Peng Jin

Small RNA regulators in bacteria: powerful tools for metabolic engineering and synthetic biology

Small RNAs, a large class of ancient posttranscriptional regulators, have recently attracted considerable attention. A plethora of small RNAs has been identified and characterized, many of which belong to the major small noncoding RNA (sRNA) or riboswitch families. It has become increasingly clear that most small RNAs play critical regulatory roles in many processes and are, therefore, considered to be powerful tools for metabolic engineering and synthetic biology. In this review, we describe recent achievements in the identification, characterization, and application of small RNAs. We give particular attention to advances in the design and synthesis of novel sRNAs and riboswitches for metabolic engineering. In addition, a novel strategy for hierarchical control of global metabolic pathways is proposed.

Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168

Low-molecular-weight hyaluronan (LMW-HA) has attracted much attention because of its many potential applications. Here, we efficiently produced specific LMW-HAs from sucrose in Bacillus subtilis. By coexpressing the identified committed genes (tuaD, gtaB, glmU, glmM, and glmS) and downregulating the glycolytic pathway, HA production was significantly increased from 1.01gL(-1) to 3.16gL(-1), with a molecular weight range of 1.40×10(6)-1.83×10(6)Da. When leech hyaluronidase was actively expressed after N-terminal engineering (1.62×10(6)UmL(-1)), the production of HA was substantially increased from 5.96gL(-1) to 19.38gL(-1). The level of hyaluronidase was rationally regulated with a ribosome-binding site engineering strategy, allowing the production of LMW-HAs with a molecular weight range of 2.20×10(3)-1.42×10(6)Da. Our results confirm that this strategy for the controllable expression of hyaluronidase, together with the optimization of the HA synthetic pathway, effectively produces specific LMW-HAs, and could also be used to produce other LMW polysaccharides.

Efficient biosynthesis of polysaccharides chondroitin and heparosan by metabolically engineered Bacillus subtilis

Chondroitin and heparosan, important polysaccharides and key precursors of chondroitin sulfate and heparin/heparan sulfate, have drawn much attention due to their wide applications in many aspects. In this study, we designed two independent synthetic pathways of chondroitin and heparosan in food-grade Bacillus subtilis, integrating critical synthases genes derived from Escherichia coli into B. subtilis genome. By RT-PCR analysis, we confirmed that synthases genes transcripted an integral mRNA chain, suggesting co-expression. In shaken flask, chondroitin and heparosan were produced at a level of 1.83gL(-1) and 1.71gL(-1), respectively. Since B. subtilis endogenous tuaD gene encodes the limiting factor of biosynthesis, overexpressing tuaD resulted in enhanced chondroitin and heparosan titers, namely 2.54gL(-1) and 2.65gL(-1). Moreover, production reached the highest peaks of 5.22gL(-1) and 5.82gL(-1) in 3-L fed-batch fermentation, respectively, allowed to double the production that in shaken flask. The weight-average molecular weight of chondroitin and heparosan from B. subtilis E168C/pP43-D and E168H/pP43-D were 114.07 and 67.70kDa, respectively. This work provided alternative safer synthetic pathways for metabolic engineering of chondroitin and heparosan in B. subtilis and a useful approach for enhancing production, which can be optimized for further improvement.

High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers

Hyaluronidases (HAases), particularly leech HAases, have attracted intense attention due to their broad applications in medical treatments and great potential for the enzymatic production of hyaluronan oligosaccharides. However, little is known about this third interesting family of HAases. Here, we applied the random amplification of cDNA ends polymerase chain reaction (RACE-PCR) approach to identify the first leech HAase-encoding gene. By combining protein engineering and high-density culture, we achieved high-level production (8.42 × 105 U ml−1) in the yeast Pichia pastoris secretory expression system. Compared with the commercial bovine testicular HAase, the recombinant leech HAase exhibited superior enzymatic properties. Furthermore, analysis of the hydrolytic process suggested that this novel enzyme adopts a nonprocessive endolytic mode, yielding a narrow-spectrum of specific HA oligosaccharides with different incubation times. Large-scale production of this novel leech HAase will not only greatly promote medical applications but also facilitate the enzymatic production of specific HA oligosaccharides.

Enzymatic production of specifically distributed hyaluronan oligosaccharides

High-molecular-mass hyaluronan (HA) was controllably depolymerized in pure aqueous solution with recombinant leech hyaluronidase (HAase). The HAase concentration per unit HA and hydrolysis time played important roles in molecular mass distribution. By modulating the concentrations of HAase and controlling the hydrolysis time, any molar-mass-defined HA oligomers could be efficiently and specifically produced on a large scale (40 g/L), such as HA oligosaccharides with weight-average molar mass of 4000, 10,000, and 30,000Da and end hydrolysates containing only HA6 and HA4. High performance liquid chromatography-size exclusion chromatography, polyacrylamide gel electrophoresis, capillary zone electrophoresis, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry confirmed low polydispersity of the produced molar-mass-defined HA oligosaccharides. Therefore, large-scale production of defined HA oligosaccharides with narrow molecular mass distribution will significantly promote progress in related research and its potential applications.

Directed evolution combined with synthetic biology strategies expedite semi-rational engineering of genes and genomes

Owing to our limited understanding of the relationship between sequence and function and the interaction between intracellular pathways and regulatory systems, the rational design of enzyme-coding genes and de novo assembly of a brand-new artificial genome for a desired functionality or phenotype are difficult to achieve. As an alternative approach, directed evolution has been widely used to engineer genomes and enzyme-coding genes. In particular, significant developments toward DNA synthesis, DNA assembly (in vitro or in vivo), recombination-mediated genetic engineering, and high-throughput screening techniques in the field of synthetic biology have been matured and widely adopted, enabling rapid semi-rational genome engineering to generate variants with desired properties. In this commentary, these novel tools and their corresponding applications in the directed evolution of genomes and enzymes are discussed. Moreover, the strategies for genome engineering and rapid in vitro enzyme evolution are also proposed.

DATEL: A Scarless and Sequence-Independent DNA Assembly Method Using Thermostable Exonucleases and Ligase

DNA assembly is a pivotal technique in synthetic biology. Here, we report a scarless and sequence-independent DNA assembly method using thermal exonucleases (Taq and Pfu DNA polymerases) and Taq DNA ligase (DATEL). Under the optimized conditions, DATEL allows rapid assembly of 2-10 DNA fragments (1-2 h) with high accuracy (between 74 and 100%). Owing to the simple operation system with denaturation-annealing-cleavage-ligation temperature cycles in one tube, DATEL is expected to be a desirable choice for both manual and automated high-throughput assembly of DNA fragments, which will greatly facilitate the rapid progress of synthetic biology and metabolic engineering.

DNA Assembly with the DATEL Method

Simple and reliable DNA assembly methods have become a critical technique in synthetic biology. Here, we present a protocol of the recently developed DATEL (scarless and sequence-independent DNA assembly method using thermostable exonuclease and ligase) method for the construction of genetic circuits and biological pathways from multiple DNA parts in one tube. DATEL is expected to be an applicable choice for both manual and automated high-throughput assembly of DNA fragments, which will greatly facilitate the rapid progress of synthetic biology and metabolic engineering.

Combinatorial Evolution of DNA with RECODE

In past decades, DNA engineering protocols have led to the rapid development of synthetic biology. To engineer the natural proteins, many directed evolution methods based on molecular biology have been presented for generating genetic diversity or obtaining specific properties. Here, we provide a simple (PCR operation), efficient (larger amount of products), and powerful (multiple point mutations, deletions, insertions, and combinatorial multipoint mutagenesis) RECODE method, which is capable of reediting the target DNA flexibly to restructure regulatory regions and remodel enzymes by using the combined function of the thermostable DNA polymerase and DNA ligase in one pot. RECODE is expected to be an applicable choice to create diverse mutant libraries for rapid evolution and optimization of enzymes and synthetic pathways.

Scarless assembly of unphosphorylated DNA fragments with a simplified DATEL method

ABSTRACT Efficient assembly of multiple DNA fragments is a pivotal technology for synthetic biology. A scarless and sequence-independent DNA assembly method (DATEL) using thermal exonucleases has been developed recently. Here, we present a simplified DATEL (sDATEL) for efficient assembly of unphosphorylated DNA fragments with low cost. The sDATEL method is only dependent on Taq DNA polymerase and Taq DNA ligase. After optimizing the committed parameters of the reaction system such as pH and the concentration of Mg2+ and NAD+, the assembly efficiency was increased by 32-fold. To further improve the assembly capacity, the number of thermal cycles was optimized, resulting in successful assembly 4 unphosphorylated DNA fragments with an accuracy of 75%. sDATEL could be a desirable method for routine manual and automated assembly.