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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.

Androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD) are valuable steroid pharmaceutical intermediates obtained by soybean phytosterol biotransformation by Mycobacterium Cyclodextrins (CDs) are generally believed to be carriers for phytosterol delivery and can improve the production of AD and ADD due to their effects on steroid solubilization and alteration in cell wall permeability for steroids. To better understand the mechanisms of CD promotion, we performed proteomic quantification of the effects of hydroxypropyl-β-CD (HP-β-CD) on phytosterol metabolism in Mycobacterium neoaurum TCCC 11978 C2. Perturbations are observed in steroid catabolism and glucose metabolism by adding HP-β-CD in a phytosterol bioconversion system. AD and ADD, as metabolic products of phytosterol, are toxic to cells, with inhibited cell growth and biocatalytic activity. Treatment of mycobacteria with HP-β-CD relieves the inhibitory effect of AD(D) on the electron transfer chain and cell growth. These results demonstrate the positive relationship between HP-β-CD and phytosterol metabolism and give insight into the complex functions of CDs as mediators of the regulation of sterol metabolism. IMPORTANCE Phytosterols from soybean are low-cost by-products of soybean oil production and, owing to their good bioavailability in mycobacteria, are preferred as the substrates for steroid drug production via biotransformation by Mycobacterium However, the low level of production of steroid hormone drugs due to the low aqueous solubility (below 0.1 mmol/liter) of phytosterols limits the commercial use of sterol-transformed strains. To improve the bioconversion of steroids, cyclodextrins (CDs) are generally used as an effective carrier for the delivery of hydrophobic steroids to the bacterium. CDs improve the biotransformation of steroids due to their effects on steroid solubilization and alterations in cell wall permeability for steroids. However, studies have rarely reported the effects of CDs on cell metabolic pathways related to sterols. In this study, the effects of hydroxypropyl-β-CD (HP-β-CD) on the expression of enzymes related to steroid catabolic pathways in Mycobacterium neoaurum were systematically investigated. These findings will improve our understanding of the complex functions of CDs in the regulation of sterol metabolism and guide the application of CDs to sterol production., (Copyright © 2020 American Society for Microbiology.)

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.