Industrial Biomanufacturing Research Articles

Data-Driven Synthetic Cell Factories Development for Industrial Biomanufacturing.

Revolutionary breakthroughs in artificial intelligence (AI) and machine learning (ML) have had a profound impact on a wide range of scientific disciplines, including the development of artificial cell factories for biomanufacturing. In this paper, we review the latest studies on the application of data-driven methods for the design of new proteins, pathways, and strains. We first briefly introduce the various types of data and databases relevant to industrial biomanufacturing, which are the basis for data-driven research. Different types of algorithms, including traditional ML and more recent deep learning methods, are also presented. We then demonstrate how these data-based approaches can be applied to address various issues in cell factory development using examples from recent studies, including the prediction of protein function, improvement of metabolic models, and estimation of missing kinetic parameters, design of non-natural biosynthesis pathways, and pathway optimization. In the last section, we discuss the current limitations of these data-driven approaches and propose that data-driven methods should be integrated with mechanistic models to complement each other and facilitate the development of synthetic strains for industrial biomanufacturing.

Development of an engineered methanotroph-based microbial platform for biocatalytic conversion of methane to phytohormone for sustainable agriculture

Methane is an important target for reducing greenhouse gases emission, owing to its abundance and highly global warming potential. Bioconversion of methane to value-added products, therefore, has become a promising and sustainable approach for industrial biomanufacturing. Herein, we metabolically engineered an obligate methanotroph Methylotuvimicrobium alcaliphilum 20Z to overproduce L-tryptophan and its derivative indole 3-acetic acid (IAA), a phytohormone, from methane. The engineered RS00 strain initially produced tryptophan from methane with a titer of 39.6 mg/L at 144 h of cultivation. The highest titer of IAA of 10.5 mg/L from methane was obtained in RS01 strain under Ptac promoter. Moreover, xylose, a carbon source derived from lignocellulosic biomass used as co-substrate, could enhance the titers of tryptophan and IAA in RS00 and RS01 strains by 60% and 82%, respectively. Remarkably, we proposed a proof-of-concept for the development of methanotroph-based plant growth-promoting bacteria (PGPB) which can utilize and convert methane into phytohormones to induce beneficial adaptation and growth promotion of plants. In this study, under saline-alkaline conditions, the germination percentage and elongation of the shoots and roots of wheat seeds treated with the IAA-producing strain were significantly improved, being 114%, 199% and 362% higher than those of control treatments, respectively. Our findings provide a novel methanotrophic platform for the conversion of renewable sources to value-added products. Additionally, the strategies and concepts presented here could be further expanded and applied to other methanotrophic hosts to develop biofertilizers that promote plant growth while mitigating methane and reducing chemical fertilizer use for sustainable agriculture production.

Rewiring the microbial metabolic network for efficient utilization of mixed carbon sources.

Carbon sources represent the most dominant cost factor in the industrial biomanufacturing of products. Thus, it has attracted much attention to seek cheap and renewable feedstocks, such as lignocellulose, crude glycerol, methanol, and carbon dioxide, for biosynthesis of value-added compounds. Co-utilization of these carbon sources by microorganisms not only can reduce the production cost but also serves as a promising approach to improve the carbon yield. However, co-utilization of mixed carbon sources usually suffers from a low utilization rate. In the past few years, the development of metabolic engineering strategies to enhance carbon source co-utilization efficiency by inactivation of carbon catabolite repression has made significant progress. In this article, we provide informative and comprehensive insights into the co-utilization of two or more carbon sources including glucose, xylose, arabinose, glycerol, and C1 compounds, and we put our focus on parallel utilization, synergetic utilization, and complementary utilization of different carbon sources. Our goal is not only to summarize strategies of co-utilization of carbon sources, but also to discuss how to improve the carbon yield and the titer of target products.

Production of myo‐inositol: Recent advance and prospective

Myo-inositol and its derivatives have been extensively used in the pharmaceutics, cosmetics, and food and feed industries. In recent years, compared with traditional chemical acid hydrolysis, biological methods have been taken as viable and cost-effective ways to myo-inositol production from cheap raw materials. In this review, we provide a thorough overview of the development, progress, current status, and future direction of myo-inositol production (e.g., chemical acid hydrolysis, microbial fermentation, and in vitro enzymatic biocatalysis). The chemical acid hydrolysis of phytate suffers from serious phosphorous pollution and intricate product separation, resulting in myo-inositol production at a high cost. For microbial fermentation, creative strategies have been provided for the efficient myo-inositol biosynthesis by synergetic utilization of glucose and glycerol in Escherichia coli. in vitro cascade enzymatic biocatalysis is a multienzymatic transformation of various substrates to myo-inositol. Here, the different in vitro pathways design, the source of selected enzymes, and the catalytic condition optimization have been summarized and analyzed. Also, we discuss some important existing challenges and suggest several viewpoints. The development of in vitro enzymatic biosystems featuring low cost, high volumetric productivity, flexible compatibility, and great robustness could be one of the promising strategies for future myo-inositol industrial biomanufacturing.

Digital Twin in biomanufacturing: challenges and opportunities towards its implementation

The domain of industrial biomanufacturing is enthusiastically embracing the concept of Digital Twin, owing to its promises of increased process efficiency and resource utilisation. However, Digital Twin in biomanufacturing is not yet clearly defined and this sector of the industry is falling behind the others in terms of its implementation. On the other hand, some of the benefits of Digital Twin seem to overlap with the more established practices of process control and optimization, and the term is vaguely used in different scenarios. In an attempt to clarify this issue, we investigate this overlap for the specific case of fermentation operation, a central step in many biomanufacturing processes. Based on this investigation, a framework built upon a five-step pathway starting from a basic steady-state process model is proposed to develop a fully-fledged Digital Twin. For demonstration purposes, the framework is applied to a bench-scale second-generation ethanol fermentation process as a case study. It is proposed that the success or failure of a fully-fledged Digital Twin implementation is determined by key factors that comprise the role of modelling, human operator actions, and other propositions of economic value.

Omics-Driven Biotechnology for Industrial Applications.

Biomanufacturing is a key component of biotechnology that uses biological systems to produce bioproducts of commercial relevance, which are of great interest to the energy, material, pharmaceutical, food, and agriculture industries. Biotechnology-based approaches, such as synthetic biology and metabolic engineering are heavily reliant on “omics” driven systems biology to characterize and understand metabolic networks. Knowledge gained from systems biology experiments aid the development of synthetic biology tools and the advancement of metabolic engineering studies toward establishing robust industrial biomanufacturing platforms. In this review, we discuss recent advances in “omics” technologies, compare the pros and cons of the different “omics” technologies, and discuss the necessary requirements for carrying out multi-omics experiments. We highlight the influence of “omics” technologies on the production of biofuels and bioproducts by metabolic engineering. Finally, we discuss the application of “omics” technologies to agricultural and food biotechnology, and review the impact of “omics” on current COVID-19 research.

Extended Metabolic Biosensor Design for Dynamic Pathway Regulation of Cell Factories.

SummaryTranscription factor-based biosensors naturally occur in metabolic pathways to maintain cell growth and to provide a robust response to environmental fluctuations. Extended metabolic biosensors, i.e., the cascading of a bio-conversion pathway and a transcription factor (TF) responsive to the downstream effector metabolite, provide sensing capabilities beyond natural effectors for implementing context-aware synthetic genetic circuits and bio-observers. However, the engineering of such multi-step circuits is challenged by stability and robustness issues. In order to streamline the design of TF-based biosensors in metabolic pathways, here we investigate the response of a genetic circuit combining a TF-based extended metabolic biosensor with an antithetic integral circuit, a feedback controller that achieves robustness against environmental fluctuations. The dynamic response of an extended biosensor-based regulated flavonoid pathway is analyzed in order to address the issues of biosensor tuning of the regulated pathway under industrial biomanufacturing operating constraints.

Unlocking the biosynthesis of sesquiterpenoids from methane via the methylerythritol phosphate pathway in methanotrophic bacteria, using α-humulene as a model compound

Isoprenoids are an abundant and diverse class of natural products with various applications in the pharmaceutical, cosmetics and biofuel industries. A methanotroph-based biorefinery is an attractive scenario for the production of a variety of value-added compounds from methane, because methane is a promising alternative feedstock for industrial biomanufacturing. In this study, we metabolically engineered Methylotuvimicrobium alcaliphilum 20Z for de novo synthesis of a sesquiterpenoid from methane, using α-humulene as a model compound, via optimization of the native methylerythritol phosphate (MEP) pathway. Expression of codon-optimized α-humulene synthase from Zingiber zerumbet in M. alcaliphilum 20Z resulted in an initial yield of 0.04 mg/g dry cell weight. Overexpressing key enzymes (IspA, IspG, and Dxs) for debottlenecking of the MEP pathway increased α-humulene production 5.2-fold compared with the initial strain. Subsequently, redirecting the carbon flux through the Embden–Meyerhof–Parnas pathway resulted in an additional 3-fold increase in α-humulene production. Additionally, a genome-scale model using flux scanning based on enforced objective flux method was used to identify potential overexpression targets to increase flux towards isoprenoid production. Several target reactions from cofactor synthesis pathways were probed and evaluated for their effects on α-humulene synthesis, resulting in α-humulene yield up to 0.75 mg/g DCW with 18.8-fold enhancement from initial yield. This study first demonstrates production of a sesquiterpenoid from methane using methanotrophs as the biocatalyst and proposes potential strategies to enhance production of sesquiterpenoid and related isoprenoid products in engineered methanotrophic bacteria.

Efficient isopropanol biosynthesis by engineered Escherichia coli using biologically produced acetate from syngas fermentation

The shortage of food based feedstocks is a challenge in industrial biomanufacturing. In this study, genetically modified Escherichia coli strains were used to produce isopropanol as the mainly product from acetate, a cost-effective nonfood-based substrate. The isopropanol biosynthesis pathway was constructed by combining genes from Clostridium acetobutylicum (thlA, adc), E. coli (atoDA) and Clostridium beijerinckii (adh). E. coli MG1655 harboring the isopropanol biosynthesis pathway successfully produced isopropanol and low amounts of acetone from pure acetate. The enhancement of the acetate assimilation pathway coupled with cofactor engineering strategy further improved the production of isopropanol to 18.5 mM with a yield of 0.26 mol/mol. With simple treatment, two kinds of biologically produced acetate were utilized to generate 16.7 and 24.5 mM isopropanol with yields of 0.25 and 0.56 mol/mol, respectively. Engineered E. coli with an optimized isopropanol biosynthesis pathway can efficiently utilize biologically produced acetate to synthesize isopropanol.

Bioproduction of Isoprenoids and Other Secondary Metabolites Using Methanotrophic Bacteria as an Alternative Microbial Cell Factory Option: Current Stage and Future Aspects

Methane is a promising carbon feedstock for industrial biomanufacturing because of its low price and high abundance. Recent advances in metabolic engineering and systems biology in methanotrophs have made it possible to produce a variety of value-added compounds from methane, including secondary metabolites. Isoprenoids are one of the largest family of secondary metabolites and have many useful industrial applications. In this review, we highlight the current efforts invested to methanotrophs for the production of isoprenoids and other secondary metabolites, including riboflavin and ectoine. The future outlook for improving secondary metabolites production (especially of isoprenoids) using metabolic engineering of methanotrophs is also discussed.

Comparison of physics‐based and data‐driven modelling techniques for dynamic optimisation of fed‐batch bioprocesses

The development of digital bioprocessing technologies is critical to operate modern industrial bioprocesses. This study conducted the first investigation on the efficiency of using physics-based and data-driven models for the dynamic optimisation of long-term bioprocess. More specifically, this study exploits a predictive kinetic model and a cutting-edge data-driven model to compute open-loop optimisation strategies for the production of microalgal lutein during a fed-batch operation. Light intensity and nitrate inflow rate are used as control variables given their key impact on biomass growth and lutein synthesis. By employing different optimisation algorithms, several optimal control sequences were computed. Due to the distinct model construction principles and sophisticated process mechanisms, the physics-based and the data-driven models yielded contradictory optimisation strategies. The experimental verification confirms that the data-driven model predicted a closer result to the experiments than the physics-based model. Both models succeeded in improving lutein intracellular content by over 40% compared to the highest previous record; however, the data-driven model outperformed the kinetic model when optimising total lutein production and achieved an increase of 40-50%. This indicates the possible advantages of using data-driven modelling for optimisation and prediction of complex dynamic bioprocesses, and its potential in industrial bio-manufacturing systems.

Metabolic engineering of Escherichia coli carrying the hybrid acetone-biosynthesis pathway for efficient acetone biosynthesis from acetate

BackgroundThe shortage of food based feedstocks has been one of the stumbling blocks in industrial biomanufacturing. The acetone bioproduction from the traditional acetone–butanol–ethanol fermentation is limited by the non-specificity of products and competitive utilization of food-based substrates. Using genetically modified Escherichia coli to produce acetone as sole product from the cost-effective non-food based substrates showed great potential to overcome these problems.ResultsA novel acetone biosynthetic pathway were constructed based on genes from Clostridium acetobutylicum (thlA encoding for thiolase, adc encoding for acetoacetate decarboxylase, ctfAB encoding for coenzyme A transferase) and Escherichia coli MG1655 (atoB encoding acetyl-CoA acetyltransferase, atoDA encoding for acetyl-CoA: acetoacetyl-CoA transferase subunit α and β). Among these constructs, one recombinant MG1655 derivative containing the hybrid pathway consisting of thlA, atoDA, and adc, produced the highest level of acetone from acetate. Reducing the gluconeogenesis pathway had little effect on acetone production, while blocking the TCA cycle by knocking out the icdA gene enhanced the yield of acetone significantly. As a result, acetone concentration increased up to 113.18 mM in 24 h by the resting cell culture coupling with gas-stripping methods.ConclusionsAn engineered E. coli strain with optimized hybrid acetone biosynthetic pathway can utilize acetate as substrate efficiently to synthesize acetone without other non-gas byproducts. It provides a potential method for industrial biomanufacturing of acetone by engineered E. coli strains from non-food based substrate.

Expanding lysine industry: industrial biomanufacturing of lysine and its derivatives.

L-Lysine is widely used as a nutrition supplement in feed, food, and beverage industries as well as a chemical intermediate. At present, great efforts are made to further decrease the cost of lysine to make it more competitive in the markets. Furthermore, lysine also shows potential as a feedstock to produce other high-value chemicals for active pharmaceutical ingredients, drugs, or materials. In this review, the current biomanufacturing of lysine is first presented. Second, the production of novel derivatives from lysine is discussed. Some chemicals like L-pipecolic acid, cadaverine, and 5-aminovalerate already have been obtained at a lab scale. Others like 6-aminocaproic acid, valerolactam, and caprolactam could be produced through a biological and chemical coupling pathway or be synthesized by a hypothetical pathway. This review demonstrates an active and expansive lysine industry, and these green biomanufacturing strategies could also be applied to enhance the competitiveness of other amino acid industry.

Microbial synthesis of pyrogallol using genetically engineered Escherichia coli

Pyrogallol is a simple phenolic compound that serves as an attractive chemical with broad applications in food, agricultural, dyeing, printing, cosmetic, photography, chemical and pharmaceutical industries owing to its antioxidant, antibacterial, antiseptic, anticancer, oxygen-absorbing and strong reducing properties. Currently, pyrogallol is commercially produced by thermal decarboxylation of gallic acid under high temperature and pressure. However, this process is limited by the inaccessible raw material, the strict reaction conditions and the relatively low product yield. Here, we report establishment of a novel and efficient biosynthetic pathway for the synthesis of pyrogallol. First, we identified and characterized an efficient 2,3-dihydroxybenzoic acid (2,3-DHBA) 1-monoxygenase from a series of oxygenases and hydroxylases based on the structural similarity in the substrates and products, which enabled non-natural production of pyrogallol from 2,3-DHBA. Then, over-expression of 2,3-DHBA synthase and 2,3-DHBA 1-monoxygenase achieved synthesis of pyrogallol in Escherichia coli, with a titer of 201.52mg/L at 24h. Further optimizations by enhancement of the carbon flux through the shikimate pathway, modular optimization of the pathway and alleviation of the pyrogallol autoxidation boosted pyrogallol titer to 1035.75mg/L in shake flask experiments. This work constructed an efficient microbial platform for gram per liter level production of pyrogallol, indicating the great potential for industrial biomanufacturing of pyrogallol.

An in vitro synthetic biology platform for the industrial biomanufacturing of myo-inositol from starch.

Myo-Inositol (vitamin B8) is widely used in the drug, cosmetic, and food & feed industries. Here, we present an in vitro non-fermentative enzymatic pathway that converts starch to inositol in one vessel. This in vitro pathway is comprised of four enzymes that operate without ATP or NAD+ supplementation. All enzyme BioBricks are carefully selected from hyperthermophilic microorganisms, that is, alpha-glucan phosphorylase from Thermotoga maritima, phosphoglucomutase from Thermococcus kodakarensis, inositol 1-phosphate synthase from Archaeoglobus fulgidus, and inositol monophosphatase from T. maritima. They were expressed efficiently in high-density fermentation of Escherichia coli BL21(DE3) and easily purified by heat treatment. The four-enzyme pathway supplemented with two other hyperthermophilic enzymes (i.e., 4-α-glucanotransferase from Thermococcus litoralis and isoamylase from Sulfolobus tokodaii) converts branched or linear starch to inositol, accomplishing a very high product yield of 98.9 ± 1.8% wt./wt. This in vitro (aeration-free) biomanufacturing has been successfully operated on 20,000-L reactors. Less costly inositol would be widely added in heath food, low-end soft drink, and animal feed, and may be converted to other value-added biochemicals (e.g., glucarate). This biochemical is the first product manufactured by the in vitro synthetic biology platform on an industrial scale. Biotechnol. Bioeng. 2017;114: 1855-1864. © 2017 Wiley Periodicals, Inc.

Establishing a novel biosynthetic pathway for the production of 3,4-dihydroxybutyric acid from xylose in Escherichia coli

3-Hydroxy-γ-butyrolactone (3HBL) is an attractive building block owing to its broad applications in pharmaceutical industry. Currently, 3HBL is commercially produced by chemical routes using petro-derived carbohydrates, which involves hazardous materials and harsh processing conditions. Only one biosynthetic pathway has been reported for synthesis of 3HBL and its hydrolyzed form 3,4-dihydroxybutyric acid (3,4-DHBA) using glucose and glycolic acid as the substrates and coenzyme A as the activator, which involves multiple steps (>10 steps) and suffers from low productivity and yield. Here we established a novel five-step biosynthetic pathway for 3,4-DHBA generation from D-xylose based on the non-phosphorylative D-xylose metabolism, which led to efficient production of 3,4-DHBA in Escherichia coli. Pathway optimization by incorporation of efficient enzymes for each step and host strain engineering by knocking out competing pathways enabled 1.27g/L 3,4-DHBA produced in shake flasks, which is the highest titer reported so far. The novel pathway established in engineered E. coli strain demonstrates a new route for 3,4-DHBA biosynthesis from xylose, and this engineered pathway has great potential for industrial biomanufacturing of 3,4-DHBA and 3HBL.

Industrial biomanufacturing: The future of chemical production.

The current model for industrial chemical manufacturing employs large-scale megafacilities that benefit from economies of unit scale. However, this strategy faces environmental, geographical, political, and economic challenges associated with energy and manufacturing demands. We review how exploiting biological processes for manufacturing (i.e., industrial biomanufacturing) addresses these concerns while also supporting and benefiting from economies of unit number. Key to this approach is the inherent small scale and capital efficiency of bioprocesses and the ability of engineered biocatalysts to produce designer products at high carbon and energy efficiency with adjustable output, at high selectivity, and under mild process conditions. The biological conversion of single-carbon compounds represents a test bed to establish this paradigm, enabling rapid, mobile, and widespread deployment, access to remote and distributed resources, and adaptation to new and changing markets.

Predicting the Dynamics and Heterogeneity of Genomic DNA Content within Bacterial Populations across Variable Growth Regimes.

For many applications in microbial synthetic biology, optimizing a desired function requires careful tuning of the degree to which various genes are expressed. One challenge for predicting such effects or interpreting typical characterization experiments is that in bacteria such as E. coli, genome copy number varies widely across different phases and rates of growth, which also impacts how and when genes are expressed from different loci. While such phenomena are relatively well-understood at a mechanistic level, our quantitative understanding of such processes is essentially limited to ideal exponential growth. In contrast, common experimental phenomena such as growth on heterogeneous media, metabolic adaptation, and oxygen restriction all cause substantial deviations from ideal exponential growth, particularly as cultures approach the higher densities at which industrial biomanufacturing and even routine screening experiments are conducted. To meet the need for predicting and explaining how gene dosage impacts cellular functions outside of exponential growth, we here report a novel modeling strategy that leverages agent-based simulation and high performance computing to robustly predict the dynamics and heterogeneity of genomic DNA content within bacterial populations across variable growth regimes. We show that by feeding routine experimental data, such as optical density time series, into our heterogeneous multiphasic growth simulator, we can predict genomic DNA distributions over a range of nonexponential growth conditions. This modeling strategy provides an important advance in the ability of synthetic biologists to evaluate the role of genomic DNA content and heterogeneity in affecting the performance of existing or engineered microbial functions.

Surface Finish Machining of Medical Parts Using Plasma Electrolytic Polishing

Biomanufactured parts, for implant or generally in-vivo use, have high requirements for surface properties. Depending on their field of application, not only smooth, but also well-defined surfaces, i.e. roughness parameters, must be set to allow for the respective functionalities such as optimal cell growth or minimal friction and wear. While for many parts mechanical polishing procedures are state-of-the-art, which are time-consuming and limited in their flexibility, established electrolytic polishing processes often incorporate aggressive media such as acids. Plasma electrolytic Polishing (PeP), a high-voltage process combining anode dissolution and plasma-chemical processes, can be considered a suitable alternative. The paper focusses on current investigations of PeP and shows application scenarios, also pointing out necessary development directions to introduce PeP to industrial biomanufacturing process chains.

SILK.IT project: Silk Italian Technology for industrial biomanufacturing

Silk fibroin is a natural protein that constitutes the core fibre of the silkworm cocoon. Recently, a great deal of attention is being paid to the use of the regenerated silk fibroin (RSF) solution that can be obtained by water-processing of the cocoon fibre. Indeed, thanks to its chemical and physical properties it has been shown that substrates obtained by RSF can be used as biomaterials for several biomedical and technological applications. In this context, silk fibroin could be exploited as raw material that can become a platform for eco-sustainable manufacturing.The project SILK.IT, coordinated by the CNR-ISOF, within the framework of the Flagship Project “Factory of the Future”, a research programme approved by the Interministerial Committee for Economic Planning (CIPE), aims to support this assumption by developing methods and protocols to use this silk fibroin as new material for advanced biotechnologies and sustainable manufacturing.The main goal of the project is to establish and control the whole-chain underpinning the silk fibroin-based technology, and to promote its up-scaling from the laboratory to industrial scale, targeting bio-photonics for biomedical application.The recent results obtained by the project are herein reported as key building block towards proposed achievement to establish a sustainable and process-controlled silk-based bio-manufacturing of the future.