Plants and prokaryotic cells have been extensively deployed for the biosynthesis of several molecules including the antibodies, hormones, enzymes, and vaccines.^1-6 An increased scale of bioproduction and bioaccumulation in the endoplasmic reticulum of plant cells lead to a substantially higher production of industrially important foreign genes, unlike the microorganisms or mammalian cells.^7,8 Generally, in plants when a stress condition arises, the transcription factors play a key role at molecular level and bind to their recognition sequences, upstream to the stress responsive genes. For a highly versatile biosynthetic production of a broad spectrum of biomolecules, their industrial application has been booming up in the last two decades.^9,10 However, the industrial stress conditions often leads to the improper growth in plants, and thus prove to be the major factor that decreases the overall yield.¹¹ Natural adaptation of plants is time consuming and often fails to yield a gain-of-function mutation in terms of an enzymatic yield or turnover. Although both bacterial and plant systems have been extensively used for the industrial production of several biomolecules, the prokaryotic systems have been preferred over plants for their short doubling time and ease of batch scale-up, especially when proteins are biologically active in native forms, with no requirement of additional posttranslational modifications.^4,6

Although the transcriptional rate is preliminarily aided by the strength of promoter and growth stage of a cell and cis- and trans-acting factors,¹² the cis elements are found to be the key regulatory switches, acting as binding sites for one or more trans-acting factors.^13,14 A trans-acting factor usually binds several different genes and controls their temporal and spatial expression patterns.^15,16 The cis-acting regulatory segments are present in the introns and 5′ and 3′ untranslated segments, or coding regions of precursor RNAs and mature mRNAs, and are specifically recognized by atleast one trans-acting factor for selectively regulating the posttranscriptional gene expression.¹⁷ The cis elements are found to play a major role in the post-transcription and post-translation of prokaryotic systems.

As cis elements and length of spacers between them predominantly modulate the expression of a gene under varied levels of biotic stresses, a set of continuous/discontinuous set of cis elements often regulate the expression of a gene,^18-20 and hence the cis elements must be extensively excavated.^18-20 Besides the variant copy number and different sequence, the relative localization and mutual distance network of the cis elements, cognate to the gene, are highly variable, and it leads to a substantial change in the yield of the encoded protein.²¹ These segments are functionally diverse, and have multiple repeats in a gene,²² and are usually analyzed through sequence information of the conserved transcription factor binding sites and their unique organization within a gene. Due to differences in the set of cis elements, a tissue-specific gene expression profile has been observed at different developmental phases.²³

Directed evolution strategy has been extensively used for many applications including the functional improvement of several plant/bacterial genes for producing vaccine or pharmaceuticals,^24,25 strain improvement,^26,27 and building variants with improved activity.^28,29 The cis elements often function as an insulator, silencer, enhancer, and promoter in plants,¹⁷ gram-positive bacteria,³⁰ thermophilic archaea,³¹ and photosynthetic bacteria.³² Although the synthetic plant/bacterial gene cassettes, with a customized minimal set of most productive cis elements, can be constructed and experimentally modulated under varied stress conditions to increase their expression level,³³ their unambiguous identification is highly difficult, expensive and painstaking. Hence, the researchers often produce the functionally improved gene copies through the directed evolution algorithms without screening and optimizing the best promoter(s) sequences, applicable for a gene.

As the biologically favored cis element network would increase the expression level of cloned genes, promoter engineering should also be considered as the alternative strategy to map the best set of cis elements in the known set of functionally similar and evolutionarily closer genes. To simplify the computational methodology of extracting the naturally encoded set of cis elements in a large dataset of naturally available alternative gene copies, and to subsequently extract the most frequent set of cis elements and design its alternative copies, a PERL script is hereby developed to efficiently span the available sequence space, available in the genome databases. It would allow us to easily construct the functionally active protein variant with most active set of cis elements and would be significantly useful in reliably pacing up the computational promoter design methodologies.