Table of Content

30 April 2021, Volume 2 Issue 2

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2021, 2(2):  0. 

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Invited Review



Advances in synthetic biomanufacturing

Yuanyuan ZHANG, Yan ZENG, Qinhong WANG

2021, 2(2):  145-160.  doi:10.12211/2096-8280.2020-052

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Synthetic biomanufacturing is a paradigm for material processing and synthesis via synthetic biology. It is expected to completely transform the traditional production mode for medicines, chemicals, food, energy, materials and agriculture in the future, trigger new industrial revolution, lead to new economic growth and reshape carbon-based civilization. In particular, the COVID-19 global spread that has accelerated the reshaping process of the world's economic and social development. In the foreseeable future, human life and production patterns will undergo profound changes in medicines and healthcare, food and agriculture, energy and materials, etc., during which demands for new technologies will promote evolution in the field of biotechnology, and the biomanufacturing industry in the post COVID-19 era is facing unprecedented opportunities for revitalization and new challenges. According to the analysis of the research report from the Ministry of Economy, Trade and Industry of Japan, engineered biological cells and their combination with information & artificial intelligence technologies will become the main driving force for the "post-fourth industrial revolution".Synthetic biomanufacturing has the characteristics of cleanliness, efficiency and renewableness that can reduce the impact of industrial economy on the ecological environment. Here, in this review, we summarize the progress of synthetic biomanufacturing with respect to bulk fermentation products, fine and pharmaceutical chemicals, renewable chemicals and bio-based polymeric materials, natural products, foods and the utilization of C₁ raw materials. The technological progress, status and potential of industrial applications of many important bio-based products via synthetic biomanufacturing are analyzed and discussed. The development of synthetic biomanufacturing shows great potentials for building up the ecological route of industrial economy and addressing current issues of economic sustainability in terms of limited substrate, high cost, and poor viability, and to form whole new industry chain with sustainable growth. In the future, with the development of synthetic biology, and the integration of new technologies such as artificial intelligence and big data, more and more bio-based products can be produced via synthetic biomanufacturing. The formation of bioeconomy can be promoted, and the sustainable development of human society will be better served.



Synthetic biology boosts biological depolymerization and upgrading of waste plastics

Xiujuan QIAN, Jiawei LIU, Rui XUE, Haojie LIU, Xiaohong WEN, Lu YANG, Anming XU, Bin XU, Fengxue XIN, Jie ZHOU, Weiliang DONG, Min JIANG

2021, 2(2):  161-180.  doi:10.12211/2096-8280.2020-087

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Characterics of high molecular weight, high hydrophobicity and high chemical bond energy make petroleum-based synthetic plastics resist to abiotic and microbial degradation. "White pollution" caused by accumulated waste plastics in the environment has become a global challenge. Currently, landfill and incineration are the simplest and most commonly used methods for eliminating plastic wastes, given that only 20% of plastic wastes is recycled, but landfill and incineration cause more serious secondary hazards, such as pollution to groundwater, soil, air and ocean. Therefore, developing a green and efficient technology for recycling and reutilization plastic wastes is the key for solving plastic pollution, to boost a plastic recycling economy.Applications of microorganisms/enzymes to degrade plastics into oligomers or monomers, which can be further transformed into high-value added chemicals, have provided a new approach for such a purpose due to their mild and environmentally friendly proceses. This article comprehensively reviews the development of biodepolymerization and biotransformation for waste plastics, including mining plastic degrading microorganisms and enzymes, designing and constructing microbial consortia/enzyme cocktails, analyzing of plastics depolymerization mechanism, and transforming plastics degradants into high value-added products, such as chemicals, energy products, and materials. However, the lack of degradation enzyme components, low degradation efficiency and difficulty for utilizing the degradants limit the development of waste plastics biogradation. With advances in synthetic biology, emerging technologies, such as high-throughput screening, evolutionary metabolism, and bioinformatics to analyze the catalytic mechanism of key degradation enzymes, orientedly designing and modification of degradation enzymes, study of the mutualism relationship and mechanism in the microbial consortia, constructing metabolic pathways for different plastic degradants, will open windows for waste plastics biodegradation, providing environmentally friendly， economically competitive and technically feasible technologies to develop circular economy for the re-utilization of waste plastics in China.



Development and application of synthetic microbiome

Zhaoyong XU, Haiyang HU, Ping XU, Hongzhi TANG

2021, 2(2):  181-193.  doi:10.12211/2096-8280.2020-062

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In recent years, the construction of artificial bacterial communities, usually referred to "synthetic” or “engineered” microbiomes, with better attributes and stability, has been a hot topic. The archetype synthetic community integrates different strains by balancing their nutrition, which occurs when strains produce and consume essential nutrients in a complementary fashion. Other key features of synthetic communities include: reciprocal interactions of metabolic pathways. On the one hand, complete metabolic pathways may develop only at the population level, and the metabolic burden on any single strain can reduce. On the other hand, growth inhibition can relief, since metabolites of one strain promote the growth of another, and reduction of the mutation rate guarantees a stable community. When combined, these features ensure intercellular interactions, spatiotemporal organization, community robustness, and biocontainment of synthetic communities. There are two general approaches for engineering microbiomes. One is "bottom-up", through which genetic circuits involving different strains are designed artificially. This approach can be more controllable, but the precise knowledge of metabolic pathway details is needed. Another approach, known as "top-down", involves the careful optimization of a core consortium from a group of natural microbiomes which can be easier to carry out, but is less designable. Synthetic microbiomes can be expected to handle complex tasks more efficiently, stably, and safely, which show a series of special characters compared with one single strain, such as apparent metabolic burden reduction and offering a platform with excellent compatibility for the expression of diverse genes. By now, the synthetic microbiome approach has already been applied to industrial production and environmental remediation, such as the biosynthesis of biofuels, chemical products, and biomedicines, and for the bioremediation of petroleum contamination and residues of petroleum derivatives and pesticides. Synthetic microbiomes open a new direction for applications of microbial technology with improved stability and compatibility. This approach could be used to enhance the roles of particularly valuable strains, helping to extend their applications to more complicated tasks in extreme environments.



Recent advances in metabolic engineering of clostridia for n-butanol production

Zhiqiang WEN, Xiaoman SUN, Qingzhuo WANG, Yanan LI, Wenzheng LIU, Yu JIANG, Sheng YANG

2021, 2(2):  194-221.  doi:10.12211/2096-8280.2020-080

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n-Butanol is a bulk chemical and renewable and alternative vehicle fuel. It can be produced from biomass by some microorganisms, which has potential to replace the unsustainable and environment-hazardous petroleum refining methods. In this work, we review and compare various metabolic pathways and chassis cells for n-butanol production, and highlight that clostridia are natural cell factories for n-butanol production with obvious advantages in butanol titer and productivity. However, strains from this species are still unsatisfactory, which includes inefficient strain genetic modification, low n-butanol yield/ratio, rigid n-butanol synthesis pathway, and low substrate utilization spectrum.Fortunately, synthetic biotechnology has significantly accelerated the development of genetic manipulation tools, and many of them including TargeTron, allelic-exchange, CRISPR/Cas system mediated gene and base editing tools have been developed and applied in clostridia. Various genetic operations such as insertion, deletion, substitution, site mutation, and regulation of target gene expression can be efficiently implemented in clostridia, which laid a foundation for its metabolic engineering. As a result, significant progress has been made in increasing n-butanol titer/yield/ratio, reconstructing and refining the n-butanol synthesis pathway in clostridial chassis, as well as enhancing pentose utilization. The enhancement of the n-butanol pathway and the weakening or deletion of pathways for producing by-products such as acetone, acetic acid, butyric acid have increased butanol titer and ratio.In addition, unconventional clostridia including cellulolytic and gas-fermenting strains have been metabolically engineered for homo-butanol fermentation through decoupling with acetone production. Moreover, genetic manipulation tools also facilitate the reconstruction of the clostridial pentose (xylose and arabinose) transport/metabolism pathway and analysis of carbon catabolite repression (CCR) mechanism, which greatly improved the utilization of pentose. In this article, we review the above metabolic engineering strategies and important milestones of n-butanol production, and address the current bottlenecks and future trends. With the driving of synthetic biotechnology, the cost of n-butanol production by clostridia will be reduced, making it eventually competitive in the market.



Research progresses and future prospects of synthetic methylotrophic cell factory for methanol assimilation

Hui ZHANG, Yaomeng YUAN, Chong ZHANG, Song YANG, Xinhui XING

2021, 2(2):  222-233.  doi:10.12211/2096-8280.2020-048

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Methanol is an important and attractive non-sugar carbon source for industry biotechnology due to its advantages of available source, easy storage, convenient transportation and competitive price. The widely-studied microorganisms such as Escherichia coli, Corynebacterium glutamicum and Saccharomyces cerevisiae have a long research history, clear genetic background and a number of matured genetic tools, showing their potential in engineering cell factories for bioproduction. With the accumulated knowledge of native methylotrophs in recent years, these traditional industrial microorganisms have been engineered as methylotrophic cell factories (MeCFs) capable of using methanol as the major carbon and energy source. In this artical, we review the recent research progress including genes involved in the methanol oxidation, assimilation and regulatory elements of MeCFs. We also summarized the strategies based on the adaptive laboratory evolution which applies sugar as the co-substrate to construct the ribulose monophosphate (RuMP) cycle, as well as strategies for designing and tuning methanol assimilation pathway. In the synthetic MeCFs, the construction of the RuMP cycle requires the introduction of three heterologous enzymes, i.e., methanol dehydrogenase encoded by mdh, 3-hexulose 6-phosphate synthase encoded by hps and 6-phospho 3-hexuloisomerase encoded by phi. These enzymes can be further engineered to improve activities through protein engineering and directed evolution. Meanwhile, genes involved in methanol oxidation and assimilation pathways can be finely tuned to exhibit dynamic expression. To further improve methanol utilization of the synthetic MeCFs, the co-substrate for supporting growth has been developed to propel methanol assimilation, which rationally designs a methanol dependent growth model, and thus provides an ideal starting point for subsequent long-term adaptive laboratory evolution experiments. At the end, we discuss the challenges of engineering the synthetic MeCFs, and summarize the future prospects for improving the efficiency of methanol utilization through the combined strategies of genome-wide targeted gene editing and adaptive laboratory evolution.



Construction and engineering application of bacterial quorum sensing elements

Ailin ZHOU, Yi LIU, Fang BA, Chao ZHONG

2021, 2(2):  234-246.  doi:10.12211/2096-8280.2020-066

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Quorum sensing refers to the specific communication among bacteria which could synchronize individual behaviors to collective benefits. This mechanism relies on the production, detection, and response to extracellular signaling molecules called autoinducers. Based on the understanding of scientific fundamentals underlying the activation and inhibition of natural quorum sensing systems, researchers attempt to introduce quorum sensing into engineering applications as modules or gene parts through synthetic biology, and employ it in fields such as medicine, industry and environment. In this review, strategies and methods used in the construction of quorum sensing system are briefly discussed, and the promising applications of engineered bacteria with quorum sensing for dynamic metabolic reflux, oscillation, and microbial consortia are also highlighted. The construction of quorum sensing elements requires the grasp of its essence, which involves the development of new elements and the optimization of existing ones. By simulating natural quorum sensing systems, and optimizing and modularizing quorum sensing elements, researchers construct libraries for quorum sensing elements, making them possible to employ quorum sensing under different circumstances. Moreover, by introducing quorum sensing, various feedback loops initially possessed by a single bacterium could be extended to the whole population. With the construction of such multicellular quorum sensing systems, more complex functions could be initiated, such as dynamic regulation of metabolic flux for boosting fermentation efficiency, robust production of drugs by collective oscillation and so on. In addition, microbial consortia containing could be manipulated by introducing exogenous quorum sensing systems, providing new tools for microbial co-culture and new ideas for the construction of biological systems with higher complexity. In the future, machine learning will be applied for designing complex quorum sensing circuits and accurately predicting the behavior of exogenous quorum sensing systems in certain microbial population.



Genome design and synthesis: from replication to rational design

Junyi WANG, Xiaole WU, Yueyang CAO, Bingzhi LI

2021, 2(2):  247-255.  doi:10.12211/2096-8208.2020-051

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With the advancement of related technologies for gene assembly, artificial genome assembly capabilities have made breakthroughs continuously, and synthetic genomics has been developed as a research hotspot in recent years. The chemical synthesis of genomes for several viruses and prokaryotes have been completed gradually, and the synthesis of eukaryotic genomes has also been explored. In the study of synthetic genomics, researchers continue to explore principles for genome design and increase its scale and depth, from the successful replication of virus and bacteriophage genomes, the substantial simplification in the Mycoplasma mycoides JCVI-syn3.0 genome to exploring multiple genome design principles in the eukaryotic Saccharomyces cerevisiae. This article summarizes the related progress of artificial genome design, with its main contents focused on: the change of artificial genome codons, the addition of artificial tags, the insertion of artificial sites and the research of genome simplification. In 2016, Church's group used synonymous codons to replace seven codons within the entire genome of E. coli, but the E. coli strain could not survive. In 2019, Chin's research group completed the synthesis of E. coli containing 61 codons using the method of complete genome synthesis, and the E. coli strain can survive normally. Four "watermark" sequences were introduced into the synthetic M. mycoides genome, and a large number of PCR tags were introduced into the synthetic Saccharomyces cerevisiae genome to distinguish between synthetic and wild-type genomes. A highlight of the synthetic yeast chromosome design in the Sc2.0 is the insertion of a reverse symmetric artificial site-loxPsym sequence after the stop codon of each non-essential gene. As a result, a SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxPsym-mediated Evolution) system that can rapidly perform genome rearrangement including deletion, duplication, inversion and translocation was formed in the synthetic yeast. The system has been continuously improved in applications, gradually making it an effective mean to optimize the host, increase the yield and enhance stress tolerance of the strain. In addition, the prospect of the rational design of genomes and rules for genome simplification is also discussed.



Research progress of genome editing technologies for industrial filamentous fungi

Qian LIU, Jingen LI, Chenyang ZHANG, Fangya LI, Chaoguang TIAN

2021, 2(2):  256-273.  doi:10.12211/2096-8280.2020-073

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Filamentous fungi, which are a large and diverse group of multicellular eukaryotic microorganisms existing in nature universally, have played pivotal roles in biotechnology as the prominent producers of enzymes, organic acids and antibiotics. Filamentous fungi are also important decomposers that contribute to the biological carbon cycle of plant biomass. Genetic engineering is a powerful approach for researchers not only to elucidate the gene function in filamentous fungi, but also to improve their production levels and minimize unwanted by-product formation. However, the efficiency of homologous integration in filamentous fungi is very low using classical genetic approaches. Due to the complicated growth and lifestyle of filamentous fungi, the development of genetic tools and gene editing is relatively slow, which hinders the basic research and biotechnological development of filamentous fungi. In recent years, genome editing technologies based on programmable nucleases have been developed as the powerful gene engineering tools in a wide variety of organisms. The most rapidly developed and wildly used technology is the class of RNA-guided Cas9 nuclease known as the adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats). The Cas9-sgRNA complex binds to the corresponding target site of the protospacer in a genome, and the specifically induces double-strand breaks. These breaks can be used as a basis for site-specific mutagenesis mediated by non-homologous end-joining or for the introduction of precise mutation or integration via homology-directed repair. CRISPR-Cas systems have recently enabled a wide range of applications for genome editing in many organisms. Remarkably, and in just the past few years, the CRISPR-Cas9 system has emerged as a more efficient strategy for gene editing in filamentous fungi. In this review, we describe the research progress of three most widely used genome editing systems, including the development of CRISPR-Cas technology and its applications in filamentous fungi, especially recent advances in industrial filamentous fungi. Finally, we give the perspectives for the CRISPR-Cas technology and its derivative systems for genomic editing in filamentous fungi.



Progress and challenge of the CRISPR-Cas system in gene editing for filamentous fungi

Han XIAO, Yixin LIU

2021, 2(2):  274-286.  doi:10.12211/2096-8280.2020-078

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Filamentous fungi are a group of microorganisms that play important roles in producing proteins (enzymes) and secondary metabolites as well as treating environmental pollutants. The basic and applied research on filamentous fungi, including identification of gene function and activation of silent gene cluster, relies heavily on gene editing. However, the apical growth, heterokaryosis, low efficiency of homologous recombination, and the lack of selective marker pose challenges for establishing gene editing platforms in filamentous fungi. In recent years, the RNA-mediated Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein (Cas) system has been widely employed in engineering filamentous fungi. Due to its simplicity and target specificity, the CRISPR-Cas system has assisted gene insertion, gene deletion, base conversion and transcriptional activation in this species. The edited targets can be single gene encoding a marker or enzyme with known or unknown function, and multiple genes as well, and the editing scale varies from one base to 48 kb. Furthermore, the CRISPR-Cas system allows precise modification at target site by introducing the cleverly designed homologous recombination donor and disrupting key genes in the non-homologous end joining (NHEJ). In this review, we comment research progress of the CRISPR-Cas system in gene editing for filamentous fungi that has been achieved in the past three years, with main focus on the delivery of CRISPR-Cas, in vivo expression of Cas protein and guide RNA (gRNA), the design of homologous recombination arms, and host modifications. The low efficiencies in both gene transformation and editing are still main challenges for CRISPR-Cas assisted gene editing in filamentous fungi , which is expected to be addressed by breakthroughs in fundamentals such as interactions between genotype and phenotype to discover genetic determinants.



Advances in yeast based adaptive laboratory evolution

Yi LI, Zhenquan LIN, Zihe LIU

2021, 2(2):  287-301.  doi:10.12211/2096-8280.2020-077

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Saccharomyces cerevisiae, as an eukaryotic model organism and microbial cell factory, has been widely used in metabolic engineering, system biology and synthetic biology. However, due to the limited knowledge of the complex cellular metabolism and inherent regulatory networks, it is difficult to obtain desired phenotypes through rational engineering. Among reported engineering strategies, evolutional engineering plays an important role in the construction of robust microbial cell factories, particularly when optimizing the whole metabolic or genome-scale network. Adaptive laboratory evolution mimics natural evolution in the laboratory via iterative cycles of culture and selection to isolate desirable phenotypes, such as tolerance of high salt concentration, low pH, high temperature condition as well as toxicities from substrate at excess and product accumulated to high titers. With recent advances in genome editing, synthetic biology and systems biology, development in evolution engineering has made great progress, including the computer-aided system and automatic continuous evolution technology. This review focuses on recent technological advances in evolution engineering tools for S. cerevisiae. Firstly, recently developed genome evolution strategies are discussed, including the recombinase-based genome-scale engineering system SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) and SCRaMbLE-in, oligo-mediated YOGE (yeast oligo-mediated genome engineering), oligo-based genome-scale engineering eMAGE (eukaryotic multiplex automated genome engineering), CRISPR mediated genome-scale engineering systems CHAnGE (CRISPR/Cas9-and homology-directed-repair-assisted genome-scale engineering), and MAGESTIC (multiplexed accurate genome editing with short, trackable, integrated cellular barcodes), Target-AID (target-activation induced cytidine deaminase). Then, yeast-based automated continuous evolution, such as OrthoRep (orthogonal error-prone replication), ICE (in vivo continuous evolution), eVOLVER (a scalable and automated continuous culture device), and ACE (automated continuous evolution, pairs OrthoRep with eVOLVER), are further addressed. These rapid editing strategies at the genome level can generate genetically diverse cell populations to identify key factors and synergies in a short period of time. Finally, we prospect future challenges and opportunities of evolution engineering approaches in advancing yeast-based microbial cell factories. Strategies learned from yeast will also guide the development of other microbial cell factories.