Enhancing fluxes through the mevalonate pathway in Saccharomyces cerevisiae by engineering the HMGR and β‐alanine metabolism

Abstract

Mevalonate (MVA) pathway is the core for terpene and sterol biosynthesis, whose metabolic flux influences the synthesis efficiency of such compounds. Saccharomyces cerevisiae is an attractive chassis for the native active MVA pathway. Here, the truncated form of Enterococcus faecalis MvaE with only 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity was found to be the most effective enzyme for MVA pathway flux using squalene as the metabolic marker, resulting in 431-fold and 9-fold increases of squalene content in haploid and industrial yeast strains respectively. Furthermore, a positive correlation between MVA metabolic flux and b-alanine metabolic activity was found based on a metabolomic analysis. An industrial strain SQ3-4 with high MVA metabolic flux was constructed by combined engineering HMGR activity, NADPH regeneration, cytosolic acetyl-CoA supply and b-alanine metabolism. The strain was further evaluated as the chassis for terpenoids production. Strain SQ3-4-CPS generated from expressing b-caryophyllene synthase in SQ3-4 produced 11.86 AE 0.09 mg l À1 b-caryophyllene, while strain SQ3-5 resulted from down-regulation of ERG1 in SQ3-4 produced 408.88 AE 0.09 mg l À1 squalene in shake flask cultivations. Strain SQ3-5 produced 4.94 g l À1 squalene in fed-batch fermentation in cane molasses medium, indicating the promising potential for cost-effective production of squalene.

Compartmentalization engineering of yeasts to overcome precursor limitations and cytotoxicity in terpenoid production

“…Similarly, mitochondria have nearly 20-30 times higher acetyl-CoA content than the cytosol (Duran et al, 2020). As shown in Figure 2C, rational organelle compartmentalization can greatly improve the utilization efficiency of the acetyl-CoA pools in different organelles (Son et al, 2022a;Lu et al, 2022). Dong et al (2021) compartmentalized the whole MVA pathway into mitochondria, leading to a 3.7-fold improvement in the production of α-santalene.…”

Cofactor Engineering for Efficient Production of α-Farnesene by Rational Modification of NADPH and ATP Regeneration Pathway in Pichia pastoris

“…On the other hand, yeast and bacteria use NADH more than NADPH as a reduced cofactor of catabolism [ 4 ]. Thus, increasing the intracellular NADPH level or eliminating NADPH consumption is a common strategy to facilitate NADPH-dependent products, including terpenoid [ 14 , 15 ]. For example, Liu et al compared the effects of six native enzymes involved in NAPDH regeneration in Yarrowia lipolytica and found that mannitol dehydrogenase benefits the increase in squalene production [ 16 ].…”

PhiReX 2.0: A Programmable and Red Light-Regulated CRISPR-dCas9 System for the Activation of Endogenous Genes in Saccharomyces cerevisiae

“…Further, dosedependent regulation of gene expression will help to optimize flux through a pathway by adjusting the endogenous gene expression levels flexibly and in a cloning free manner. The fact that the adjustment of endogenous protein levels has a positive influence on product yield has already been described several times, e.g., for terpene production, 19 overproduction of Design of sgRNA Entry and Multimerization Vectors. The expression of sgRNAs is based on the previously published tRNA-HDV architecture, 23 which we modified to allow Golden Gate-based cloning of up to four sgRNA cassettes into a single plasmid (see Figure 2 and the Methods section for details).…”

“…Further, dose-dependent regulation of gene expression will help to optimize flux through a pathway by adjusting the endogenous gene expression levels flexibly and in a cloning free manner. The fact that the adjustment of endogenous protein levels has a positive influence on product yield has already been described several times, e.g., for terpene production, overproduction of triacylglycerols (TAGs), , and high-level production of aromatic chemicals …”