Synthetic Biology Toolbox and Chassis Development in Bacillus subtilis

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Review

Synthetic Biology Toolbox and Chassis Development in Bacillus subtilis Yanfeng Liu,1,2,* Long Liu,1,2 Jianghua Li,1,2 Guocheng Du,1,2,* and Jian Chen2,3 Based on technical advances in the sequencing and synthesis of genetic components as well as the genome, significant progress has recently been made in developing synthetic biology toolboxes and chassis for the model Gram-positive bacterium Bacillus subtilis. In this review, we discuss recently developed synthetic biology toolboxes, including gene expression toolsets and genome editing tools. Next, advances in the B. subtilis chassis and its applications are discussed in comparison to those of other model microorganisms. Finally, future directions for the integrative use of B. subtilis synthetic biology tools and the development of an advanced chassis for efficient biomanufacturing are discussed. These factors are expected to become a major driving force for facilitating biotechnological applications of B. subtilis.

Highlights Recent development of synthetic biology toolboxes for B. subtilis, including gene expression regulatory toolboxes and genome-wide editing tools, provides powerful tools for precise gene expression control and efficient genome editing. Advances of B. subtilis chassis and their applications help to understand fundamental cellular processes and techniques for improving production of biomolecules or heterologous enzymes. Comparing B. subtilis chassis development with E. coli and S. cerevisiae chassis may provide potential directions for B. subtilis chassis construction.

B. subtilis and Its Background in Synthetic Biology B. subtilis is a Gram-positive model bacterium commonly used for studies in genetics and cellular metabolism [1–5]. In addition, B. subtilis has long been used as a cell factory for microbial production of enzymes, vitamins, and functional sugars due to its high secretory function and production abilities, coupled with relaxed fermentation media requirements [6–8]. Moreover, B. subtilis is favored for use in nutraceutical production, owing to its generally regarded as safe (GRAS) status, and it was one of the earlies species used in synthetic biology research, specifically for genome minimization (see Glossary) in order to understand essential cellular processes. In one early example, a genome-reduced B. subtilis was generated by deleting 7.7% of the genome without affecting either cell growth or replication [9]. Further application of genome-reduced B. subtilis for heterologous protein production revealed the biotechnological potential of the synthetic biology chassis, which was developed as a host for enzyme or biochemical production based on synthetic biology strategies [10–13]. However, in comparison with Escherichia coli and Saccharomyces cerevisiae, the most widely used prokaryotic and eukaryotic production hosts of metabolic engineering and synthetic biology, biotechnological research on B. subtilis has historically been constrained by limitations imposed by the lack of genetic regulatory elements and efficient genome-wide engineering approaches [14,15]. Recent developments with regard to B. subtilis synthetic biology toolboxes have significantly increased the efficacy of genetic manipulation and chassis construction [16–23]. Synthetic biology tools and methods are expected to be the major driving force for the further development of B. subtilis in basic and applied research. Previous reviews of B. subtilis have mainly focused on metabolic engineering of B. subtilis to produce industrially important enzymes and biochemicals, or advances in systems biology of B. subtilis for elucidating metabolic mechanisms and identifying metabolic engineering targets [7,24]. Progress in the B. subtilis synthetic biology toolbox and chassis, which are of importance for metabolic engineering and synthetic biology in balancing synthetic pathway and genetic circuit construction, has not been systematically discussed, especially for using

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1

Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China 2 Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China 3 National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China

*Correspondence: [email protected] (Y. Liu) and [email protected] (G. Du).

https://doi.org/10.1016/j.tibtech.2018.10.005 © 2018 Elsevier Ltd. All rights reserved.

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such tools to develop advanced chassis cells for expanding scientific and industrial applications. Here, we discuss current progress and future perspectives of synthetic biology toolboxes for B. subtilis and its use as a chassis.

Advances in Synthetic Toolboxes for B. subtilis Developed Genetic Regulatory Elements for Gene Expression Modulation The needs for gene expression regulatory elements with gradient strength to regulate gene expression facilitate development of appropriate genetic regulatory elements in B. subtilis (Box 1). To bridge the gap between the requirements for B. subtilis genetic regulatory elements and the limited number of experimentally characterized promoters and available plasmids, researchers have focused on the following three aspects: characterizing native promoters as regulatory tools; establishing synthetic promoters, ribosome-binding site (RBS) sequences, and proteolysis tags to combinatorically control gene expression; and developing modular plasmids and selection of genomic loci for efficient chromosomal integration of gene for expression [16–23,25] (Figure 1). Two experimentally characterized native promoter collections are currently available for B. subtilis. These were developed by measuring the fluorescence abundance of different promoters that regulate the expression of the green fluorescent protein coding sequence [16,17] (Table 1). The first promoter collection contains 84 promoters with three orders of magnitude range in expression strength. The second promoter collection further refined the dynamic expression patterns of promoters and clustered 114 native promoters into four categories based on their active phases, such as exponential and stationary phases, which provide guidance for selecting promoters for dynamic control. Synthetic promoters that are theoretically more orthogonal to endogenous cellular regulation with reduced interactions with intrinsic transcription were developed based on de novo sequence design and synthesis [18–20]. The first established synthetic promoter library of B. subtilis was based on the synthesis of de novo sequences with conserved
35,
10,
16, and UP elements [18]. Furthermore, tandem synthetic promoters with improved strength, compared to the commonly used strong constitutive promoter P43 (2.77-fold), were developed. To elucidate effects of nonconserved sequences, from UP elements to spacer sequences upstream to the first codons, on gene expression, approximately 12 000 synthetic expression modules were constructed and systematically analyzed [19]. The dynamic range of synthetic expression modules covers five orders of magnitude variation, including 32 synthetic expression modules with significant enhanced expression. Sequences at the 50 end of mRNA between the
10 region and the RBS sequence were demonstrated to be the dominant effectors of gene expression. To expand gene expression regulation from transcription to multiregulatory levels, including transcription, translation, and post-translation levels, a synthetic gene expression toolbox was developed [20]. A promoter mutant library, a RBS sequence library, and Box 1. Genetic Regulatory Elements Genetic regulatory elements provide an important basis for synthetic biology; these include promoters, RBS sequences, terminators, and other biological components for controlling gene expression. Assembling various genetic regulatory elements in a combinatorial manner allows modulating specific gene expression in a wide dynamic range. Therefore, genetic regulatory elements are mainly used for the following two purposes: precisely controlling key genes expression to balance synthetic pathways for efficient production of biomolecule; and optimizing the dynamic response range of genetic circuits via stepwise substitution of genetic regulatory elements. Two categories of genetic regulatory elements are used: native genetic regulatory elements that are characterized endogenous genetic regulatory elements; and synthetized genetic regulatory elements obtained via the engineering of endogenous genetic regulatory elements and de novo synthetized genetic regulatory elements based on de novo design.

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Glossary Fine-tuning gene expression: modulation of abundance or activities of key pathway enzymes to obtain optimal and balanced relative activities of pathway enzymes in metabolic pathways for improve metabolic flux. Genome minimization: a strategy for obtaining an engineered host with reduced genome size by large-scale deletion; for example, to understand the fundamental cellular processes of living cells and construct a cell factory to improve the production of enzymes or biomolecules. Synthetic biology chassis: engineered hosts for enzyme or biochemical production based on synthetic biology strategies. Synthetic pathways and synthetic devices can be introduced into the synthetic biology chassis cell to test feasibility and efficacy of designed synthetic pathways and synthetic devices. Synthetic biology toolbox: consists of genetic regulatory elements and genetic engineering approaches, which provides a basis for constructing synthetic pathways, genetic circuits and synthetic biology chassis.

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Proteolysis tag

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Characterizing genec regulatory elements

Idenfying nave genec regulatory element in B. sublis gfp

Construcng a system to characterize genec regulatory elements

Designing and synthesizing synthec genec regulatory elements

Comparing and clustering genec regulatory elements

Figure 1. Characterization of Bacillus subtilis Genetic Regulatory Elements. The DNA sequences of genetic regulatory elements were obtained from B. subtilis genome mining and de novo design. The strength and dynamic patterns of genetic regulatory elements were subsequently characterized in vivo. Abbreviations: RBS, ribosome-binding site.

various proteolysis tags were assembled to regulate gene transcription, translation, and protein degradation synergistically, leading to a five-orders-of-magnitude dynamic ranges. Integrating target genes into the chromosome for expression overcomes plasmid instability. This effect is useful in B. subtilis, especially when used in industrial biotechnology. However, a lack of sufficient integration plasmids and complicated integration plasmid construction hamper the multiple gene expression as well as genetic circuit integration into B. subtilis chromosome. To this end, a series of modular integration plasmids were developed via the Biobrick and Golden Gate DNA assembly method, which includes Bacillus Biobrick box, Bacillus Biobrick box 2.0, and Bacillus SEVA siblings [21–23]. The effects of B. subtilis chromosomal loci on gene expression were further systematically investigated, and the results indicated that expression levels gradually decreased from replication origin to replication terminus with a fivefold difference. This provides guidance for choosing chromosome positions for fine-tuning gene expression in synthetic pathways [25]. Besides systematically developed synthetic toolsets, several conditionally dependent inducible promoters and antibiotic free expression were developed, for use as complementary tools for fine-tuning gene expression in B. subtilis [26–28]. A synthetic cross-species promoter was developed to promote gene expression in E. coli, S. cerevisiae, and B. subtilis [29], in order to reduce the redesigning process where genetic circuits or synthetic pathways need to be characterized in the three model microorganisms. In addition, construction of synthetic pathways and genetic circuits requires efficient gene assembly methods. The ordered gene assembly in B. subtilis (OGAB) method for the assembly of large numbers of DNA fragments was previously developed. This method is based on the high transformation efficiency of multimeric plasmids, consisting of tandem repeat linear DNA fragments that can be cyclized inside B. subtilis [30]. To enhance the efficiency of multimeric plasmid preparation for OGAB, Trends in Biotechnology, Month Year, Vol. xx, No. yy

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Table 1. Gene Expression Regulatory Elements and Genome-wide Engineering Tools for B. subtilis Names

Features and limitations

Applications

Refs

Gene expression regulatory elements Native promoter library-1

Features: (1) 84 promoters with 3 orders of magnitude in the variation of maximal expression strength Limitations: (1) Lack of promoters for dynamic regulation of gene expression (2) Long sequences of promoters causing difficulties in regard to genetic manipulation

(1) Enhancing heterologous protein expression (2) Static regulation of gene expression level for metabolic engineering (3) Genetic circuit construction

Native promoter library-2

Features: (1) 114 promoters classified into 4 categories based on their active phase from exponential phase to stationary phase Limitations: (1) Long sequences of promoters (300 bp) that are inconvenient for use in genetic manipulation

(1) Enhancing heterologous protein expression (2) Static and dynamic regulation of gene expression level for metabolic engineering (3) Genetic circuit construction

[17]

Synthetic promoter library-1

Features: (1) 32 synthetic promoters with 900-fold differences strength, which consisted of short promoter sequences (60 bp) for convenient genetic manipulation Limitations: (1) Lack of promoters for dynamic regulation of gene expression

(1) Enhancing heterologous protein expression (2) Static regulation of gene expression level for metabolic engineering (3) Genetic circuit construction

[20]

Synthetic promoter library-2

Features: (1) 220 synthetic promoters with 140-fold differences strength, which consisted of short sequences (54–220 bp) for convenient genetic manipulation Limitations: (1) Lack of promoters for dynamic regulation of gene expression

heterologous protein (1) Enhancing expression (2) Static regulation of gene expression level for metabolic engineering (3) Genetic circuit construction

[18]

Synthetic expression modules from UP element to spacer sequence between RBS and the first codons

Features: (1) 12 000 synthetic expression modules with 5 orders of magnitude in variation of expression strength, including 32 synthetic expression modules for significant enhanced expression Limitations: (1) Lack of modules for dynamic regulation of gene expression

heterologous protein (1) Enhancing expression (2) Static regulation of gene expression level for metabolic engineering (3) Genetic circuit construction

[19]

RBS sequence library

Features: (1) 31 synthetic RBS sequences with 800-fold strength differences Limitations: (1) Effects of 50 end of the coding sequence on translation were not considered

(1) Enhancing heterologous protein expression (2) Static regulation of gene expression level for metabolic engineering (3) Genetic circuit construction

[20]

Synthetic proteolysis tag library

Features: (1) 22 synthetic proteolysis tags with 100-fold strength differences Limitations: (1) Lack of proteolysis tags for dynamic regulation of gene expression

(1) Enhancing heterologous protein expression (2) Static regulation of gene expression level for metabolic engineering (3) Genetic circuit construction

[20]

[16]

Modular integration plasmids Bacillus Biobrick box

Features: (1) 3 compatible empty integration vectors, 2 reporter vectors and 6 promoters with 3 orders of magnitude strength differences Limitations: (1) Maximum number of synthetic device is limited to 3

(1) Chromosomal expression and fine tuning of multiple genes (2) Characterizing strength of synthetic elements for gene expression regulation

[21]

Bacillus Biobrick box 2.0

Features: (1) 11 integration vectors, 3 replicative vectors, and 7 different codonoptimized gene sequences encoding fluorescent proteins Limitations: (1) Maximum number of synthetic devices is limited to 5

(1) Chromosomal expression and fine tuning of multiple genes (2) Characterizing strength of synthetic elements for gene expression regulation

[22]

4

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Table 1. (continued) Names

Features and limitations

Applications

Bacillus SEVA siblings

Features: (1) The backbone plasmid for integration expression with customized integration loci and 7 Bacillus species resistance markers Limitations: (1) Maximum number of synthetic devices is limited to be 7

(1) Chromosomal expression and fine tuning of multiple genes (2) Characterizing strength of synthetic elements for gene expression regulation

Refs [23]

Genome-wide engineering tools CRISPR-Cas9 genome editing system-1

Features: (1) A single plasmid for Cas9 and sgRNAs expression Limitations: (1) Potential instability of Cas9 nuclease and sgRNAs expression

(1) Large chromosomal regions deletion (2) Point mutation in chromosome

[35]

CRISPR-Cas9 genome editing system-2

Features: (1) Chromosomally expressing Cas9 nuclease and sgRNAs Limitations: (1) Integrating Cas9 nuclease and sgRNAs into the chromosome is a prerequisite for genome editing

(1) Introducing single and double gene mutations (2) Gene insertion

[36]

CRISPRi gene expression repression system-1

Features: (1) Chromosomally expressing dCas9 nuclease and sgRNAs Limitations: (1) Integrating dCas9 nuclease and sgRNAs into the chromosome prerequisite for genome editing

(1) gene expression repression

[3,36]

the equimolar DNA fragment preparation and assembly method was developed by designing DNA fragments with an identical length, cloned into the same backbone plasmid with type IIS restriction enzyme sites [31]. A maximum of 50 DNA fragments averaging 1 kb in length can be successfully assembled in one step. Achieving efficient translation modulation and high level chromosomal integration expression multiple genes are promising steps for future research. Modifying the 50 end of a coding sequence significantly affects translational initiation rates, and this may be potentially developed as an efficient strategy for regulating gene translation [32,33]. In addition, the maximum number of chromosomally integrated genetic parts is generally limited by the maximum number of available selective markers based on current integration systems. Introducing recombinase to evict selective markers, leading to a marker-free chromosomal integration system, may help to overcome the bottleneck caused by the maximum number available for DNA fragment integration [34]. Moreover, the chromosomal integration expression level is usually lower than plasmid expression because of the lower copy number. To enhance integration expression levels, gene expression regulatory elements, such as promoters and RBS sequences, need to be used in various combinations. Genome-wide Editing and Regulating Tools The natural competence of B. subtilis for linear DNA absorption and its high homology recombination efficiency make it possible to engineer the genome of B. subtilis. Despite the development of homology recombination-based gene deletion, insertion, and nucleotide substitution, simultaneous large-scale editing of multiple genes is still challenging. The CRISPR-Cas genome editing system has revolutionized the field of genome editing and has been successfully applied to B. subtilis [3,35–38]. Two types of CRISPR-Cas9-based B. subtilis genome editing methods have been developed: expressing Cas9 nuclease and single guide RNAs (sgRNAs) via a plasmid expression system, and chromosomally expressing Trends in Biotechnology, Month Year, Vol. xx, No. yy

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Cas9 nuclease and sgRNAs [35–38]. The CRISPR-Cas9-based genome editing approach was successfully extended to engineering undomesticated and rhizospheric B. subtilis, overcoming difficulties associated with its low genome engineering efficiency [39,40]. Replacing Cas9 nuclease with dCas9 nuclease enabled the development of genome-wide gene expression repression by CRISPR interference (CRISPRi), which has been demonstrated by systematically investigating the effects of knockdown of essential genes on specific cell growth rates, survival rates during the stationary phase, and morphological changes [3,36]. Currently used CRISPR systems in B. subtilis focus on gene deletion or repression. However, there is room to develop more versatile genome editing and modulation tools. For example, bifunctional or trifunctional dynamic pathway control methods were established in E. coli or S. cerevisiae by using CRISPRi and antisense RNAs in a combinatorial manner or by using orthogonal CRISPRi, CRISPR-based transcriptional activation (CRISPRa) and the gene deletion (CRISPRd) system [41,42]. Therefore, development of multifunctional dynamic pathway regulation tools for B. subtilis should in principle be feasible and may be necessary for enhancing pathway engineering efficiency further. Moreover, development of a highly precise base-editing approach with low off-target effects, based on Cas9 and cytidine deaminase fusion, may significantly enhance genome editing efficiency without the need to introduce DNA templates [43,44]. Global transcriptional regulators are of great importance for B. subtilis to coordinate its gene expression network for adapting to dynamic environmental changes [43,44]. The most important global transcriptional regulators in B. subtilis for controlling carbon and nitrogen metabolism are CcpA and CodY, respectively [45]. Therefore, changing the expression level or DNA binding activity of CcpA and CodY can globally perturb the carbon and nitrogen metabolism of B. subtilis. These global transcriptional regulators have been engineered to enhance heterologous protein expression levels by expressing mutant variants of both CodY and CcpA, demonstrating an efficient approach for genome-wide engineering [45]. The Subtiwiki database, one of the most complete and comprehensive databases for B. subtilis, provides integrative information about transcriptional regulatory networks and protein–protein interaction networks, which is a powerful tool for guiding transcriptional regulatory network engineering [46].

Progress in B. subtilis Chassis Development and Comparison with E. coli and S. cerevisiae Advances in B. subtilis Chassis Construction Constructing a chassis cell is foundational for understanding essential cellular functions and interactions, and a chassis cell also provides a potential advanced host for improved heterologous protein and biochemical production. Current developments in B. subtilis chassis construction focus on the following three aspects: large-scale genome reduction; elimination of carbon catabolite repression for multiple carbon source co-utilization; and cellular component engineering for specific product synthesis (Figure 2). In this section, we summarize the progress of the B. subtilis chassis in regard to the three aspects stated above, and discuss the gaps between current progress and the ultimate goal for chassis cell construction. To identify fundamental cellular processes and essential functions, and to divert more cellular resources and energy from dispensable cellular processes to the synthesis of target products, genome minimization has been investigated thought de novo synthesis of the genome based on a design-build-test cycle and large-scale genome reduction based on model microorganisms. A representative example of genome minimization via de novo synthesis is the synthetic 6

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nagP-L

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Genome reducon by marker-free gene deleon

B. sublis chassis with 36% genome reducon

CcpA Carbon catabolite repression cre sites

B. sublis wild-type strain

Removal of carbon catabolite repression OUT

IN Engineering cell membrane and protein secreon pathways

B. sublis chassis for mulple carbon source co-ulizaon

B. sublis chassis for specific biochemical or enzyme producon

Figure 2. Construction of a B. subtilis Chassis. A B. subtilis chassis was attained via genome reduction, carbon catabolite repression removal, cell membrane engineering, and protein secretion pathway engineering.

genome of Mycoplasma mycoides JCVI-syn3.0, which is currently the smallest synthetic genome (531 kb) that enables independent bacterium replication [49]. Large-scale genome reduction has been used to minimize the genomes of laboratory and industrially important model microorganisms, such as E. coli and B. subtilis [50,51]. Currently, the minimal genome size of B. subtilis has been reduced to 2.68 Mb with 1605 gene deletions, which is a deletion of 36.5% of the original genome [51] (Table 2). The reduced genome contains 18% genes with unknown functions, which is still far from the ultimate goal to understand all essential processes and build a chassis cell with only essential processes. Moreover, the reduced cell growth rate of B. subtilis with the current minimal genome raises another issue for its biotechnological application, which is the potential for decreased productivity of the targets. Therefore, the contributions of genes with unknown functions to the fundamental cellular processes need to be further investigated, and the growth defects of genome-minimized B. subtilis still need to be eliminated. Engineering chassis cells to efficiently co-utilize multiple carbon sources is important for industrial applications [52–57]. A potential application of co-utilizing multiple carbon sources is developing lignocellulosic hydrolysates as substrates for biochemical production for reducing biochemical production costs. Another application is using multiple carbon sources as substrates for balancing the multiple precursor supply for product biosynthesis in a modular fashion. Deleting the gene encoding CcpA – the major global transcriptional factor for regulating carbon catabolite repression – and selecting a mutant that restored its growth rate enabled broad carbon source utilization by B. subtilis [52]. Removing the carbon catabolite repression in the gluconate catabolic pathway enabled glucose and gluconate co-utilization by introducing point mutagenesis in cre-binding sites in promoter Pgnt and gene gntR sequences while constitutively expressing gntR [53]. Noticeably, the physiology of chassis cells that can co-utilize multiple carbon sources suggest a that central carbon metabolism may be perturbed by multiple carbon substrate input. Therefore, the effects of multiple carbon source co-utilization on metabolic flux and metabolite pool size need to be further elucidated. Moreover, to enhance carbon yield Trends in Biotechnology, Month Year, Vol. xx, No. yy

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Table 2. Comparison of Chassis Cells Developed from B. subtilis, E. coli, and S. cerevisiae Names

Features and limitations

Applications and guidance for B. subtilis chassis construction

B. subtilis MBG874, obtained from genome reduction

Features: (1) Deletion of 0.88 Mb genomic DNA, which is 20.7% of B. subtilis 168 genome Limitations: (1) Reduced growth rate (30%) compare with wild-type strain B. subtilis 168

Applications: (1) Unraveling essential cellular process (2) Increasing cellulase and protease production (1.7- and 2.5-fold, respectively)

[12]

B. subtilis PS38, obtained from genome reduction

Features: (1) Deletion of 1.54 Mb genomic DNA, which is 36.5% of B. subtilis 168 genome Limitations: (1) Increase of doubling time (36%) compared with B. subtilis 168

Applications: (1) Unraveling essential cellular processes (2) Potentially used for increasing heterologous proteins and biochemical production Guidance:

[43]

B. subtilis with reduced carbon catabolite repression for multiple carbon source co-utilization

Features: (1) Deletion of the gene encoding CcpA, the major global transcriptional factor for regulating carbon catabolite repression (2) DNA topoisomerase I (TopA) with mutation V44D or S478P. (3) Growth rate is comparable with wild type B. subtilis 168 Limitations: (1) Potential reorganization of central carbon metabolism with varied metabolite pool sizes, which may affect different biochemical production capabilities

Applications: (1) Biosynthesis of chemicals with mixture of multiple carbon source, such as lignocellulosic hydrolysates

[44]

B. subtilis that can co-utilize glucose and gluconate

Features: (1) Introducing point mutagenesis in cre-binding site of the coding sequence of promoter Pgnt and gene gntR to abolish carbon catabolite repression (2) Expressing gntR under the control of a constitutive promoter

Applications: (1) Gluconate inducible gene express system (2) Biosynthesis of biochemical with precursor from both glucose and gluconate

[45]

B. subtilis with cell membrane engineering

Features: (1) Overexpression of pgsA and clsA to strengthen the cardiolipin synthetic pathway to improve cardiolipin content in the membrane to enhance the functional expression of hyaluronan synthase (2) Repression of ftsZ expression for redistribution of cardiolipin along the lateral membrane Limitations: (1) Membrane engineering to modify cardiolipin content and distribution is only applicable for functional expression of hyaluronan synthase

Applications: (1) Facilitating functional expression of hyaluronan synthase of S. equisimilis for enhancing hyaluronic acid production

[52]

Features: (1) Deletion of 1.58 Mb genomic DNA, which is 35% of E. coli K-12 genome (2) No auxotrophy Limitations: (1) Reduced cell yield in minimal medium

Applications: (1) Provides guidance for understanding the essential cellular processes of E. coli (2) Used as a host and platform strain for metabolic engineering Guidance for B. subtilis: (1) Avoiding effects of genome reduction on reduced cell growth

[42]

Refs

B. subtilis

E. coli E. coli DGF-298, which was obtained from genome reduction

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Table 2. (continued) Names

Features and limitations

Applications and guidance for B. subtilis chassis construction

E. coli simultaneously utilizes glucose and glycerol

Features: (1) Blocks glucose catabolic pathway and alleviates carbon catabolite repression by deleting zwf and pgi genes (2) Deleted pykA, pykF, and gldA genes to improve intracellular phosphoenolpyruvic acid concentration Limitations: (1) Only generalized to product using glucose as C6 building block (2) Reduced growth rate

Applications: (1) Trehalose biosynthesis 200% improvement in yield Guidance for B. subtilis: (1) Providing strategies for alleviating carbon catabolite repression

[47]

E. coli that simultaneously utilizes glucose and acetate

Features: (1) Overexpressing native ackA and pta genes (2) pflB gene deleted Limitations: (1) Generally only applicable for products with both glucose and acetate as direct carbon precursor

Applications: (1) Isobutyl acetate production with increased carbon yield compared with using sole carbon source Guidance for B. subtilis: (1) Providing strategies for balancing precursor supply via modular pathway engineering

[49]

E. coli with nonoxidative glycolysis

Features: (1) Introducing phosphoketolase reaction, which enabled complete conversion of C6 or C5 sugar into acetylcoenzyme A without carbon loss Limitations: (1) Energy and redox cannot be generated by nonoxidative glycolysis

Applications: (1) Biosynthesis of acetyl-CoA derived products (2) CO2 fixation and other one-carbon assimilation Guidance for B. subtilis: (1) Applicable for refactoring acetylcoenzyme A synthetic pathway without carbon loss

[50]

E. coli with nonphosphorylative metabolism

Features: (1) Co-utilizing lignocellulosic sugars (D-xylose, L-arabinose, or D-galacturonate) into the important TCA cycle intermediate 2-ketoglutarate Limitations: (1) Inefficient co-utilization of sugars (2) By-product acetate accumulation

Applications: (1) Produces 1,4-butanediol with D-xylose, Larabinose or D-galacturonate as substrates Guidance for B. subtilis: (1) Applicable for constructing 2ketoglutarate derivate compound pathway with fewer steps and improved yield

[51]

E. coli with 57-codon genome (under construction)

Features: (1) Codons AGA, AGG, AGC, AGU, UUA, UUG, and UAG replaced by synonymous codons Limitations: (1) Doubling time is 1.5 fold of E. coli wild-type strain

Applications: (1) Provides guidance for synthetic genome design and construction (2) Provides codons AGA, AGG, AGC, AGU, UUA, UUG, and UAG for introducing nonnatural amino acids Guidance for B. subtilis: (1) Applicable for biocontainment (2) Avoiding effects of genome redesign on reduced doubling time

[55]

Features: (1) 6.5 chromosomes of S. cerevisiae (II, III, V, VI, IX-R, X, and XII) were designed and synthetized with stop codon recoding, loxPsym sites insertion, repeat elements deletion

Applications: (1) Facilitates understanding of chromosome structure and function (2) Enabling inducible evolution based on recombination between loxPsym sites

[56]

Refs

S. cerevisiae S. cerevisiae synthetic genome

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Table 2. (continued) Names

Features and limitations

Applications and guidance for B. subtilis chassis construction

Refs

Guidance for B. subtilis: (1) Providing methods for chromosome synthesis and assembly for reduced B. subtilis genome reduction (2) Introducing loxPsym sites in B. subtilis genome for inducible evolution S. cerevisiae with single-locus glycolysis

Features: (1) Entire 13 glycolytic genes were assembled into one DNA fragment (35 kb) and integrated into chromosome IX or V Limitations: (1) Growth rate of S. cerevisiae with single-locus glycolysis is 17% lower

Applications: (1) Glycolytic pathway swapping with a complete glycolysis genes from Saccharomyces kudriavzevii and a combinatory glycolysis consisting of S. cerevisiae, S. kudriavzevii, and Homo sapiens enzymes Guidance for B. subtilis: (1) Providing engineering strategies for engineering cellular properties in a modular manner

[59]

S. cerevisiae with improved protein secretion capacity

Features: (1) Mutant with 5-fold increase of protein secretion capacity, obtained by microfluidic screening of mutant library generated via UV mutagenesis Limitations: (1) Only a-amylase was used as an example, general applicability needs to be further demonstrated

Applications: (1) Improving a-amylase production (2) Providing engineering targets for enhancing protein secretion capacity Guidance for B. subtilis: (1) Providing high-throughput screening strategies for improving protein secretion capacity

[54]

S. cerevisiae that coutilizes glucose and xylose efficiently

Features: (1) Multiple integrations of RuXylA (2) Overexpression of XKS1, TAL1, TKL1, RPE1, RKI1, and MGT transporter encoding gene (3) Deletion of PHO13 GRE3 (4) Evolved on xylose and hydrolysates

Applications: (1) Ethanol production using lignocellulosic hydrolysates as substrates Guidance for B. subtilis: (1) Providing strategies for improving efficiency of glucose and xylose coutilization

[48]

from multiple carbon sources, nonoxidative glycolysis or the phosphoketolase pathway, which generates acetyl coenzyme A from phosphosugars without carbon loss, may be introduced [58,59]. In addition to the above-mentioned general B. subtilis chassis, B. subtilis chassis for specific product synthesis have also been developed. For instance, to enhance hyaluronic acid production, pgsA and clsA were overexpressed while repressing ftsZ expression to enhance the cardiolipin synthetic pathway, which regulates cardiolipin content in the membrane and redistributes cardiolipin along the lateral membrane. This approach enhances hyaluronan production by improving functional expression of hyaluronan synthase from Streptococcus equisimilis [60]. Protein secretion pathways can also be engineered for heterologous protein expression. However, this engineering is only possible on a case-by-case basis because of the incomplete understanding of the protein secretion mechanism [24]. Therefore, generating efficient chassis cells with improved heterologous protein synthesis and secretion capabilities may require techniques such as model-based engineering target prediction; high-throughput microfluidic screening could also be a promising direction [61,62]. 10

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Trends in Chassis Establishment in E. coli and S. cerevisiae E. coli and S. cerevisiae are prokaryotic and eukaryotic model microorganisms that have seen more systematic investigation on chassis construction, so they can provide important guidance for B. subtilis chassis construction. For example, advances in efficient DNA synthesis and large DNA fragment assembly have enabled de novo synthesis of chromosomes of E. coli and S. cerevisiae [63,64]. CRISPR-based genome editing methods provide powerful tools for efficient multiple target engineering, which have been applied in chassis establishment for E. coli and S. cerevisiae [65]. E. coli and S. cerevisiae chassis construction, fueled by improved DNA synthesis and manipulation capabilities, will ideally inspire further advances in B. subtilis chassis construction. Table 2 compares the guidance and applicability of E. coli and S. cerevisiae chassis construction in B. subtilis and shows some common issues faced in using the chassis in biotechnological applications. The E. coli synthetic genome is being de novo synthesized and assembled with 57 codons, replacing codons AGA, AGG, AGC, AGU, UUA, UUG, and UAG with synonymous codons. Once completed, E. coli with 57 codons can be used to introduce non-natural amino acids for biocontainment and other biotechnological applications [63]. By contrast, 6.5 synthetic chromosomes (II, III, V, VI, IX-R, X, and XII) of the 16 chromosomes of S. cerevisiae have been designed and synthesized with stop codon recoding, inserting loxPsyms sites, and deleting repeat elements, thus facilitating a better understanding of chromosome structure and function. Moreover, inserting loxPsym sites has facilitated inducible evolution based on recombination between loxPsym sites [64]. S. cerevisiae with single-locus glycolysis was constructed using successive gene assembly, DNA fragment integration into the genome, and gene deletion aided by CRISPR-based genome editing [65]. An entire set of 13 glycolytic genes was assembled into one DNA fragment (35 kb) and integrated into chromosome IX or V. Glycolytic pathway swapping was conducted to demonstrate the convenience of engineering glycolysis in a modular and combinatorial manner with glycolysis genes from different organisms. However, decreased growth rates were observed in almost all chassis cells, which is an undesirable property if these cells are to be used in practical applications. Therefore, it is necessary to develop techniques to restore the cell growth of chassis cells and further improve such cell growth rates in order to reduce the fermentation period, which will enable convenient use of chassis cells in scientific and industrial applications.

Future Perspectives Integrating Synthetic Regulatory Elements for Genetic Circuit Construction and Application High-throughput DNA sequencing and high-throughput screening platforms enable the efficient identification and characterization of genetic regulatory elements, so these obstacles should not pose a significant limitation for synthetic toolbox development. Unfortunately, B. subtilis lacks a platform that integrates already-identified genetic regulatory elements carrying key information, which may guide the selection of genetic regulatory elements for genetic circuit design or synthetic pathway fine tuning. For the purpose of establishing a comparative and dynamic pattern traceable database for genetic regulatory elements, uniform benchmarks should first be selected for various elements such as promoters, RBS sequences, and terminators. Next, the strength of already established genetic regulatory elements should be calibrated and categorized. Finally, newly characterized genetic regulatory elements should be updated into the database, based on validated strength and dynamic patterns (Figure 3, Key Figure). Identified metabolite-responsive transcriptional factors and riboswitches are increasingly expanding the ability to construct various genetic circuits and apply such circuits in the Trends in Biotechnology, Month Year, Vol. xx, No. yy

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Key Figure

Recent Advances and Perspectives of Bacillus subtilis Synthetic Biology Toolboxes and Chassis Development Advances of synthec biology toolboxes and chassis cells

Genome minimizaon Mulple carbon source co-ulizaon

Promoter RBS Proteolysis tags ofo

Protein secreon pathways or cell membrane engineering

CRISPR-based genome eding

Gene assembly

Chassis cell

Perspecves of synthec biology toolboxes and chassis cells AAGGCCTT GGCCTTAA GCTAGCTA CTGAAGTC GCTAGCTA TTGGAACC

Integrave database of genec regulatory elements

Target gene NagC gfp

OUTPUT1 OUTPUT2 OUTPUT3

NagC binding site

Genec circuit for dynamic control

x P X or P

x

Algorithm NOT AND OR nagC

Chassis cell with improved growth rate X or P

INPUT1 INPUT2 INPUT3

B. sublis for basic and applied synthec biology research

Time

P

Time

Decoupling cell growth and producon

Figure 3. Progress of B. subtilis synthetic regulatory elements facilitates the precise control of key gene expression and of genetic circuit optimization. Precise genome editing and genome-wide gene expression regulation were realized by modular plasmid development, CRISPR-Cas9 genome editing system and CRISPRi gene expression repression system. B. subtilis chassis cells were obtained via genome minimization, carbon catabolite repression removal, cell membrane engineering, and protein secretion pathway engineering. In the future, integrating information about various genetic regulatory elements and application of genetic circuit will confer dynamic control ability to engineered B. subtilis to enhance its use in basic and applied synthetic biology. An advanced B. subtilis chassis with improved growth rate and decoupled growth and production still needs to be developed. Abbreviations: RBS, ribosome-binding site.

dynamic control of metabolic pathways [66]. Key to establishing genetic circuit with a desired dynamic strength and metabolite-responsive range is the use of proper genetic regulatory elements [67,68]. Construction and application of promising genetic circuits are currently underexploited in B. subtilis. For instance, target product synthetic capacity can be coupled with cell growth by introducing a target product responsive genetic circuit to regulate the expression of key cell growth genes, and selectively confer growth advantages to cells with high productivity. Therefore, mutants with beneficial genetic mutation may be conveniently selected based on cell growth differences. Moreover, conferring growth advantages to high producers may enrich a high-production subpopulation in a heterogeneous population during fermentation for enhanced production [69,70]. Further development and application of 12

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genetic circuits with advanced properties, such as multiple-input-responsive features, may improve engineered cells to maintain constant cell growth and production during changes in industrial conditions [71]. Developing Advanced Chassis for Scientific and Industrial Applications So far, the genome-reduced B. subtilis chassis has not been widely used in biotechnological applications. One major reason is that the cell growth rates of almost all constructed chassis cells decrease, thus prolonging cell cultivation periods and decreasing the productivity of target products. Is it possible to develop a B. subtilis chassis cell that grows faster than wild-type B. subtilis? Moreover, introducing heterologous pathways usually decreases growth rates, caused mainly by metabolic burden due to heterologous gene expression and non-native compound synthesis. Enhancing the growth rate of B. subtilis is theoretically possible, because B. subtilis is in a suboptimal growth condition due to the investment of valuable cellular resources to promote the expression of enzymes that counter potential environmental changes at the expense of growth optimality [72]. Moreover, central metabolic enzymes are expressed in excess amounts during growth with glucose as the carbon source, depleting cellular resources and potentially reducing cell growth [73]. Therefore, reallocating cellular resources from unnecessary enzymes that are only used as a response for environmental perturbation and excess central metabolic enzyme expression toward important pathways for cell growth will likely enhance the cell growth rate. In addition, laboratory-adaptive evolution in combination with microfluidic-based high-throughput screening may be implemented to efficiently identify evolved strains with improved growth rates [74]. Once established, a chassis cell with enhanced growth can be used as a production host for target compound production with improved productivity.

Outstanding Questions Large numbers of native and synthetic genetic regulatory elements in B. subtilis have been characterized. How can we integrate already identified genetic regulatory elements with key information to guide genetic regulatory element selection for fine tuning synthetic pathways? How can we make full use of the genetic regulatory elements for genetic circuit construction to improve production of engineered B. subtilis via dynamic control? B. subtilis chassis cells developed via large-scale genome reduction have reduced growth rates compared to wild-type B. subtilis, which hampers their scientific and industrial application. Is it possible to develop a B. subtilis chassis cell that grows faster than wild-type B. subtilis? How can we improve the cell growth rate of B. subtilis? How can we efficiently decouple cell growth from biosynthesis of target products in order to divert more cellular energy and resources from cell growth to production?

To alleviate the metabolic burden on chassis cells, two strategies can be used: decoupling cell growth and production via dynamic control to reduce competition for precursors shared between cell growth and product synthesis, and introducing economic pathways with less cellular resource consumption based on protein cost calculations. First, cell growth may be decoupled from production via genetic circuits that regulate the expression of key cell growth genes, such as the genes involved in central carbon metabolism [75,76]. Second, introducing pathways with reduced protein costs may enhance the economy of cellular resource utilization with minimal effect of the heterologous pathways on cell growth [77,78].

Concluding Remarks Significant advances in synthetic biology toolsets and chassis of B. subtilis have been made, but further advances are still possible (see Outstanding Questions). For example, an integrative database of synthetic biology elements in B. subtilis needs to be established as a platform to guide the selection of proper tools required for the construction of genetic circuits for dynamic control. Additionally, further development of an advanced B. subtilis chassis with an improved growth rate will boost its potential for scientific and industrial applications. Progress in synthetic biology toolboxes for B. subtilis may further facilitate efficient B. subtilis chassis construction. Development of an advanced B. subtilis chassis cell may in turn improve the characterization of regulatory elements and expand the biotechnology application potential of B. subtilis. Acknowledgments We are grateful to Dr. Elad Noor from ETH Zurich for constructive advice on the manuscript. This work is financially supported by the National Natural Science Foundation of China (31600068, 31622001, 31671845, 21676119), the Natural Science Foundation of Jiangsu Province (BK20160176), the China Postdoctoral Science Foundation

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(2016M600363, 2017T100327), the 111 Project (No. 111-2-06), the Fundamental Research Funds for the Central Universities (JUSRP11725), National First-class Discipline Program of Light Industry Technology and Engineering (LITE2018-16).

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