Convenient synthesis and delivery of a megabase-scale designer accessory chromosome empower biosynthetic capacity.

Synthetic biology confers new functions to hosts by introducing exogenous genetic elements, yet rebuilding complex traits that are based on large-scale genetic information remains challenging. Here, we developed a CRISPR/Cas9-mediated haploidization method that bypasses the natural process of meiosis. Based on the programmed haploidization in yeast, we further developed an easy-to-use method designated HAnDy (Haploidization-based DNA Assembly and Delivery in yeast) that enables efficient assembly and delivery of large DNA, with no need for any fussy in vitro manipulations. Using HAnDy, a de novo designed 1.024 Mb synthetic accessory chromosome (synAC) encoding 542 exogenous genes was parallelly assembled and then directly transferred to six phylogenetically diverse yeasts. The synAC significantly promotes hosts' adaptations and increases the scope of the metabolic network, which allows the emergence of valuable compounds. Our approach should facilitate the assembly and delivery of large-scale DNA for expanding and deciphering complex biological functions.

Enhancement of polymyxin B1 production by an artificial microbial consortium of Paenibacillus polymyxa and recombinant Corynebacterium glutamicum producing precursor amino acids.

Polymyxin B, produced by Paenibacillus polymyxa, is used as the last line of defense clinically. In this study, exogenous mixture of precursor amino acids increased the level and proportion of polymyxin B1 in the total of polymyxin B analogs of P. polymyxa CJX518-AC (PPAC) from 0.15 g/L and 61.8 % to 0.33 g/L and 79.9 %, respectively. The co-culture of strain PPAC and recombinant Corynebacterium glutamicum-leu01, which produces high levels of threonine, leucine, and isoleucine, increased polymyxin B1 production to 0.64 g/L. When strains PPAC and C. glu-leu01 simultaneously inoculated into an optimized medium with 20 g/L peptone, polymyxin B1 production was increased to 0.97 g/L. Furthermore, the polymyxin B1 production in the co-culture of strains PPAC and C. glu-leu01 increased to 2.21 g/L after optimized inoculation ratios and fermentation medium with 60 g/L peptone. This study provides a new strategy to improve polymyxin B1 production.

An Engineered Microbial Consortium Provides Precursors for Fengycin Production by Bacillus subtilis.

Fengycin has great potential for applications in biological control because of its biosafety and degradability. In this study, the addition of exogenous precursors increased fengycin production by Bacillus subtilis. Corynebacterium glutamicum was engineered to produce high levels of precursors (Thr, Pro, Val, and Ile) to promote the biosynthesis of fengycin. Furthermore, recombinant C. glutamicum and Yarrowia lipolytica providing amino acid and fatty acid precursors were co-cultured to improve fengycin production by B. subtilis in a three-strain artificial consortium, in which fengycin production was 2100 mg·L-1. In addition, fengycin production by the consortium in a 5 L bioreactor reached 3290 mg·L-1. Fengycin had a significant antifungal effect on Rhizoctonia solani, which illustrates its potential as a food preservative. Taken together, this work provides a new strategy for improving fengycin production by a microbial consortium and metabolic engineering.

Global regulator IrrE on stress tolerance: a review.

Stress tolerance is a vital attribute for all living beings to cope with environmental adversities. IrrE (also named PprI) from Deinococcus radiodurans enhances resistance to extreme radiation stress by functioning as a global regulator, mediating the transcription of genes involved in deoxyribonucleic acid (DNA) damage response (DDR). The expression of IrrE augmented the resilience of various species to heat, radiation, oxidation, osmotic stresses and inhibitors, encompassing bacterial, fungal, plant, and mammalian cells. Moreover, IrrE was employed in a global regulator engineering strategy to broaden its applications in stress tolerance. The regulatory impacts of heterologously expressed IrrE have been investigated at the molecular and systems level, including the regulation of genes, proteins, modules, or pathways involved in DNA repair, detoxification proteins, protective molecules, native regulators and other aspects. In this review, we discuss the regulatory role and mechanism of IrrE in the antiradiation response of D. radiodurans. Furthermore, the applications and regulatory effects of heterologous expression of IrrE to enhance abiotic stress tolerance are summarized in particular.

Unleashing the capacity of Rhodococcus for converting lignin into lipids.

Bioconversion of bioresources/wastes (e.g., lignin, chemical pulping byproducts) represents a promising approach for developing a bioeconomy to help address growing energy and materials demands. Rhodococcus, a promising microbial strain, utilizes numerous carbon sources to produce lipids, which are precursors for synthesizing biodiesel and aviation fuels. However, compared to chemical conversion, bioconversion involves living cells, which is a more complex system that needs further understanding and upgrading. Various wastes amenable to bioconversion are reviewed herein to highlight the potential of Rhodococci for producing lipid-derived bioproducts. In light of the abundant availability of these substrates, Rhodococcus' metabolic pathways converting them to lipids are analyzed from a "beginning-to-end" view. Based on an in-depth understanding of microbial metabolic routes, genetic modifications of Rhodococcus by employing emerging tools (e.g., multiplex genome editing, biosensors, and genome-scale metabolic models) are presented for promoting the bioconversion. Co-solvent enhanced lignocellulose fractionation (CELF) strategy facilitates the generation of a lignin-derived aromatic stream suitable for the Rhodococcus' utilization. Novel alkali sterilization (AS) and elimination of thermal sterilization (ETS) approaches can significantly enhance the bioaccessibility of lignin and its derived aromatics in aqueous fermentation media, which promotes lipid titer significantly. In order to achieve value-added utilization of lignin, biodiesel and aviation fuel synthesis from lignin and lipids are further discussed. The possible directions for unleashing the capacity of Rhodococcus through synergistically modifying microbial strains, substrates, and fermentation processes are proposed toward a sustainable biological lignin valorization.

Yeast Metabolic Engineering for Biosynthesis of Caffeic Acid-Derived Phenethyl Ester and Phenethyl Amide.

Caffeic acid (CA)-derived phenethyl ester (CAPE) and phenethyl amide (CAPA) are extensively investigated bioactive compounds with therapeutic applications such as antioxidant, anti-inflammatory, and anticarcinogenic properties. To construct microbial cell factories for production of CAPE or CAPA is a promising option given the limitation of natural sources for product extraction and the environmental toxicity of the agents used in chemical synthesis. We reported the successful biosynthesis of caffeic acid in yeast previously. Here in this work, we further constructed the downstream synthetic pathways in yeast for biosynthesis of CAPE and CAPA. After combinatorial engineering of yeast chassis based on the rational pathway engineering method and library-based SCRaMbLE method, we finally obtained the optimal strains that respectively produced 417 µg/L CAPE and 1081 µg/L CAPA. Two screened gene targets of ΔHAM1 and ΔYJL028W were discovered to help improve the product synthesis capacity. This is the first report of the de novo synthesis of CAPA from glucose in an engineered yeast chassis. Future work on enzyme and chassis engineering will further support improving the microbial cell factories for the production of CA derivatives.

Enhanced single-base mutation diversity by the combination of cytidine deaminase with DNA-repairing enzymes in yeast.

The occurrence of random mutations can increase the diversity of the genome and promote the evolutionary process of organisms. High efficiency mutagenesis techniques significantly accelerate the evolutionary process. In this work, we describe a targeted mutagenesis system named MutaT7trans to significantly increase mutation rate and generate mutations across all four nucleotides in yeast. We constructed different DNA-repairing enzyme-PmCDA1-T7 RNA polymerase (T7 RNAP) fusion proteins, achieved targeted mutagenesis by flanking the target gene with T7 promoters, and tuned the mutation spectra by introducing different DNA-repairing enzymes. With this mutagenesis tool, the proportion of non-C â†’ T mutations was 10-11-fold higher than the cytidine deaminase-based evolutionary tools, and the transversion mutation frequency was also elevated. The mutation rate of the target gene was significantly increased to 5.25 × 10-3 substitutions per base (s. p. b.). We also demonstrated that MutaT7trans could be used to evolve the CrtE, CrtI, and CrtYB gene in the ß-carotene biosynthesis process and generate different types of mutations.

Artificial mixed microbial system for polycyclic aromatic hydrocarbons degradation.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants with major risks to human health. Biological degradation is environmentally friendly and the most appealing remediation method for a wide range of persistent pollutants. Meanwhile, due to the large microbial strain collection and multiple metabolic pathways, PAH degradation via an artificial mixed microbial system (MMS) has emerged and is regarded as a promising bioremediation approach. The artificial MMS construction by simplifying the community structure, clarifying the labor division, and streamlining the metabolic flux has shown tremendous efficiency. This review describes the construction principles, influencing factors, and enhancement strategies of artificial MMS for PAH degradation. In addition, we identify the challenges and future opportunities for the development of MMS toward new or upgraded high-performance applications.

Ethylenediamine pretreatment simultaneously improved carbohydrate hydrolysis and lignin valorization.

Lignocellulosic biomass (LCB) is the promising feedstock for value-added products, which would contribute to the bioeconomy and sustainable development. The efficient pretreatment is still required in the biorefinery of LCB. To make a simultaneous utilization of carbohydrates and lignin, a novel easy-recycled ethylenediamine (EDA) pretreatment was designed and evaluated in the present study. The results highlighted that this pretreatment yielded 96% glucose and 70% xylose in enzymatic hydrolysis. It simultaneously promoted the depolymerization of lignin into small molecules and functionalized the yielded lignin with Schiff base and amide structures. These animated-lignins showed a pH-responsive behavior and the excellent flocculation capacity by reducing more than 90% turbidity of kaolin suspensions. Therefore, easy-recycled EDA pretreatment hold the promise to simultaneously enhance the enzymatic hydrolysis of carbohydrates and endowed the new functionality of lignin toward downstream valorization, which improved the process feasibility and potentially enable the sustainability of LCB utilization.

YLC-assembly: large DNA assembly via yeast life cycle.

As an enabling technique of synthetic biology, the scale of DNA assembly largely determines the scale of genetic manipulation. However, large DNA assembly technologies are generally cumbersome and inefficient. Here, we developed a YLC (yeast life cycle)-assembly method that enables in vivo iterative assembly of large DNA by nesting cell-cell transfer of assembled DNA in the cycle of yeast mating and sporulation. Using this method, we successfully assembled a hundred-kilobase (kb)-sized endogenous yeast DNA and a megabase (Mb)-sized exogenous DNA. For each round, over 104 positive colonies per 107 cells could be obtained, with an accuracy ranging from 67% to 100%. Compared with other Mb-sized DNA assembly methods, this method exhibits a higher success rate with an easy-to-operate workflow that avoid in vitro operations of large DNA. YLC-assembly lowers the technical difficulty of Mb-sized DNA assembly and could be a valuable tool for large-scale genome engineering and synthetic genomics.

Genome engineering on size reduction and complexity simplification: A review.

BACKGROUND: Genome simplification is an important topic in the field of life sciences that has attracted attention from its conception to the present day. It can help uncover the essential components of the genome and, in turn, shed light on the underlying operating principles of complex biological systems. This has made it a central focus of both basic and applied research in the life sciences. With the recent advancements in related technologies and our increasing knowledge of the genome, now is an opportune time to delve into this topic. AIM OF REVIEW: Our review investigates the progress of genome simplification from two perspectives: genome size reduction and complexity simplification. In addition, we provide insights into the future development trends of genome simplification. KEY SCIENTIFIC CONCEPTS OF REVIEW: Reducing genome size requires eliminating non-essential elements as much as possible. This process has been facilitated by advances in genome manipulation and synthesis techniques. However, we still need a better and clearer understanding of living systems to reduce genome complexity. As there is a lack of quantitative and clearly defined standards for this task, we have opted to approach the topic from various perspectives and present our findings accordingly.

Surfactant-assisted ethylenediamine for the deconstruction and conversion of corn stover biomass.

Lignocellulosic biomass is a promising feedstock to produce sustainable fuels and energy toward a green bioeconomy. A surfactant-assisted ethylenediamine (EDA) was developed for the deconstruction and conversion of corn stover in this study. The effects of surfactants on the whole conversion process of corn stover was also evaluated. The results showed that xylan recovery and lignin removal in solid fraction were significantly enhanced by surfactant-assisted EDA. The glucan and xylan recoveries in solid fraction reached 92.1% and 65.7%, respectively, while the lignin removal was 74.5% by sodium dodecyl sulfate (SDS)-assisted EDA. SDS-assisted EDA also improved the sugar conversion in 12 h enzymatic hydrolysis at low enzyme loadings. The ethanol production and glucose consumption of washed EDA pretreated corn stover in simultaneous saccharification and co-fermentation were improved with the addition of 0.001 g/mL SDS. Therefore, surfactant-assisted EDA showed the potential to improve the bioconversion performance of biomass.

Enhancing fengycin production in the co-culture of Bacillus subtilis and Corynebacterium glutamicum by engineering proline transporter.

Fengycin possesses antifungal activity but has limited application due to its low yields. Amino acid precursors play a crucial role in fengycin synthesis. Herein, the overexpression of alanine, isoleucine, and threonine transporter-related genes in Bacillus subtilis increased fengycin production by 34.06%, 46.66%, and 7.83%, respectively. Particularly, fengycin production in B. subtilis reached 871.86 mg/L with the addition of 8.0 g/L exogenous proline after enhancing the expression of the proline transport-related gene opuE. To overcome the metabolic burden caused by excessive enhancement of gene expression for supplying precursors, B. subtilis and Corynebacterium glutamicum which produced proline, were co-cultured, which further improved fengycin production. Fengycin production in the co-culture of B. subtilis and C. glutamicum in shake flasks reached 1554.74 mg/L after optimizing the inoculation time and ratio. The fengycin level in the fed-batch co-culture was 2309.96 mg/L in a 5.0-L bioreactor. These findings provide a new strategy for improving fengycin production.

Cross-species microbial genome transfer: a Review.

Synthetic biology combines the disciplines of biology, chemistry, information science, and engineering, and has multiple applications in biomedicine, bioenergy, environmental studies, and other fields. Synthetic genomics is an important area of synthetic biology, and mainly includes genome design, synthesis, assembly, and transfer. Genome transfer technology has played an enormous role in the development of synthetic genomics, allowing the transfer of natural or synthetic genomes into cellular environments where the genome can be easily modified. A more comprehensive understanding of genome transfer technology can help to extend its applications to other microorganisms. Here, we summarize the three host platforms for microbial genome transfer, review the recent advances that have been made in genome transfer technology, and discuss the obstacles and prospects for the development of genome transfer.

Dynamics of synthetic yeast chromosome evolution shaped by hierarchical chromatin organization.

Synthetic genome evolution provides a dynamic approach for systematically and straightforwardly exploring evolutionary processes. Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is an evolutionary system intrinsic to the synthetic yeast genome that can rapidly drive structural variations. Here, we detect over 260 000 rearrangement events after the SCRaMbLEing of a yeast strain harboring 5.5 synthetic yeast chromosomes (synII, synIII, synV, circular synVI, synIXR and synX). Remarkably, we find that the rearrangement events exhibit a specific landscape of frequency. We further reveal that the landscape is shaped by the combined effects of chromatin accessibility and spatial contact probability. The rearrangements tend to occur in 3D spatially proximal and chromatin-accessible regions. The enormous numbers of rearrangements mediated by SCRaMbLE provide a driving force to potentiate directed genome evolution, and the investigation of the rearrangement landscape offers mechanistic insights into the dynamics of genome evolution.

Machine learning-aided scoring of synthesis difficulties for designer chromosomes.

Designer chromosomes are artificially synthesized chromosomes. Nowadays, these chromosomes have numerous applications ranging from medical research to the development of biofuels. However, some chromosome fragments can interfere with the chemical synthesis of designer chromosomes and eventually limit the widespread use of this technology. To address this issue, this study aimed to develop an interpretable machine learning framework to predict and quantify the synthesis difficulties of designer chromosomes in advance. Through the use of this framework, six key sequence features leading to synthesis difficulties were identified, and an eXtreme Gradient Boosting model was established to integrate these features. The predictive model achieved high-quality performance with an AUC of 0.895 in cross-validation and an AUC of 0.885 on an independent test set. Based on these results, the synthesis difficulty index (S-index) was proposed as a means of scoring and interpreting synthesis difficulties of chromosomes from prokaryotes to eukaryotes. The findings of this study emphasize the significant variability in synthesis difficulties between chromosomes and demonstrate the potential of the proposed model to predict and mitigate these difficulties through the optimization of the synthesis process and genome rewriting.

Biological valorization of lignin to flavonoids.

Lignin is the most affluent natural aromatic biopolymer on the earth, which is the promising renewable source for valuable products to promote the sustainability of biorefinery. Flavonoids are a class of plant polyphenolic secondary metabolites containing the benzene ring structure with various biological activities, which are largely applied in health food, pharmaceutical, and medical fields. Due to the aromatic similarity, microbial conversion of lignin derived aromatics to flavonoids could facilitate flavonoid biosynthesis and promote the lignin valorization. This review thereby prospects a novel valorization route of lignin to high-value natural products and demonstrates the potential advantages of microbial bioconversion of lignin to flavonoids. The biodegradation of lignin polymers is summarized to identify aromatic monomers as momentous precursors for flavonoid synthesis. The biosynthesis pathways of flavonoids in both plants and strains are introduced and compared. After that, the key branch points and important intermediates are clearly discussed in the biosynthesis pathways of flavonoids. Moreover, the most significant enzyme reactions including Claisen condensation, cyclization and hydroxylation are demonstrated in the biosynthesis pathways of flavonoids. Finally, current challenges and potential future strategies are also discussed for transforming lignin into various flavonoids. The holistic microbial conversion routes of lignin to flavonoids could make a sustainable production of flavonoids and improve the feasibility of lignin valorization.

Structural Variations and Adaptations of Synthetic Chromosome Ends Driven by SCRaMbLE in Haploid and Diploid Yeasts.

Variations and adaptations of chromosome ends play an important role in eukaryotic karyotype evolution. Traditional experimental studies of the adaptations of chromosome ends mainly rely on the strategy of introducing defects; thus, the adaptation methods of survivors may vary depending on the initial defects. Here, using the SCRaMbLE strategy, we obtained a library of haploid and diploid synthetic strains with variations in chromosome ends. Analysis of the SCRaMbLEd survivors revealed four routes of adaptation: homologous recombination between nonhomologous chromosome arms (haploids) or homologous chromosome arms (diploids), site-specific recombination between intra- or interchromosomal ends, circularization of chromosomes, and loss of whole chromosomes (diploids). We also found that circularization of synthetic chromosomes can be generated by SCRaMbLE. Our study of various adaptation routes of chromosome ends provides insight into eukaryotic karyotype evolution from the viewpoint of synthetic genomics.

Mutualistic microbial community of Bacillus amyloliquefaciens and recombinant Yarrowia lipolytica co-produced lipopeptides and fatty acids from food waste.

Bioconversion is an important method for transforming food waste (FW) into high value-added products, rendering it harmless, and recycling resources. An artificial microbial consortium (AMC) was constructed to produce FW-based lipopeptides in order to investigate the strategy of FW bioconversion into value-added products. Exogenous fatty acids as a precursor significantly improved the lipopeptide production of Bacillus amyloliquefaciens HM618. To enhance fatty acid synthesis and efflux in AMC, the recombinant Yarrowia lipolytica YL21 (strain YL21) was constructed by screening 12 target genes related to fatty acids to replace exogenous fatty acids in order to improve lipopeptide production. The levels of fengycin, surfactin, and iturin A in the AMC of strains HM618 and YL21 reached 76.19, 192.80, and 31.32 mg L-1, increasing 7.24-, 12.13-, and 3.23-fold compared to the results from the pure culture of strain HM618 in flask with Landy medium, respectively. Furthermore, free fatty acids were almost undetectable in the co-culture of strains HM618 and YL21, although its level was around 1.25 g L-1 in the pure culture of strain YL21 with Landy medium. Interestingly, 470.24 mg L-1 of lipopeptides and 18.11 g L-1 of fatty acids were co-produced in this AMC in a bioreactor with FW medium. To our knowledge, it is the first report of FW biotransformation into co-produce of lipopeptides and fatty acids in the AMC of B. amyloliquefaciens and Y. lipolytica. These results provide new insights into the biotransformation potential of FW for value-added co-products by AMC.

Directed evolution of the fusion enzyme for improving astaxanthin biosynthesis in Saccharomyces cerevisiae.

The accumulation of the intermediate zeaxanthin and canthaxanthin in the astaxanthin biosynthesis pathway catalyzed by ß-carotene hydroxylase (crtZ) and ß-carotene ketolase (crtW) decreases the content of the astaxanthin. Here, we exploited directed evolution of the fusion of crtZ and crtW for improving astaxanthin biosynthesis in Saccharomyces cerevisiae. The results demonstrated that the fusion enzyme of crtZ-crtW with 2 X GGGGS peptides linker can effectively reduce the accumulation of intermediates and improves the content of astaxanthin. Compared with the control strain, the fusion enzyme of ketase and hydroxylase reduced zeaxanthin and canthaxanthin by 7 and 14 times and increased astaxanthin by 1.6 times, respectively. Moreover, 9 variant fusion mutants with improved astaxanthin production were generated through directed evolution. Combining these dominant mutants generated a variant, L95S + I206L, which increased the astaxanthin content of 3.8 times than the control strain. The AlphaFold2 assisted structural analysis indicated that these two mutations alter the interaction between the substrate and the enzymes pocket. Our research provided an efficient idea to reduce the accumulation of the intermediate products in complex biosynthesis pathway.