Transportation engineering for enhanced production of plant natural products in microbial cell factories.

Plant natural products (PNPs) exhibit a wide range of biological activities and have essential applications in various fields such as medicine, agriculture, and flavors. Given their natural limitations, the production of high-value PNPs using microbial cell factories has become an effective alternative in recent years. However, host metabolic burden caused by its massive accumulation has become one of the main challenges for efficient PNP production. Therefore, it is necessary to strengthen the transmembrane transport process of PNPs. This review introduces the discovery and mining of PNP transporters to directly mediate PNP transmembrane transportation both intracellularly and extracellularly. In addition to transporter engineering, this review also summarizes several auxiliary strategies (such as small molecules, environmental changes, and vesicles assisted transport) for strengthening PNP transportation. Finally, this review is concluded with the applications and future perspectives of transportation engineering in the construction and optimization of PNP microbial cell factories.

[Engineering of microorganisms for high production of amino acids].

Amino acids as the building blocks of proteins are widely applied in food, medicine, feed, and chemical industries. Amino acid production by microbial cell factories from renewable resources is praised for the environmental friendliness, mild reaction conditions, and high product purity, which helps to achieve the goal of carbon neutrality. Researchers have employed the methods of metabolic engineering and synthetic biology to engineer Escherichia coli and Corynebacterium glutamicum and optimized the culture conditions to construct the microbial cell factories with high performance for producing branched chain amino acids, amino acids of the aspartic acid and glutamic acid families, and aromatic amino acids. We review the engineering process of microbial cell factories for high production of amino acids, in the hope of providing a reference for the creation of high-performance microbial cell factories.

Relieving metabolic burden to improve robustness and bioproduction by industrial microorganisms.

Metabolic burden is defined by the influence of genetic manipulation and environmental perturbations on the distribution of cellular resources. The rewiring of microbial metabolism for bio-based chemical production often leads to a metabolic burden, followed by adverse physiological effects, such as impaired cell growth and low product yields. Alleviating the burden imposed by undesirable metabolic changes has become an increasingly attractive approach for constructing robust microbial cell factories. In this review, we provide a brief overview of metabolic burden engineering, focusing specifically on recent developments and strategies for diminishing the burden while improving robustness and yield. A variety of examples are presented to showcase the promise of metabolic burden engineering in facilitating the design and construction of robust microbial cell factories. Finally, challenges and limitations encountered in metabolic burden engineering are discussed.

Microbial synthesis of anthocyanins and pyranoanthocyanins: current bottlenecks and potential solutions.

Anthocyanins (ACNs) are secondary metabolites found in plants. Due to their impressive biological activities, ACNs have gained significant popularity and extensive application within the food, pharmaceutical, and nutraceutical industries. A derivative of ACNs: pyranoanthocyanins (PACNs) possesses more stable properties and interesting biological activities. However, conventional methods for the production of ACNs, including chemical synthesis and plant extraction, involve organic solvents. Microbial synthesis of ACNs from renewable biomass, such as amino acids or flavonoids, is considered a sustainable and environmentally friendly method for large-scale production of ACNs. Recently, the construction of microbial cell factories (MCFs) for the efficient biosynthesis of ACNs and PACNs has attracted much attention. In this review, we summarize the cases of microbial synthesis of ACNs, and analyze the bottlenecks in reconstructing the metabolic pathways for synthesizing PACNs in microorganisms. Consequently, there is an urgent need to investigate the mechanisms behind the development of MCFs for PACNs synthesis. Such research also holds significant promise for advancing the production of food pigments. Meanwhile, we propose potential solutions to the bottleneck problem based on metabolic engineering and enzyme engineering. Finally, the development prospects of natural food and biotechnology are discussed.

Transcending membrane barriers: advances in membrane engineering to enhance the production capacity of microbial cell factories.

Microbial cell factories serve as pivotal platforms for the production of high-value natural products, which tend to accumulate on the cell membrane due to their hydrophobic properties. However, the limited space of the cell membrane presents a bottleneck for the accumulation of these products. To enhance the production of intracellular natural products and alleviate the burden on the cell membrane caused by product accumulation, researchers have implemented various membrane engineering strategies. These strategies involve modifying the membrane components and structures of microbial cell factories to achieve efficient accumulation of target products. This review summarizes recent advances in the application of membrane engineering technologies in microbial cell factories, providing case studies involving Escherichia coli and yeast. Through these strategies, researchers have not only improved the tolerance of cells but also optimized intracellular storage space, significantly enhancing the production efficiency of natural products. This article aims to provide scientific evidence and references for further enhancing the efficiency of similar cell factories.

Multiplex Marker-Less Genome Integration in Pichia pastoris Using CRISPR/Cas9.

Pichia pastoris is known for its excellent protein expression ability. As an industrial methyl nutritional yeast, it can effectively utilize methanol as the sole carbon source, serving as a potential platform for C1 biotransformation. Unfortunately, the lack of synthetic biology tools in P. pastoris limits its broad applications, particularly when multigene pathways should be manipulated. Here, the CRISPR/Cas9 system is established to efficiently integrate multiple heterologous genes to construct P. pastoris cell factories. In this protocol, with the 2,3-butanediol (BDO) biosynthetic pathway as a representative example, the procedures to construct P. pastoris cell factories are detailed using the established CRISPR-based multiplex genome integration toolkit, including donor plasmid construction, competent cell preparation and transformation, and transformant verification. The application of the CRISPR toolkit is demonstrated by the construction of engineered P. pastoris for converting methanol to BDO. This lays the foundation for the construction of P. pastoris cell factories harboring multi-gene biosynthetic pathways for the production of high-value compounds.

Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing.

BACKGROUND: The abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers. RESULTS: The aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-ß-xylanase and ß-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA ß-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L- 1 after 48 h under oxygen limited condition in bioreactor fermentations. CONCLUSION: This work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast's expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.

Engineering Escherichia coli for high-yielding 2,5-Dimethylpyrazine synthesis from L-Threonine by reconstructing metabolic pathways and enhancing cofactors regeneration.

2,5-Dimethylpyrazine (2,5-DMP) is important pharmaceutical raw material and food flavoring agent. Recently, engineering microbes to produce 2,5-DMP has become an attractive alternative to chemical synthesis approach. In this study, metabolic engineering strategies were used to optimize the modified Escherichia coli BL21 (DE3) strain for efficient synthesis of 2,5-DMP using L-threonine dehydrogenase (EcTDH) from Escherichia coli BL21, NADH oxidase (EhNOX) from Enterococcus hirae, aminoacetone oxidase (ScAAO) from Streptococcus cristatus and L-threonine transporter protein (EcSstT) from Escherichia coli BL21, respectively. We further optimized the reaction conditions for synthesizing 2,5-DMP. In optimized conditions, the modified strain can convert L-threonine to obtain 2,5-DMP with a yield of 2897.30 mg/L. Therefore, the strategies used in this study contribute to the development of high-level cell factories for 2,5-DMP.

Parageobacillus thermoglucosidasius as an emerging thermophilic cell factory.

Parageobacillus thermoglucosidasius is a thermophilic and facultatively anaerobic microbe, which is emerging as one of the most promising thermophilic model organisms for metabolic engineering. The use of thermophilic microorganisms for industrial bioprocesses provides the advantages of increased reaction rates and reduced cooling costs for bioreactors compared to their mesophilic counterparts. Moreover, it enables starch or lignocellulose degradation and fermentation to occur at the same temperature in a Simultaneous Saccharification and Fermentation (SSF) or Consolidated Bioprocessing (CBP) approach. Its natural hemicellulolytic capabilities and its ability to convert CO to metabolic energy make P. thermoglucosidasius a potentially attractive host for bio-based processes. It can effectively degrade hemicellulose due to a number of hydrolytic enzymes, carbohydrate transporters, and regulatory elements coded from a genomic cluster named Hemicellulose Utilization (HUS) locus. The growing availability of effective genetic engineering tools in P. thermoglucosidasius further starts to open up its potential as a versatile thermophilic cell factory. A number of strain engineering examples showcasing the potential of P. thermoglucosidasius as a microbial chassis for the production of bulk and fine chemicals are presented along with current research bottlenecks. Ultimately, this review provides a holistic overview of the distinct metabolic characteristics of P. thermoglucosidasius and discusses research focused on expanding the native metabolic boundaries for the development of industrially relevant strains.

Evaluating oleaginous yeasts for enhanced microbial lipid production using sweetwater as a sustainable feedstock.

BACKGROUND: Yeasts exhibit promising potential for the microbial conversion of crude glycerol, owing to their versatility in delivering a wide range of value-added products, particularly lipids. Sweetwater, a methanol-free by-product of the fat splitting process, has emerged as a promising alternative feedstock for the microbial utilization of crude glycerol. To further optimize sweetwater utilization, we compared the growth and lipid production capabilities of 21 oleaginous yeast strains under different conditions with various glycerol concentrations, sweetwater types and pH. RESULTS: We found that nutrient limitation and the unique carbon composition of sweetwater boosted significant lipid accumulation in several strains, in particular Rhodosporidium toruloides NRRL Y-6987. Subsequently, to decipher the underlying mechanism, the transcriptomic changes of R. toruloides NRRL Y-6987 were further analyzed, indicating potential sugars and oligopeptides in sweetwater supporting growth and lipid accumulation as well as exogenous fatty acid uptake leading to the enhanced lipid accumulation. CONCLUSION: Our comparative study successfully demonstrated sweetwater as a cost-effective feedstock while identifying R. toluroides NRRL Y-6987 as a highly promising microbial oil producer. Furthermore, we also suggested potential sweetwater type and strain engineering targets that could potentially enhance microbial lipid production.

Advances in the metabolic engineering of Saccharomyces cerevisiae and Yarrowia lipolytica for the production of ß-carotene.

ß-Carotene is one kind of the most important carotenoids. The major functions of ß-carotene include the antioxidant and anti-cardiovascular properties, which make it a growing market. Recently, the use of metabolic engineering to construct microbial cell factories to synthesize ß-carotene has become the latest model for its industrial production. Among these cell factories, yeasts including Saccharomyces cerevisiae and Yarrowia lipolytica have attracted the most attention because of the: security, mature genetic manipulation tools, high flux toward carotenoids using the native mevalonate pathway and robustness for large-scale fermentation. In this review, the latest strategies for ß-carotene biosynthesis, including protein engineering, promoters engineering and morphological engineering are summarized in detail. Finally, perspectives for future engineering approaches are proposed to improve ß-carotene production.

Building Yarrowia lipolytica Cell Factories for Advanced Biomanufacturing: Challenges and Solutions.

Microbial cell factories have shown great potential for industrial production with the benefit of being environmentally friendly and sustainable. Yarrowia lipolytica is a promising and superior non-model host for biomanufacturing due to its cumulated advantages compared to model microorganisms, such as high fluxes of metabolic precursors (acetyl-CoA and malonyl-CoA) and its naturally hydrophobic microenvironment. However, although diverse compounds have been synthesized in Y. lipolytica cell factories, most of the relevant studies have not reached the level of industrialization and commercialization due to a number of remaining challenges, including unbalanced metabolic flux, conflict between cell growth and product synthesis, and cytotoxic effects. Here, various metabolic engineering strategies for solving the challenges are summarized, which is developing fast and extremely conducive to rational design and reconstruction of robust Y. lipolytica cell factories for advanced biomanufacturing. Finally, future engineering efforts for enhancing the production efficiency of this platform strain are highlighted.

Carotenoid Cleavage Dioxygenase 1 and Its Application for the Production of C13-Apocarotenoids in Microbial Cell Factories: A Review.

C13-apocarotenoids are naturally derived from the C9-C10 (C9'-C10') double-bond cleavage of carotenoids by carotenoid cleavage dioxygenases (CCDs). As high-value flavors and fragrances in the food and cosmetic industries, the sustainable production of C13-apocarotenoids is emerging in microbial cell factories by the carotenoid cleavage dioxygenase 1 (CCD1) subfamily. However, the commercialization of microbial-based C13-apocarotenoids is still limited by the poor performance of CCD1, which severely constrains its conversion efficiency from precursor carotenoids. This review focuses on the classification of CCDs and their cleavage modes for carotenoids to generate corresponding apocarotenoids. We then emphatically discuss the advances for C13-apocarotenoid biosynthesis in microbial cell factories with various strategies, including optimization of CCD1 expression, improvement of CCD1's catalytic activity and substrate specificity, strengthening of substrate channeling, and development of oleaginous microbial hosts, which have been verified to increase the conversion rate from carotenoids. Lastly, the current challenges and future directions will be discussed to enhance CCDs' application for C13-apocarotenoids biomanufacturing.

Multisite Mutation of the Escherichia coli cAMP Receptor Protein: Enhancing Xylitol Biosynthesis by Activating Xylose Catabolism and Improving Strain Tolerance.

The bioproduction of xylitol from hemicellulose hydrolysate has good potential for industrial development. However, xylitol productivity has always been limited due to corncob hydrolysate toxicity and glucose catabolic repression. To address these challenges, this work selected the S83 and S128 amino acid residues of the cyclic AMP receptor protein (CRP) as the modification target. By introducing multisite mutation in CRP, this approach successfully enhanced xylose catabolism and improved the strain's tolerance to corncob hydrolysate. The resulting mutant strain, designated as CPH (CRP S83H-S128P), underwent fermentation in a 20 L bioreactor with semicontinuous feeding of corncob hydrolysate. Remarkably, xylitol yield and xylitol productivity for 41 h fermentation were 175 and 4.32 g/L/h, respectively. Therefore, multisite CRP mutation was demonstrated as an efficient global regulatory strategy to effectively improve xylitol productivity from lime-pretreated corncob hydrolysates.

Recent Advances in the Biosynthesis of Natural Sugar Substitutes in Yeast.

Natural sugar substitutes are safe, stable, and nearly calorie-free. Thus, they are gradually replacing the traditional high-calorie and artificial sweeteners in the food industry. Currently, the majority of natural sugar substitutes are extracted from plants, which often requires high levels of energy and causes environmental pollution. Recently, biosynthesis via engineered microbial cell factories has emerged as a green alternative for producing natural sugar substitutes. In this review, recent advances in the biosynthesis of natural sugar substitutes in yeasts are summarized. The metabolic engineering approaches reported for the biosynthesis of oligosaccharides, sugar alcohols, glycosides, and rare monosaccharides in various yeast strains are described. Meanwhile, some unresolved challenges in the bioproduction of natural sugar substitutes in yeast are discussed to offer guidance for future engineering.

Intracellular accumulation of c-di-GMP and its regulation on self-flocculation of the bacterial cells of Zymomonas mobilis.

Zymomonas mobilis is an emerging chassis for being engineered to produce bulk products due to its unique glycolysis through the Entner-Doudoroff pathway with less ATP produced for lower biomass accumulation and higher product yield. When self-flocculated, the bacterial cells are more productive, since they can self-immobilize within bioreactors for high density, and are more tolerant to stresses for higher product titers, but this morphology needs to be controlled properly to avoid internal mass transfer limitation associated with their strong self-flocculation. Herewith we explored the regulation of cyclic diguanosine monophosphate (c-di-GMP) on self-flocculation of the bacterial cells through activating cellulose biosynthesis. While ZMO1365 and ZMO0919 with GGDEF domains for diguanylate cyclase activity catalyze c-di-GMP biosynthesis, ZMO1487 with an EAL domain for phosphodiesterase activity catalyzes c-di-GMP degradation, but ZMO1055 and ZMO0401 contain the dual domains with phosphodiesterase activity predominated. Since c-di-GMP is synthesized from GTP, the intracellular accumulation of this signal molecule through deactivating phosphodiesterase activity is preferred for activating cellulose biosynthesis to flocculate the bacterial cells, because such a strategy exerts less perturbance on intracellular processes regulated by GTP. These discoveries are significant for not only engineering unicellular Z. mobilis strains with the self-flocculating morphology to boost production but also understanding mechanism underlying c-di-GMP biosynthesis and degradation in the bacterium.

Recent progress in CRISPR-based bioengineering of microbial cell factories for important nutraceuticals synthesis.

Nutraceuticals are defined as food or food components with therapeutic capabilities that have few side effects and are regarded as a natural therapy for preventing the onset of several life-threatening illnesses. The use of microbial cell factories to produce nutraceuticals is considered to be sustainable and promising for meeting market demand. Among the diverse strategies for optimizing microbial cell factories, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system has emerged as a valuable tool for gene integration, deletion, activation, and downregulation. With the advent of multiplexed and precise CRISPR strategies, optimized microbial cell factories are revolutionizing the yield of nutraceuticals. This review focuses on the development of highly adaptable CRISPR strategies to optimize the production in microbial cell factories of some important nutraceuticals (belonging to the class of carotenoids, flavonoids, stilbenoids, polysaccharides, and nonprotein amino acids). Further, we highlighted current challenges related to the efficiency of CRISPR strategies and addressed potential future directions to fully harness CRISPR strategies to make nutraceutical synthesis in microbial cell factories an industrially favorable method.

Valorization of waste streams and C1 gases for sustainable food nutrients and value-added compounds production: Acetate as a promising intermediate.

Resource recovery from waste streams and C1 gaseous substrates (CO2, CO and CH4) are of extensive interest due to the insufficient utilization and threats to the environment. From a perspective of sustainability, valorization of waste streams and C1 gases into target energy-rich value-added products in a sustainable way offers tempting approaches for simultaneously alleviating the environmental problems and achieving a circular carbon economy, while it still suffers from the complicated compositions of feedstocks or the low solubility of gaseous feeds. Recently, a C2 feedstock-based biomanufacturing serving acetate as potential next-generation platform has received much attention, where different gaseous or cellulosic wastes are recycling into acetate and then be further processed into a wide range of valuable long-chain compounds. The different alternative waste-processing technologies that are being developed to generate acetate from various wastes or gaseous substrates are summarized, in which gas fermentation and electrochemical reduction from CO2 represent the most promising routes for achieving high acetate yield. The recent advances and innovations in metabolic engineering for acetate bioconversion into various bioproducts ranging from food nutrients to value-added compounds were then highlighted. The challenges and promising strategies to reinforce microbial acetate conversion were also proposed, which conferred a new horizon for future food and chemical manufacturing with reduced carbon footprint.

Designing Intracellular Compartments for Efficient Engineered Microbial Cell Factories.

With the rapid development of synthetic biology, various kinds of microbial cell factories (MCFs) have been successfully constructed to produce high-value-added compounds. However, the complexity of metabolic regulation and pathway crosstalk always cause issues such as intermediate metabolite accumulation, byproduct generation, and metabolic burden in MCFs, resulting in low efficiencies and low yields of industrial biomanufacturing. Such issues could be solved by spatially rearranging the pathways using intracellular compartments. In this review, design strategies are summarized and discussed based on the types and characteristics of natural and artificial subcellular compartments. This review systematically presents information for the construction of efficient MCFs with intracellular compartments in terms of four aspects of design strategy goals: (1) improving local reactant concentration; (2) intercepting and isolating competing pathways; (3) providing specific reaction substances and environments; and (4) storing and accumulating products.