Elsevier

Metabolic Engineering

Volume 55, September 2019, Pages 131-141
Metabolic Engineering

Synthetic N-terminal coding sequences for fine-tuning gene expression and metabolic engineering in Bacillus subtilis

https://doi.org/10.1016/j.ymben.2019.07.001Get rights and content

Highlights

  • N-terminal coding sequences (NCSs) from Bacillus subtilis exhibited broad dynamic range for gene expression regulation.

  • Synthetic NCSs were developed guided by statistical analysis, which expanded dynamic range of gene expression regulation.

  • Synthetic and native NCSs were used to fine-tune N-acetylneuraminic acid pathway aided by kinetic modeling.

Abstract

N-terminal coding sequences (NCSs) of genes significantly influence gene expression at the translation level and are important for fine-tuning gene expression in bacteria, however, engineering NCSs to fine-tune metabolic pathways is challenging. Here, we developed a statistics-guided native and synthetic NCSs engineering approach to fine-tune gene expression in the industrially important microorganism Bacillus subtilis. This method is based on experimentally characterizing and statistically analyzing 96 rationally selected NCSs from B. subtilis endogenous genes. These NCSs exhibited a magnitude difference of greater than 4 orders in their ability to drive gene expression in 4 different dynamic patterns, including growth-coupled, growth-delayed, consistent expression, and inhibitory patterns. Synthetic and native NCSs were used to fine-tune expression of key enzymes, identified via pathway analysis and kinetic modeling, in the biosynthetic pathway of the useful nutraceutical N-acetylneuraminic acid (NeuAc). We observed a 3.21-fold improvement in NeuAc biosynthesis, indicating that NCSs can provide a synthetic biology toolbox to fine-tune gene expression for metabolic engineering.

Introduction

Precise control of gene expression at desired levels and with the desired dynamic patterns is of great importance for metabolic engineering and synthetic biology, especially for fine-tuning metabolic pathways and optimizing genetic circuits (Brophy and Voigt, 2014; Chae et al., 2017; McNerney et al., 2015; Nielsen and Keasling, 2016; Woolston et al., 2013). Therefore, a series of synthetic or engineered genetic regulatory elements for controlling gene expression at the transcriptional, translational, and protein degradation levels was established via experimental characterization or computational design, including promoters, terminators, RBS sequences, small regulatory RNAs, and proteolysis tags (Alper et al., 2005; Brockman and Prather, 2015, 2014; Curran et al., 2014; Guiziou et al., 2016; Na et al., 2013; Redden and Alper, 2015; Salis et al., 2009). In addition to the previously mentioned regulatory elements, N-terminal coding sequences (NCSs) strongly impact gene expression at the level of translation by influencing the efficiency of ribosome binding to mRNA and ribosome extension at the initial stage of translation, which is an important mechanism for fine-tuning endogenous gene expression in bacteria (Cambray et al., 2018; Goodman et al., 2013; Kudla et al., 2009; Sauer et al., 2018).

NCSs have been engineered for the modulation of gene expression, however, this engineering was carried out in a brute-force manner by fusing a limited number of NCSs to the 5’ end of genes of interest (Chaves et al., 2017; Sauer et al., 2018; Zhou et al., 2013). Additionally, the effect of NCS variation on translational strength cannot be completely predicted by current computational tools (Sauer et al., 2018). There is also a lack of an experimentally characterized synthetic NCS libraries and rational design methods, and these limitations hamper the use of NCSs as regulatory tools for fine-tuning metabolic pathways. Rational NCSs design and development for their use as regulatory tools fine-tuning gene expression and metabolic engineering a real challenge.

In this study, we chose Bacillus subtilis, the model gram-positive bacterium that is used for the industrial production of important enzymes and biochemicals, as a system in which to develop a series of native and synthetic NCSs for fine-tuning gene expression. We selected 96 endogenous NCSs with a potential wide range of regulatory effects based on transcriptomic and proteomic data. Next, the effects of different NCSs on gene expression were systematically verified and divided into 4 classes based on their dynamic patterns for driving gene expression. A series of synthetic NCSs was then designed to expand the dynamic range of gene expression regulation based on a proposed synthetic NCS design method. Finally, NCS engineering was used for the static and dynamic regulation of N-acetylneuraminic acid (NeuAc) biosynthesis aided by kinetic modeling for pathway debottlenecking. We showed that NCS engineering using native and synthetic NCSs can be used for fine-tuning gene expression in B. subtilis for metabolic engineering and synthetic biology.

Section snippets

Plasmids and strains

The plasmids used in this study included pP43NMK (Zhang et al., 2005) and pHT01 (Nguyen et al., 2007); and the strains included B. subtilis 168 wild type B. subtilis No. 6 (Zhang et al., 2018). All constructed plasmids and strains are listed in Supplementary Table S1.

We used GFP to characterize NCS intensity. We first ligated the GFP gene downstream of the promoter P43 using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China) to obtain the plasmid pP43NMK-gfp. Then, we transformed

Transcriptomics and proteomics-based selection and characterization of B. subtilis native NCSs

To select native NCSs exhibiting a wide range of strength for driving gene translation, a rational selection strategy was implemented based on the reported transcriptomic and proteomic data of B. subtilis present during the exponential growth phase (Brinsmade et al., 2014; Buescher et al., 2012; Maaβ et al., 2014). Genes that show abundant expression can potentially harbor NCSs that efficiently drive gene translation. Therefore, the translational strength of each gene in B. subtilis was

Discussion

In this study, synthetic NCSs were developed as gene expression regulatory elements in B. subtilis. Compared to the other currently available gene expression regulatory elements, including promoters, terminators, RBS sequences, and spacer sequences, there are many advantages in using NCSs for regulating gene expression and metabolic pathways. First, genetic manipulation through NCS engineering is convenient, as NCSs developed in this study are as short as 45 bp, and primers can be used to

Acknowledgements

This work is financially supported by the National Key Research and Development Program of China (2018YFA0900303), the National Natural Science Foundation of China (31600068, 31622001, 31671845, 21676119), the Natural Science Foundation of Jiangsu Province (BK20160176), the China Postdoctoral Science Foundation (2016M600363, 2017T100327), the 111 Project (No. 111-2-06), the Fundamental Research Funds for the Central Universities (JUSRP11725), and National First-class Discipline Program of Light

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