Stringency of Synthetic Promoter Sequences inClostridiumRevealed and Circumvented by Tuning Promoter Library Mutation Rates

ABSTRACTCollections of characterized promoters of different strengths are key resources for synthetic biology, but are not well established for many important organisms, including industrially-relevantClostridiumspp. When generating promoters, reporter constructs are used to measure expression, but classical fluorescent reporter proteins are oxygen-dependent and hence inactive in anaerobic bacteria likeClostridium. We directly compared oxygen-independent reporters of different types inClostridium acetobutylicumand found that glucuronidase (GusA) fromE. coliperformed best. Using GusA, a library of synthetic promoters was first generated by a typical approach entailing complete randomization of a constitutive thiolase gene promoter (Pthl) except for the consensus -35 and -10 elements. In each synthetic promoter, the chance of each degenerate position matching Pthlwas 25%. Surprisingly, none of the synthetic promoters from this library were functional inC. acetobutylicum, even though they functioned as expected inE. coli. Next, instead of complete randomization, we specified lower promoter mutation rates using oligonucleotide primers synthesized using custom mixtures of nucleotides. Using these primers, two promoter libraries were constructed in which the chance of each degenerate position matching Pthlwas 79% or 58%, instead of 25% as before. Synthetic promoters from these ‘stringent’ libraries functioned well inC. acetobutylicum, covering a wide range of strengths. The promoters functioned similarly in the distantly-related speciesClostridium sporogenes, and allowed predictable metabolic engineering ofC. acetobutylicumfor acetoin production. Besides generating the desired promoters and demonstrating their useful properties, this work indicates an unexpected ‘stringency’ of promoter sequences inClostridium, not reported previously.GRAPHICAL ABSTRACT

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Model-driven promoter strength prediction based on a fine-tuned synthetic promoter library in Escherichia coli

10.1101/2020.06.25.170365 ◽

2020 ◽

Author(s):

Mei Zhao ◽

Shenghu Zhou ◽

Longtao Wu ◽

Yu Deng

Keyword(s):

Metabolic Pathways ◽

Regulatory Elements ◽

Strength Prediction ◽

Promoter Strength ◽

Synthetic Promoter ◽

Promoter Sequences ◽

Model Driven ◽

Promoter Library ◽

Mutant Promoter ◽

The Relationship

AbstractPromoters are one of the most critical regulatory elements controlling metabolic pathways. However, in recent years, researchers have simply perfected promoter strength, but ignored the relationship between the internal sequences and promoter strength. In this context, we constructed and characterized a mutant promoter library of Ptrc through dozens of mutation-construction-screening-characterization engineering cycles. After excluding invalid mutation sites, we established a synthetic promoter library, which consisted of 3665 different variants, displaying an intensity range of more than two orders of magnitude. The strongest variant was 1.52-fold stronger than a 1 mM isopropyl-β-D-thiogalactoside driven PT7 promoter. Our synthetic promoter library exhibited superior applicability when expressing different reporters, in both plasmids and the genome. Different machine learning models were built and optimized to explore relationships between the promoter sequences and transcriptional strength. Finally, our XgBoost model exhibited optimal performance, and we utilized this approach to precisely predict the strength of artificially designed promoter sequences. Our work provides a powerful platform that enables the predictable tuning of promoters to achieve the optimal transcriptional strength.

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Precise Prediction of Promoter Strength Based on a De Novo Synthetic Promoter Library Coupled with Machine Learning

ACS Synthetic Biology ◽

10.1021/acssynbio.1c00117 ◽

2021 ◽

Author(s):

Mei Zhao ◽

Zhenqi Yuan ◽

Longtao Wu ◽

Shenghu Zhou ◽

Yu Deng

Keyword(s):

Machine Learning ◽

De Novo ◽

Promoter Strength ◽

Synthetic Promoter ◽

Promoter Library ◽

Strength Based ◽

Precise Prediction

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Model-driven generation of artificial yeast promoters

10.1101/748616 ◽

2019 ◽

Cited By ~ 1

Author(s):

Benjamin J. Kotopka ◽

Christina D. Smolke

Keyword(s):

Model Organism ◽

Inducible Promoter ◽

Design Strategies ◽

Multiple Sequence ◽

Promoter Sequences ◽

Model Driven ◽

Large Sets ◽

Promoter Library ◽

Small Set ◽

Rapid Generation

AbstractPromoters play a central role in controlling gene regulation; however, a small set of promoters is used for most genetic construct design in the yeast Saccharomyces cerevisiae. Generating and utilizing models that accurately predict protein expression from promoter sequences would enable rapid generation of novel useful promoters and facilitate synthetic biology efforts in this model organism. We measured the gene expression activity of over 675,000 unique sequences in a constitutive promoter library, and over 327,000 sequences in an inducible promoter library. Training an ensemble of convolutional neural networks jointly on the two datasets enabled very high (R2 > 0.79) predictive accuracies on multiple sequence-activity prediction tasks. We developed model-guided design strategies which yielded large, sequence-diverse sets of novel promoters exhibiting activities similar to current best-in-class sequences. In addition to providing large sets of new promoters, our results show the value of model-guided design as an approach for generating useful DNA parts.

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Model-based reconstruction of synthetic promoter library in Corynebacterium glutamicum

Biotechnology Letters ◽

10.1007/s10529-018-2539-y ◽

2018 ◽

Vol 40 (5) ◽

pp. 819-827 ◽

Cited By ~ 7

Author(s):

Shuanghong Zhang ◽

Dingyu Liu ◽

Zhitao Mao ◽

Yufeng Mao ◽

Hongwu Ma ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Synthetic Promoter ◽

Model Based ◽

Promoter Library

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Design, construction and characterisation of a synthetic promoter library for fine-tuned gene expression in actinomycetes

Metabolic Engineering ◽

10.1016/j.ymben.2013.07.006 ◽

2013 ◽

Vol 19 ◽

pp. 98-106 ◽

Cited By ~ 96

Author(s):

Theresa Siegl ◽

Bogdan Tokovenko ◽

Maksym Myronovskyi ◽

Andriy Luzhetskyy

Keyword(s):

Gene Expression ◽

Synthetic Promoter ◽

Promoter Library ◽

Design Construction

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A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum

Microbiology ◽

10.1099/mic.0.28599-0 ◽

2006 ◽

Vol 152 (4) ◽

pp. 1011-1019 ◽

Cited By ~ 88

Author(s):

Ida Rud ◽

Peter Ruhdal Jensen ◽

Kristine Naterstad ◽

Lars Axelsson

Keyword(s):

Gene Expression ◽

Lactobacillus Plantarum ◽

Protein Production ◽

Consensus Sequence ◽

Fine Tuning ◽

Stable Gene ◽

Synthetic Promoter ◽

Synthetic Promoters ◽

Promoter Library ◽

Wide Range

A synthetic promoter library (SPL) for Lactobacillus plantarum has been developed, which generalizes the approach for obtaining synthetic promoters. The consensus sequence, derived from rRNA promoters extracted from the L. plantarum WCFS1 genome, was kept constant, and the non-consensus sequences were randomized. Construction of the SPL was performed in a vector (pSIP409) previously developed for high-level, inducible gene expression in L. plantarum and Lactobacillus sakei. A wide range of promoter strengths was obtained with the approach, covering 3–4 logs of expression levels in small increments of activity. The SPL was evaluated for the ability to drive β-glucuronidase (GusA) and aminopeptidase N (PepN) expression. Protein production from the synthetic promoters was constitutive, and the most potent promoters gave high protein production with levels comparable to those of native rRNA promoters, and production of PepN protein corresponding to approximately 10–15 % of the total cellular protein. High correlation was obtained between the activities of promoters when tested in L. sakei and L. plantarum, which indicates the potential of the SPL for other Lactobacillus species. The SPL enables fine-tuning of stable gene expression for various applications in L. plantarum.

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Engineering of Bacillus subtilis 168 for Increased Nisin Resistance

Applied and Environmental Microbiology ◽

10.1128/aem.00943-09 ◽

2009 ◽

Vol 75 (21) ◽

pp. 6688-6695 ◽

Cited By ~ 17

Author(s):

Mette E. Hansen ◽

Romilda Wangari ◽

Egon B. Hansen ◽

Ivan Mijakovic ◽

Peter R. Jensen

Keyword(s):

Bacillus Subtilis ◽

Lactococcus Lactis ◽

Resistance Genes ◽

Food Industry ◽

Cost Effective ◽

Heterologous Production ◽

Host Organism ◽

Synthetic Promoter ◽

Bacillus Subtilis 168 ◽

Promoter Library

ABSTRACT Nisin is a natural bacteriocin produced commercially by Lactococcus lactis and widely used in the food industry as a preservative because of its broad host spectrum. Despite the low productivity and troublesome fermentation of L. lactis, no alternative cost-effective host has yet been found. Bacillus subtilis had been suggested as a potential host for the biosynthesis of nisin but was discarded due to its sensitivity to the lethal action of nisin. In this study, we have reevaluated the potential of B. subtilis as a host organism for the heterologous production of nisin. We applied transcriptome and proteome analyses of B. subtilis and identified eight genes upregulated in the presence of nisin. We demonstrated that the overexpression of some of these genes boosts the natural defenses of B. subtilis, which allows it to sustain higher levels of nisin in the medium. We also attempted to overcome the nisin sensitivity of B. subtilis by introducing the nisin resistance genes nisFEG and nisI from L. lactis under the control of a synthetic promoter library.

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Synthetic promoter design for new microbial chassis

Biochemical Society Transactions ◽

10.1042/bst20160042 ◽

2016 ◽

Vol 44 (3) ◽

pp. 731-737 ◽

Cited By ~ 34

Author(s):

James Gilman ◽

John Love

Keyword(s):

Synthetic Biology ◽

De Novo ◽

Saturation Mutagenesis ◽

Small Scale ◽

Synthetic Promoter ◽

Promoter Sequences ◽

Drive Gene Expression ◽

Judicious Choice ◽

Drive Gene ◽

Flanking Regions

The judicious choice of promoter to drive gene expression remains one of the most important considerations for synthetic biology applications. Constitutive promoter sequences isolated from nature are often used in laboratory settings or small-scale commercial production streams, but unconventional microbial chassis for new synthetic biology applications require well-characterized, robust and orthogonal promoters. This review provides an overview of the opportunities and challenges for synthetic promoter discovery and design, including molecular methodologies, such as saturation mutagenesis of flanking regions and mutagenesis by error-prone PCR, as well as the less familiar use of computational and statistical analyses for de novo promoter design.

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