Background: The sesquiterpene germacrene D is a highly promising product due to its wide variety of insecticidal activities and ability to serve as a precursor for many other sesquiterpenes. Biosynthesis of high value compounds through genome mining for synthases and metabolic engineering of microbial factories, especially Saccharomyces cerevisiae, has been proven to be an effective strategy. However, there have been no studies on the de novo synthesis of germacrene D from carbon sources in microbes. Hence, the construction of the S. cerevisiae cell factory to achieve high production of germacrene D is highly desirable. Results: We identified five putative sesquiterpene synthases (AcTPS1 to AcTPS5) from Acremonium chrysogenum and the major product of AcTPS1 characterized by in vivo, in vitro reaction and NMR detection was revealed to be (–)-germacrene D. After systematically comparing twenty-one germacrene D synthases, AcTPS1 was found to generate the highest amount of (–)-germacrene D and was integrated into the terpene precursor-enhancing yeast strain, achieving 376.2 mg/L of (–)-germacrene D. Iterative engineering was performed to improve the production of (–)-germacrene D, including increasing the copy numbers of AcTPS1, tHMG1 and ERG20, and downregulating or knocking out other inhibitory factors (such as erg9, rox1, dpp1). Finally, the optimal strain LSc81 achieved 1.94 g/L (–)-germacrene D in shake-flask fermentation and 7.9 g/L (–)-germacrene D in a 5-L bioreactor, which is the highest reported (–)-germacrene D titer achieved to date. Conclusion: We successfully achieved high production of (–)-germacrene D in S. cerevisiae through terpene synthase mining and metabolic engineering, providing an impressive example of microbial overproduction of high-value compounds.

High-energy-density liquid fuels (HED fuels) are essential for volume-limited aerospace vehicles and could serve as energetic additives for conventional fuels. Terpene-derived HED biofuel is an important research field for green fuel synthesis. The direct extraction of terpenes from natural plants is environmentally unfriendly and costly. Designing efficient synthetic pathways in microorganisms to achieve high yields of terpenes shows great potential for the application of terpene-derived fuels. This review provides an overview of the current research progress of terpene-derived HED fuels, surveying terpene fuel properties and the current status of biosynthesis. Additionally, we systematically summarize the engineering strategies for biosynthesizing terpenes, including mining and engineering terpene synthases, optimizing metabolic pathways and cell-level optimization, such as the subcellular localization of terpene synthesis and adaptive evolution. This article will be helpful in providing insight into better developing terpene-derived HED fuels.

Cephalosporin C (CPC) produced by Acremonium chrysogenum is one of the most important drugs for treatment of bacterial infectious diseases. As the major stimulant, methionine is widely used in the industrial production of CPC. In this study, we found methionine stimulated CPC production through enhancing the accumulation of endogenous S-adenosylmethionine (SAM). To overcome the methionine dependent stimulation of CPC production, the methionine cycle of A. chrysogenum was reconstructed by metabolic engineering. Three engineered strains were obtained by overexpressing the SAM synthetase gene AcsamS and the cystathionine-γ-lyase gene mecB, and disrupting a SAM dependent methyltransferase gene Acppm1, respectively. Overexpression of AcsamS resulted in fourfold increase of CPC production which reached to 129.7 µg/mL. Disruption of Acppm1 also increased CPC production (up to 135.5 µg/mL) through enhancing the accumulation of intracellular SAM. Finally, an optimum recombinant strain (Acppm1DM-mecBOE) was constructed through overexpressing mecB in the Acppm1 disruption mutant. In this strain, CPC production reached to the maximum value (142.7 µg/mL) which was 5.5-fold of the wild-type level and its improvement was totally independent of methionine stimulation. In this study, we constructed a recombinant strain in which the improvement of CPC production was totally independent of methionine stimulation. This work provides an economic route for improving CPC production in A. chrysogenum through metabolic engineering.

γ-bisabolene is a monocyclic sesquiterpene with various biological activities; it has also been approved as a food additive. Additionally, the hydrogenated form of bisabolene is considered as a potential alternative to D2 diesel. Saccharomyces cerevisiae has the ability to produce a large amount of acetyl-CoA in both cytosol and peroxisomes, which serves as a precursor in terpene biosynthesis. In this study, AcTPS5 was identified as a new γ-bisabolene synthase. By expressing AcTPS5 and the mevalonate pathway in peroxisomes, γ-bisabolene titer was achieved at 125.0 mg/L. Deleting the peroxisome autophagy gene atg36 further improved γ-bisabolene production to 216.9 mg/L. The implementation of dual cytoplasmic–peroxisomal engineering further boosted γ-bisabolene production to 296.4 mg/L. Finally, through increasing the acetyl-CoA supply and down-regulating the expression of ERG9, γ-bisabolene production was achieved at 584.14 mg/L in shake-flask fermentation and 2.69 g/L in fed-batch fermentation, which is the highest reported production of γ-bisabolene to date. The strategy presented in this study provides an efficient approach for terpene production in S. cerevisiae.

Tricyclene, a tricyclic monoterpene naturally occurring in plant essential oils, holds potential for the development of medicinal and fuel applications. In this study, we successfully synthesized tricyclene in E. coli by introducing the heterologous mevalonate (MVA) pathway along with Abies grandis geranyl diphosphate synthase (GPPS) and Nicotiana sylvestris tricyclene synthase (TS) XP_009791411. Initially, the shake-flask fermentation at 30 ^◦C yielded a tricyclene titer of 0.060 mg/L. By increasing the copy number of the TS-coding gene, we achieved a titer of 0.103 mg/L. To further enhance tricyclene production, optimal truncation in the N-terminal region of TS XP_009791411 resulted in an impressive highest titer of 47.671 mg/L, approximately a 794.5-fold improvement compared to its wild-type counterpart. To the best of our knowledge, this is the highest titer of the heterologous synthesis of tricyclene in E. coli. The SDS-PAGE analysis revealed that lowering induction temperature and truncating the random coil N-terminal region effectively improved TS solubility, which was closely associated with tricyclene production levels. Furthermore, by truncating other TSs, the titers of tricyclene were improved to different degrees.

Vibrio natriegens (V. natriegens) is an emerging synthetic biology chassis characterized by rapid growth, and its potential for the synthesis of sesquiterpenes (such as pentalenene) has not been developed. In this study, heterologous pentalenene biosynthesis was successfully established in V. natriegens via metabolic engineering. The optimization of gene dosage and culture conditions led to an increase in pentalenene yield from 0.75 mg/L to 39.4 mg/L, representing the highest titer reported in V. natriegens to date, though still markedly lower than yields achieved in conventional microbial hosts. Transcriptome analysis demonstrated that the exogenous mevalonate (MVA) pathway effectively activated terpenoid precursor synthesis, as evidenced by the up-regulation of key pathway genes. However, the endogenous methylerythritol 4-phosphate (MEP) pathway remained inactive, and genes involved in oxidative phosphorylation, the pentose phosphate pathway, and thiamine biosynthesis were down-regulated, leading to limited availability of ATP, NADPH, and acetyl-CoA. Competition for cofactors, particularly NADPH, further constrained precursor supply and pathway efficiency. This study confirmed the potential of V. natriegens as a pentalenene production platform and revealed its metabolic bottleneck, providing a theoretical basis for subsequent engineering optimization.