Bacterial genome reduction for optimal chassis of synthetic biology: a review.

Bacteria with streamlined genomes, that harbor full functional genes for essential metabolic networks, are able to synthesize the desired products more effectively and thus have advantages as production platforms in industrial applications. To obtain streamlined chassis genomes, a large amount of effort has been made to reduce existing bacterial genomes. This work falls into two categories: rational and random reduction. The identification of essential gene sets and the emergence of various genome-deletion techniques have greatly promoted genome reduction in many bacteria over the past few decades. Some of the constructed genomes possessed desirable properties for industrial applications, such as: increased genome stability, transformation capacity, cell growth, and biomaterial productivity. The decreased growth and perturbations in physiological phenotype of some genome-reduced strains may limit their applications as optimized cell factories. This review presents an assessment of the advancements made to date in bacterial genome reduction to construct optimal chassis for synthetic biology, including: the identification of essential gene sets, the genome-deletion techniques, the properties and industrial applications of artificially streamlined genomes, the obstacles encountered in constructing reduced genomes, and the future perspectives.

Biodegradation of polyurethane by the microbial consortia enriched from landfill.

Polyurethanes (PU) are one of the most used categories of plastics and have become a significant source of environmental pollutants. Degrading the refractory PU wastes using environmentally friendly strategies is in high demand. In this study, three microbial consortia from the landfill leachate were enriched using PU powder as the sole carbon source. The consortia efficiently degraded polyester PU film and accumulated high biomass within 1 week. Scanning electron microscopy, Fourier transform infrared spectroscopy, and contact angle analyses showed significant physical and chemical changes to the PU film after incubating with the consortia for 48 h. In addition, the degradation products adipic acid and butanediol were detected by high-performance liquid chromatography in the supernatant of the consortia. Microbial composition and extracellular enzyme analyses revealed that the consortia can secrete esterase and urease, which were potentially involved in the degradation of PU. The dominant microbes in the consortia changed when continuously passaged for 50 generations of growth on the PU films. This work demonstrates the potential use of microbial consortia in the biodegradation of PU wastes. KEY POINTS: • Microbial consortia enriched from landfill leachate degraded polyurethane film. • Consortia reached high biomass within 1 week using polyurethane film as the sole carbon source. • The consortia secreted potential polyurethane-degrading enzymes.

Unique Raoultella species isolated from petroleum contaminated soil degrades polystyrene and polyethylene.

Polyolefin plastics, such as polyethylene (PE) and polystyrene (PS), are the most widely used synthetic plastics in our daily life. However, the chemical structure of polyolefin plastics is composed of carbon-carbon (C-C) bonds, which is extremely stable and makes polyolefin plastics recalcitrant to degradation. The growing accumulation of plastic waste has caused serious environmental pollution and has become a global environmental concern. In this study, we isolated a unique Raoultella sp. DY2415 strain from petroleum-contaminated soil that can degrade PE and PS film. After 60 d of incubation with strain DY2415, the weight of the UV-irradiated PE (UVPE) film and PS film decreased by 8% and 2%, respectively. Apparent microbial colonization and holes on the surface of the films were observed by scanning electron microscopy (SEM). Furthermore, the Fourier transform infrared spectrometer (FTIR) results showed that new oxygen-containing functional groups such as -OH and -CO were introduced into the polyolefin molecular structure. Potential enzymes that may be involved in the biodegradation of polyolefin plastics were analyzed. These results demonstrate that Raoultella sp. DY2415 has the ability to degrade polyolefin plastics and provide a basis for further investigating the biodegradation mechanism.

Valorization of Polyethylene Terephthalate to Muconic Acid by Engineering Pseudomonas Putida.

Plastic waste is rapidly accumulating in the environment and becoming a huge global challenge. Many studies have highlighted the role of microbial metabolic engineering for the valorization of polyethylene terephthalate (PET) waste. In this study, we proposed a new conceptual scheme for upcycling of PET. We constructed a multifunctional Pseudomonas putida KT2440 to simultaneously secrete PET hydrolase LCC, a leaf-branch compost cutinase, and synthesize muconic acid (MA) using the PET hydrolysate. The final product MA and extracellular LCC can be separated from the supernatant of the culture by ultrafiltration, and the latter was used for the next round of PET hydrolysis. A total of 0.50 g MA was produced from 1 g PET in each cycle of the whole biological processes, reaching 68% of the theoretical conversion. This new conceptual scheme for the valorization of PET waste should have advantages over existing PET upcycling schemes and provides new ideas for the utilization of other macromolecular resources that are difficult to decompose, such as lignin.

Identification of phage recombinase function unit in genus Corynebacterium.

Phage recombinase function unit (PRFU) plays a key role in the life cycle of phage. Repurposing this system such as lambda-Redαß or Rac-RecET for recombineering has gained success in Escherichia coli. Previous studies have showed that most PRFUs only worked well in its native hosts but poorly in the distant species. Thus, identification of new PRFUs in specific species is necessary for the development of its corresponding genetic engineering tools. Here, we present a thorough study of PRFUs in the genomes of genus Corynebacterium. We first used a database to database searching method to facilitate accurate prediction of novel PRFUs in 423 genomes. A total number of 60 sets of unique PRFUs were identified and divided into 8 types based on evolution affinities. Recombineering ability of the 8 representative PRFUs was experimentally verified in the Corynebacterium glutamicum ATCC 13032 strain. In particular, PRFU from C. aurimucosum achieved highest efficiency in both ssDNA and dsDNA mediated recombineering, which is expected to greatly facilitate genome engineering in genus Corynebacterium. These results will provide new insights for the study and application of PRFUs. KEY POINTS: • First report of bioinformatic mining and systematic analysis of Phage recombinase function unit (PRFU) in Corynebacterium genomes. • Recombineering ability of the representative PRFUs was experimentally verified in Corynebacterium glutamicum ATCC 13032 strain. • PRFU with the highest recombineering efficiency at 10-2 magnitude was identified from Corynebacterium aurimucosum.

The phage T4 DNA ligase in vivo improves the survival-coupled bacterial mutagenesis.

BACKGROUND: Microbial mutagenesis is an important avenue to acquire microbial strains with desirable traits for industry application. However, mutagens either chemical or physical used often leads narrow library pool due to high lethal rate. The T4 DNA ligase is one of the most widely utilized enzymes in modern molecular biology. Its contribution to repair chromosomal DNA damages, therefore cell survival during mutagenesis will be discussed. RESULTS: Expression of T4 DNA ligase in vivo could substantially increase cell survival to ionizing radiation in multiple species. A T4 mediated survival-coupled mutagenesis approach was proposed. When polyhydroxybutyrate (PHB)-producing E. coli with T4 DNA ligase expressed in vivo was subjected to ionizing radiation, mutants with improved PHB production were acquired quickly owing to a large viable mutant library generated. Draft genome sequence analysis showed that the mutants obtained possess not only single nucleotide variation (SNV) but also DNA fragment deletion, indicating that T4 DNA ligase in vivo may contribute to the repair of DNA double strand breaks. CONCLUSIONS: Expression of T4 DNA ligase in vivo could notably enhance microbial survival to excess chromosomal damages caused by various mutagens. Potential application of T4 DNA ligase in microbial mutagenesis was explored by mutating and screening PHB producing E. coli XLPHB strain. When applied to atmospheric and room temperature plasma (ARTP) microbial mutagenesis, large survival pool was obtained. Mutants available for subsequent screening for desirable features. The use of T4 DNA ligase we were able to quickly improve the PHB production by generating a larger viable mutants pool. This method is a universal strategy can be employed in wide range of bacteria. It indicated that traditional random mutagenesis became more powerful in combine with modern genetic molecular biology and has exciting prospect.

Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system.

BACKGROUND: Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is a recently developed powerful tool for gene regulation. In Escherichia coli, the type I CRISPR system expressed endogenously shall be easy for internal regulation without causing metabolic burden in compared with the widely used type II system, which expressed dCas9 as an additional plasmid. RESULTS: By knocking out cas3 and activating the expression of CRISPR-associated complex for antiviral defense (Cascade), we constructed a native CRISPRi system in E. coli. Downregulation of the target gene from 6 to 82% was demonstrated using green fluorescent protein. Regulation of the citrate synthase gene (gltA) in the TCA cycle affected host metabolism. The effect of metabolic flux regulation was demonstrated by the poly-3-hydroxbutyrate (PHB) accumulation in vivo. CONCLUSION: By regulating native gltA in E. coli using an engineered endogenous type I-E CRISPR system, we redirected metabolic flux from the central metabolic pathway to the PHB synthesis pathway. This study demonstrated that the endogenous type I-E CRISPR-Cas system is an easy and effective method for regulating internal metabolic pathways, which is useful for product synthesis.

Novel technologies provide more engineering strategies for amino acid-producing microorganisms.

Traditionally, amino acid-producing strains were obtained by random mutagenesis and subsequent selection. With the development of genetic and metabolic engineering techniques, various microorganisms with high amino acid production yields are now constructed by rational design of targeted biosynthetic pathways. Recently, novel technologies derived from systems and synthetic biology have emerged and open a new promising avenue towards the engineering of amino acid production microorganisms. In this review, these approaches, including rational engineering of rate-limiting enzymes, real-time sensing of end-products, pathway optimization on the chromosome, transcription factor-mediated strain improvement, and metabolic modeling and flux analysis, were summarized with regard to their application in microbial amino acid production.

Construction of an L-serine producing Escherichia coli via metabolic engineering.

L-Serine is a nonessential amino acid, but plays a crucial role as a building block for cell growth. Currently, L-serine production is mainly dependent on enzymatic or cellular conversion. In this study, we constructed a recombinant Escherichia coli that can fermentatively produce L-serine from glucose. To accumulate L-serine, sdaA encoding the L-serine dehydratase, iclR encoding the isocitrate lyase regulator, and arcA encoding the aerobic respiration control protein were deleted in turn. In batch fermentation, the engineered E. coli strain YF-5 exhibited obvious L-serine accumulation but poor cell growth. To restore cell growth, aceB encoding the malate synthase was knocked out, and the engineered strain was then transformed with plasmid that overexpressed serA (FR) , serB, and serC genes. The resulting strain YF-7 produced 4.5 g/L L-serine in batch cultivation and 8.34 g/L L-serine in fed-batch cultivation.

In Vivo DNA Assembly Using the PEDA Method.

Simple and efficient DNA assembly methods have been widely used in synthetic biology. Here, we provide the protocol for the recently developed PEDA (phage enzyme-assisted in vivo DNA assembly) method for direct in vivo assembly of individual DNA parts in multiple microorganisms, such as Escherichia coli, Ralstonia eutropha, Pseudomonas putida, Lactobacillus plantarum, and Yarrowia lipolytica. PEDA allows in vivo assembly of DNA fragments with homologous sequences as short as 5 bp, and the efficiency is comparable to the prevailing in vitro DNA assembly, which will broadly boost the rapid progress of synthetic biology.

[Preface to the special issue: biotechnology of plastic waste degradation and valorization].

Synthetic plastics have been widely used in various fields of the national economy and are the pillar industry. However, irregular production, plastic product use, and plastic waste piling have caused long-term accumulation in the environment, contributing considerably to the global solid waste stream and environmental plastic pollution, which has become a global problem to be solved. Biodegradation has recently emerged as a viable disposal method for a circular plastic economy and has become a thriving research area. In recent years, important breakthroughs have been made in the screening, isolation, and identification of plastic-degrading microorganisms/enzyme resources and their further engineering, which provide new ideas and solutions for treating microplastics in the environment and the closed-loop bio-recycling of waste plastics. On the other hand, the use of microorganisms (pure cultures or consortia) to further transform different plastic degradants into biodegradable plastics and other compounds with high added value is of great significance, promoting the development of a plastic recycling economy and reducing the carbon emission of plastics in their life cycle. We edited a Special Issue on the topic of "Biotechnology of Plastic Waste Degradation and Valorization", focusing on the researches progress in three aspects: Mining microbial and enzyme resources for plastic biodegradation, Design and engineering of plastic depolymerase, and biological high-value transformation of plastic degradants. In total, 16 papers have been collected in this issue including reviews, comments, and research articles, which provide reference and guidance for further development of plastic waste degradation and valorization biotechnology.

Upcycling of PET oligomers from chemical recycling processes to PHA by microbial co-cultivation.

Polyethylene terephthalate (PET) is the most widely consumed polyester plastic and can be recycled by many chemical processes, of which glycolysis is most cost-effective and commercially viable. However, PET glycolysis produces oligomers due to incomplete depolymerization, which are undesirable by-products and require proper disposal. In this study, the PET oligomers from chemical recycling processes were completely bio-depolymerized into monomers and then used for the biosynthesis of biodegradable plastics polyhydroxyalkanoates (PHA) by co-cultivation of two engineered microorganisms Escherichia coli BL21 (DE3)-LCCICCG and Pseudomonas putida KT2440-ΔRDt-ΔZP46C-M. E. coli BL21 (DE3)-LCCICCG was used to secrete the PET hydrolase LCCICCG into the medium to directly depolymerize PET oligomers. P. putida KT2440-ΔRDt-ΔZP46C-M that mastered the metabolism of aromatic compounds was engineered to accelerate the hydrolysis of intermediate products mono-2-(hydroxyethyl) terephthalate (MHET) by expressing IsMHETase, and biosynthesize PHA using ultimate products terephthalate and ethylene glycol depolymerized from the PET oligomers. The population ratios of the two microorganisms during the co-cultivation were characterized by fluorescent reporter system, and revealed the collaboration of the two microorganisms to bio-depolymerize and bioconversion of PET oligomers in a single process. This study provides a biological strategy for the upcycling of PET oligomers and promotes the plastic circular economy.

[Advances in biodegradation of polyolefin plastics].

Polyolefin plastics are a group of polymers with C-C backbone that have been widely used in various areas of daily life. Due to their stable chemical properties and poor biodegradability, polyolefin plastic waste continues to accumulate worldwide, causing serious environmental pollution and ecological crises. In recent years, biological degradation of polyolefin plastics has attracted considerable attention. The abundant microbial resources in the nature offer the possibility of biodegradation of polyolefin plastic waste, and microorganisms capable of degrading polyolefin have been reported. This review summarizes the research progress on the biodegradation microbial resources and the biodegradation mechanisms of polyolefin plastics, presents the current challenges in the biodegradation of polyolefin plastics, and provides an outlook on future research directions.

Heme biosensor-guided in vivo pathway optimization and directed evolution for efficient biosynthesis of heme.

BACKGROUND: Heme has attracted much attention because of its wide applications in medicine and food. The products of genes hemBCDEFY convert 5-aminolevulinic acid to protoporphyrin IX (PPIX; the immediate precursor of heme); protoporphyrin ferrochelatase (FECH) inserts Fe2+ into PPIX to generate heme. Biosynthesis of heme is limited by the need for optimized expression levels of multiple genes, complex regulatory mechanisms, and low enzymatic activity; these problems need to be overcome in metabolic engineering to improve heme synthesis. RESULTS: We report a heme biosensor-guided screening strategy using the heme-responsive protein HrtR to regulate tcR expression in Escherichia coli, providing a quantifiable link between the intracellular heme concentration and cell survival in selective conditions (i.e., the presence of tetracycline). This system was used for rapid enrichment screening of heme-producing strains from a library with random ribosome binding site (RBS) variants and from a FECH mutant library. Through up to four rounds of iterative evolution, strains with optimal RBS intensities for the combination of hemBCDEFY were screened; we obtained a PPIX titer of 160.8 mg/L, the highest yield yet reported in shaken-flask fermentation. A high-activity FECH variant was obtained from the saturation mutagenesis library. Fed-batch fermentation of strain SH20C, harboring the optimized hemBCDEFY and the FECH mutant, produced 127.6 mg/L of heme. CONCLUSION: We sequentially improved the multigene biosynthesis pathway of PPIX and performed in vivo directed evolution of FECH, based on a heme biosensor, which demonstrated the effectiveness of the heme biosensor-based pathway optimization strategy and broadens our understanding of the mechanism of heme synthesis.

A synthetic population-level oscillator in non-microfluidic environments.

Synthetic oscillators have become a research hotspot because of their complexity and importance. The construction and stable operation of oscillators in large-scale environments are important and challenging. Here, we introduce a synthetic population-level oscillator in Escherichia coli that operates stably during continuous culture in non-microfluidic environments without the addition of inducers or frequent dilution. Specifically, quorum-sensing components and protease regulating elements are employed, which form delayed negative feedback to trigger oscillation and accomplish the reset of signals through transcriptional and post-translational regulation. We test the circuit in devices with 1 mL, 50 mL, 400 mL of medium, and demonstrate that the circuit could maintain stable population-level oscillations. Finally, we explore potential applications of the circuit in regulating cellular morphology and metabolism. Our work contributes to the design and testing of synthetic biological clocks that function in large populations.

Reduction of the Bacterial Genome by Transposon-Mediated Random Deletion.

Genome reduction is an important strategy in synthetic biology for constructing functional chassis cells or minimal genomes. However, the limited knowledge of complex gene functions and interactions makes genome reduction by rational design encounter a bottleneck. Here, we present an iterative and random genome reduction method for Escherichia coli, named "transposon-mediated random deletion (TMRD)". TMRD generates random double-strand breaks (DSBs) in the genome by combining Tn5 transposition with the CRISPR/Cas9 system and allows genomic deletions of various sizes at random positions during DSB repair through the intracellular alternative end-joining mechanism. Using E. coli MG1655 as the original strain, a pool of cells with multiple random genomic deletions were obtained after five reduction cycles. The growth rates of the obtained cells were comparable to that of MG1655, while the electroporation efficiency increased by at least 2 magnitudes. TMRD can generate a small E. coli library carrying multiple and random genomic deletions while enriching the cells with environmental fitness in the population. TMRD has the potential to be widely applied in the construction of minimal genomes or chassis cells for metabolic engineering.

Identification of genome integration sites for developing a CRISPR-based gene expression toolkit in Yarrowia lipolytica.

With the rapid development of synthetic biology, the oleaginous yeast Yarrowia lipolytica has become an attractive microorganism for chemical production. To better optimize and reroute metabolic pathways, we have expanded the CRISPR-based gene expression toolkit of Y. lipolytica. By sorting the integration sites associated with high expression, new neutral integration sites associated with high expression and high integration efficiency were identified. Diverse genetic components, including promoters and terminators, were also characterized to expand the expression range. We found that in addition to promoters, the newly characterized terminators exhibited large variations in gene expression. These genetic components and integration sites were then used to regulate genes involved in the lycopene biosynthesis pathway, and different levels of lycopene production were achieved. The CRISPR-based gene expression toolkit developed in this study will facilitate the genetic engineering of Y. lipolytica.

Phage Enzyme-Assisted Direct In Vivo DNA Assembly in Multiple Microorganisms.

The assembly of DNA fragments is extremely important for molecular biology. Increasing numbers of studies have focused on streamlining the laborious and costly protocols via in vivo DNA assembly. However, the existing methods were mainly developed for Escherichia coli or Saccharomyces cerevisiae, whereas there are few direct in vivo DNA assembly methods for other microorganisms. The use of shuttle vectors and tedious plasmid extraction and transformation procedures make DNA cloning in other microorganisms laborious and inefficient, especially for DNA library construction. In this study, we developed a "phage enzyme-assisted in vivo DNA assembly" (PEDA) method via combinatorial expression of T5 exonuclease and T4 DNA ligase. PEDA facilitated the in vivo assembly of DNA fragments with homologous sequences as short as 5 bp, and it is applicable to multiple microorganisms, such as Ralstonia eutropha, Pseudomonas putida, Lactobacillus plantarum, and Yarrowia lipolytica. The cloning efficiency of optimized PEDA is much higher than that of the existing in vivo DNA assembly methods and comparable to that of in vitro DNA assembly, making it extremely suitable for DNA library cloning. Collectively, PEDA will boost the application of in vivo DNA assembly in various microorganisms.

Computational design of a cutinase for plastic biodegradation by mining molecular dynamics simulations trajectories.

Polyethylene terephthalate (PET) has caused serious environmental concerns but could be degraded at high temperature. Previous studies show that cutinase from Thermobifida fusca KW3 (TfCut2) is capable of degrading and upcycling PET but is limited by its thermal stability. Nowadays, Popular protein stability modification methods rely mostly on the crystal structures, but ignore the fact that the actual conformation of protein is complex and constantly changing. To solve these problems, we developed a computational approach to design variants with enhanced protein thermal stability by mining Molecular Dynamics simulation trajectories using Machine Learning methods (MDL). The optimal classification accuracy and the optimal Pearson correlation coefficient of MDL model were 0.780 and 0.716, respectively. And we successfully designed variants with high ΔT m values using MDL method. The optimal variant S121P/D174S/D204P had the highest ΔT m value of 9.3 °C, and the PET degradation ratio increased by 46.42-fold at 70℃, compared with that of wild type TfCut2. These results deepen our understanding on the complex conformations of proteins and may enhance the plastic recycling and sustainability at glass transition temperature.

Random genome reduction coupled with polyhydroxybutyrate biosynthesis to facilitate its accumulation in Escherichia coli.

Genome reduction has been emerged as a powerful tool to construct ideal chassis for synthetic biology. Random genome reduction couple genomic deletion with growth and has the potential to construct optimum genome for a given environment. Recently, we developed a transposon-mediated random deletion (TMRD) method that allows the random and continuous reduction of Escherichia coli genome. Here, to prove its ability in constructing optimal cell factories, we coupled polyhydroxybutyrate (PHB) accumulation with random genome reduction and proceeded to reduce the E. coli genome. Five mutants showed high biomass and PHB yields were selected from 18 candidates after ten rounds of genome reduction. And eight or nine genomic fragments (totally 230.1-270.0 Kb) were deleted in their genomes, encompassing 4.95%-5.82% of the parental MG1655 genome. Most mutants displayed better growth, glucose utilization, protein expression, and significant increase of electroporation efficiency compared with MG1655. The PHB content and concentration enhanced up to 13.3%-37.2% and 60.2%-102.9% when batch fermentation was performed in M9-glucose medium using the five mutants. Particularly, in mutant H16, lacking 5.28% of its genome, the increase of biomass and PHB concentration were more than 50% and 100% compared with MG1655, respectively. This work expands the strategy for creating streamlined chassis to improve the production of high value-added products.