Balancing Cell Growth and Product Synthesis for Efficient Microbial Cell Factories

Systems metabolic engineering is facilitating the development of high-performing microbial cell factories for producing chemicals and materials. However, constructing an efficient microbial cell factory still requires exploring and selecting various host strains, as well as identifying the best-suited metabolic engineering strategies, which demand significant time, effort, and costs. Here, we comprehensively evaluate the capacities of various microbial cell factories and propose strategies for systems metabolic engineering steps, including host strain selection, metabolic pathway reconstruction, and metabolic flux optimization. We analyze the metabolic capacities of five representative industrial microorganisms as cell factories for the production of 235 different bio-based chemicals and suggest the most suitable host strain for the corresponding chemical production. To improve the innate metabolic capacity by constructing more efficient metabolic pathways, heterologous metabolic reactions, and cofactor exchanges are systematically analyzed. Additionally, we present metabolic engineering strategies, which include up- and down-regulation target reactions, for the improved production of chemicals. Altogether, this study will serve as a comprehensive resource for the systems metabolic engineering of microorganisms in the bio-based production of chemicals.

BackgroundAdvanced DNA synthesis, biosensor assembly, and genetic circuit development in synthetic biology and metabolic engineering have reinforced the application of filamentous bacteria, yeasts, and fungi as promising chassis cells for chemical production, but their industrial application remains a major challenge that needs to be solved.ResultsAs important chassis strains, filamentous microorganisms can synthesize important enzymes, chemicals, and niche pharmaceutical products through microbial fermentation. With the aid of metabolic engineering and synthetic biology, filamentous bacteria, yeasts, and fungi can be developed into efficient microbial cell factories through genome engineering, pathway engineering, tolerance engineering, and microbial engineering. Mutant screening and metabolic engineering can be used in filamentous bacteria, filamentous yeasts (Candida glabrata, Candida utilis), and filamentous fungi (Aspergillus sp., Rhizopus sp.) to greatly increase their capacity for chemical production. This review highlights the potential of using biotechnology to further develop filamentous bacteria, yeasts, and fungi as alternative chassis strains.ConclusionsIn this review, we recapitulate the recent progress in the application of filamentous bacteria, yeasts, and fungi as microbial cell factories. Furthermore, emphasis on metabolic engineering strategies involved in cellular tolerance, metabolic engineering, and screening are discussed. Finally, we offer an outlook on advanced techniques for the engineering of filamentous bacteria, yeasts, and fungi.

Systems metabolic engineering, industrial biotechnology and microbial cell factories

Recent advances in systems metabolic engineering tools and strategies

Author for correspondence: Institut de Biotecnologia i de Biomedicina, Universitat Autonoma de Barcelona, Bellaterra, 08193 Barcelona, Spain and Department de Genetica i de Microbiologia, Universitat Autonoma de Barcelona, Bellaterra, 08193 Barcelona, Spain and CIBER en Bioingenieria, Biomateriales y Nanomedicina, Bellaterra, 08193 Barcelona, Spain antoni.villaverde@uab.cat Microbial biofabrication for nanomedicine: biomaterials, nanoparticles and beyond

Chapter 6 - Metabolic engineering for microbial cell factories

Synthetic biology has promoted a conceptual shift in metabolic engineering for the microbial production of industrial chemicals toward a sustainable economy. Engineering principles from synthetic biology and metabolic engineering are integrated to redesign cellular metabolism to create microbial cell factories with emerging and programmable functionalities. Combining metabolic engineering with programmed spatial control is a promising approach that enables deep rewiring of microbial cell factory metabolism for the efficient production of bio-based chemicals. In this review, we discuss metabolic compartmentalization approaches for programmable control of cellular metabolism, including intracellular or intercellular partitioning-based organization of biosynthetic pathways. We also examine the designs and applications of cellular compartments and their analogs, highlighting selected examples for creating efficient and sustainable microbial cell factories.

Combining protein and metabolic engineering to construct efficient microbial cell factories

Robust production of N-acetyl-glucosamine in engineered Escherichia coli from glycerol-glucose mixture

Microbial cell factories (MCFs) are of considerable interest to convert low value renewable substrates to biofuels and high value chemicals. This review highlights the progress of computational models for the rational design of an MCF to produce a target bio-commodity. In particular, the rational design of an MCF involves: (i) product selection, (ii) de novo biosynthetic pathway identification (i.e., rational, heterologous, or artificial), (iii) MCF chassis selection, (iv) enzyme engineering of promiscuity to enable the formation of new products, and (v) metabolic engineering to ensure optimal use of the pathway by the MCF host. Computational tools such as (i) de novo biosynthetic pathway builders, (ii) docking, (iii) molecular dynamics (MD) and steered MD (SMD), and (iv) genome-scale metabolic flux modeling all play critical roles in the rational design of an MCF. Genome-scale metabolic flux models are of considerable use to the design process since they can reveal metabolic capabilities of MCF hosts. These can be used for host selection as well as optimizing precursors and cofactors of artificial de novo biosynthetic pathways. In addition, recent advances in genome-scale modeling have enabled the derivation of metabolic engineering strategies, which can be implemented using the genomic tools reviewed here as well.

Carbon competition between cell growth and product synthesis is the bottleneck in efficient N-acetyl glucosamine (GlcNAc) production in microbial cell factories. In this study, a xylose-induced T7 RNA polymerase-PT7 promoter system was introduced in Escherichia coli W3110 to control the GlcNAc synthesis. Meanwhile, an arabinose-induced CRISPR interference (CRISPRi) system was applied to adjust cell growth by attenuating the transcription of key growth-related genes. By designing proper sgRNAs, followed by elaborate adjustment of the addition time and concentration of the two inducers, the carbon flux between cell growth and GlcNAc synthesis was precisely redistributed. Comparative metabolomics analysis results confirmed that the repression of pfkA and zwf significantly attenuated the TCA cycle and the synthesis of related amino acids, saving more carbon for the GlcNAc synthesis. Finally, the simultaneous repression of pfkA and zwf in strain GLA-14 increased the GlcNAc titer by 47.6% compared with that in E. coli without the CRISPRi system in a shake flask. GLA-14 could produce 90.9 g/L GlcNAc within 40 h in a 5 L bioreactor, with a high productivity of 2.27 g/L/h. This dynamic strategy for rebalancing cell growth and product synthesis could be applied in the fermentative production of other chemicals derived from precursors synthesized via central carbon metabolism.

<p indent="0mm">Microbial cell factories are among the key subjects in synthetic biology research. Microbial cell factories produce bulk chemicals and natural products through renewable biomass resources. These green and clean biological manufacturing routes can supplement nonrenewable petrochemical resources and rare plant resources; thus, these routes can partially replace petrochemical manufacturing and plant extraction routes, which consume high amounts of energy and generate high pollution levels. The green routes provide an important method for society to address resource, energy and environmental problems. To successfully commercialize microbial cell factories, the regulatory mechanisms that microorganisms use to efficiently produce chemicals were investigated from two perspectives, including carbon metabolism and energy metabolism. Regarding carbon metabolism, a synthetic biology platform for enzyme mining was established, and the synthetic pathways of several triterpenoids were characterized using this platform. Furthermore, some cell factories that produce triterpenoids, such as hawthorn acid, corosolic acid and trichoparic acid, were created by <italic>Saccharomyces cerevisiae</italic>. In addition, precision pathway regulation technologies, including simultaneous modulation of multiple genes in chromosomes and glycosylase base editor (GBE), were developed and could improve the rate of xylose utilization by 3-fold compared with that of the control. The rate-limiting steps of the synthetic pathways for a series of chemicals were identified, and the rate-limiting enzymes were activated to increase the rate of chemical production by 2.5- to 600-fold; thus, the adaptation problems between enzyme parts and synthetic pathways were solved. When considering energy metabolism, four new modules of generating energy through glucose metabolism were designed according to the requirements of synthetic products; thus, the imbalance problems between the supply and the necessity of reducing equivalents in the synthetic pathway were solved. Based on the knowledge of the regulatory mechanisms for synthetic metabolism, efficient microbial cell factories were constructed by our research team to produce a series of chemicals. Technologies to microbially produce 14 chemicals have been licensed. Among these chemicals, four have been successfully commercialized, and the annual production of each chemical can reach over <sc>10000 t.</sc> In addition, one company realized IPO. All these factors have promoted the industrial application of microbial cell factories. The future directions of research on microbial cell factories are discussed at the end of this paper. Most of the synthetic pathways of these cell factories are based on existing biosynthetic pathways in nature. However, for the vast majority of chemicals, the biosynthetic pathways are often unknown or do not occur in nature. How to microbially synthesize these chemicals is among the difficulties encountered when constructing a microbial cell factory. The principle of designing new pathways and new enzymes, the regulation of energy metabolism based on nonnatural coenzymes and the mechanism of chemical stress on cells can be studied in substance metabolism, energy metabolism, and physiological metabolism, respectively.

Innovative Tools and Strategies for Optimizing Yeast Cell Factories

BackgroundMenaquinone-7 (MK-7) is a valuable vitamin K2 produced by Bacillus subtilis. Although many strategies have been adopted to increase the yield of MK-7 in B. subtilis, the effectiveness of these common approaches is not high because long metabolic synthesis pathways and numerous bypass pathways competing for precursors with MK-7 synthesis. Regarding the modification of bypass pathways, studies of common static metabolic engineering method such as knocking out genes involved in side pathway have been reported previously. Since byproductsphenylalanine(Phe), tyrosine (Tyr), tryptophan (Trp), folic acid, dihydroxybenzoate, hydroxybutanone in the MK-7 synthesis pathway are indispensable for cell growth, the complete knockout of the bypass pathway restricts cell growth, resulting in limited increase in MK-7 synthesis. Dynamic regulation via quorum sensing (QS) provides a cost-effective strategy to harmonize cell growth and product synthesis, eliminating the need for pricey inducers. SinR, a transcriptional repressor, is crucial in suppressing biofilm formation, a process closely intertwined with MK-7 biosynthesis. Given this link, we targeted SinR to construct a dynamic regulatory system, aiming to modulate MK-7 production by leveraging SinR’s regulatory influence.ResultsA modular PhrC-RapC-SinR QS system is developed to dynamic regulate side pathway of MK-7. In this study, first, we analyzed the SinR-based gene expression regulation system in B. subtilis 168 (BS168). We constructed a promoter library of different abilities, selected suitable promoters from the library, and performed mutation screening on the selected promoters. Furthermore, we constructed a PhrC-RapC-SinR QS system to dynamically control the synthesis of Phe, Tyr, Trp, folic acid, dihydroxybenzoate, hydroxybutanone in MK-7 synthesis in BS168. Cell growth and efficient synthesis of the MK-7 production can be dynamically balanced by this QS system. Using this system to balance cell growth and product fermentation, the MK-7 yield was ultimately increased by 6.27-fold, from 13.95 mg/L to 87.52 mg/L.ConclusionIn summary, the PhrC-RapC-SinR QS system has been successfully integrated with biocatalytic functions to achieve dynamic metabolic pathway control in BS168, which has potential applicability to a large number of microorganisms to fine-tune gene expression and enhance the production of metabolites.

Metabolic engineering of an auto-regulated Corynebacterium glutamicum chassis for biosynthesis of 5-aminolevulinic acid