Unlocking Nature's Biosynthetic Power-Metabolic Engineering for the Fermentative Production of Chemicals.

Fermentation as a production method for chemicals is especially attractive, as it is based on cheap renewable raw materials and often exhibits advantages in terms of costs and sustainability. The tremendous development of technology in bioscience has resulted in an exponentially increasing knowledge about biological systems and has become the main driver for innovations in the field of metabolic engineering. Progress in recombinant DNA technology, genomics, and computational methods open new, cheaper, and faster ways to metabolically engineer microorganisms. Existing biosynthetic pathways for natural products, such as vitamins, organic acids, amino acids, or secondary metabolites, can be discovered and optimized efficiently, thereby enabling competitive commercial production processes. Novel biosynthetic routes can now be designed by the rearrangement of nature's unlimited number of enzymes and metabolic pathways in microbial strains. This expands the range of chemicals accessible by biotechnology and has yielded the first commercial products, while new fermentation technologies targeting novel active ingredients, commodity chemicals, and CO2 -fixation methods are on the horizon.

Novel routes towards bioplastics from plants: elucidation of the methylperillate biosynthesis pathway from Salvia dorisiana trichomes.

Plants produce a large variety of highly functionalized terpenoids. Functional groups such as partially unsaturated rings and carboxyl groups provide handles to use these compounds as feedstock for biobased commodity chemicals. For instance, methylperillate, a monoterpenoid found in Salvia dorisiana, may be used for this purpose, as it carries both an unsaturated ring and a methylated carboxyl group. The biosynthetic pathway of methylperillate in plants is still unclear. In this work, we identified glandular trichomes from S. dorisiana as the location of biosynthesis and storage of methylperillate. mRNA from purified trichomes was used to identify four genes that can encode the pathway from geranyl diphosphate towards methylperillate. This pathway includes a (-)-limonene synthase (SdLS), a limonene 7-hydroxylase (SdL7H, CYP71A76), and a perillyl alcohol dehydrogenase (SdPOHDH). We also identified a terpene acid methyltransferase, perillic acid O-methyltransferase (SdPAOMT), with homology to salicylic acid OMTs. Transient expression in Nicotiana benthamiana of these four genes, in combination with a geranyl diphosphate synthase to boost precursor formation, resulted in production of methylperillate. This demonstrates the potential of these enzymes for metabolic engineering of a feedstock for biobased commodity chemicals.

Toward Genome-Based Metabolic Engineering in Bacteria.

Prokaryotes modified stably on the genome are of great importance for production of fine and commodity chemicals. Traditional methods for genome engineering have long suffered from imprecision and low efficiencies, making construction of suitable high-producer strains laborious. Here, we review the recent advances in discovery and refinement of molecular precision engineering tools for genome-based metabolic engineering in bacteria for chemical production, with focus on the λ-Red recombineering and the clustered regularly interspaced short palindromic repeats/Cas9 nuclease systems. In conjunction, they enable the integration of in vitro-synthesized DNA segments into specified locations on the chromosome and allow for enrichment of rare mutants by elimination of unmodified wild-type cells. Combination with concurrently developing improvements in important accessory technologies such as DNA synthesis, high-throughput screening methods, regulatory element design, and metabolic pathway optimization tools has resulted in novel efficient microbial producer strains and given access to new metabolic products. These new tools have made and will likely continue to make a big impact on the bioengineering strategies that transform the chemical industry.

Metabolic engineering to enhance the value of plants as green factories.

The promise of plants to serve as the green factories of the future is ever increasing. Plants have been used traditionally for construction, energy, food and feed. Bioactive compounds primarily derived from specialized plant metabolism continue to serve as important scaffold molecules for pharmaceutical drug production. Yet, the past few years have witnessed a growing interest on plants as the ultimate harvesters of carbon and energy from the sun, providing carbohydrate and lipid biofuels that would contribute to balancing atmospheric carbon. How can the metabolic output from plants be increased even further, and what are the bottlenecks? Here, we present what we perceive to be the main opportunities and challenges associated with increasing the efficiency of plants as chemical factories. We offer some perspectives on when it makes sense to use plants as production systems because the amount of biomass needed makes any other system unfeasible. However, there are other instances in which plants serve as great sources of biological catalysts, yet are not necessarily the best-suited systems for production. We also present emerging opportunities for manipulating plant genomes to make plant synthetic biology a reality.

Combination of Genome-Scale Models and Bioreactor Dynamics to Optimize the Production of Commodity Chemicals.

The current production of a number of commodity chemicals relies on the exploitation of fossil fuels and hence has an irreversible impact on the environment. Biotechnological processes offer an attractive alternative by enabling the manufacturing of chemicals by genetically modified microorganisms. However, this alternative approach poses some important technical challenges that must be tackled to make it competitive. On the one hand, the design of biotechnological processes is based on trial-and-error approaches, which are not only costly in terms of time and money, but also result in suboptimal designs. On the other hand, the manufacturing of chemicals by biological processes is almost exclusively carried out by batch or fed-batch cultures. Given that batch cultures are expensive and not easy to scale, technical means must be developed to make continuous cultures feasible and efficient. In order to address these challenges, we have developed a mathematical model able to integrate in a single model both the genome-scale metabolic model for the organism synthesizing the chemical of interest and the dynamics of the bioreactor in which the organism is cultured. Such a model is based on the use of Flexible Nets, a modeling formalism for dynamical systems. The integration of a microscopic (organism) and a macroscopic (bioreactor) model in a single net provides an overall view of the whole system and opens the door to global optimizations. As a case study, the production of citramalate with respect to the substrate consumed by E. coli is modeled, simulated and optimized in order to find the maximum productivity in a steady-state continuous culture. The predicted computational results were consistent with the wet lab experiments.

Biosynthesis of Commodity Chemicals From Oil Palm Empty Fruit Bunch Lignin.

Lignin is one of the most abundant natural resources that can be exploited for the bioproduction of value-added commodity chemicals. Oil palm empty fruit bunches (OPEFBs), byproducts of palm oil production, are abundant lignocellulosic biomass but largely used for energy and regarded as waste. Pretreatment of OPEFB lignin can yield a mixture of aromatic compounds that can potentially serve as substrates to produce commercially important chemicals. However, separation of the mixture into desired individual substrates is required, which involves expensive steps that undermine the utility of OPEFB lignin. Here, we report successful engineering of microbial hosts that can directly utilize heterogeneous mixtures derived from OPEFB lignin to produce commodity chemicals, adipic acid and levulinic acid. Furthermore, the corresponding bioconversion pathway was placed under a genetic controller to autonomously activate the conversion process as the cells are fed with a depolymerized OPEFB lignin mixture. This study demonstrates a simple, one-pot biosynthesis approach that directly utilizes derivatives of agricultural waste to produce commodity chemicals.

State-of-the-Art Genetic Modalities to Engineer Cyanobacteria for Sustainable Biosynthesis of Biofuel and Fine-Chemicals to Meet Bio-Economy Challenges.

In recent years, metabolic engineering of microorganisms has attained much research interest to produce biofuels and industrially pertinent chemicals. Owing to the relatively fast growth rate, genetic malleability, and carbon neutral production process, cyanobacteria has been recognized as a specialized microorganism with a significant biotechnological perspective. Metabolically engineering cyanobacterial strains have shown great potential for the photosynthetic production of an array of valuable native or non-native chemicals and metabolites with profound agricultural and pharmaceutical significance using CO2 as a building block. In recent years, substantial improvements in developing and introducing novel and efficient genetic tools such as genome-scale modeling, high throughput omics analyses, synthetic/system biology tools, metabolic flux analysis and clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (CRISPR/cas) systems have been made for engineering cyanobacterial strains. Use of these tools and technologies has led to a greater understanding of the host metabolism, as well as endogenous and heterologous carbon regulation mechanisms which consequently results in the expansion of maximum productive ability and biochemical diversity. This review summarizes recent advances in engineering cyanobacteria to produce biofuel and industrially relevant fine chemicals of high interest. Moreover, the development and applications of cutting-edge toolboxes such as the CRISPR-cas9 system, synthetic biology, high-throughput "omics", and metabolic flux analysis to engineer cyanobacteria for large-scale cultivation are also discussed.

Commodity Chemicals From Engineered Modular Type I Polyketide Synthases.

Reduced polyketides are a subclass of natural products that have a variety of medical, veterinary, and agricultural applications and are well known for their structural diversity. Although these compounds do not resemble each other, they are all made by a class of enzymes known as modular polyketide synthases (PKSs). The commonality of PKS domains/modules that compose PKSs and the understanding of the relationship between the sequence of the PKS and the structure of the compound it produces render modular PKSs as excellent targets for engineering to produce novel compounds with predicted structures. Here, we describe experimental protocols and considerations for modular PKS engineering and two case studies to produce commodity chemicals by engineered PKSs.

Methyl Perillate as a Highly Functionalized Natural Starting Material for Terephthalic Acid.

Renewable commodity chemicals can be generated from plant materials. Often abundant materials such as sugars are used for this purpose. However, these lack appropriate functionalities and, therefore, they require extensive chemical modifications before they can be used as commodity chemicals. The plant kingdom is capable of producing an almost endless variety of compounds, including compounds with highly appropriate functionalities, but these are often not available in high quantities. It has been demonstrated that it is possible to produce functionalized plant compounds on a large scale by fermentation in microorganisms. This opens up the potential to exploit plant compounds that are less abundant, but functionally resemble commodity chemicals more closely. To elaborate this concept, we demonstrate the suitability of a highly functionalized plant compound, methyl perillate, as a precursor for the commodity chemical terephthalic acid.

Rapid Thermostabilization of Bacillus thuringiensis Serovar Konkukian 97-27 Dehydroshikimate Dehydratase through a Structure-Based Enzyme Design and Whole Cell Activity Assay.

Thermostabilization of an enzyme with complete retention of catalytic efficiency was demonstrated on recombinant 3-dehydroshikimate dehydratase (DHSase or wtAsbF) from Bacillus thuringiensis serovar konkukian 97-27 (hereafter, B. thuringiensis 97-27). The wtAsbF is relatively unstable at 37 °C, in vitro (t1/237 = 15 min), in the absence of divalent metal. We adopted a structure-based design to identify stabilizing mutations and created a combinatorial library based upon predicted mutations at specific locations on the enzyme surface. A diversified asbF library (∼2000 variants) was expressed in E. coli harboring a green fluorescent protein (GFP) reporter system linked to the product of wtAsbF activity (3,4-dihydroxybenzoate, DHB). Mutations detrimental to DHSase function were rapidly eliminated using a high throughput fluorescence activated cell sorting (FACS) approach. After a single sorting round and heat screen at 50 °C, a triple AsbF mutant (Mut1), T61N, H135Y, and H257P, was isolated and characterized. The half-life of Mut1 at 37 °C was >10-fold higher than the wtAsbF (t1/237 = 169 min). Further, the second-order rate constants for both wtAsbF and Mut1 were approximately equal (9.9 × 105 M-1 s-1, 7.8 × 105 M-1 s-1, respectively), thus demonstrating protein thermostability did not come at the expense of enzyme thermophilicity. In addition, in vivo overexpression of Mut1 in E. coli resulted in a ∼60-fold increase in functional enzyme when compared to the wild-type enzyme under the identical expression conditions. Finally, overexpression of the thermostable AsbF resulted in an approximate 80-120% increase in DHB accumulation in the media relative to the wild-type enzyme.

Synthetic Biology for Specialty Chemicals.

In this review, we address recent advances in the field of synthetic biology and describe how those tools have been applied to produce a wide variety of chemicals in microorganisms. Here we classify the expansion of the synthetic biology toolbox into three different categories based on their primary function in strain engineering-for design, for construction, and for optimization. Next, focusing on recent years, we look at how chemicals have been produced using these new synthetic biology tools. Advances in producing fuels are briefly described, followed by a more thorough treatment of commodity chemicals, specialty chemicals, pharmaceuticals, and nutraceuticals. Throughout this review, an emphasis is placed on how synthetic biology tools are applied to strain engineering. Finally, we discuss organism and host strain diversity and provide a future outlook in the field.

Integrated supply chain design for commodity chemicals production via woody biomass fast pyrolysis and upgrading.

This study investigates the optimal supply chain design for commodity chemicals (BTX, etc.) production via woody biomass fast pyrolysis and hydroprocessing pathway. The locations and capacities of distributed preprocessing hubs and integrated biorefinery facilities are optimized with a mixed integer linear programming model. In this integrated supply chain system, decisions on the biomass chipping methods (roadside chipping vs. facility chipping) are also explored. The economic objective of the supply chain model is to maximize the profit for a 20-year chemicals production system. In addition to the economic objective, the model also incorporates an environmental objective of minimizing life cycle greenhouse gas emissions, analyzing the trade-off between the economic and environmental considerations. The capital cost, operating cost, and revenues for the biorefinery facilities are based on techno-economic analysis, and the proposed approach is illustrated through a case study of Minnesota, with Minneapolis-St. Paul serving as the chemicals distribution hub.

A synthetic biochemistry system for the in vitro production of isoprene from glycolysis intermediates.

The high yields required for the economical production of chemicals and fuels using microbes can be difficult to achieve due to the complexities of cellular metabolism. An alternative to performing biochemical transformations in microbes is to build biochemical pathways in vitro, an approach we call synthetic biochemistry. Here we test whether the full mevalonate pathway can be reconstituted in vitro and used to produce the commodity chemical isoprene. We construct an in vitro synthetic biochemical pathway that uses the carbon and ATP produced from the glycolysis intermediate phosphoenolpyruvate to run the mevalonate pathway. The system involves 12 enzymes to perform the complex transformation, while providing and balancing the ATP, NADPH, and acetyl-CoA cofactors. The optimized system produces isoprene from phosphoenolpyruvate in ∼100% molar yield. Thus, by inserting the isoprene pathway into previously developed glycolysis modules it may be possible to produce isoprene and other acetyl-CoA derived isoprenoids from glucose in vitro.

From physiology to systems metabolic engineering for the production of biochemicals by lactic acid bacteria.

The lactic acid bacteria (LAB) are a functionally related group of low-GC Gram-positive bacteria known essentially for their roles in bioprocessing of foods and animal feeds. Due to extensive industrial use and enormous economical value, LAB have been intensively studied and a large body of comprehensive data on their metabolism and genetics was generated throughout the years. This knowledge has been instrumental in the implementation of successful applications in the food industry, such as the selection of robust starter cultures with desired phenotypic traits. The advent of genomics, functional genomics and high-throughput experimentation combined with powerful computational tools currently allows for a systems level understanding of these food industry workhorses. The technological developments in the last decade have provided the foundation for the use of LAB in applications beyond the classic food fermentations. Here we discuss recent metabolic engineering strategies to improve particular cellular traits of LAB and to design LAB cell factories for the bioproduction of added value chemicals.