Wenhai Xiao

β-Myrcene is a key acyclic monoterpene that has been extensively applied in the agricultural pesticide, fragrance, and flavor industries; it also serves as a critical monoterpene precursor of high-value compounds. Our lab previously successfully achieved β-myrcene biosynthesis in the peroxisomes of Saccharomyces cerevisiae. However, the titer remained limited by the supply and utilization of precursors in peroxisomes. In this study, an efficient and stable β-myrcene biosynthetic pathway was constructed through the multicopy integration of the fusion protein MS-ERG20^ww (β-myrcene synthase) with peroxisome localization into Ty1 loci. This approach improved the supply and utilization of geranyl pyrophosphate (GPP) precursors, resulting in a 2-fold increase in the β-myrcene titer to 82.53 ± 3.78 mg/L. A new mevalonate (MVA) pathway was subsequently constructed for β-myrcene production by localizing truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) to peroxisomes, resulting in a 1.2-fold increase in the titer. In addition, KAR2 (ER chaperones) and GSH2 (reduced glutathione synthase) overexpression in chassis increased the β-myrcene titer to 176.40 ± 17.56 mg/L. Finally, 15-L fed-batch fermentation optimization increased the β-myrcene titer to 4.7 g/L, which represents the highest level yet reported. This work provides a comprehensive framework for engineering yeast cell factories, highlighting the ability of integrated metabolic engineering to enhance high-value monoterpene production.

• The NPP orthogonal pathway was introduced, and the well-matched key enzyme of SsCarTS was selected and truncated to increase the yield of 3-carene. • The appropriate subcellular localization into endoplasmic reticulum for the truncated SsCarT improved the production efficiency of 3-carene. • The molecular chaperone of PDI1 was coexpressed with the SsCarTS to further increase the yield of 3-carene. • Obtained a 3-carene yield of 506.7 mg/L in a 5-L fed-batch culture system, which is the highest titre reported in microbes.

Utilizing synthetic biology techniques to construct recombinant engineered cells for the synthesis of paclitaxel and its key precursors has emerged as an effective method to address the supply–demand imbalance and protect rare plant resources. Taxadiene, a critical precursor of paclitaxel, exhibits limited yield in eukaryotic systems, constraining its biosynthetic potential. Previous research has demonstrated that glycosylation modifications in Saccharomyces cerevisiae substantially impact the regulation of exogenous protein expression. In this study, we found that knocking out endogenous protein glycosylation genes in the chassis significantly improved the expression of heterologous proteins, especially the key taxadiene synthase (TS), and thereby increased the yield of taxadiene. In particular, we identified that the deletion of the glycosyltransferase gene mnn11 increased taxadiene production levels by 65.2 %. The subsequent multi-copy integration of the key enzyme taxadiene synthase further elevated taxadiene production levels in shake flasks by more than 3-fold, reaching 113.5 mg/L. Moreover, the enhancement of geranylgeranyl diphosphate synthesis-related expression modules resulted in a 2.69-fold increase in taxadiene yield, to 420.4 mg/L. Following the optimization of fed-batch fermentation conditions, taxadiene production levels of up to 1.26 g/L were achieved, representing a 63-fold improvement over that obtained with the initial strain. Our findings provide valuable insights into enhancing heterologous taxadiene production efficiency by blocking endogenous protein glycosylation modifications in S. cerevisiae, establishing a critical precedent for improving compatibility between natural product synthesis and microbial cell factories.

Progesterone is a widely used therapeutic hormone and a common precursor for the synthesis of pharmaceutical steroids in both mammals and plants. It has been successfully produced in heterologous microorganisms, but the market demand has not been met. The low progesterone yield, possibly due to product stress, may put pressure on the growth of strains and limit product synthesis efficiency. In this study, key pathways and genes that cause changes in amino acid and lipid metabolism and protein transport were identified through omics analysis. The expression of transporters leads to increased progesterone production and alleviated growth inhibition. Two related genes (gene IDs: 2912325 and 2908366) encoding the transporters glpF and SNQ2 improved production by 29.2 % and 51.7 %, respectively. Isoenzymes of native and exogenous transporters were screened and overexpressed. YCF1 from Saccharomyces cerevisiae exhibited the greatest benefit, increasing progesterone synthesis by 69.6 %. Our findings help reveal the impact of product stress on cellular metabolism and processes, providing research directions and literature support for the synthesis of other products.

Parthenolide is confirmed to be an important component of the anticancer drug—ACT001. However, parthenolide biosynthesis in Saccharomyces cerevisiae (S. cerevisiae) was greatly hindered by the low conversion rate of its precursor, costunolide. In this study, the Position Specific Scoring Matrix (PSSM) was used to analyze the sequence evolutionary information of parthenolide synthase from Tanacetum parthenium (TpPTS), and a series of mutants were designed and validated. Notably, when the mutant of TpPTS—Y22G was introduced in S. cerevisiae, the parthenolide titer increased by 110 % compared to that of the TpPTS wild-type. Considering TpPTS as an endoplasmic reticulum-localized cytochrome P450 and the importance of heme supply, endoplasmic-associated molecular chaperone HRD1 (hydroxymethyl glutaryl-coenzyme A reductase degradation protein 1) and heme biosynthesis gene HEM2 (aminolevulinate dehydratase) were overexpressed in S. cerevisiae to improve TpPTS expression and catalytic activity. As a result, a titer of 27.08 mg/L parthenolide was achieved in a shake flask, which was further increased by 209 %. Additionally, the conversion rate of costunolide to parthenolide increased from 20.4 % to 51.8 % compared to the initial strain yYTQ001. Eventually, a parthenolide titer of 99.71 mg/L was achieved in a 5-L bioreactor. Our research provides effective strategies and valuable references for engineering rate-limiting cytochrome P450 enzymes to improve sesquiterpenes production in S. cerevisiae.

Citronellol is a typical acyclic monoterpene alcohol widely used for fragrance and seasoning in the food industry and as a fungicide of plant origin in agriculture. Microbial cell factories offer a promising green and sustainable approach to efficient citronellol production. Inadequate supply of precursor geranyl pyrophosphate and cofactor NADPH, and monoterpene cytotoxicity are considered major bottlenecks in citronellol production. Additional copies of the mevalonate pathway (ERG10, ERG13, ERG12, ERG19) and peroxisome-localized (ERG8, ERG20^ww, tCrGES, CrIS) genes were integrated for citronellol biosynthesis based on our former citronellol overproduction strain, resulting in a 1.5-fold increase in citronellol production. Moreover, overexpression of nonoxidative pathway genes (TAL1 and TKL1) of the pentose phosphate pathway promotes the NADPH supply, resulting in a 16% increase in citronellol yield. Screening of endogenous transporter proteins and integration of PDR1 increased citronellol production to 3.38 g/L. Ultimately, 10.556 g/L citronellol was attained in Saccharomyces cerevisiae via 100 L of fed-batch fermentation, which is the highest titer in yeast so far. In summary, the titer of citronellol was systematically increased through modification of chassis cells, and the findings provide guidance for the biosynthesis of other monoterpenes.

Enzymatic halogenation reactions are gaining attention because of their mild reaction conditions. S-Adenosylmethionine (SAM), a critical cofactor in biological systems, is utilized by SAM-dependent halogenases to produce 5'-halogenated adenosines for antibiotic biosynthesis.^1-3 Both SAM-dependent halogenases and hydroxide adenosyltransferase (HATase) belong to the domain of unknown function 62 (DUF-62) family, but HATase uses water as a substrate to produce adenosine.^3-5 Recently, we transformed a HATase into a halogenase by protein engineering. However, whether this protein could be further tuned for regioselective control of SAM halogenation remains unclear. To our knowledge, regioselective control of SAM halogenation has never been achieved with any SAM-dependent halogenases so far. Here, we identified key residues influencing regioselective cleavage of SAM with a HATase from Thermotoga maritima MSB8. Multiple rounds of directed evolution, aided by cell surface display in E. coli, generated HATase mutants that convert SAM to CH₃I and S-adenosylhomocysteine (SAH). Computational studies revealed that mutations altered the halide orientation, leading to changes in regioselectivity for SAM cleavage. Notably, these identified mutation sites conferred regioselective halogenation capability to other HATases. Our study unveils the potential of the DUF-62 superfamily for regioselective cleavage of SAM and expands the halide methyltransferase repertoire through engineered HATases.

Yarrowia lipolytica, a safe yeast, efficiently metabolizes lipids for the production of food additives and agricultural products. Boosting its growth and lipid utilization capabilities is crucial to enhancing the overall efficiency Y. lipolytica. Herein, an integrated strategy was implemented to enhance lipid uptake, accumulation and metabolism and systematically promote the growth and lipid utilization of the commonly used Y. lipolytica Po1f strain. The engineered strain had a specific growth rate of 0.32 h⁻¹ and a lipid content of 67.66 % (g/g DCW), which were 54 % and 26 % greater than those of the original strain. β-Carotene was used to verify the production of lipophilic natural compounds, and the highest yield was obtained 48 h earlier using the engineered strain compared to the original strain when consuming same carbon source. These findings show promise in using engineered Y. lipolytica for rapid growth and improved lipid utilization to boost efficiency of lipophilic product production.

Zeaxanthin, as an important natural pigment and nutrient, is applied in food and cosmetics industries. Zeaxanthin is converted from β-carotenoid by the β-carotene hydroxylase (CrtZ). Limited by the thermostability of CrtZ, zeaxanthin and its derivatives tend to be fermented in Saccharomyces cerevisiae at low temperatures. In this study, CrtZ mutants with improved thermostability and catalytic efficiency were designed via the position-specific scoring matrix (PSSM), and the beneficial mutants were verified in vitro. Moreover, molecular dynamics simulations revealed the thermostability mechanisms of the mutants. Subsequently, CrtZ mutant M83L was introduced into the S. cerevisiae chassis, and the zeaxanthin titer increased by 121.2% compared with that of the CrtZ, reaching 156.8 mg/L in shake flask fermentation at 30 °C. Furthermore, the oxidoreductase RFNR/FD3 system was introduced to match the overexpressed M83L, and the zeaxanthin titer further increased by 138.9% (reaching 374.6 mg/L). Ultimately, 814.6 mg/L zeaxanthin was produced in S. cerevisiae in 5.0 L fed-batch fermentation at 30 °C, which is the highest reported titer in S. cerevisiae. This study not only provides a useful strategy to increase the thermostability of key enzymes but also describes an efficient platform for the biosynthesis of zeaxanthin and its high-value derivatives.