Abstract
BackgroundMicrobes are extensively engineered to produce compounds of biotechnological or pharmaceutical interest. However, functional integration of synthetic pathways into the respective host cell metabolism and optimization of heterologous gene expression for achieving high product titers is still a challenging task. In this manuscript, we describe the optimization of a tetracistronic operon for the microbial production of the plant-derived phenylpropanoid p-coumaryl alcohol in Escherichia coli.ResultsBasis for the construction of a p-coumaryl alcohol producing strain was the development of Operon-PLICing as method for the rapid combinatorial assembly of synthetic operons. This method is based on the chemical cleavage reaction of phosphorothioate bonds in an iodine/ethanol solution to generate complementary, single-stranded overhangs and subsequent hybridization of multiple DNA-fragments. Furthermore, during the assembly of these DNA-fragments, Operon-PLICing offers the opportunity for balancing gene expression of all pathway genes on the level of translation for maximizing product titers by varying the spacing between the Shine-Dalgarno sequence and START codon. With Operon-PLICing, 81 different clones, each one carrying a different p-coumaryl alcohol operon, were individually constructed and screened for p-coumaryl alcohol formation within a few days. The absolute product titer of the best five variants ranged from 48 to 52 mg/L p-coumaryl alcohol without any further optimization of growth and production conditions.ConclusionsOperon-PLICing is sequence-independent and thus does not require any specific recognition or target sequences for enzymatic activities since all hybridization sites can be arbitrarily selected. In fact, after PCR-amplification, no endonucleases or ligases, frequently used in other methods, are needed. The modularity, simplicity and robustness of Operon-PLICing would be perfectly suited for an automation of cloning in the microtiter plate format.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0274-9) contains supplementary material, which is available to authorized users.
Highlights
Microbes are extensively engineered to produce compounds of biotechnological or pharmaceutical interest
We developed Operon-phosphorothioate-based ligase-independent gene cloning (PLICing) for the rapid assembly of synthetic pathways and included the possibility to simultaneously tune (“balance”) the expression of all pathway genes on the level of translation
Model pathway for the development of this method was a four-step, hybrid plant/bacterial pathway to convert the amino acid l-tyrosine to the monolignol p-coumaryl alcohol (Figure 1). This plant natural product is an important precursor of pharmaceutically interesting lignans and key building block of the plant polymer lignin. p-Coumaryl alcohol synthesis from l-tyrosine starts with a deamination step catalyzed by a tyrosine ammonia lyase (TAL, EC 4.3.1.23) to form p-coumaric acid
Summary
Microbes are extensively engineered to produce compounds of biotechnological or pharmaceutical interest. Functional integration of synthetic pathways into the respective host cell metabolism and optimiza‐ tion of heterologous gene expression for achieving high product titers is still a challenging task. In this manuscript, we describe the optimization of a tetracistronic operon for the microbial production of the plant-derived phenylpropa‐ noid p-coumaryl alcohol in Escherichia coli. Tuning of gene expression from such synthetic pathways can be achieved on the level of transcription or translation. Combinatorial assembly and subsequent evaluation of many different pathway variants can be a very time consuming and expensive task depending on the cloning method employed. Among the more prominent methods for the assembly of pathways are polymerase cycling assembly (PCA) [12], Gibson assembly [13], the sequence and ligation independent cloning (SLIC) method [14] or USERfusion [15]
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