Isopentenol Utilization Pathway for the Production of Linalool in Escherichia coli Using an Improved Bacterial Linalool/Nerolidol Synthase

Abstract

Linalool is a monoterpenoid used as a fragrance ingredient, and is a promising source for alternative fuels. Synthetic biology offers attractive alternative production methods compared to extraction from natural sources and chemical synthesis. Linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus is a bifunctional enzyme, producing linalool as well as the sesquiterpenoid nerolidol when expressed in engineered Escherichia coli harbouring a precursor terpenoid pathway such as the mevalonate (MVA) pathway. Here we identified two residues important for substrate selection by bLinS, L72 and V214, where the introduction of bulkier residues results in variants with reduced nerolidol formation. Terpenoid production using canonical precursor pathways is usually limited by numerous and highly regulated enzymatic steps. Here we compared the canonical MVA pathway to the non‐canonical isopentenol utilization (IU) pathway to produce linalool using the optimised bLinS variant. The IU pathway uses isoprenol and prenol to produce linalool in only five steps. Adjusting substrate, plasmid system, inducer concentration, and cell strain directs the flux towards monoterpenoids. Our integrated approach, combining enzyme engineering with flux control using the artificial IU pathway, resulted in high purity production of the commercially attractive monoterpenoid linalool, and will guide future efforts towards efficient optimisation of terpenoid production in engineered microbes.

Optimization of microbial cell factories for astaxanthin production: Biosynthesis and regulations, engineering strategies and fermentation optimization strategies

“…In addition to the metabolic engineering strategies mentioned above, introduction of non-native pathways to enhance the precursors supply might be of great influence on increasing astaxanthin production. For example, IPP enhancement has been achieved in several organisms via introducing exogenous isoprenol utilizing pathway (IUP), which led to enhanced isoprenoids production [ [232] , [233] , [234] ]. Additionally, other promising membrane stress management strategies have to be adopted to relief the burden of astaxanthin accumulation on the cells.…”

The beauty of biocatalysis: sustainable synthesis of ingredients in cosmetics

“…One example of linalool production, combines a limonene synthase (blinS ( Streptomyces clavuligerus )) that was engineered to reduce substrate promiscuity, with an alternative pathway that produced the terpene precursors GPP or FPP, namely the isopentenol utilisation (IU) pathway, in E. coli . 390 The IU pathway uses isoprenol and prenol as substrates and using the IU pathway flux can be directed away from larger terpednoids such as nerolidol and towards the monoterpene linalool. Steps that would be highly regulated in the MVA pathway are bypassed.…”

Alternative metabolic pathways and strategies to high-titre terpenoid production inEscherichia coli

“…This might involve the engineering of enzymes that are more resistant to feedback control, or enzymes with improved catalytic efficiency, or new specicities towards desired target activities/products. 77,137,168,173,186,198,257,285,286 The use of alternative and articial pathways with this ever-expanding enzyme resource and pathway engineering tools should extend current capabilities, thereby allowing high-titre production of a wide range of terpenoids in E. coli and other microbial species.…”

“… 129 The pathway has been coupled to different downstream operons to produce several mono-, sesqui- and diterpenoids and lycopene, achieving a maximum titre of 9 mg g −1 DCW lycopene and 4.5 mg L −1 taxadiene, 129 and 33.4 mg L −1 linalool. 137 In a different study, Shigella flexneri non-specific acid phosphatase was used to catalyse the initial phosphorylation, and combined with T. acidophilum IPK and E. coli IDI to produce 190 mg L −1 lycopene. 133 The highest reported terpenoid titres with this shorter 3-step pathway were obtained using E. coli hydroxyethylthiazole kinase, archaeal Methanothermobacter thermautotrophicus IPK and Streptomyces sp.…”