Construction Of Cell Factories Research Articles

Evaluation of Aspergillus niger Six Constitutive Strong Promoters by Fluorescent-Auxotrophic Selection Coupled with Flow Cytometry: A Case for Citric Acid Production.

Aspergillus niger is an important industrial workhorse for the biomanufacturing of organic acids, proteins, etc. Well-controlled genetic regulatory elements, including promoters, are vital for strain engineering, but available strong promoters for A. niger are limited. Herein, to efficiently assess promoters, we developed an accurate and intuitive fluorescent-auxotrophic selection workflow based on mCherry, pyrG, CRISPR/Cas9 system, and flow cytometry. With this workflow, we characterized six endogenous constitutive promoters in A. niger. The endogenous glyceraldehyde-3-phosphate dehydrogenase promoter PgpdAg showed a 2.28-fold increase in promoter activity compared with the most frequently used strong promoter PgpdAd from A. nidulans. Six predicted conserved motifs, including the gpdA-box, were verified to be essential for the PgpdAg activity. To demonstrate its application, the promoter PgpdAg was used for enhancing the expression of citrate exporter cexA in a citric acid-producing isolate D353.8. Compared with the cexA controlled by PgpdAd, the transcription level of the cexA gene driven by PgpdAg increased by 2.19-fold, which is consistent with the promoter activity assessment. Moreover, following cexA overexpression, several genes involved in carbohydrate transport and metabolism were synergically upregulated, resulting in up to a 2.48-fold increase in citric acid titer compared with that of the parent strain. This study provides an intuitive workflow to speed up the quantitative evaluation of A. niger promoters and strong constitutive promoters for fungal cell factory construction and strain engineering.

Expanding the promoter toolbox for metabolic engineering of methylotrophic yeasts.

Methylotrophic yeasts have been widely recognized as a promising host for production of recombinant proteins and value-added chemicals. Promoters for controlled gene expression are critical for construction of efficient methylotrophic yeasts cell factories. Here, we summarized recent advances in characterizing and engineering promoters in methylotrophic yeasts, such as Komagataella phaffii and Ogataea polymorpha. Constitutive and inducible promoters controlled by methanol or other inducers/repressors were introduced to demonstrate their applications in production of proteins and chemicals. Furthermore, efforts of promoter engineering, including site-directed mutagenesis, hybrid promoter, and transcription factor regulation to expand the promoter toolbox were also summarized. This mini-review also provides useful information on promoters for the application of metabolic engineering in methylotrophic yeasts. KEY POINTS: • The characteristics of six methylotrophic yeasts and their promoters are described. • The applications of Komagataella phaffii and Ogataea polymorpha inmetabolic engineeringare expounded. • Threepromoter engineering strategies are introduced in order to expand the promoter toolbox.

Assessment of fluorescent protein candidates for multi-color flow cytometry analysis of Saccharomyces cerevisiae

Transcription factor-based biosensors represent promising tools in the construction and evaluation of efficient cell factories for the sustainable production of fuels, chemicals and pharmaceuticals. They can notably be designed to follow the production of a target compound or to monitor key cellular properties, such as stress or starvation. In most cases, the biosensors are built with fluorescent protein (FP) genes as reporter genes because of the direct correlation between promoter activity and fluorescence level that can be measured using, for instance, flow cytometry or fluorometry. The expansion of available FPs offers the possibility of using several FPs - and biosensors – in parallel in one host, with simultaneous detection using multicolor flow cytometry. However, the technique is currently limited by the unavailability of combinations of FP whose genes can be successfully expressed in the host and whose fluorescence can be efficiently distinguished from each other.In the present study, the broad collection of available FPs was explored and four different FPs were successfully expressed in the yeast Saccharomyces cerevisiae: yEGFP, mEGFP, CyOFP1opt and mBeRFPopt. After studying their fluorescence signals, population heterogeneity and possible interactions, we recommend two original combinations of FPs for bi-color flow cytometry: mEGFP together with either CyOFP1opt or mBeRFPopt, as well as the combination of all three FPs mEGFP, CyOFP1opt and mBeRFPopt for tri-color flow cytometry. These combinations will allow to perform different types of bi-color or possibly tri-color flow cytometry and FACS experiments with yeast, such as phenotype evaluation, screening or sorting, by single-laser excitation with a standard 488 nm blue laser.

Construction and application of microbial cell factories for unnatural amino acids

Unnatural amino acids are widely used in medicine, pesticide, material, and other industries and the green and efficient synthesis has attracted a lot of attention. In recent years, with the rapid development of synthetic biology, microbial cell factories have become a promising means for biosynthesis of unnatural amino acids. This study reviewed the construction and application of microbial cell factories for unnatural amino acid, including the synthetic pathway reconstruction, design/modification of key enzymes and their coordinated regulation with precursors, blocking of competitive alternative pathways, and construction of cofactor circulation systems. Meanwhile, on the basis of the new principles for designing the microbial cell factories, new biosynthetic pathways adapted to cells and the production environment, as well as new biomanufacturing system established based on cell adaptive evolution and intelligent fermentation regulation, we looked forward to the further construction and application of microbial cell factories for industrial bio-production.

Biomanufacturing driven by engineered microbes

This article summarized the reviews and research articles published in Chinese Journal of Biotechnology in the field of biomanufacturing in 2021. The article covered major chassis cells such as Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, filamentous fungi, non-model bacteria and non-conventional yeasts. Moreover, this article summarized the advances in the production of amino acids, organic acids, vitamins, higher alcohols, natural compounds (terpenoids, flavonoids, alkaloids), antibiotics, enzymes and enzyme-catalyzed products, biopolymers, as well as the utilization of biomass and one-carbon materials. The key technologies used in the construction of cell factories, such as regulation, evolution, and high-throughput screening, were also included. This article may help the readers better understand the R & D trend in biomanufacturing driven by engineered microbes.

Thermostable CRISPR/Cas9 genome editing system and its application in construction of cell factories with thermophilic bacteria: a review

The diverse thermophilic strains of Thermoanaerobacter, serving as unique platforms with a broad range of application in biofuels and chemicals, have received wide attention from scholars and practitioners. Although biochemical experiments and genome sequences have been reported for a variety of Thermoanaerobacter strains, an efficient genetic manipulation system remains to be established for revealing the biosynthetic pathways of Thermoanaerobacter. In line with this demand, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems for editing, regulating and targeting genomes have been well developed in thermophiles. Here, we reviewed and discussed the current status, associated challenges, and future perspectives of the construction of thermostable CRISPR/Cas9 genome editing systems for some representative Thermoanaerobacter species. The establishment, optimization, and application of thermostable CRISPR/Cas genome editing systems would potentially provide a foundation for further genetic modification of thermophilic bacteria.

Recent advances in construction and regulation of yeast cell factories.

The past decade has witnessed the rapid progress in development of synthetic biology, and advances in construction of yeast cell factories open vast opportunities for green and sustainable production of chemicals. Focusing on the progress in yeast engineering for production of plant natural products in the last 5 years, this review introduces different yeast chassis used for cell factory construction, including Saccharomyces cerevisiae, Yarrowia lipolytica and Komagataella phaffii, together with the emerging genome editing tools. The metabolic regulation strategies developed for yeast engineering are highlighted, such as subcellular pathway localization dynamic regulation, and transporter engineering. C1-based chemical bioproduction by engineered yeast is also covered. Finally, the existing challenges and future prospects in creating efficient yeast cell factories are summarized.

Biotransformation Enables Innovations Toward Green Synthesis of Steroidal Pharmaceuticals.

Steroids have been widely used in birth-control, prevention, and treatment of various diseases, representing the largest sector after antibiotics in the global pharmaceutical market. The steroidal active pharmaceutical ingredients (APIs) have been produced via partial synthetic processes first mainly from sapogenins, which was converted into 16-dehydropregnenolone by the famous "Marker Degradation". Traditional mutation and screening, and process engineering have resulted in the industrial production of 4-androstene-3,17-dione (AD), androst-1,4-diene-3,17-dione (ADD), 9α-hydroxy-androsta-4-ene-3,17-dione (9α-OH-AD), and so on, which serve as the key intermediates for the synthesis of steroidal APIs. Recently, genetic and metabolic engineering have generated highly efficient microbial strains for the production of these precursors, leading to the replacement of sapogenins with phytosterols as the starting materials. Further advances in synthetic biology hold promise in the design and construction of microbial cell factories for the industrial production of steroidal intermediates and/or APIs from simple carbon sources such as glucose. Integration of biotransformation into the synthesis of steroidal APIs can greatly reduce the number of reaction steps, achieve lower waste discharge and higher production efficiency, thus enabling a greener steroidal pharmaceutical industry.

Construction of cell factories for high production of ginsenoside Rh_2 in Saccharomyces cerevisiae

Ginsenoside Rh_2 is a rare active ingredient in precious Chinese medicinal materials such as Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Panacis Quinquefolii Radix. It has important pharmacological activities such as anti-cancer and improving human immunity. However, due to the extremely low content of ginsenoside Rh_2 in the source plants, the traditional way of obtaining it has limitations. This study intended to apply synthetic biological technology to develop a cell factory of Saccharomyces cerevisiae to produce Rh_2 by low-cost fermentation. First, we used the high protopanaxadiol(PPD)-yielding strain LPTA as the chassis strain, and inserted the Panax notoginseng enzyme gene Pn1-31, together with yeast UDP-glucose supply module genes[phosphoglucose mutase 1(PGM1), α-phosphoglucose mutase(PGM2), and uridine diphosphate glucose pyrophosphorylase(UGP1)], into the EGH1 locus of yeast chromosome. The engineered strain LPTA-RH2 produced 17.10 mg·g~(-1) ginsenoside Rh_2. This strain had low yield of Rh_2 while accumulated much precursor PPD, which severely restricted the application of this strain. In order to further improve the production of ginsenoside Rh_2, we strengthened the UDP glucose supply module and ginsenoside Rh_2 synthesis module by engineered strain LPTA-RH2-T. The shaking flask yield of ginsenoside Rh_2 was increased to 36.26 mg·g~(-1), which accounted for 3.63% of the dry weight of yeast cells. Compared with those of the original strain LPTA-RH2, the final production and the conversion efficiency of Rh_2 increased by 112.11% and 65.14%, respectively. This study provides an important basis for further obtaining the industrial-grade cell factory for the production of ginsenoside Rh_2.

Directed evolution of an EamB transporter for improved L-cysteine tolerance and production in Escherichia coli.

Engineering transporter for improved efflux efficiency provides a powerful strategy to alleviate product-associated cytotoxicity and promote microbial production of desired compounds. However, the scarcity of efficient transporters limits its application in the construction of microbial cell factories. Here, we sought to improve the transport performance of the EamB transporter, a L-cysteine exporter from Escherichia coli. A total of four EamB variants (A31V, I83M, G156A, and N157M) were firstly obtained by random mutagenesis and screening, and two other improved mutant (G156S and N157S) were also identified by site-specific saturation mutagenesis. The transport assays revealed that the G156S and N157S mutants had increased L-cysteine export capacity relative to the native EamB transporter. A combinatorial mutagenesis approach was used to generate the best mutant G156S/N157S, which conferred cells optimal resistance to L-cysteine and highest yields of L-cysteine in shake flask fermentation. Taken together, our results offer several EamB mutants with improved efflux properties, highlighting the potential of these exporters in L-cysteine fermentative production.

Enhancing Homologous Recombination Efficiency in Pichia pastoris for Multiplex Genome Integration Using Short Homology Arms.

There is a growing interest in establishing the methylotrophic yeast Pichia pastoris as microbial cell factories for producing fuels, chemicals, and natural products, particularly with methanol as the feedstock. Although CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based genome editing technology has been established for the integration of multigene biosynthetic pathways, long (500-1000 bp) homology arms are generally required, probably due to low homologous recombination (HR) efficiency in P.pastoris. To achieve efficient genome integration of heterologous genes with short homology arms, we aimed to enhance HR efficiency by introducing the recombination machinery from Saccharomyces cerevisiae. First, we overexpressed HR related genes, including RAD52, RAD59, MRE11, and SAE2, and evaluated their effects on genome integration efficiency. Then, we constructed HR efficiency enhanced P.pastoris, which enabled single-, two-, and three-loci integration of heterologous gene expression cassettes with ∼40 bp homology arms with efficiencies as high as 100%, ∼98%, and ∼81%, respectively. Finally, we demonstrated the construction of β-carotene producing strain and the optimization of betaxanthin producing strain in a single step. The HR efficiency enhanced P.pastoris strains can be used for the construction of robust cell factories, and our machinery engineering strategy can be employed for the modification of other nonconventional yeasts.

Modular, synthetic chromosomes as new tools for large scale engineering of metabolism

The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range of pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.

Systems Metabolic Engineering of Methanotrophic Bacteria for Biological Conversion of Methane to Value-Added Compounds.

Methane is considered the carbon source with the highest potential in industrial biotechnology because of its abundance and sustainability. The biological conversion of methane into chemicals or fuels can not only reduce greenhouse gas emissions, but can also substitute edible substrates used in biorefineries. Methanotrophs that can utilize methane as the sole energy and carbon source play a significant role in the ecology of methane. Studies on metabolically-engineered methanotrophs have attracted extensive attention in recent years. In this chapter, the approaches and strategies of methanotrophic cell factory construction are summarized based on synthetic biology tools, systematic manipulation, metabolic modeling, and carbon flux enhancement. Finally, the challenges and opportunities for methane bioconversion by methanotrophs are discussed based on industrial applications.

Mapping of Nonhomologous End Joining-Mediated Integration Facilitates Genome-Scale Trackable Mutagenesis in Yarrowia lipolytica.

Genome-scale mutagenesis, phenotypic screening, and tracking the causal mutations is a powerful approach for genetic analysis. However, classic mutagenesis approaches require extensive effort to identify causal mutations. It is desirable to demonstrate a powerful approach for rapid trackable mutagenesis. Here, we mapped the distribution of nonhomologous end joining (NHEJ)-mediated integration for the first time and demonstrated that it can be used for constructing the genome-scale trackable mutagenesis library in Yarrowia lipolytica. The sequencing of 9.15 × 105 insertions showed that NHEJ-mediated integration inserted DNA randomly across the chromosomes, and the transcriptional regulatory regions exhibited integration preference. The insertions were located in both nucleosome-occupancy regions and nucleosome-free regions. Using NHEJ-mediated integration to construct the genome-scale mutagenesis library, the new targets that improved β-carotene biosynthesis and acetic acid tolerance were identified rapidly. This mutagenesis approach is readily applicable to other organisms with strong NHEJ preference and will contribute to cell factory construction.

Metabolic engineering of Escherichia coli for production of hydroxytyrosol

Hydroxytyrosol is an important fine chemical and is widely used in food and medicine as a natural antioxidant. Production of hydroxytyrosol through synthetic biology is of important significance. Here we cloned and functionally characterized a hydroxylase encoding gene HpaBC from Escherichia coli BL21, and both subunits of this enzyme can be successfully expressed to convert the tyrosol into hydroxytyrosol. A HpaBC gene integration expression cassette under the tac promoter was constructed, and integrated into the genome of a tyrosol hyper-producing E. coli YMG5A*R using CRISPR-Cas9 technology. Meanwhile, the pathway for production of acetic acid was deleted, resulting in a recombinant strain YMGRD1H1. Shake flask fermentation showed that strain YMGRD1H1 can directly use glucose to produce hydroxytyrosol, reaching a titer of 1.81 g/L, and nearly no by-products were detected. A titer of 2.95 g/L was achieved in a fed-batch fermentation conducted in a 5 L fermenter, which is the highest titer for the de novo synthesis of hydroxytyrosol from glucose reported to date. Production of hydroxytyrosol by engineered E. coli lays a foundation for further construction of hydroxytyrosol cell factories with industrial application potential, adding another example for microbial manufacturing of aromatic compounds.

Developing iterative and quantified transgenic manipulations of non-conventional filamentous fungus Talaromyces pinophilus Li-93

Talaromyces pinophilus is a filamentous fungus that can be employed for the remarkable high production of biomass-degrading enzymes used in biorefining and the modification of natural products. However, the genetic tools of T. pinophilus have not been advanced sufficiently, thus limiting its utilization and exploitation. Here, we conducted a systematic study on genetic tools exploitation of T. pinophilus Li-93, and established a marker recycle transformation system to meet the requirements of iterative manipulations of transgenic modification. Furthermore, to enable the quantitative expression of functional genes, a series of promoters with different strengths were mined from transcriptomic data and characterized by detecting with the fluorescent sensor. These promoters may be employed in the fine transcriptional regulation of functional genes and the construction of cell factories, to produce valuable industrial products in T. pinophilus. In conclusion, our work provides useful genetic modification tools in T. pinophilus and lays the foundation for exploiting the significant industrial value of T. pinophilus.

Characterizing methanol metabolism-related promoters for metabolic engineering of Ogataea polymorpha.

Promoters play an important role in regulating gene expression, and construction of microbial cell factories requires multiple promoters for balancing the metabolic pathways. However, there are only a limited number of characterized promoters for gene expression in the methylotrophic yeast Ogataea polymorpha, which hampers the extensive harnessing of this important yeast toward a cell factory. Here we characterized the promoters of methanol utilization pathway, precursor supply pathway, and reactive oxygen species (ROS) defense system, by using a green fluorescence protein variant (GFPUV) as a quantification signal. Finally, the characterized promoters were used for tuning a fatty alcohol biosynthetic pathway in O. polymorpha and realized fatty alcohol production from methanol. This promoter box should be helpful for gene expression and pathway optimization in the methylotrophic yeast O. polymorpha. KEY POINTS : • 22 promoters related to methanol metabolism were characterized in O. polymorpha. • Promoter truncation resulted shorter and compact promoters. • Promoters with various strengths were used for regulating a fatty alcohol biosynthesis from methanol.

Definitive screening accelerates Taxol biosynthetic pathway optimization and scale up in Saccharomyces cerevisiae cell factories.

Recent technological advancements in synthetic and systems biology have enabled the construction of microbial cell factories expressing diverse heterologous pathways in unprecedentedly short time scales. However, the translation of such laboratory scale breakthroughs to industrial bioprocesses remains a major bottleneck. In this study, an accelerated bioprocess development approach was employed to optimize the biosynthetic pathway of the blockbuster chemotherapy drug, Taxol. Statistical design of experiments approaches were coupled with an industrially relevant high-throughput microbioreactor system to optimize production of key Taxol intermediates, Taxadien-5α-ol and Taxadien-5α-yl-acetate, in engineered yeast cell factories. The optimal factor combination was determined via data driven statistical modelling and validated in 1 L bioreactors leading to a 2.1-fold improvement in taxane production compared to a typical defined media. Elucidation and mitigation of nutrient limitation enhanced product titers a further two-fold and titers of the critical Taxol precursors, Taxadien-5α-ol and Taxadien-5α-yl-acetate were improved to 34 and 11mg L-1 , representing a three-fold improvement compared to the highest literature titers in S. cerevisiae. Comparable titers were obtained when the process was scaled up a further five-fold using 5 L bioreactors. The results of this study highlight the benefits of a holistic design of experiments guided approach to expedite early stage bioprocess development.

Bioprospecting microbial hosts to valorize lignocellulose biomass – Environmental perspectives and value-added bioproducts

Current biorefinery approaches comprehend diverse biomass feedstocks and various conversion techniques to produce a variety of high-value biochemicals and biofuels. Lignocellulose is among the most abundant, bio-renewable, and sustainable bioresources on earth. It is regarded as a prodigious alternative raw feedstock to produce a large number of chemicals and biofuels. Producing biofuels and platform chemicals from lignocellulosic biomasses represent advantages in terms of energy and environmental perspectives. Lignocellulose is a main structural constituent of non-woody and woody plants consisting of lignin, cellulose, and hemicellulose. Efficient exploitation of all these components is likely to play a considerable contribution to the economic viability of the processes since lignocellulosic biomass often necessitate pretreatment for liberating fermentable sugars and added value products that might serve as feedstocks for microbial strains to produce biofuels and biochemicals. Developing robust microbial culture and advancements in metabolic engineering approaches might lead to the rapid construction of cell factories for the effective biotechnological transformation of biomass feedstocks to produce biorefinery products. In this comprehensive review, we discuss the recent progress in the valorization of agro-industrial wastes as prospective microbial feedstocks to produce a spectrum of high-value products, such as microbial pigments, biopolymers, industrial biocatalysts, biofuels, biologically active compounds, bioplastics, biosurfactants, and biocontrol agents with therapeutic and industrial potentialities. Lignocellulosic biomass architecture, compositional aspects, revalorization, and pretreatment strategies are outlined for efficient conversion of lignocellulosic biomass. Moreover, metabolic engineering approaches are briefly highlighted to develop cell factories to make the lignocellulose biorefinery platforms appealing.

Advances and Opportunities of CRISPR/Cas Technology in Bioengineering Non-conventional Yeasts.

Non-conventional yeasts have attracted a growing interest on account of their excellent characteristics. In recent years, the emerging of CRISPR/Cas technology has improved the efficiency and accuracy of genome editing. Utilizing the advantages of CRISPR/Cas in bioengineering of non-conventional yeasts, quite a few advancements have been made. Due to the diversity in their genetic background, the ways for building a functional CRISPR/Cas system of various species non-conventional yeasts were also species-specific. Herein, we have summarized the different strategies for optimizing CRISPR/Cas systems in different non-conventional yeasts and their biotechnological applications in the construction of cell factories. In addition, we have proposed some potential directions for broadening and improving the application of CRISPR/Cas technology in non-conventional yeasts.