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.

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.

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.

Combining nucleotide variations and structure variations for improving astaxanthin biosynthesis.

BACKGROUND: Mutational technology has been used to achieve genome-wide variations in laboratory and industrial microorganisms. Genetic polymorphisms of natural genome evolution include nucleotide variations and structural variations, which inspired us to suggest that both types of genotypic variations are potentially useful in improving the performance of chassis cells for industrial applications. However, highly efficient approaches that simultaneously generate structural and nucleotide variations are still lacking. RESULTS: The aim of this study was to develop a method of increasing biosynthesis of astaxanthin in yeast by Combining Nucleotide variations And Structure variations (CNAS), which were generated by combinations of Atmospheric and room temperature plasma (ARTP) and Synthetic Chromosome Recombination and Modification by LoxP-Mediated Evolution (SCRaMbLE) system. CNAS was applied to increase the biosynthesis of astaxanthin in yeast and resulted in improvements of 2.2- and 7.0-fold in the yield of astaxanthin. Furthermore, this method was shown to be able to generate structures (deletion, duplication, and inversion) as well as nucleotide variations (SNPs and InDels) simultaneously. Additionally, genetic analysis of the genotypic variations of an astaxanthin improved strain revealed that the deletion of YJR116W and the C2481G mutation of YOL084W enhanced yield of astaxanthin, suggesting a genotype-to-phenotype relationship. CONCLUSIONS: This study demonstrated that the CNAS strategy could generate both structure variations and nucleotide variations, allowing the enhancement of astaxanthin yield by different genotypes in yeast. Overall, this study provided a valuable tool for generating genomic variation diversity that has desirable phenotypes as well as for knowing the relationship between genotypes and phenotypes in evolutionary processes.

Advances in biosynthesis of scopoletin.

Scopoletin is a typical example of coumarins, which can be produced in plants. Scopoletin acts as a precursor for pharmaceutical and health care products, and also possesses promising biological properties, including antibacterial, anti-tubercular, anti-hypertensive, anti-inflammatory, anti-diabetic, and anti-hyperuricemic activity. Despite the potential benefits, the production of scopoletin using traditional extraction processes from plants is unsatisfactory. In recent years, synthetic biology has developed rapidly and enabled the effective construction of microbial cell factories for production of high value-added chemicals. Herein, this review summarizes the progress of scopoletin biosynthesis in artificial microbial cell factories. The two main pathways of scopoletin biosynthesis are summarized firstly. Then, synthetic microbial cell factories are reviewed as an attractive improvement strategy for biosynthesis. Emerging techniques in synthetic biology and metabolic engineering are introduced as innovative tools for the efficient synthesis of scopoletin. This review showcases the potential of biosynthesis of scopoletin in artificial microbial cell factories.

Directed genome evolution driven by structural rearrangement techniques.

Directed genome evolution simulates the process of natural evolution at the genomic level in the laboratory to generate desired phenotypes. Here we review the applications of recent technological advances in genome writing and editing to directed genome evolution, with a focus on structural rearrangement techniques. We highlight how these techniques can be used to generate diverse genotypes, and to accelerate the evolution of phenotypic traits. We also discuss the perspectives of directed genome evolution.

Stress-driven dynamic regulation of multiple tolerance genes improves robustness and productive capacity of Saccharomyces cerevisiae in industrial lignocellulose fermentation.

Yeast productivity in lignocellulosic ethanol fermentation is clearly impeded by stress. Enhancing the robustness of xylose-fermenting yeast is important for improving lignocellulosic ethanol production. In this study, the glutathione biosynthesis pathway and acetic acid degradation pathway were strengthened to enhance yeast tolerance to stress due to elevated reactive oxygen species (ROS) and acetic acid. Dynamic feedback regulation of the anti-stress genetic circuits was achieved using stress-driven promoters discovered from the transcriptome to maintain low intracellular ROS, relieve the metabolic burden, and ultimately improve the robustness and ethanol production of yeast. The cell growth, xylose utilization and ethanol production of the engineered strain were enhanced under both stress and nonstress conditions. The engineered strain showed 49.5% and 17.5% higher ethanol productivity in laboratory media and industrial lignocellulosic media, respectively, at 36 °C compared with the parent strain. This study provides novel insights on the rational design and construction of feedback genetic circuits for dynamically improving yeast robustness.

Discovering and genotyping genomic structural variations by yeast genome synthesis and inducible evolution.

Genomic structural variations (SVs) promote the evolution of Saccharomyces cerevisiae, and play an important role in phenotypic diversities. Yeast genomic structures can be remodeled by design and bottom-up synthesis. The synthesis of yeast genome creates novel copy number variations (CNVs) and SVs and develops new strategies to discover gene functions. Further, an inducible evolution system SCRaMbLE, consisted of 3,932 loxPsym sites, was incorporated on synthetic yeast genome. SCRaMbLE enables genomic rearrangements at will and rapidly generates chromosomal number variations, and massive SVs under customized conditions. The impacts of genetic variations on phenotypes can be revealed by genome analysis and chromosome restructuring. Yeast genome synthesis and SCRaMbLE provide a new research paradigm to explore the genotypic mechanisms of phenotype diversities, and can be used to improve biological traits and optimize industrial chassis.

In vitro and in vivo recombination of heterologous modules for improving biosynthesis of astaxanthin in yeast.

BACKGROUND: Astaxanthin is a kind of tetraterpene and has strong antioxygenic property. The biosynthesis of astaxanthin in engineered microbial chassis has greater potential than its chemical synthesis and extraction from natural producers in an environmental-friendly way. However, the cost-offsetting production of astaxanthin in engineered microbes is still constrained by the poor efficiency of astaxanthin synthesis pathway as a heterologous pathway. RESULTS: To address the bottleneck of limited production of astaxanthin in microbes, we developed in vitro and in vivo recombination methods respectively in engineered yeast chassis to optimize the combination of heterologous ß-carotene ketolase (crtW) and hydroxylase (crtZ) modules that were selected from different species. As a result, the in vitro and in vivo recombination methods enhanced the astaxanthin yield respectively to 2.11-8.51 folds and 3.0-9.71 folds compared to the initial astaxanthin pathway, according to the different combination of particular genes. The highest astaxanthin producing strain yQDD022 was constructed by in vivo method and produced 6.05 mg g-1 DCW of astaxanthin. Moreover, it was proved that the in vivo recombination method showed higher DNA-assembling efficiency than the in vitro method and contributed to higher stability to the engineered yeast strains. CONCLUSIONS: The in vitro and in vivo recombination methods of heterologous modules provide simple and efficient ways to improve the astaxanthin yield in yeast. Both the two methods enable high-throughput screening of heterologous pathways through recombination of certain crtW and crtZ derived from different species. This study not only exploited the underlying optimal combination of crtZ and crtW for astaxanthin synthesis, but also provided a general approach to evolve a heterologous pathway for the enhanced accumulation of desired biochemical products.

Control of the polymyxin analog ratio by domain swapping in the nonribosomal peptide synthetase of Paenibacillus polymyxa.

Polymyxins are used as the last-line therapy against multidrug-resistant bacteria. However, their further clinical development needs to solve problems related to the presence of heterogeneous analogs, but there is still no platform or methods that can regulate the biosynthesis of polymyxin analogs. In this study, we present an approach to swap domains in the polymyxin gene cluster to regulate the production of different analogs. Following adenylation domain swapping, the proportion of polymyxin B1 increased from 41.36 to 52.90%, while that of B1-1 decreased from 18.25 to 3.09%. The ratio of polymyxin B1 and B3 following starter condensation domain swapping changed from 41.36 and 16.99 to 55.03 and 6.39%, respectively. The two domain-swapping strains produced 62.96% of polymyxin B1, 6.70% of B3 and 3.32% of B1-1. This study also revealed the presence of overflow fluxes between acetoin, 2,3-butanediol and polymyxin. To our best knowledge, this is the first report of engineering the polymyxin synthetase gene cluster in situ to regulate the relative proportions of polymyxin analogs. This research paves a way for regulating lipopeptide analogs and will facilitate the development of novel lipopeptide derivatives.

SCRaMbLE generates evolved yeasts with increased alkali tolerance.

BACKGROUND: Strains with increased alkali tolerance have a broad application in industrial, especially for bioremediation, biodegradation, biocontrol and production of bio-based chemicals. A novel synthetic chromosome recombination and modification by LoxP-mediated evolution (SCRaMbLE) system has been introduced in the synthetic yeast genome (Sc 2.0), which enables generation of a yeast library with massive structural variations and potentially drives phenotypic evolution. The structural variations including deletion, inversion and duplication have been detected within synthetic yeast chromosomes. RESULTS: Haploid yeast strains harboring either one (synV) or two (synV and synX) synthetic chromosomes were subjected to SCRaMbLE. Seven of evolved strains with increased alkali tolerance at pH 8.0 were generated through multiple independent SCRaMbLE experiments. Various of structural variations were detected in evolved yeast strains by PCRTag analysis and whole genome sequencing including two complex structural variations. One possessed an inversion of 20,743 base pairs within which YEL060C (PRB1) was deleted simultaneously, while another contained a duplication region of 9091 base pairs in length with a deletion aside. Moreover, a common deletion region with length of 11,448 base pairs was mapped in four of the alkali-tolerant strains. We further validated that the deletion of YER161C (SPT2) within the deleted region could increase alkali tolerance in Saccharomyces cerevisiae. CONCLUSIONS: SCRaMbLE system provides a simple and efficient way to generate evolved yeast strains with enhanced alkali tolerance. Deletion of YER161C (SPT2) mapped by SCRaMbLE can improve alkali tolerance in S. cerevisiae. This study enriches our understanding of alkali tolerance in yeast and provides a standard workflow for the application of SCRaMbLE system to generate various phenotypes that may be interesting for industry and extend understanding of phenotype-genotype relationship.

Synthetic cell-cell communication in a three-species consortium for one-step vitamin C fermentation.

OBJECTIVES: A three-species consortium for one-step fermentation of 2-keto-L-gulonic acid (2-KGA) was constructed to better strengthen the cell-cell communication. And the programmed cell death module based on the LuxI/LuxR quorum-sensing (QS) system was established in Gluconobacter oxydans to reduce the competition that between G. oxydans and Ketogulonicigenium vulgare. RESULTS: By constructing and optimizing the core region of the promoter, which directly regulated the expression of lethal ccdB genes in QS system, IR3C achieved the best lethal effect. The consortium of IR3C- K. vulgare-Bacillus megaterium (abbreviated as 3C) achieved the highest 2-KGA titer (68.80 ± 4.18 g/l), and the molar conversion rate was 80.7% within 36 h in 5 l fermenter. Metabolomic analysis on intracellular small molecules of consortia 3C and 1C showed that most amino acids (such as glycine, leucine, methionine and proline) and TCA cycle intermediates (such as succinic acid, fumaric acid and malic acid) were significantly affected. These results further validated that the programmed cell death module based on the LuxI/LuxR QS system in G. oxydans could also faciliate better growth and higher production of consortium 3C for one-step fermentation. CONCLUSIONS: We successfully constructed a novel three-species consortia for one-step vitamin C fermentation by strengthening the cell-cell communication. This will be very useful for probing the rational design principles of more complex multi-microbial consortia.

Integrated proteomic and metabolomic analysis of a reconstructed three-species microbial consortium for one-step fermentation of 2-keto-L-gulonic acid, the precursor of vitamin C.

Microbial consortia, with the merits of strong stability, robustness, and multi-function, played critical roles in human health, bioenergy, and food manufacture, etc. On the basis of 'build a consortium to understand it', a novel microbial consortium consisted of Gluconobacter oxydans, Ketogulonicigenium vulgare and Bacillus endophyticus was reconstructed to produce 2-keto-L-gulonic acid (2-KGA), the precursor of vitamin C. With this synthetic consortium, 73.7 g/L 2-KGA was obtained within 30 h, which is comparable to the conventional industrial method. A combined time-series proteomic and metabolomic analysis of the fermentation process was conducted to further investigate the cell-cell interaction. The results suggested that the existence of B. endophyticus and G. oxydans together promoted the growth of K. vulgare by supplying additional nutrients, and promoted the 2-KGA production by supplying more substrate. Meanwhile, the growth of B. endophyticus and G. oxydans was compromised from the competition of the nutrients by K. vulgare, enabling the efficient production of 2-KGA. This study provides valuable guidance for further study of synthetic microbial consortia.

Convergent engineering of syntrophic Escherichia coli coculture for efficient production of glycosides.

Synthetic microbial coculture to express heterologous biosynthetic pathway for de novo production of medicinal ingredients is an emerging strategy for metabolic engineering and synthetic biology. Here, taking efficient production of salidroside as an example of glycosides, we design and construct a syntrophic Escherichia coli-E. coli coculture composed of the aglycone (AG) strain and the glycoside (GD) strain, which convergently accommodate biosynthetic pathways of tyrosol and salidroside, respectively. To accomplish this the phenylalanine-deficient AG strain was engineered to utilize xylose preferentially and to overproduce precursor tyrosol, while the tyrosine-deficient GD strain was constructed to consume glucose exclusively and to enhance another precursor UDP-glucose availability for synthesis of salidroside. The AG and GD strains in the synthetic consortium are obligatory cooperators through crossfeeding of tyrosine and phenylalanine and compatible in glucose and xylose mixture. Through balancing the metabolic pathway strength, we show that the syntrophic coculture was robust and stable, and produced 6.03 g/L of salidroside. It was the de novo production of salidroside for the first time in E. coli coculture system, which would be applicable for production of other important glycosides and natural products.

Gene repression via multiplex gRNA strategy in Y. lipolytica.

BACKGROUND: The oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory due to their biochemical characteristics and native capacity to accumulate lipid-based chemicals. To create heterogenous biosynthesis pathway and manipulate metabolic flux in Y. lipolytica, numerous studies have been done for developing synthetic biology tools for gene regulation. CRISPR interference (CRISPRi), as an emerging technology, has been applied for specifically repressing genes of interest. RESULTS: In this study, we established CRISPRi systems in Y. lipolytica based on four different repressors, that was DNase-deactivated Cpf1 (dCpf1) from Francisella novicida, deactivated Cas9 (dCas9) from Streptococcus pyogenes, and two fusion proteins (dCpf1-KRAB and dCas9-KRAB). Ten gRNAs that bound to different regions of gfp gene were designed and the results indicated that there was no clear correlation between the repression efficiency and targeting sites no matter which repressor protein was used. In order to rapidly yield strong gene repression, a multiplex gRNAs strategy based on one-step Golden-brick assembly technology was developed. High repression efficiency 85% (dCpf1) and 92% (dCas9) were achieved in a short time by making three different gRNAs towards gfp gene simultaneously, which avoided the need of screening effective gRNA loci in advance. Moreover, two genes interference including gfp and vioE and three genes repression including vioA, vioB and vioE in protodeoxy-violaceinic acid pathway were also realized. CONCLUSION: Taken together, successful CRISPRi-mediated regulation of gene expression via four different repressors dCpf1, dCas9, dCpf1-KRAB and dCas9-KRAB in Y. lipolytica is achieved. And we demonstrate a multiplexed gRNA targeting strategy can efficiently achieve transcriptional simultaneous repression of several targeted genes and different sites of one gene using the one-step Golden-brick assembly. This timesaving method promised to be a potent transformative tool valuable for metabolic engineering, synthetic biology, and functional genomic studies of Y. lipolytica.

Identification and manipulation of a novel locus to improve cell tolerance to short-chain alcohols in Escherichia coli.

Escherichia coli KO11 is a popular ethanologenic strain, but is more sensitive to ethanol than other producers. Here, an ethanol-tolerant mutant EM was isolated from ultraviolet mutagenesis library of KO11. Comparative genomic analysis added by piecewise knockout strategy and complementation assay revealed EKO11_3023 (espA) within the 36.6-kb deletion from KO11 was the only locus responsible for ethanol sensitivity. Interestingly, when espA was deleted in strain W (the parent strain of KO11), ethanol tolerance was dramatically elevated to the level of espA-free hosts [e.g., MG1655 and BL21(DE3)]. And overexpression of espA in strains MG1655 and BL21(DE3) led to significantly enhanced ethanol sensitivity. In addition to ethanol, deletion of espA also improved cell tolerance to other short-chain (C2-C4) alcohols, including methanol, isopropanol, n-butanol, isobutanol and 2-butanol. Therefore, espA was responsible for short-chain alcohol sensitivity of W-strains compared to other cells, which provides a potential engineering target for alcohols production.

Improvement of sulfamethoxazole (SMX) elimination and inhibition of formations of hydroxylamine-SMX and N4-acetyl-SMX by membrane bioreactor systems.

Sulfamethoxazole (SMX) has frequently been detected in aquatic environments. In natural environment, not only individual microorganism but also microbial consortia are involved in some biotransformation of pollutants. The competition for space under consortia causing cell-cell contact inhibition changes the cellular behaviors. Herein, the membrane bioreactor system (MBRS) was applied to improve SMX elimination thorough exchanging the cell-free broths (CFB). The removal efficiency of SMX was increased by more than 24% whether under the pure culture of A. faecalis or under the co-culture of A. faecalis and P. denitrificans with MBRS. Meanwhile, MBRS significantly inhibited the formation of HA-SMX, and Ac-SMX from parent compound. Additionally, the cellular growth under MBRS was obviously enhanced, indicating that the increases in the cellular growth under MBRS are possibly related to the decreases in the levels of HA-SMX and Ac-SMX compared to that without MBRS. The intracellular NADH/NAD+ ratios of A. faecalis under MBRS were increased whether thorough itself-recycle of CFB or exchanging CFB between the pure cultures of A. faecalis and P. denitrificans, suggesting that the enhancement in the bioremoval efficiencies of SMX under MBRS by A. faecalis is likely related to the increases in the NADH/NAD+ ratio. Taken together, the regulation of cell-to-cell communication is preferable strategy to improve the bioremoval efficiency of SMX.

Design and chemical synthesis of eukaryotic chromosomes.

Following the discovery of the DNA double helix structure and the advancement of genome sequencing, we have entered a promising stage with regard to genome writing. Recently, a milestone breakthrough was achieved in the chemical synthesis of designer yeast chromosomes. Here, we review the systematic approaches to the de novo synthesis of designer eukaryotic chromosomes, with an emphasis on technologies and methodologies that enable design, building, testing and debugging. The achievement of chemically synthesized genomes with customized genetic features offers an opportunity to rebuild genome organization, remold biological functions and promote life evolution, which will be of great benefit for application in medicine and industrial manufacturing.

Manipulation of GES and ERG20 for geraniol overproduction in Saccharomyces cerevisiae.

Manipulation of monoterpene synthases to maximize flux towards targeted products from GPP (geranyl diphosphate) is the main challenge for heterologous monoterpene overproduction, in addition to cell toxicity from compounds themselves. In our study, by manipulation of the key enzymes geraniol synthase (GES) and farnesyl diphosphate synthase (Erg20), geraniol (a valuable acyclic monoterpene alcohol) overproduction was achieved in Saccharomyces cerevisiae with truncated 3-hydroxy-3-methylglutaryl-coenzyme reductase (tHMGR) and isopentenyl diphosphate isomerase (IDI1) overexpressed. The expressions of all above engineered genes were under the control of Gal promoter for alleviating product toxicity. Geraniol production varied from trace amount to 43.19mg/L (CrGES, GES from Catharanthus roseus) by screening of nine GESs from diverse species. Further through protein structure analysis and site-directed mutation in CrGES, it was firstly demonstrated that among the high-conserved amino acid residues located in active pocket, Y436 and D501 with strong affinity to diphosphate function group, were critical for the dephosphorylation (the core step for geraniol formation). Moreover, the truncation position of the transit peptide from the N-terminus of CrGES was found to influence protein expression and activity significantly, obtaining a titer of 191.61mg/L geraniol in strain with CrGES truncated at S43 (t3CrGES). Furthermore, directed by surface electrostatics distribution of t3CrGES and Erg20WW (Erg20F96W-N127W), co-expression of the reverse fusion of Erg20ww/t3CrGES and another copy of Erg20WW promoted the geraniol titer to 523.96mg/L at shakes flask level, due to enhancing GPP accessibility led by protein interaction of t3CrGES-Erg20WW and the free Erg20WW. Eventually, a highest reported titer of 1.68g/L geraniol in eukaryote cells was achieved in 2.0L fed-batch fermentation under carbon restriction strategy. Our research opens large opportunities for other microbial production of monoterpenes. It also sets a good reference for desired compounds overproduction in microorganisms in terms of manipulation of key enzymes by protein engineering and metabolic engineering.

Reconstruction of amino acid biosynthetic pathways increases the productivity of 2-keto-L-gulonic acid in Ketogulonicigenium vulgare-Bacillus endophyticus consortium via genes screening.

Defect in the amino acid biosynthetic pathways of Ketogulonicigenium vulgare, the producing strain for 2-keto-L-gulonic acid (2-KGA), is the key reason for its poor growth and low productivity. In this study, five different strains were firstly reconstructed by expressing absent genes in threonine, proline and histidine biosynthetic pathways for better 2-KGA productivity. When mono-cultured in the shake flasks, the strain SyBE_Kv02080002 expressing hsk from Gluconobacter oxydans in threonine biosynthetic pathway achieved the highest biomass and the titer increased by 25.13%. When co-cultured with Bacillus endophyticus, the fermentation cycle decreased by 28.57% than that of the original consortium in 5-L fermenter. Furthermore, reconstruction of threonine biosynthetic pathway resulted in up-regulation of genes encoding sorbosone dehydrogenase and idonate-dehydrogenase, which increased the 2-KGA productivity in SyBE_Kv02080002. This study shows that reconstruction of absent biosynthetic pathways in bacteria is an effective way to enhance the productivity of target products.