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Due to the diverse structures and broad functions of terpenes, microbial biosynthesis of these compounds has been favored as a sustainable alternative to phytoextraction and chemosynthesis. Here, systematic metabolic engineering strategies were explored in the oleaginous yeast Yarrowia lipolytica for hyperproducing sesquiterpene germacrene A which serves as an important intermediate of numerous anticarcinogens. By identifying the most efficient germacrene A synthase to date, reconstructing the endogenous mevalonate pathway and extending the cytosolic acetyl-CoA pool by regulating lipid metabolism, the resulting strain overproduced 2.794 g L−1 germacrene A in shake flasks, which represented a 38-fold improvement over the initial strain. The engineered strain was subsequently capable of producing 39 g L−1 germacrene A at a yield of 0.181 g g−1 glucose during optimized bioreactor fermentation, with this being the highest sesquiterpene production level reported to date for Y. lipolytica. These results demonstrate that reprogramming the metabolism of the host cell by systematic metabolic engineering plays an essential role in diverting its inherent metabolic fluxes for sesquiterpene biosynthesis and these approaches can be extensively applied for synthesizing natural terpenes. [ABSTRACT FROM AUTHOR]

Terpenoids, known for their structural and functional diversity, are highly valued, especially in food, cosmetics, and cleaning products. Microbial biosynthesis has emerged as a sustainable and environmentally friendly approach for the production of terpenoids. However, the natural enzymes involved in the synthesis of terpenoids have problems such as low activity, poor specificity, and insufficient stability, which limit the biosynthesis efficiency. Enzyme engineering plays a pivotal role in the microbial synthesis of terpenoids. By modifying the structures and functions of key enzymes, researchers have significantly improved the catalytic activity, specificity, and stability of enzymes related to terpenoid synthesis, providing strong support for the sustainable production of terpenoids. This article reviews the strategies for the modification of key enzymes in microbial synthesis of terpenoids, including improving enzyme activity and stability, changing specificity, and promoting mass transfer through multi-enzyme collaboration. Additionally, this article looks forward to the challenges and development directions of enzyme engineering in the microbial synthesis of terpenoids.

Over the past 30 years, Yarrowia lipolytica, Kluyveromyces, Pichia, Candida, Hansenula and other non-conventional yeasts have attracted wide attention because of their desirable phenotypes, such as rapid growth, capability of utilizing multiple substrates, and stress tolerance. A variety of synthetic biology tools are being developed for exploitation of their unique phenotypes, making them potential cell factories for the production of recombinant proteins and renewable bio-based chemicals. This review summarizes the gene editing tools and the metabolic engineering strategies recently developed for non-conventional yeasts. Moreover, the challenges and future perspectives for developing non-conventional yeasts into efficient cell factories for the production of useful products through metabolic engineering are discussed.

Background: Cofactor engineering is involved in the modification of enzymes related to nicotinamide adenine dinucleotides (NADH and NAD + ) metabolism, which results in a significantly altered spectrum of metabolic products. Cofactor engineering plays an important role in metabolic engineering but is rarely reported in the sterols biotransformation process owing to its use of multi-catabolic enzymes, which promote multiple consecutive reactions. Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are important steroid medicine intermediates that are obtained via the nucleus oxidation and the side chain degradation of phytosterols by Mycobacterium. Given that the biotransformation from phytosterols to AD (D) is supposed to be a NAD + -dependent process, this work utilized cofactor engineering in Mycobacterium neoaurum and investigated the effect on cofactor and phytosterols metabolism., Results: Through the addition of the coenzyme precursor of nicotinic acid in the phytosterols fermentation system, the intracellular NAD + /NADH ratio and the AD (D) production of M. neoaurum TCCC 11978 (MNR M3) were higher than in the control. Moreover, the NADH: flavin oxidoreductase was identified and was supposed to exert a positive effect on cofactor regulation and phytosterols metabolism pathways via comparative proteomic profiling of MNR cultured with and without phytosterols. In addition, the NADH: flavin oxidoreductase and a water-forming NADH oxidase from Lactobacillus brevis, were successfully overexpressed and heterologously expressed in MNR M3 to improve the intracellular ratio of NAD + /NADH. After 96 h of cultivation, the expression of these two enzymes in MNR M3 resulted in the decrease in intracellular NADH level (by 51 and 67%, respectively) and the increase in NAD + /NADH ratio (by 113 and 192%, respectively). Phytosterols bioconversion revealed that the conversion ratio of engineered stains was ultimately improved by 58 and 147%, respectively. The highest AD (D) conversion ratio by MNR M3N2 was 94% in the conversion system with soybean oil as reaction media to promote the solubility of phytosterols., Conclusions: The ratio of NAD + /NADH is an important factor for the transformation of phytosterols. Expression of NADH: flavin oxidoreductase and water-forming NADH oxidase in MNR improved AD (D) production. Besides the manipulation of key enzyme activities, which included in phytosterols degradation pathways, maintenance the balance of redox also played an important role in promoting steroid biotransformation. The recombinant MNR strain may be useful in industrial production.