state as key rea-sons for the excellent R-enantioselectivity of Mu27 towards OPBE.The success in this study provides a viable approach for rational design of alcohol dehydrogenases with high enantioselectivity towards unnatural substrates.