Plasticity engineering of plant monoterpene synthases and application for microbial production of monoterpenoids.

Plant monoterpenoids with structural diversities have extensive applications in food, cosmetics, pharmaceuticals, and biofuels. Due to the strong dependence on the geographical locations and seasonal annual growth of plants, agricultural production for monoterpenoids is less effective. Chemical synthesis is also uneconomic because of its high cost and pollution. Recently, emerging synthetic biology enables engineered microbes to possess great potential for the production of plant monoterpenoids. Both acyclic and cyclic monoterpenoids have been synthesized from fermentative sugars through heterologously reconstructing monoterpenoid biosynthetic pathways in microbes. Acting as catalytic templates, plant monoterpene synthases (MTPSs) take elaborate control of the monoterpenoids production. Most plant MTPSs have broad substrate or product properties, and show functional plasticity. Thus, the substrate selectivity, product outcomes, or enzymatic activities can be achieved by the active site mutations and domain swapping of plant MTPSs. This makes plasticity engineering a promising way to engineer MTPSs for efficient production of natural and non-natural monoterpenoids in microbial cell factories. Here, this review summarizes the key advances in plasticity engineering of plant MTPSs, including the fundamental aspects of functional plasticity, the utilization of natural and non-natural substrates, and the outcomes from product isomers to complexity-divergent monoterpenoids. Furthermore, the applications of plasticity engineering for improving monoterpenoids production in microbes are addressed.

Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach.

BACKGROUND: 3-Phenylpropanol with a pleasant odor is widely used in foods, beverages and cosmetics as a fragrance ingredient. It also acts as the precursor and reactant in pharmaceutical and chemical industries. Currently, petroleum-based manufacturing processes of 3-phenypropanol is environmentally unfriendly and unsustainable. In this study, we aim to engineer Escherichia coli as microbial cell factory for de novo production of 3-phenypropanol via retrobiosynthesis approach. RESULTS: Aided by in silico retrobiosynthesis analysis, we designed a novel 3-phenylpropanol biosynthetic pathway extending from L-phenylalanine and comprising the phenylalanine ammonia lyase (PAL), enoate reductase (ER), aryl carboxylic acid reductase (CAR) and phosphopantetheinyl transferase (PPTase). We screened the enzymes from plants and microorganisms and reconstructed the artificial pathway for conversion of 3-phenylpropanol from L-phenylalanine. Then we conducted chromosome engineering to increase the supply of precursor L-phenylalanine and combined the upstream L-phenylalanine pathway and downstream 3-phenylpropanol pathway. Finally, we regulated the metabolic pathway strength and optimized fermentation conditions. As a consequence, metabolically engineered E. coli strain produced 847.97 mg/L of 3-phenypropanol at 24 h using glucose-glycerol mixture as co-carbon source. CONCLUSIONS: We successfully developed an artificial 3-phenylpropanol pathway based on retrobiosynthesis approach, and highest titer of 3-phenylpropanol was achieved in E. coli via systems metabolic engineering strategies including enzyme sources variety, chromosome engineering, metabolic strength balancing and fermentation optimization. This work provides an engineered strain with industrial potential for production of 3-phenylpropanol, and the strategies applied here could be practical for bioengineers to design and reconstruct the microbial cell factory for high valuable chemicals.

Combining Metabolic and Monoterpene Synthase Engineering for de Novo Production of Monoterpene Alcohols in Escherichia coli.

The monoterpene alcohols acyclic nerol and bicyclic borneol are widely applied in the food, cosmetic, and pharmaceutical industries. The emerging synthetic biology enables microbial production to be a promising alternative for supplying monoterpene alcohols in an efficient and sustainable approach. In this study, we combined metabolic and plant monoterpene synthase engineering to improve the de novo production of nerol and borneol in prene-overproducing Escherichia coli. We engineered the growth-orthogonal neryl diphosphate (NPP) as the universal precursor of monoterpene alcohol biosynthesis and coexpressed nerol synthase (GmNES) from Glycine max to generate nerol or coexpressed the truncated bornyl diphosphate synthase (LdtBPPS) from Lippia dulcis for borneol production. Further, through site-directed mutation of LdtBPPS based on the structural simulation, we screened multiple variants that markedly elevated the production of acyclic nerol or bicyclic borneol, of which the LdtBPPSS488T mutant outperformed the wild-type LdtBPPS on borneol synthesis and the LdtBPPSF612A variant was superior to GmNES on nerol production. Subsequently, we overexpressed the endogenous Nudix hydrolase NudJ to facilitate the dephosphorylation of precursors and boosted the production of nerol and borneol from glucose. Finally, after the optimization of the fermentation process, the engineered strain ENO2 produced 966.55 mg/L nerol, and strain ENB57 generated 87.20 mg/L borneol in a shake flask, achieving the highest reported titers of nerol and borneol in microbes to date. This work shows a combinatorial engineering strategy for microbial production of natural terpene alcohols.

Integrative Biosynthetic Gene Cluster Mining to Optimize a Metabolic Pathway to Efficiently Produce l-Homophenylalanine in Escherichia coli.

Mining biosynthetic genes for the exploration of hybrid metabolic pathways is a promising approach in heterologous production of natural and unnatural products. Here, we developed an integrative biosynthetic gene cluster (BGC) mining strategy to engineer the biosynthesis of l-homophenylalanine (l-Hph), an important intermediate for the synthesis of angiotensin-converting enzyme inhibitors. We assembled the putative l-Hph BGCs and integrated phylogenetic analysis with target metabolite abundance mapping to prioritize candidate BGCs. To obtain an effective l-Hph pathway, various combinations of candidate genes from different species were screened in an iterative design-build-test stepwise manner. After the pathway was strength balanced and the metabolic flux was enhanced, engineered Escherichia coli produced 1.41 g/L of l-Hph from glucose in feeding shake-flask fermentation. Our cluster mining strategy enabled optimization of the target metabolic pathway, and it would be promising for production of other valuable products in the postgenomic era.

Orthogonal Engineering of Biosynthetic Pathway for Efficient Production of Limonene in Saccharomyces cerevisiae.

Limonene, a plant-derived natural cyclic monoterpene, is widely used in the pharmaceutical, food, and cosmetics industries. The conventional limonene biosynthetic (CLB) pathway in engineered Saccharomyces cerevisiae consists of heterologous limonene synthase (LS) using endogenous substrate geranyl diphosphate (GPP) and suffers from poor production of limonene. In this study, we report on an orthogonal engineering strategy in S. cerevisiae for improving the production of limonene. We reconstructed the orthogonal limonene biosynthetic (OLB) pathway composed of SlNDPS1 that catalyzes IPP and DMAPP to NPP ( cis-GPP) and plant LS that converts NPP to limonene. We find that the OLB pathway is more efficient for production of limonene than the CLB pathway. When expression of the competing gene ERG20 was chromosomally regulated by the glucose-sensing promoter HXT1, the OLB pathway-harboring strain produced 917.7 mg/L of limonene in fed-batch fermentation, a 6-fold increase of the CLB pathway, representing the highest titer reported to date. Orthogonal engineering exhibits great potential for production of terpenoids in S. cerevisiae.