Enabling commercial success of industrial biotechnology.

Commercial-scale research translation has been muted.

A bio-inspired cell-free system for cannabinoid production from inexpensive inputs.

Moving cannabinoid production away from the vagaries of plant extraction and into engineered microbes could provide a consistent, purer, cheaper and environmentally benign source of these important therapeutic molecules, but microbial production faces notable challenges. An alternative to microbes and plants is to remove the complexity of cellular systems by employing enzymatic biosynthesis. Here we design and implement a new cell-free system for cannabinoid production with the following features: (1) only low-cost inputs are needed; (2) only 12 enzymes are employed; (3) the system does not require oxygen and (4) we use a nonnatural enzyme system to reduce ATP requirements that is generally applicable to malonyl-CoA-dependent pathways such as polyketide biosynthesis. The system produces ~0.5 g l-1 cannabigerolic acid (CBGA) or cannabigerovarinic acid (CBGVA) from low-cost inputs, nearly two orders of magnitude higher than yeast-based production. Cell-free systems such as this may provide a new route to reliable cannabinoid production.

Biosynthetic strategies to produce xylitol: an economical venture.

Xylitol is a natural five-carbon sugar alcohol with potential for use in food and pharmaceutical industries owing to its insulin-independent metabolic regulation, tooth rehardening, anti-carcinogenic, and anti-inflammatory, as well as osteoporosis and ear infections preventing activities. Chemical and biosynthetic routes using D-xylose, glucose, or biomass hydrolysate as raw materials can produce xylitol. Among these methods, microbial production of xylitol has received significant attention due to its wide substrate availability, easy to operate, and eco-friendly nature, in contrast with high-energy consuming and environmental-polluting chemical method. Though great advances have been made in recent years for the biosynthesis of xylitol from xylose, glucose, and biomass hydrolysate, and the yield and productivity of xylitol are substantially improved by metabolic engineering and optimizing key metabolic pathway parameters, it is still far away from industrial-scale biosynthesis of xylitol. In contrary, the chemical synthesis of xylitol from xylose remains the dominant route. Economic and highly efficient xylitol biosynthetic strategies from an abundantly available raw material (i.e., glucose) by engineered microorganisms are on the hard way to forwarding. However, synthetic biology appears as a novel and promising approach to develop a super yeast strain for industrial production of xylitol from glucose. After a brief overview of chemical-based xylitol production, we critically analyzed and comprehensively summarized the major metabolic strategies used for the enhanced biosynthesis of xylitol in this review. Towards the end, the study is wrapped up with current challenges, concluding remarks, and future prospects for designing an industrial yeast strain for xylitol biosynthesis from glucose.

Enhanced production of recombinant Staphylococcus simulans lysostaphin using medium engineering.

Staphylococcus aureus, among other staphylococcal species, developed multidrug resistance and causes serious health risks that require complex treatments. Therefore, the development of novel and effective strategies to combat these bacteria has been gaining importance. Since Staphylococcus simulans lysostaphin is a peptidoglycan hydrolase effective against staphylococcal species, the enzyme has a significant potential for biotechnological applications. Despite promising results of lysostaphin as a bacteriocin capable of killing staphylococcal pathogens, it is still not widely used in healthcare settings due to its high production cost. In this study, medium engineering techniques were applied to improve the expression yield of recombinant lysostaphin in E. coli. A new effective inducible araBAD promoter system and different mediums were used to enhance lysostaphin production. Our results showed that the composition of autoinduction media enhanced the amount of lysostaphin production 5-fold with the highest level of active lysostaphin at 30 °C. The production cost of 1000 U of lysostaphin was determined as 4-fold lower than the previously proposed technologies. Therefore, the currently developed bench scale study has a great potential as a large-scale fermentation procedure to produce lysostaphin efficiently.

Modulation of culture medium confers high-specificity production of isopentenol in Bacillus subtilis.

Enthusiasm for mining isoprenoid-based flavors, pharmaceuticals, and nutraceuticals from GRAS (Generally Regarded as Safe) status microbial hosts has increased in the past few years due to the limitations associated with their plant-based extraction and chemical synthesis. Bacillus subtilis, a well-known GRAS microbe, is a promising alternative due to its fast growth rate and the ability to metabolize complex carbon sources. The study focused on the high-specificity production of isopentenol in B. subtilis by modulating the culture medium. Media modulation led to a 2.5 folds improvement in isopentenol titer in the wild-type strain. In the recombinant strain, optimization of physico-chemical factors, coupled with overexpression of the nudF enzyme resulted in a maximum isopentenol titer of ∼6 mg/L in a shake flask. The recombinant strain produced ∼5 mg/L isoprenol (∼80% of the total isopentenol production) and ∼1.8 mg/L prenol (∼65% of the total isopentenol production) by utilizing sorbitol and pyruvate as the carbon sources, respectively. Replacement of glucose with sorbitol and pyruvate reduced the production of the undesired metabolites and enhanced high-specificity production of isopentenol. Upon replacement of the carbon source with a low-cost substrate, a non-detoxified rice-straw hydrolysate, the engineered strain produced 2.19 mg/L isopentenol. This proof-of-concept study paves the path for the high-specificity production and cost-effective recovery of isopentenol from industrially competent microbial strains with engineered isoprenoid pathways.

A framework for the identification of promising bio-based chemicals.

Recent progress in metabolic engineering and synthetic biology enables the use of microorganisms for the production of chemicals-"bio-based chemicals." However, it is still unclear which chemicals have the highest economic prospect. To this end, we develop a framework for the identification of such promising ones. Specifically, we first develop a genome-scale constraint-based metabolic modeling approach, which is used to identify a candidate pool of 209 chemicals (together with the estimated yield, productivity, and residence time for each) from the intersection of the high-production-volume chemicals and the KEGG and MetaCyc databases. Second, we design three screening criteria based on a chemical's profit margin, market volume, and market size. The total process cost, including the downstream separation cost, is systematically incorporated into the evaluation. Third, given the three aforementioned criteria, we identify 32 products as economically promising if the maximum yields can be achieved, and 22 products if the maximum productivities can be achieved. The breakeven titer that renders zero profit margin for each product is also presented. Comparisons between extracellular and intracellular production, as well as Escherichia coli and Saccharomyces cerevisiae systems are also discussed. The proposed framework provides important guidance for future studies in the production of bio-based chemicals. It is also flexible in that the databases, yield estimations, and criteria can be modified to customize the screening.

Cofactor Regeneration Using Permeabilized Escherichia coli Expressing NAD(P)+-Dependent Glycerol-3-Phosphate Dehydrogenase.

Oxidoreductases are effective biocatalysts, but their practical use is limited by the need for large quantities of NAD(P)H. In this study, a whole-cell biocatalyst for NAD(P)H cofactor regeneration was developed using the economical substrate glycerol. This cofactor regeneration system employs permeabilized Escherichia coli cells in which the glpD and gldA genes were deleted and the gpsA gene, which encodes NAD(P)+-dependent glycerol-3-phosphate dehydrogenase, was overexpressed. These manipulations were applied to block a side reaction (i.e., the conversion of glycerol to dihydroxyacetone) and to switch the glpD-encoding enzyme reaction to a gpsA-encoding enzyme reaction that generates both NADH and NADPH. We demonstrated the performance of the cofactor regeneration system using a lactate dehydrogenase reaction as a coupling reaction model. The developed biocatalyst involves an economical substrate, bifunctional regeneration of NAD(P)H, and simple reaction conditions as well as a stable environment for enzymes, and is thus applicable to a variety of oxidoreductase reactions requiring NAD(P)H regeneration.

An economically and environmentally acceptable synthesis of chiral drug intermediate L-pipecolic acid from biomass-derived lysine via artificially engineered microbes.

Deficiency in petroleum resources and increasing environmental concerns have pushed a bio-based economy to be built, employing a highly reproducible, metal contaminant free, sustainable and green biomanufacturing method. Here, a chiral drug intermediate L-pipecolic acid has been synthesized from biomass-derived lysine. This artificial bioconversion system involves the coexpression of four functional genes, which encode L-lysine α-oxidase from Scomber japonicus, glucose dehydrogenase from Bacillus subtilis, Δ1-piperideine-2-carboxylase reductase from Pseudomonas putida, and lysine permease from Escherichia coli. Besides, a lysine degradation enzyme has been knocked out to strengthen the process in this microbe. The overexpression of LysP improved the L-pipecolic acid titer about 1.6-folds compared to the control. This engineered microbial factory showed the highest L-pipecolic acid production of 46.7 g/L reported to date and a higher productivity of 2.41 g/L h and a yield of 0.89 g/g. This biotechnological L-pipecolic acid production is a simple, economic, and green technology to replace the presently used chemical synthesis.

Engineering NOG-pathway in Escherichia coli for poly-(3-hydroxybutyrate) production from low cost carbon sources.

Poly-(3-hydroxybutyrate) (P3HB) is a polyester with biodegradable and biocompatible characteristics suitable for bio-plastics and bio-medical use. In order to reduce the raw material cost, cheaper carbon sources such as xylose and glycerol were evaluated for P3HB production. We first conducted genome-scale metabolic network analysis to find the optimal pathways for P3HB production using xylose or glycerol respectively as the sole carbon sources. The results indicated that the non-oxidative glycolysis (NOG) pathway is important to improve the product yields. We then engineered this pathway into E. coli by introducing foreign phophoketolase enzymes. The results showed that the carbon yield improved from 0.19 to 0.24 for xylose and from 0.30 to 0.43 for glycerol. This further proved that the introduction of NOG pathway can be used as a general strategy to improve P3HB production.

Complete Biosynthesis of Anthocyanins Using E. coli Polycultures.

Fermentation-based chemical production strategies provide a feasible route for the rapid, safe, and sustainable production of a wide variety of important chemical products, ranging from fuels to pharmaceuticals. These strategies have yet to find wide industrial utilization due to their inability to economically compete with traditional extraction and chemical production methods. Here, we engineer for the first time the complex microbial biosynthesis of an anthocyanin plant natural product, starting from sugar. This was accomplished through the development of a synthetic, 4-strain Escherichia coli polyculture collectively expressing 15 exogenous or modified pathway enzymes from diverse plants and other microbes. This synthetic consortium-based approach enables the functional expression and connection of lengthy pathways while effectively managing the accompanying metabolic burden. The de novo production of specific anthocyanin molecules, such as calistephin, has been an elusive metabolic engineering target for over a decade. The utilization of our polyculture strategy affords milligram-per-liter production titers. This study also lays the groundwork for significant advances in strain and process design toward the development of cost-competitive biochemical production hosts through nontraditional methodologies.IMPORTANCE To efficiently express active extensive recombinant pathways with high flux in microbial hosts requires careful balance and allocation of metabolic resources such as ATP, reducing equivalents, and malonyl coenzyme A (malonyl-CoA), as well as various other pathway-dependent cofactors and precursors. To address this issue, we report the design, characterization, and implementation of the first synthetic 4-strain polyculture. Division of the overexpression of 15 enzymes and transcription factors over 4 independent strain modules allowed for the division of metabolic burden and for independent strain optimization for module-specific metabolite needs. This study represents the most complex synthetic consortia constructed to date for metabolic engineering applications and provides a new paradigm in metabolic engineering for the reconstitution of extensive metabolic pathways in nonnative hosts.

Building a bio-based industry in the Middle East through harnessing the potential of the Red Sea biodiversity.

The incentive for developing microbial cell factories for production of fuels and chemicals comes from the ability of microbes to deliver these valuable compounds at a reduced cost and with a smaller environmental impact compared to the analogous chemical synthesis. Another crucial advantage of microbes is their great biological diversity, which offers a much larger "catalog" of molecules than the one obtainable by chemical synthesis. Adaptation to different environments is one of the important drives behind microbial diversity. We argue that the Red Sea, which is a rather unique marine niche, represents a remarkable source of biodiversity that can be geared towards economical and sustainable bioproduction processes in the local area and can be competitive in the international bio-based economy. Recent bioprospecting studies, conducted by the King Abdullah University of Science and Technology, have established important leads on the Red Sea biological potential, with newly isolated strains of Bacilli and Cyanobacteria. We argue that these two groups of local organisms are currently most promising in terms of developing cell factories, due to their ability to operate in saline conditions, thus reducing the cost of desalination and sterilization. The ability of Cyanobacteria to perform photosynthesis can be fully exploited in this particular environment with one of the highest levels of irradiation on the planet. We highlight the importance of new experimental and in silico methodologies needed to overcome the hurdles of developing efficient cell factories from the Red Sea isolates.

Application of recombinant Pediococcus acidilactici BD16 (fcs +/ech +) for bioconversion of agrowaste to vanillin.

Biotechnological production of vanillin is gaining momentum as the natural synthesis of vanillin that is very expensive. Ferulic acid (FA), a costly compound, is used as the substrate to produce vanillin biotechnologically and the making process is still expensive. Therefore, this study investigated the practical use of an agrobiomass waste, rice bran, and provides the first evidence of a cost-effective production of vanillin within 24 h of incubation using recombinant Pediococcus acidilactici BD16 (fcs +/ech +). Introduction of two genes encoding feruloyl CoA synthetase and enoyl CoA hydratase into the native strain increased vanillin yield to 4.01 g L-1. Bioconversion was monitored through the transformation of phenolic compounds. A hypothetical metabolic pathway of rice bran during the vanillin bioconversion was proposed with the inserted pathway from ferulic acid to vanillin and compared with that of other metabolic engineered strains. These results could be a gateway of using recombinant lactic acid bacteria for industrial production of vanillin from agricultural waste.

Screening of novel bacteria for the 2,3-butanediol production.

Biotechnologically produced 2,3-butanediol (2,3-BDO) is a potential starting material for industrial bulk chemicals such as butadiene or methyl ethyl ketone which are currently produced from fossil feedstocks. So far, the highest 2,3-BDO concentrations have been obtained with risk group 2 microorganisms. In this study, three risk group 1 microorganisms are presented that are so far unknown for an efficient production of 2,3-BDO. The strains Bacillus atrophaeus NRS-213, Bacillus mojavensis B-14698, and Bacillus vallismortis B-14891 were evaluated regarding their ability to produce high 2,3-BDO concentrations with a broad range of different carbon sources. A maximum 2,3-BDO concentration of 60.4 g/L was reached with the strain B. vallismortis B-14891 with an initial glucose concentration of 200 g/L within 55 h in a batch cultivation. Besides glucose, B. vallismortis B-14891 converts 14 different substrates that can be obtained from residual biomass sources to 2,3-BDO. Therefore B. vallismortis B-14891 is a promising candidate for the large-scale production of 2,3-BDO with low-cost substrates.

Biosensor-based engineering of biosynthetic pathways.

Biosynthetic pathways provide an enzymatic route from inexpensive renewable resources to valuable metabolic products such as pharmaceuticals and plastics. Designing these pathways is challenging due to the complexities of biology. Advances in the design and construction of genetic variants has enabled billions of cells, each possessing a slightly different metabolic design, to be rapidly generated. However, our ability to measure the quality of these designs lags by several orders of magnitude. Recent research has enabled cells to report their own success in chemical production through the use of genetically encoded biosensors. A new engineering discipline is emerging around the creation and application of biosensors. Biosensors, implemented in selections and screens to identify productive cells, are paving the way for a new era of biotechnological progress.

Multi-scale exploration of the technical, economic, and environmental dimensions of bio-based chemical production.

In recent years, bio-based chemicals have gained traction as a sustainable alternative to petrochemicals. However, despite rapid advances in metabolic engineering and synthetic biology, there remain significant economic and environmental challenges. In order to maximize the impact of research investment in a new bio-based chemical industry, there is a need for assessing the technological, economic, and environmental potentials of combinations of biomass feedstocks, biochemical products, bioprocess technologies, and metabolic engineering approaches in the early phase of development of cell factories. To address this issue, we have developed a comprehensive Multi-scale framework for modeling Sustainable Industrial Chemicals production (MuSIC), which integrates modeling approaches for cellular metabolism, bioreactor design, upstream/downstream processes and economic impact assessment. We demonstrate the use of the MuSIC framework in a case study where two major polymer precursors (1,3-propanediol and 3-hydroxypropionic acid) are produced from two biomass feedstocks (corn-based glucose and soy-based glycerol) through 66 proposed biosynthetic pathways in two host organisms (Escherichia coli and Saccharomyces cerevisiae). The MuSIC framework allows exploration of tradeoffs and interactions between economy-scale objectives (e.g. profit maximization, emission minimization), constraints (e.g. land-use constraints) and process- and cell-scale technology choices (e.g. strain design or oxygenation conditions). We demonstrate that economy-scale assessment can be used to guide specific strain design decisions in metabolic engineering, and that these design decisions can be affected by non-intuitive dependencies across multiple scales.

Pilot scale demonstration of D-lactic acid fermentation facilitated by Ca(OH)2 using a metabolically engineered Escherichia coli.

In this study, a genetically engineered Escherichia coli strain, HBUT-D (ΔpflB Δpta ΔfrdABCD ΔadhE Δald ΔcscR), was initially evaluated on a laboratory scale (7 L) in a glucose (130 g L(-1)) mineral salts medium for d-lactic acid fermentation using 6N KOH, Ca(OH)2 or NH4OH as the neutralizing agent. Fermentations neutralized by Ca(OH) 2 achieved a volumetric productivity of 6.35 g L(-1) h(-1), tripling that achieved by KOH (1.71 g L(-1) h(-1)) and NH4OH (1.5 g L(-1) h(-1)). The facilitative effect of Ca(OH)2 neutralization was then demonstrated on a pilot scale (6 ton vessel, 130 kg glucose ton(-1)), resulting in a volumetric productivity of 6 kg ton(-1) h(-1), a titer of 126 kg ton(-1), a yield of 97%, and an optical purity of 99.5%. These results demonstrated that E. coli HBUT-D is a promising strain for large scale d-lactic acid fermentation using mineral salts medium and Ca(OH)2 for neutralization.

Utilization of economical substrate-derived carbohydrates by solventogenic clostridia: pathway dissection, regulation and engineering.

Solventogenic clostridia can produce acetone, butanol and ethanol (ABE) by using different carbohydrates. For economical reasons, the utilization of cheap and renewable biomass in clostridia-based ABE fermentation has recently attracted increasing interests. With the study of molecular microbiology and development of genetic tools, the understanding of carbohydrate metabolism in clostridia has increased in recent years. Here, we review the pioneering work in this field, with a focus on dissecting the pathways and describing the regulation of the metabolism of economical substrate-derived carbohydrates by clostridia. Recent progress in the metabolic engineering of carbohydrate utilization pathways is also described.

From the first drop to the first truckload: commercialization of microbial processes for renewable chemicals.

Fermentation of carbohydrate substrates by microorganisms represents an attractive route for the manufacture of industrial chemicals from renewable resources. The technology to manipulate metabolism of bacteria and yeast, including the introduction of heterologous chemical pathways, has accelerated research in this field. However, the public literature contains very few examples of strains achieving the production metrics required for commercialization. This article presents the challenges in reaching commercial titer, yield, and productivity targets, along with other necessary strain and process characteristics. It then reviews various methods in systems biology, synthetic biology, enzyme engineering, and fermentation engineering which can be applied to strain improvement, and presents a strategy for using these tools to overcome the major hurdles on the path to commercialization.

Combining metabolic engineering and adaptive evolution to enhance the production of dihydroxyacetone from glycerol by Gluconobacter oxydans in a low-cost way.

Gluconobacter oxydans can rapidly and effectively transform glycerol to dihydroxyacetone (DHA) by membrane-bound quinoprotein sorbitol dehydrogenase (mSLDH). Two mutant strains of GDHE Δadh pBBR-PtufBsldAB and GDHE Δadh pBBR-sldAB derived from the GDHE strain were constructed for the enhancement of DHA production. Growth performances of both strains were largely improved after adaptively growing in the medium with glucose as the sole carbon source. The resulting GAT and GAN strains exhibited better catalytic property than the GDHE strain in the presence of a high concentration of glycerol. All strains of GDHE, GAT and GAN cultivated on glucose showed enhanced catalytic capacity than those grown on sorbitol, indicating a favorable prospect of using glucose as carbon source to reduce the cost in industrial production. It was also the first time to reveal that the expression level of the sldAB gene in glucose-growing strains were higher than that of the strains cultivated on sorbitol.

Synthetic biology and the development of tools for metabolic engineering.

Synthetic biology can significantly advance metabolic engineering by contributing tools (minimal hosts, vectors, genetic controllers, characterized enzymes). The development of these tools significantly reduced the costs and time to develop the antimalarial drug artemisinin, but the availability of more tools could have reduced these costs substantially.