# Plant Synthetic Promoters
## 4 Native and Synthetic Bidirectional Promoters  
Bidirectional promoters assure more coordinated expression of several genes than unidirectional promoters [236]. This was observed for the bidirectional promoter obtained from the _ZeamaysUlbiautiin-1_ (_ZUMbi1_) gene, which was utilized to regulate the expression of insect (_crys4 Ab1_ and _crys35 Ab1_) or herbicide (_aad1_) resistance and a phi-yfp reporter gene in corn (_Zea mays_) [173].  
A set of green-tissue-specific, bidirectional promoters was built by combining the unidirectional promoter POsrbcs-550 with the inversely oriented OsTub6 intron to increase both the transcription efficiency and green-tissue expression rate of GFP in the 5' direction. Another group of regulatory elements, viz. the reverse-oriented core promoter of POsrbcs-550 (POsrbcs-62), or its alternative PD540-544, the reversed first intron of _OsAct_ (_OsAct1_) and four-times repeated GEAT regulatory motif, were joined to increase the high level of the 3'-directed _Gus_ gene expression [236]. In transgenic _O. sativa_, the obtained constructs demonstrated generally predominant expression in green tissues, i.e., leaves, stems, panicles and sheaths, and low expression in roots and endosperm [236].  
Some bidirectional promoters exist naturally as 1156-bp-long fragments in the hot pepper genome, localized between two head-to-head-oriented genes: _sesquiiterpene cyclase_ (_EAS_) and a hydroxylase (_EAH_). A study of promoter deletion mutants in _Nicotiana benthamiana_ leaves, assessed by _EGFP_ and _shRLUC_ reporter genes, found the minimal promoters for the pathogen-inducible expression of both genes to be a 199-bp fragment containing the GCC-box of EAH and a downstream 226-bp fragment of _EAS_ bearing four W-boxes [237].  
The naturally occurring bidirectional histone gene promoters PHTX1, PHHX1, and PHHX2 are much shorter than monodirectional promoters and drive gene expression in a TATA-box-dependent manner [177]. Thanks to their compactness and clear regulatory mechanism in both directions, these promoters allow plant gene expression to be regulated. Therefore, these bidirectional promoters were used to construct a library of 168 synthetic BDPs in the yeast _Komagataella phaffii_ (syn. _Pichia pastoris_) to allow the rapid screening and optimization of different expression ratios [177]. Prepared synthetic promoters showed not a constitutive but rather a tight cell-cycle-regulated expression. Such tight, temporal expression control of metabolic pathway genes combined with the precise regulation of subsequent gene co-expression rate protects against excessive loads of heterologous proteins or concentrations of toxic metabolites [177]. By truncating and deleting PHHX2 fragments, BDPs of varying strength were produced. In addition, expression was made inducible by fusing shortened and bidirectionalized versions of the methanol utilization pathway promoters PDAS1-DAS2 with reversely oriented histone core promoters. Such an inducible approach was efficiently exercised in taxadiene biosynthesis, where the _CYP2D6_ and _CPR_ genes were controlled by methanol-inducible PDAS1-DAS2; _GGPPS_ was also regulated by a PGAP+CAT1 fusion promoter that is repressed in the presence of glucose, partially deregressed when the glucose is absent, and fully induced by methanol [177]. Moreover, assembly of the four carotenoid pathway biosynthesis genes _CrTE_, _CrTB_, _CrTI_, and _CrTY_ under the control of histone BDPs enables the achievement of a 14.9-fold higher \(\beta\)-carotene yield as compared to a monodirectional PGAP constitutive promoter [177].  
A related approach enabled the expression of \(\beta\)-carotene ketolase and hydroxylase genes in maize seed using a seed-specific bidirectional promoter. The obtained transgenic maize lines accumulated astaxanthin from 47.76 to 111.82 mg/kg DW in seeds, and the maximum level is approximately sixfold higher than that in previous reports (16.2-16.8 mg/kg DW) [238].