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This article provides an overview of microbial host selection, synthetic biology, genome annotation, metabolic modeling, and computational methods for predicting gene essentiality for developing a microbial chassis. This article focuses on lactic acid bacteria (LAB) as a microbial chassis and strategies for genome annotation of the LAB genome. As a case study, Lactococcus lactis is chosen based on its well-established therapeutic applications such as probiotics and oral vaccine development. In this article, we have delineated the strategies for genome annotations of lactic acid bacteria. These strategies also provide insights into streamlining genome reduction without compromising the functionality of the chassis and the potential for minimal genome chassis development. These insights underscore the potential for the development of efficient and sustainable synthetic biology systems using streamlined microbial chassis with minimal genomes. [ABSTRACT FROM AUTHOR]

Abstract Central carbon metabolism (CCM), including glycolysis, tricarboxylic acid cycle and the pentose phosphate pathway, is the most fundamental metabolic process in the activities of living organisms that maintains normal cellular growth. CCM has been widely used in microbial metabolic engineering in recent years due to its unique regulatory role in cellular metabolism. Using yeast and Escherichia coli as the representative organisms, we summarized the metabolic engineering strategies on the optimization of CCM in eukaryotic and prokaryotic microbial chassis, such as the introduction of heterologous CCM metabolic pathways and the optimization of key enzymes or regulatory factors, to lay the groundwork for the future use of CCM optimization in metabolic engineering. Furthermore, the bottlenecks in the application of CCM optimization in metabolic engineering and future application prospects are summarized.

Central carbon metabolism (CCM), including glycolysis, tricarboxylic acid cycle and the pentose phosphate pathway, is the most fundamental metabolic process in the activities of living organisms that maintains normal cellular growth. CCM has been widely used in microbial metabolic engineering in recent years due to its unique regulatory role in cellular metabolism. Using yeast and Escherichia coli as the representative organisms, we summarized the metabolic engineering strategies on the optimization of CCM in eukaryotic and prokaryotic microbial chassis, such as the introduction of heterologous CCM metabolic pathways and the optimization of key enzymes or regulatory factors, to lay the groundwork for the future use of CCM optimization in metabolic engineering. Furthermore, the bottlenecks in the application of CCM optimization in metabolic engineering and future application prospects are summarized. [ABSTRACT FROM AUTHOR]

Accumulating plastics in the biosphere implicates adverse effects, raising serious concern among scientists worldwide. Plastic waste in nature disintegrates into microplastics. Because of their minute appearance, at a scale of <5 mm, microplastics easily penetrate different pristine water bodies and terrestrial niches, posing detrimental effects on flora and fauna. The potential bioremediative application of microbial enzymes is a sustainable solution for the degradation of microplastics. Studies have reported a plethora of bacterial and fungal species that can degrade synthetic plastics by excreting plasticdegrading enzymes. Identified microbial enzymes, such as IsPETase and IsMHETase from Ideonella sakaiensis 201-F6 and Thermobifida fusca cutinase (Tfc), are able to depolymerize plastic polymer chains producing ecologically harmless molecules like carbon dioxide and water. However, thermal stability and pH sensitivity are among the biochemical limitations of the plasticdegrading enzymes that affect their overall catalytic activities. The application of biotechnological approaches improves enzyme action and production. Protein-based engineering yields enzyme variants with higher enzymatic activity and temperature-stable properties, while site-directed mutagenesis using the Escherichia coli model system expresses mutant thermostable enzymes. Furthermore, microalgal chassis is a promising model system for “green” microplastic biodegradation. Hence, the bioremediative properties of microbial enzymes are genuinely encouraging for the biodegradation of synthetic microplastic polymers. [ABSTRACT FROM AUTHOR]

Accumulating plastics in the biosphere implicates adverse effects, raising serious concern among scientists worldwide. Plastic waste in nature disintegrates into microplastics. Because of their minute appearance, at a scale of

Microbial diversity is magnificent and essential to almost all life on Earth. Microbes are an essential part of every human, allowing us to utilize otherwise inaccessible resources. It is no surprise that humans started, initially unconsciously, domesticating microbes for food production: one may call this microbial domestication 1.0. Sourdough bread is just one of the miracles performed by microbial fermentation, allowing extraction of more nutrients from flour and at the same time creating a fluffy and delicious loaf. There are a broad range of products the production of which requires fermentation such as chocolate, cheese, coffee and vinegar. Eventually, with the rise of microscopy, humans became aware of microbial life. Today our knowledge and technological advances allow us to genetically engineer microbes - one may call this microbial domestication 2.0. Synthetic biology and microbial chassis adaptation allow us to tackle current and future food challenges. One of the most apparent challenges is the limited space on Earth available for agriculture and its major tolls on the environment through use of pesticides and the replacement of ecosystems with monocultures. Further challenges include transport and packaging, exacerbated by the 24/7 on-demand mentality of many customers. Synthetic biology already tackles multiple food challenges and will be able to tackle many future food challenges. In this perspective article, we highlight recent microbial synthetic biology research to address future food challenges. We further give a perspective on how synthetic biology tools may teach old microbes new tricks, and what standardized microbial domestication could look like.

Sustainable and environment-friendly microbial fermentation processes have been developed to produce numerous chemicals. However, the high energy input required for sterilization and substantial fresh water consumption restrict the economic feasibility of traditional fermentation processes. To address these problems, Vibrio natriegens, a promising microbial chassis with low nutritional requirements, high salt tolerance and rapid growth rate can be selected as the host for chemical production. In this study, V. natriegens was metabolic engineered to produce 2,3-butanediol (2,3-BD), an important platform chemical, through non-sterilized fermentation with seawater-based minimal medium after expressing a 2,3-BD synthesis cluster and deleting two byproduct encoding genes. Under optimized fermentative conditions, 41.27 g/L 2,3-BD was produced with a productivity of 3.44 g/L/h and a yield of 0.39 g/g glucose by recombinant strain V. natriegensΔfrdAΔldhA-pETRABC. This study confirmed the feasibility of non-sterilized fermentation using seawater to replace freshwater and other valuable chemicals may also be produced through metabolic engineering of the emerging synthetic biology chassis V. natriegens.

Global warming and uneven distribution of fossil fuels worldwide concerns have spurred the development of alternative, renewable, sustainable, and environmentally friendly resources. From an engineering perspective, biosynthesis of fatty acid-derived chemicals (FACs) is an attractive and promising solution to produce chemicals from abundant renewable feedstocks and carbon dioxide in microbial chassis. However, several factors limit the viability of this process. This review first summarizes the types of FACs and their widely applications. Next, we take a deep look into the microbial platform to produce FACs, give an outlook for the platform development. Then we discuss the bottlenecks in metabolic pathways and supply possible solutions correspondingly. Finally, we highlight the most recent advances in the fast-growing model-based strain design for FACs biosynthesis.

The purple non-sulfur bacterium Rhodopseudomonas palustris TIE-1 can produce useful biochemicals such as bioplastics and biobutanol. Production of such biochemicals requires intracellular electron availability, which is governed by the availability and the transport of essential metals such as iron (Fe). Because of the distinct chemical properties of ferrous [Fe(II)] and ferric iron [Fe(III)], different transport systems are required for their transport and storage in bacteria. Although Fe(III) transport systems are well characterized, we know much less about Fe(II) transport systems except for the FeoAB system. Iron transporters can also import manganese (Mn). Here, we study Fe and Mn transport by five putative Fe transporters in TIE-1 under metal-replete, -deplete, oxic and anoxic conditions. We observe that by overexpressing feoAB, efeU, and nramp1AB, the intracellular concentration of Fe and Mn can be enhanced in TIE-1, under oxic and anoxic conditions, respectively. The deletion of a single gene/operon does not attenuate Fe or Mn uptake in TIE-1 regardless of the growth conditions used. This indicates that genetically dissimilar yet functionally redundant Fe transporters in TIE-1 can complement each other. Relative gene expression analysis shows that feoAB and efeU are expressed during Fe and Mn depletion under both oxic and anoxic conditions. The promoters of these transporter genes contain a combination of Fur and Fnr boxes suggesting that their expression is regulated by both Fe and oxygen availability. The findings from this study will help us modulate intracellular Fe and Mn concentration, ultimately improving TIE-1's ability to produce desirable biomolecules. [ABSTRACT FROM AUTHOR]

The cryptic secondary metabolite biosynthetic gene clusters (BGCs) far outnumber currently known secondary metabolites. Heterologous production of secondary metabolite BGCs in suitable chassis facilitates yield improvement and discovery of new-to-nature compounds. The two juxtaposed conventional model microorganisms, Escherichia coli , Saccharomyces cerevisiae , have been harnessed as microbial chassis to produce a bounty of secondary metabolites with the help of certain host engineering. In last decade, engineering non-model microbes to efficiently biosynthesize secondary metabolites has received increasing attention due to their peculiar advantages in metabolic networks and/or biosynthesis. The state-of-the-art synthetic biology tools lead the way in operating genetic manipulation in non-model microorganisms for phenotypic optimization or yields improvement of desired secondary metabolites. In this review, we firstly discuss the pros and cons of several model and non-model microbial chassis, as well as the importance of developing broader non-model microorganisms as alternative programmable heterologous hosts to satisfy the desperate needs of biosynthesis study and industrial production. Then we highlight the lately advances in the synthetic biology tools and engineering strategies for optimization of non-model microbial chassis, in particular, the successful applications for efficient heterologous production of multifarious complex secondary metabolites, e.g., polyketides, nonribosomal peptides, as well as ribosomally synthesized and post-translationally modified peptides. Lastly, emphasis is on the perspectives of chassis cells development to access the ideal cell factory in the artificial intelligence-driven genome era. • The model and non-model microbial chassis for complex secondary metabolites • State-of-the-art synthetic biology pave the way to multifarious microbial chassis. • The tools, construction and application of each non-model microbial chassis • The prospects of developing non-model microbial chassis are reviewed. [ABSTRACT FROM AUTHOR]

Microbial diversity is magnificent and essential to almost all life on Earth. Microbes are an essential part of every human, allowing us to utilize otherwise inaccessible resources. It is no surprise that humans started, initially unconsciously, domesticating microbes for food production: one may call this microbial domestication 1.0. Sourdough bread is just one of the miracles performed by microbial fermentation, allowing extraction of more nutrients from flour and at the same time creating a fluffy and delicious loaf. There are a broad range of products the production of which requires fermentation such as chocolate, cheese, coffee and vinegar. Eventually, with the rise of microscopy, humans became aware of microbial life. Today our knowledge and technological advances allow us to genetically engineer microbes - one may call this microbial domestication 2.0. Synthetic biology and microbial chassis adaptation allow us to tackle current and future food challenges. One of the most apparent challenges is the limited space on Earth available for agriculture and its major tolls on the environment through use of pesticides and the replacement of ecosystems with monocultures. Further challenges include transport and packaging, exacerbated by the 24/7 on-demand mentality of many customers. Synthetic biology already tackles multiple food challenges and will be able to tackle many future food challenges. In this perspective article, we highlight recent microbial synthetic biology research to address future food challenges. We further give a perspective on how synthetic biology tools may teach old microbes new tricks, and what standardized microbial domestication could look like., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ramírez Rojas, Swidah and Schindler.)

Sustainable and environment-friendly microbial fermentation processes have been developed to produce numerous chemicals. However, the high energy input required for sterilization and substantial fresh water consumption restrict the economic feasibility of traditional fermentation processes. To address these problems, Vibrio natriegens , a promising microbial chassis with low nutritional requirements, high salt tolerance and rapid growth rate can be selected as the host for chemical production. In this study, V. natriegens was metabolic engineered to produce 2,3-butanediol (2,3-BD), an important platform chemical, through non-sterilized fermentation with seawater-based minimal medium after expressing a 2,3-BD synthesis cluster and deleting two byproduct encoding genes. Under optimized fermentative conditions, 41.27 g/L 2,3-BD was produced with a productivity of 3.44 g/L/h and a yield of 0.39 g/g glucose by recombinant strain V. natriegens Δ frdA Δ ldhA -pETRABC. This study confirmed the feasibility of non-sterilized fermentation using seawater to replace freshwater and other valuable chemicals may also be produced through metabolic engineering of the emerging synthetic biology chassis V. natriegens ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Meng, Zhang, Ma, Lü, Xu, Ma and Gao.)

Natural products from fungi represent an important source of biologically active metabolites notably for therapeutic agent development. Genome sequencing revealed that the number of biosynthetic gene clusters (BGCs) in fungi is much larger than expected. Unfortunately, most of them are silent or barely expressed under laboratory culture conditions. Moreover, many fungi in nature are uncultivable or cannot be genetically manipulated, restricting the extraction and identification of bioactive metabolites from these species. Rapid exploration of the tremendous number of cryptic fungal BGCs necessitates the development of heterologous expression platforms, which will facilitate the efficient production of natural products in fungal cell factories. Host selection, BGC assembly methods, promoters used for heterologous gene expression, metabolic engineering strategies and compartmentalization of biosynthetic pathways are key aspects for consideration to develop such a microbial platform. In the present review, we summarize current progress on the above challenges to promote research effort in the relevant fields., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Global warming and uneven distribution of fossil fuels worldwide concerns have spurred the development of alternative, renewable, sustainable, and environmentally friendly resources. From an engineering perspective, biosynthesis of fatty acid-derived chemicals (FACs) is an attractive and promising solution to produce chemicals from abundant renewable feedstocks and carbon dioxide in microbial chassis. However, several factors limit the viability of this process. This review first summarizes the types of FACs and their widely applications. Next, we take a deep look into the microbial platform to produce FACs, give an outlook for the platform development. Then we discuss the bottlenecks in metabolic pathways and supply possible solutions correspondingly. Finally, we highlight the most recent advances in the fast-growing model-based strain design for FACs biosynthesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Liu, Benitez, Chen, Harrison, Khusnutdinova and Mahadevan.)

A key challenge for domesticating alternative cultivable microorganisms with biotechnological potential lies in the development of innovative technologies. Within this framework, a myriad of genetic tools has flourished, allowing the design and manipulation of complex synthetic circuits and genomes to become the general rule in many laboratories rather than the exception. More recently, with the development of novel technologies such as DNA automated synthesis/sequencing and powerful computational tools, molecular biology has entered the synthetic biology era. In the beginning, most of these technologies were established in traditional microbial models (known as chassis in the synthetic biology framework) such as Escherichia coli and Saccharomyces cerevisiae , enabling fast advances in the field and the validation of fundamental proofs of concept. However, it soon became clear that these organisms, although extremely useful for prototyping many genetic tools, were not ideal for a wide range of biotechnological tasks due to intrinsic limitations in their molecular/physiological properties. Over the last decade, researchers have been facing the great challenge of shifting from these model systems to non-conventional chassis with endogenous capacities for dealing with specific tasks. The key to address these issues includes the generation of narrow and broad host plasmid-based molecular tools and the development of novel methods for engineering genomes through homologous recombination systems, CRISPR/Cas9 and other alternative methods. Here, we address the most recent advances in plasmid-based tools for the construction of novel cell factories, including a guide for helping with "build-your-own" microbial host. [ABSTRACT FROM AUTHOR]

Engineering and modifying synthetic microbial chassis is one of the best ways not only to unravel the fundamental principles of life but also to enhance applications in the health, medicine, agricultural, veterinary, and food industries. The two primary strategies for constructing a microbial chassis are the top-down approach (genome reduction) and the bottom-up approach (genome synthesis). Research programs on this topic have been funded in several countries. The 'Minimum genome factory' (MGF) project was launched in 2001 in Japan with the goal of constructing microorganisms with smaller genomes for industrial use. One of the best examples of the results of this project is E. coli MGF-01, which has a reduced-genome size and exhibits better growth and higher threonine production characteristics than the parental strain [1]. The 'cell factory' project was carried out from 1998 to 2002 in the Fifth Framework Program of the EU (European Union), which tried to comprehensively understand microorganisms used in the application field. One of the outstanding results of this project was the elucidation of proteins secreted by Bacillus subtilis , which was summarized as the 'secretome' [2]. The GTL (Genomes to Life) program began in 2002 in the United States. In this program, researchers aimed to create artificial cells both in silico and in vitro , such as the successful design and synthesis of a minimal bacterial genome by John Craig Venter's group [3]. This review provides an update on recent advances in engineering, modification and application of synthetic microbial chassis, with particular emphasis on the value of learning about chassis as a way to better understand life and improve applications.