Metabolic pathway optimization using ribosome binding site variants and combinatorial gene assembly

Abstract

The genes encoding the mevalonate-based farnesyl pyrophosphate (FPP) biosynthetic pathway were encoded in two operons and expressed in Escherichia coli to increase the production of sesquiterpenes. Inefficient translation of several pathway genes created bottlenecks and led to the accumulation of several pathway intermediates, namely, mevalonate and FPP, and suboptimal production of the sesquiterpene product, amorphadiene. Because of the difficulty in choosing ribosome binding sites (RBSs) to optimize translation efficiency, a combinatorial approach was used to choose the most appropriate RBSs for the genes of the lower half of the mevalonate pathway (mevalonate to amorphadiene). RBSs of various strengths, selected based on their theoretical strengths, were cloned 5' of the genes encoding mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and amorphadiene synthase. Operons containing one copy of each gene and all combinations of RBSs were constructed and tested for their impact on growth, amorphadiene production, enzyme level, and accumulation of select pathway intermediates. Pathways with one or more inefficiently translated enzymes led to the accumulation of pathway intermediates, slow growth, and low product titers. Choosing the most appropriate RBS combination and carbon source, we were able to reduce the accumulation of toxic metabolic intermediates, improve growth, and improve the production of amorphadiene approximately fivefold. This work demonstrates that balancing flux through a heterologous pathway and maintaining steady growth are key determinants in optimizing isoprenoid production in microbial hosts.

Engineering Methylobacterium extorquens for de novo synthesis of the sesquiterpenoid α-humulene from methanol

“…RBS optimization has the advantage of low cloning effort and maintaining the existing plasmid system. Improvement of expression of MVA pathway genes on translational level has already been realized by RBS optimization leading to increased terpenoid production in E. coli (Nowroozi et al, 2014). Hence, to prevent prenyl phosphate accumulation in M. extorquens, TIR of α-humulene synthase RBS was increased 146 fold in plasmids pFS57 (zssI 225k ), pFS58 (zssI 225k -ERG20) and pFS59 (zssI 225k -ERG20-MVA) (see Table 2) to obtain higher levels of ZSSI and thus more conversion of toxic FPP to α-humulene.…”

“…The most prominent example for high-level sesquiterpenoid production by heterologous overexpression of the MVA pathway in E. coli is dedicated to the production of the artemisinin precursor amorphadiene. Product concentrations of up to 300 mg/L OD À 1 were achieved by flux optimization including promoter, ribosome binding site (RBS) and codon improvements (Anthony et al, 2009;Martin et al, 2003;Nowroozi et al, 2014;Pitera et al, 2007). In the last years, the monocyclic sesquiterpenoid α-humulene (also called α-caryophyllene) has received increased attention due to its anti-inflammatory and potential anti-cancerogenic properties Passos et al, 2007).…”

The Need for Integrated Approaches in Metabolic Engineering

“…Often, engineered pathways have been optimized through sampling of large design spaces (Pfleger et al 2006; Lütke-Ever- Wang et al 2012a;Xu et al 2013;Nowroozi et al 2014). Such combinatorial approaches are specifically valuable for organisms with less-defined genetic and metabolic networks (Oliver et al 2014).…”

Designer Microorganisms for Optimized Redox Cascade Reactions – Challenges and Future Perspectives

“…(Figure 10). [108] Codon usage also influencest he translation efficiency andt he availability of at RNAc orresponding to its codon on the mRNAd epends on the species. Theu se of codon-optimized genes is interesting in the context of heterologous pathway design when enzymes from different (especially higher) species are used (and produced in al ower host organism).…”