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Table of Content

    31 August 2021, Volume 2 Issue 4
    Current contents in Chinese and English
    2021, 2(4):  0. 
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    Invited Review
    Great impact of Professor Daniel I.C. Wang and BPEC on development of biochemical engineering
    Zhiguo SU
    2021, 2(4):  470-481.  doi:10.12211/2096-8280.2020-095
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    Professor Daniel I.C. Wang was the founder of the Biotechnology Process Engineering Center (BPEC) at Massachusetts Institute of Technology (MIT). Under his leadership, BPEC has made remarkable achievements. It is a historical monument with great influence on the development of biochemical engineering. The historical background, outstanding contributions, innovative mechanisms, and significant impact of BPEC, led by Prof. Wang, were reviewed in this paper. (1) Leading biochemical engineering into a new era of biopharmaceuticals. With the development of genetic engineering in the 1970-1980s, a new era of modern biopharmaceuticals emerged. Professor Wang acutely felt that the production of biomacromolecule medicines by genetic engineering must solve a series of engineering science problems, which was the opportunity and challenge of biochemical engineering. He proposed to the National Science Foundation of the United States (NSF) for establishing BPEC at MIT. It was approved by the NSF in 1985. Compared with the initial concept of biochemical engineering at that time, the focus of BPEC had a great shift. The target molecules were no longer limited to microbial fermentation or bioconversion of antibiotics, chemicals and fuels. The mission of BPEC was to solve the engineering problems encountered by the large-scale production of various protein pharmaceuticals. In doing so, BPEC has successfully led biochemical engineering into the new era of biopharmaceuticals and established a unique position in the development of modern biotechnology. (2) Launching animal cell culture engineering. Animal cell culture is required for production of very complex biomacromolecules. While cell biologists were busy to scale up production by increasing the number of roller bottles, BPEC started engineering approach by design of bioreactors and control strategies. Successful bioreactor cultivation of mammalian cells was realized by minimization of shear stress and diffusion limitation. The culture nutritional composition was optimized by stoichiometric calculation. The strategy of nutritional medium addition was set up by metabolism analysis. Through intelligent cell culture control strategy and optimized reactor configuration, post-translational modification of the target protein was controlled at the optimal level to effectively avoid the loss of target protein. (3) Innovating separation and purification techniques for high quality biomacromolecules. Prof. Wang is one of the pioneers of biochemical separation engineering. He proposed biochemical separation engineering as another main direction of BPEC. Novel approaches such as affinity membrane, reversed micelles, electric-assisted separation were set up. He and his students discovered the mechanisms of protein refolding and aggregation. Using quasi-elastic light scattering technique, they were able to invent a novel technique of stabilizing refolding intermediates with polyethylene glycol. The smart work was published in Nature Biotechnology, and initiated various approaches of protein stabilization by different research groups. (4) Establishing a collaborative innovation and education system for biochemical engineering talents. Prof. Wang actively promoted the spirit of collaborative innovation, encouraged close collaborations among BPEC members, worked closely with biotechnology enterprises, and held lectures on downstream processing courses for R & D personnel of biotechnology companies in the US and other parts of the world. BPEC also attracted many young talents worldwide. Prof. Wang made the academic atmosphere of BPEC extremely active. The scientific exchange brought about brain storming to solve scientific and engineering problems. Numerous graduates and visiting scholars from BPEC have become successful professionals and entrepreneurs. (5) The influence of Prof. Wang and BPEC on biochemical engineering development in China. The influence of Prof. Wang and BPEC on biochemical engineering in China is the spirit of innovation. This spirit has been embodied to the strategic planning of the key laboratories and research directions from bioreactor to biocatalysis, from biological separation/purification to bioformulation, from equipment, media, and technologies to intelligent scale up productions. Biochemical engineering has become the support for sustainable development of life science and biotechnology in this country. Not only that, biochemical engineering researchers have integrated themselves into synthetic biology, gene editing, vaccine development and other frontier life science research, playing increasingly important roles.

    Contributions to the booming biologics industrialization —a dedicated pioneer: Professor. Daniel I. C. Wang
    Liangzhi XIE
    2021, 2(4):  482-496.  doi:10.12211/2096-8280.2021-051
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    Biologics including monoclonal antibodies and recombinant proteins have made significant impacts on the prevention and treatment of many important diseases, such as cancer, autoimmune disease and viral infections. Mammalian cell culture is a key but full-of-challenging technology for large-scale manufacturing of these high-demand and life-saving biologics. Until the early nineties, mammalian cell culture process was largely deemed as an unsolvable black box, and cell culture process scale-up was a significant challenge. As a result, mammalian cell culture process was an essential and key but bottle-neck technology for the development and manufacturing of biologics at that time. Prof. Daniel I.C. Wang, a pioneer in industrial biotechnology and founding director of MIT's Biotechnology Process Engineering Center, took the initiatives in the late 1960's to systematical study on all spectra of problems related to mammalian cell culture and its application in industrial biologics production. His research interests spanned from the microcarrier culture of adherent cells to the serum-free culture of suspension-adapted CHO cells; from batch culture, continuous culture, perfusion culture, air-lift fiber-bed culture to high-density fed-batch culture; from process technologies to engineering challenges. Several decades of Prof. Wang's leading role in the cutting-edge research and cross-discipline collaboration made significant breakthroughs in cell metabolism control, stoichiometrically-balanced culture medium and feed supplement design, serum-free medium development, inhibitory by-product control, protein glycosylation heterogeneity monitoring and control, on-line monitoring of bioreactor and cell culture process scale-up. Prof. Wang's achievements greatly advanced knowledge and understanding of the cell culture process and laid a solid foundation for the subsequent development and wide application of these novel technologies, especially the fed-batch culture process, in the commercial manufacturing of life-saving biological products and facilitated the booming of the biotech industry in the recent decades. This review article is dedicated in memorial of my Sc.D. thesis advisor Prof. Daniel. I.C. Wang!

    Remembering Professor Daniel I.C. Wang’s contribution to biorefining and my perspective on the progress
    Yi-Heng ZHANG
    2021, 2(4):  497-508.  doi:10.12211/2096-8280.2021-003
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    The goals of this article are in memory of Professor Daniel I.C. Wang's contribution to biorefining, the interaction and motivation between Professor Wang and me in this area, and I present opinions on addressing several key challenges of the next-generation biorefinery. Prof. Wang was the primary driver of innovation in both education and multidisciplinary research initiatives that have defined modern Biochemical Engineering, a frontier of chemical engineering. To meet great needs, he adjusted his scientific research directions timely throughout his career from microbial fermentation, single cell protein production, animal cell cultures to biorefineries. The industrial processes of the first-generation biorefinery plants based on food resources (e.g., corn kernel, sucrose, aged grains, used oil) have been very mature, but they potentially have negative impacts on food security and the environment, resulting in their limited development. Due to current low prices of crude oil, the second-generation biorefineries based on non-food lignocellulosic biomass are suffering from relatively high production cost, leading to their economic inviability. New biorefineries, which could be distinctive from the second generation biorefineries that produce liquid biofuels as dominant products, would convert lignocellulosic feedstock (e.g., rice straw, corn stover, energy crops) to multiple products, such as food and feed, healthy sweeteners, and other biocommodities (e.g., biofuels and biomaterials). New biorefineries could be implemented with newly-developed biochemical engineering tools, such as consolidated bioprocessing, novel enzymes, metabolic engineering, in vitro synthetic biology (i.e., multiple-enzyme molecular machines that assemble a number of natural enzymes, novel-function or engineered enzymes, coenzymes, and/or biomimetic coenzymes to form artificial pathways to implement novel biomanufacturing capacity beyond the limits of microbial fermentation and cell cultures). They could be industrial scalability, economic viability, and environmental sustainability. Also, agricultural revolution would take place by the cultivation of perennial plants in marginal lands to replace annual crops, wherein perennial plant communities have higher biomass yield per hectare with less resource management, store more carbon, maintain better water quality, utilize fertilizers more efficiently, tolerate extreme weather, and resist pests. The combination of the cultivation of nonfood perennial (energy) crops and next-generation biorefineries could address major social and economic needs of China, such as food supply, energy security, general health, and environmental conservation. Also, new biorefineries and new agricultural revolution could address challenge of our time to meet increasing need in the energy-food-water nexus without compromising the benefit of next generations.

    In memory of Prof. Daniel I.C. Wang: Engineering Yarrowia lipolytica for the production of plant-based lipids: technical constraints and perspectives for a sustainable cellular agriculture economy
    Peng XU
    2021, 2(4):  509-527.  doi:10.12211/2096-8280.2021-041
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    Developing cellular agriculture economy is one of the solutions to mitigate resource limitation, reduce greenhouse gas emission, slow down global warming as well as achieve true sustainability. Microbial cell factory has become a critical component to advance biomanufacturing due to the availability of versatile genetic tools, ease scale-up and the high conversion efficiency of low-cost renewable feedstocks. Prof. Daniel I.C. Wang was one of the trailblazers and founders of biochemical engineering, who built and led the Biotechnology Process Engineering Center (BPEC) at MIT. When I and my colleagues worked as postdoc associates and research scientists in Prof. Stephanopoulos' lab, part of our work on engineering oleaginous yeast cell factory was a direct result of the analytical, imaging and cell culture facility at BPEC. Plant-based oil and fats have an overall market value about $200 billion. Recently, the world has seen a craving for plant oil products, which negatively impacted our environment due to massive-scale deforestation and loss of ecological diversity in tropical regions. We thought oleaginous yeast could be a solution to this problem. Centering around the important genetic targets of the oleaginous species Yarrowia lipolytica, we summarized the essential metabolic engineering strategies for improving the carbon conversion efficiency (yield), lipid titer, lipid production rate (productivity) and cell growth fitness. We further analyzed technical constraints that limit our ability to build high oil-yielding yeast cell factory, including high throughput strain screening or phenotyping techniques, the incomplete understanding of the lipogenesis and underlying regulatory mechanism, as well as the lack of well-defined biochemical models to guide bioprocess optimization and scale-up. Further we discussed the technical and economic feasibility of converting sugarcane feedstock to high value plant-based lipids with metabolically engineered Y. lipolytica. Our analysis indicates that Y. lipolytica has a great potential to address the current market gap of high value plant-based fats (i.e. cocoa butter equivalent to make chocolate with a potential market of $50 billions). Engineering oleaginous yeast to provide plant-based healthy fats will help us address energy, foods, environment and resource challenges, which will surely move us one step closer to a society of low-carbon footprint and sustainable economy.

    Biological synthesis and applications of artificial protein functional materials
    Dan ZENG, Jianlin CHU, Yanru CHEN, Daidi FAN
    2021, 2(4):  528-542.  doi:10.12211/2096-8280.2020-091
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    Artificial protein functional materials have good biocompatibility, biodegradability, broad resources of raw materials, and diverse functions. As ideal biological materials, they have a wide range of application prospects in bioengineering, medicine, military and textiles. However, microbial synthesis of protein functional materials still has problems such as low expression and unstable performance, which severely restrict their efficient production and application. In recent years, there is a trend of designing artificial protein functional materials that not only possess multi-functional properties but are biomimetic, adaptive and dynamically responsive to biological processes, and such a trend puts forward new requirements for the design and engineering control of macromolecular systems, which raise a necessity for developing research methods to assemble engineered protein molecules into functional macroscopic materials. Under the guidance of engineering concepts, synthetic biology provides a strategy for developing "precise design-system construction-regulatory expression-engineering application" for artificial protein functional materials. This review comments the research progress in the biological synthesis of major protein functional materials such as spider silk protein, silk protein, human-like collagen and mussel protein, and explores the construction of artificial protein cell factory, the regulation of protein expression and their applications as tissue engineering materials and other fields. In view of the current problems of poor biocompatibility and insufficient efficacy of clinical products, artificial protein functional materials can be assembled and processed according to functional requirements, and used to fabricate artificial tendons, artificial skin, degradable hemostatic materials, artificial bones and new protein material medical products such as high-viscosity anti-fouling coating products. There are still several challenges that need to be solved urgently: (1) There are few theoretical analysis tools and models at this stage, making the design, prediction and analysis of functional proteins relatively limited, and a more comprehensive protein database needs to be established; (2) There are many expression regulators in E. coli and other microbial systems, and adaptation for the design of expression elements and the target gene, the regulation of protein synthesis pathways and the systemic nature need to be studied to improve the efficiency of protein expression; (3) In terms of engineering applications, it is also necessary to comprehensively consider material stability and biological safety. Finally, the research direction is prospected, which provides insights for scholars working in related fields.

    From single-enzyme catalysis to multienzyme cascade: inspired from Professor Daniel I.C. Wang’s pioneer work in enzyme technology
    Shuke WU, Yi ZHOU, Wen WANG, Wei ZHANG, Pengfei GAO, Zhi LI
    2021, 2(4):  543-558.  doi:10.12211/2096-8280.2021-004
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    As the founder and pioneer in the fields of bioengineering and biotechnology, Professor Daniel I. C. Wang at the Massachusetts Institute of Technology (MIT) had made tremendous contributions in many aspects in the up-, mid- and downstream of biochemical engineering for over 50 years. In the area of enzyme technology, he had impell several significant advances from the 1970s to 1990s, such as enzymatic digestion of fish protein, immobilization of methanol oxidase, cell-free multi-enzyme synthesis, and enzyme catalysis in organic medium, etc. From 2005 to 2015 through the Singapore-MIT Alliance Program, Prof. Wang had jointly supervised several PhD students with Prof. Li Zhi at the National University of Singapore, and achieved important contributions biocatalysis, including: 1) developed enzyme immobilization methods for fabrication of highly active and recyclable magnetic nano-biocatalysts; 2) explored P450 monooxygenase for asymmetric sulfur oxidation in aqueous phase-ionic liquid systems; 3) developed an NADPH regeneration system based on permeabilized whole cells; 4) successfully developed modular multi-enzyme cascade catalysis to synthesize high-value chiral compounds, which significantly expanded the realm of biocatalysis. Among them, multi-enzyme cascade catalysis has become a research hotspot in the field of enzyme technology, due to its several desirable features, including the possibility of retrosynthetic design of various synthetic routes, the facile one-pot synthesis of final products, the reduction of additional unit operations, the saving of inputs from manpower and materials, and the minimization of waste generation. In this account, we also review the latest progress of multi-enzyme cascades for the synthesis of chiral compounds (e.g., chiral amines, amino acids) and bulk chemicals (e.g., precursors for polymers), and discuss its future development directions. Last but not the least, we provide an outlook for integrating multi-enzyme cascades with synthetic biology, and thus assembling biochemical reactions together with quantitative analysis and engineering concepts, as advocated by Prof. Wang throughout his scientific career.

    Application of multi-enzyme catalytic system in the synthesis of pharmaceutical chemicals
    Heng TANG, Xin HAN, Shuping ZOU, Yuguo ZHENG
    2021, 2(4):  559-576.  doi:10.12211/2096-8280.2021-028
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    The multi-enzyme catalysis system has become a hotspot in the field of bio-catalysis in recent years. Due to the catalytic process's controllability and the ease of downstream separation, more and more intracellular metabolic pathways are being developed and applied to produce fine chemicals in vitro. With the gradual maturity of multi-enzyme catalytic system construction technology, it will bring a broad application prospect for the manufacture of chemical products and pharmaceutical products in the future. In this regard, we review recently published examples of multi-enzyme catalysis, compare relevant design principles. Thermodynamics and kinetics of the reaction process and synthesis path analysis are used to design a bio-catalysis path. The critical enzymes are then mined in the pathway. By combining various assembly strategies, enzymes with different functions are cascaded into a whole structure and function unit to form a "substrate channel." Then intermediate loss and side reaction was reduced during the efficient bioconversion process from simple substrates to complex products. This method has the advantages of improving reaction efficiency, high regional selectivity and stereoselectivity, and also reducing environmental impact. Thus, biocatalysts are now widely used to produce valuable commercial chemicals, pharmaceuticals, and fuels. The target products could be obtained by the bio-catalysis process in vivo or in vitro. High-value products that are difficult to synthesize, or in an extremely complex synthesis pathway, or a very costly synthesis process by non-biological methods can now be produced in microbial hosts. To match the corresponding products, various genes in the host cell need to be optimized and fine-tuned. However, in vitro bio-catalysis can avoid these limitations. By adding a variety of enzymes to the reaction system, the product can be obtained after completing the catalysis at one time. Thus, industrial bio-fabrication has emerged as an attractive and economically viable alternative to conventional large-scale chemical synthesis. The multi-enzyme catalysis system in the synthesis of pharmaceutical chemicals (such as antibiotics, drugs for anti-cancer, cardiovascular disease treatment, liver disease treatment and psychiatric treatment) and various active ingredients (such as D-gluconic acid, terpenoids, 5-aminolevulinic acid) were discussed. The problems existing in the multi-enzyme catalytic system and the possible solutions are also summarized.

    Research progress of cyclic lipopeptide biosynthesis
    Zhengjie HOU, Huizhong SUN, Song BAI, Xinyue CHEN, Chunyang CAO, Jingsheng CHENG
    2021, 2(4):  577-597.  doi:10.12211/2096-8280.2021-008
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    As antibiotics and bio-surfactants,cyclic lipopeptides have unique molecular structure and biological activity and are widely applied in the fields of biological control, drug development, environmental remediation and disease treatment. It has vigorous market demand and promising future. Cyclic lipopeptides are a class of antibiotics synthesized from non-ribosomal peptide pathways by microorganisms. However, the complex metabolic network and precursor requirements, specific and strict synthetic pathway, and the coexistence of multiple homologues are restricting our capability of developing the lipopeptide syntheses potential of microorganism and promoting the product value of lipopeptide of bacteria. In this paper, we summarize the types of chassis cells for producing cyclic lipopeptides. We also introduce the structural characteristics of cyclic lipopeptides based on bacterial origin, synthesis pathway of non-ribosomal peptide, and structural domain characteristics of non-ribosomal peptide synthetase. In addition, the strategies for homologues regulation and biosynthetic yield improvement through genetic and metabolic engineering methods were reviewed, as well as the development status of natural product chassis strains. The synthesis of lipopeptide products can be effectively improved by optimizing the precursor metabolism, enhancing the expression of lipopeptide synthesis gene cluster, blocking the competitive pathway of lipopeptide synthesis, and the modification of various regulatory factors. The non-ribosomal peptide synthetase structural domain can be modified to obtain higher value lipopeptide and new lipopeptide drugs. We also review the effects of mixed-culture on lipopeptide syntheses, the development status of chassis strains for producing natural product, and the application of synthetic biology for improving lipopeptide biosynthesis. With the rapid development and application of synthetic biotechnology, the quality and quantity of natural lipopeptide from microorganisms will be improved rapidly. It also boosts the development of novel cyclic lipopeptides. And a better understanding of the synthesis, modification and mechanism of action of antimicrobial peptides will restart its commercial development.

    Microbial promoter engineering strategies in synthetic biology
    Huimin YU, Yukun ZHENG, Yan DU, Miaomiao WANG, Youxiang LIANG
    2021, 2(4):  598-611.  doi:10.12211/2096-8280.2020-092
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    Synthetic biology is of vital importance to the green biomanufacturing industry and sustainable development strategies of our country. Promoter is the core-component of synthetic biology, playing a significant role in highly efficient and fine-tuning expression and regulation of target genes at the transcriptional level. Herein we summarized and discussed the key progress and future frontiers of microbial promoter engineering, particularly for prokaryotic microorganisms. Firstly, we introduced the basic DNA sequence characteristics of promoters and the regular mechanism for promoter recognition and transcription-initiation by RNA polymerase sigma factors. Inducible mechanisms for both negative and positive regulation were particularly highlighted with the typical lac operator of Escherichia coli as an example. Then, effective strategies for obtaining improved-promoters were summarized, which were roughly divided into two categories: endogenous promoter mutation and heterologous promoter replacement. For the endogenous promoter mutation, the following strategies, e.g. point mutation toward sigma factor consensus sequence, coupling optimization of -35 and -10 regions with RBS sequence, random mutation or saturation mutagenesis of UP element or spacer sequences accompanying with promoter library construction and high-throughput screening were emphasized. For the heterologous promoter replacement, strategies such as substituting the native promoter into stronger ones from other microorganisms, introducing phage-source chimeric promoters, tuning the constitutive promoter into inducible pattern and integrating positive regulator(s), were mainly discussed. We further sorted out the representative inducers for inducible promoters reported so far, including both chemical molecules and physical signals. Progress in constitutive promoters of non-model and model microbial organisms were simply summarized as well. Next, arising from the breakthrough development of dynamic metabolic regulation and artificial intelligence (AI), we proposed that the innovative research on identification and evolution of new and unique promoters with dynamic-response features and AI de novo design for promoters with novel/superior functions will be the new frontiers of promoter engineering. Finally, we analyzed the challenging scientific issues in the microbial promoter engineering, from the viewpoint of both basic research and large-scale applications; and further discussed the research priority coupling with the vigorous development of synthetic biology.

    Present situation and prospect for large-scale mammalian cell culture engineering
    Ziyu ZHU, Guan WANG, Yingping ZHUANG
    2021, 2(4):  612-634.  doi:10.12211/2096-8280.2020-094
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    With requirements for the improvement of the quantity and quality of vaccines, therapeutic protein drugs and many other biological products, large-scale cell culture technologies continue to be developed. In order to increase production and reduce cost to produce safer and more effective drugs, the development of large-scale cell culture processes is essential, while the process optimization and scale up of animal cell culture are challenging. Ongoing research and development of animal cell culture technology are urgently required to increase the expression level of target poducts, to expand the scale of cell culture and to ensure stable product quality. This article focuses on above issues and systematically summarizes the establishment of suitable large-scale culture to achieve high-density and high-efficiency production through the construction of excellent cell lines, the design of medium, particularly serum-free medium , as well as the optimization and scale-up of the culture process based on process analysis technology (PAT). Genomics and gene editing tools are frequently applied to construct an excellent cell line that supports high-density growth and secretes a large amount of therapeutic proteins. With the in-depth study of cell physiological and metabolic characteristics, serum-free culture can be designed based on multi-omics study, and through a variety of online sensing technologies to monitor and analyze the biological process, precise feedback control can thus be performed. On the other hand, the generation of a large amount of inconsistent data during the cell culture process is basically due to inefficient manual processing and judgment, and lacking of an in-depth consideration of global factors. Therefore, for the high-efficiency industrial biomanufacturing process, it is necessary to visualize a large number of process parameters, establish a database of process parameters for subsequent big data analysis, and conduct deep learning and data mining to perform real-time bioprocess intelligent analysis, diagnosis and precise control, and then realize intelligent manufacturing.

    Design and progress of synthetic consortia: a new frontier in synthetic biology
    Yu LIU, Huiling WEI, Jixiang LIU, Shaojie WANG, Haijia SU
    2021, 2(4):  635-650.  doi:10.12211/2096-8280.2021-031
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    Metabolic engineering and synthetic biology have enabled efficient bioproduct synthesis in mono-microbial culture by pathway reconstruction. In spite of that, in the demand of increasing bioproduction, mono-culture has gradually showed some limitations in the tasks of accomplishing complicated biosynthesis and achieving high efficiency. The expression of multi-step biosynthetic pathways in one cell may greatly increase the metabolic burden of the host, which may result in a huge imbalance between cell growth and gene expression and thus decrease the titer of bioproduct. Synthetic consortia have been designed as an alternative effective biosynthesis system to distribute complex functions and pathways into different cells or species. In this article, the advantages of the synthetic consortia are summarized, including providing better environment for different functional genes expression, balancing working intensity, reducing metabolic burden, plug-and-play and eliminating feedback inhibition. Based on the above advantages and taking recent advances in biosynthesis studies utilizing synthetic consortia systems as case studies, some design principles involving functional modules, synergism and robustness are put forward in this article. To be specific, it is necessary to reasonably divide and allocate functional modules into different strains in the system. With appropriate chassis strains, it will be beneficial to establish synergistic substrate utilization and mutually beneficial symbiosis between different strains, which could increase the connections between functional modules as well as the interactions between chassis strains. Moreover, the application of population control strategies will efficiently increase system robustness. Under the premise of material exchange, spatiotemporal distribution in synthetic consortia is conducive for better coordination and interaction between strains. In addition, modeling on synthetic consortia systems have been initiated to simulate bioproduct process and optimize consortia construction. In response to the current problems, some global regulation methods and prospectives for synthetic consortia are also proposed in three aspects: internal optimization, external reinforcement, and mathematical modeling.

    Creation of non-natural cofactor-dependent methanol dehydrogenase
    Junting WANG, Xiaojia GUO, Qing LI, Li WAN, Zongbao ZHAO
    2021, 2(4):  651-661.  doi:10.12211/2096-8280.2021-016
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    The C1 organic carbon compound methanol is a potential raw material for biorefinery. Recently, intensive efforts have been devoted to engineer cell factories for direct conversion of methanol into valuable metabolites. The oxidation of methanol into formaldehyde is the first committed step to provide useful substance for metabolism. While some methylotrophic microorganisms oxidize methanol with hydrogen peroxide as a co-product, many engineered systems are designed to co-produce NADH, the reduced nicotinamide adenine dinucleotide (NAD). The later route, normally catalyzed by NAD-dependent methanol dehydrogenase (MDH), is more attractive as NADH can be used as reducing power for cellular metabolism. However, NAD(H) are used by many redox enzymes and methanol oxidation-derived NADH can cause unpredictable biological effects. We recently engineered the cofactor preference of several NAD-dependent redox enzymes to favor a non-natural cofactor nicotinamide cytosine dinucleotide (NCD). By coupling these enzymes we demonstrated the construction of NCD-linked redox systems orthogonal to the natural cofactor NAD(H), which could be used for pathway-selective chemical energy transfer in Escherichia coli. By redesigning the cofactor binding pocket of MDH, it is possible to obtain mutants favoring NCD, and thus utilize methanol as a carbon source with co-producing reduced NCD for dedicated redox chemistry. In this paper, we first analyzed the cofactor-binding pocket of Bacillus stearothermophilus DSM2334 derived NAD-dependent MDH (UniProt: P42327.1). Through virtual screening and single-site mutation library screening, hot spots for cofactor binding were identified. More mutants were rationally generated based on insights into the volume of cofactor binding cavity and those were obtained with improved activities in the presence of NCD. The mutants were overexpressed in E. coli, purified, and their catalytic performance were analyzed. The results showed that the catalytic efficiency of the mutant 9D1 (MDH Y171R/I196V/V237T/N240E/K241A) with NCD reached 858 L/(mol·s), and its NCD preference was 13 000-fold higher than that of the wild-type protein. These MDH variants can be considered as new functional parts for the bioconversion of methanol and the construction of new redox metabolism.