Vitamin B6 metabolism in microbes and approaches for fermentative production

Vitamin B6 is a designation for the six vitamers pyridoxal, pyridoxine, pyridoxamine, pyridoxal 5′-phosphate (PLP), pyridoxine 5′-phosphate, and pyridoxamine. PLP, being the most important B6 vitamer, serves as a cofactor for many proteins and enzymes. In contrast to other organisms, animals and humans have to ingest vitamin B6 with their food. Several disorders are associated with vitamin B6 deficiency. Moreover, pharmaceuticals interfere with metabolism of the cofactor, which also results in vitamin B6 deficiency. Therefore, vitamin B6 is a valuable compound for the pharmaceutical and the food industry. Although vitamin B6 is currently chemically synthesized, there is considerable interest on the industrial side to shift from chemical processes to sustainable fermentation technologies. Here, we review recent findings regarding biosynthesis and homeostasis of vitamin B6 and describe the approaches that have been made in the past to develop microbial production processes. Moreover, we will describe novel routes for vitamin B6 biosynthesis and discuss their potential for engineering bacteria that overproduce the commercially valuable substance. We also highlight bottlenecks of the vitamin B6 biosynthetic pathways and propose strategies to circumvent these limitations.     

Chassis engineering for microbial production of chemicals: from natural microbes to synthetic organisms

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From microbes to fish the next revolution in food production

Increasing global population and the consequent increase in demand for food are not a new story. Agroindustrial activities such as livestock help meet this demand. Aquaculture arose decades ago and revolutionized the agroindustrial activity as a significant food generator. However, like livestock, aquaculture is based on finite resources and has been accused of being unsustainable. Abandoning aquaculture is not an option considering the food, foreign exchange, and employment it generates, and therefore must be reinvented. Among the many alternatives suggested to make aquaculture more sustainable, microorganisms have been highlighted as a direct food source for cultured fish and crustaceans, a strategy that promises to revolutionize aquaculture by eliminating waste. Considering waste, as part of a cycle, it can increase stock densities and reduce emissions of contaminants and operational costs.     

Harnessing biodiesel-producing microbes: from genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway

Microbial production routes, notably whole-cell lipase-mediated biotransformation and fatty-acids-derived biosynthesis, offer new opportunities for synthesizing biodiesel. They compare favorably to immobilized lipase and chemically catalyzed processes. Genetically modified whole-cell lipase-mediated in vitro route, together with in vivo and ex vivo microbial biosynthesis routes, constitutes emerging and rapidly developing research areas for effective production of biodiesel. This review presents recent advances in customizing microorganisms for producing biodiesel, via genetic engineering of lipases and metabolic engineering (including system regulation) of fatty-acids-derived pathways. Microbial hosts used include Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and Aspergillus oryzae. These microbial cells can be genetically modified to produce lipases under different forms: intracellularly expressed, secreted or surface-displayed. They can be metabolically redesigned and systematically regulated to obtain balanced biodiesel-producing cells, as highlighted in this study. Such genetically or metabolically modified microbial cells can support not only in vitro biotransformation of various common oil feedstocks to biodiesel, but also de novo biosynthesis of biodiesel from glucose, glycerol or even cellulosic biomass. We believe that the genetically tractable oleaginous yeast Yarrowia lipolytica could be developed to an effective biodiesel-producing microbial cell factory. For this purpose, we propose several engineered pathways, based on lipase and wax ester synthase, in this promising oleaginous host.     

Importance of systems biology in engineering microbes for biofuel production

Microorganisms have been rich sources for natural products, some of which have found use as fuels, commodity chemicals, specialty chemicals, polymers, and drugs, to name a few. The recent interest in production of transportation fuels from renewable resources has catalyzed numerous research endeavors that focus on developing microbial systems for production of such natural products. Eliminating bottlenecks in microbial metabolic pathways and alleviating the stresses due to production of these chemicals are crucial in the generation of robust and efficient production hosts. The use of systems-level studies makes it possible to comprehensively understand the impact of pathway engineering within the context of the entire host metabolism, to diagnose stresses due to product synthesis, and provides the rationale to cost-effectively engineer optimal industrial microorganisms.     

Metabolic engineering of Escherichia coli for high production of 1,5-pentanediol via a cadaverine-derived pathway

1,5-Pentanediol (1,5-PDO) is a high value-added chemical which is widely used as a monomer in the polymer industry. There are no natural organisms that could directly produce 1,5-PDO from renewable carbon sources. In this study, we report metabolic engineering of Escherichia coli for high-level production of 1,5-PDO from glucose via a cadaverine-derived pathway. In the newly proposed pathway, cadaverine can be converted to 1,5-PDO via 5-hydroxyvalerate (5-HV) by introducing only one heterologous enzyme in E. coli. Different endogenous genes of E. coli were screened and heterologous carboxylic acid reductase genes were tested to build a functional pathway. Compared to the previously reported pathways, the engineered cadaverine-based pathway has a higher theoretical yield (0.70 mol/mol glucose) and higher catalytic efficiency. By further combining strategies of pathway engineering and process engineering, we constructed an engineered E. coli strain that could produce 2.62 g/L 1,5-PDO in shake-flask and 9.25 g/L 1,5-PDO with a yield of 0.28 mol/mol glucose in fed-batch fermentation. The proposed new pathway and engineering strategies reported here should be useful for developing biological routes to produce 1,5-PDO for real application.     

Metabolic engineering for the microbial production of marine bioactive compounds

Many marine bioactive compounds have medicinal and nutritional values. These bioactive compounds have been prepared using solvent-based extraction from marine bio-resources or chemical synthesis, which are costly, inefficient with low yields, and environmentally unfriendly. Recent advances in metabolic engineering allowed to some extent more efficient production of these compounds, showing promises to meet the increasing demand of marine natural bioactive compounds. In this paper, we review the strategies and statuses of metabolic engineering applied to microbial production of marine natural bioactive compounds including terpenoids and their derivatives, omega-3 polyunsaturated fatty acids, and marine natural drugs, and provide perspectives.     

The geobiological nitrogen cycle: From microbes to the mantle

Nitrogen forms an integral part of the main building blocks of life, including DNA, RNA, and proteins. N₂ is the dominant gas in Earth's atmosphere, and nitrogen is stored in all of Earth's geological reservoirs, including the crust, the mantle, and the core. As such, nitrogen geochemistry is fundamental to the evolution of planet Earth and the life it supports. Despite the importance of nitrogen in the Earth system, large gaps remain in our knowledge of how the surface and deep nitrogen cycles have evolved over geologic time. Here, we discuss the current understanding (or lack thereof) for how the unique interaction of biological innovation, geodynamics, and mantle petrology has acted to regulate Earth's nitrogen cycle over geologic timescales. In particular, we explore how temporal variations in the external (biosphere and atmosphere) and internal (crust and mantle) nitrogen cycles could have regulated atmospheric pN₂. We consider three potential scenarios for the evolution of the geobiological nitrogen cycle over Earth's history: two in which atmospheric pN₂ has changed unidirectionally (increased or decreased) over geologic time and one in which pN₂ could have taken a dramatic deflection following the Great Oxidation Event. It is impossible to discriminate between these scenarios with the currently available models and datasets. However, we are optimistic that this problem can be solved, following a sustained, open‐minded, and multidisciplinary effort between surface and deep Earth communities.     

Metabolic engineering of Bacillus subtilis for ethanol production: lactate dehydrogenase plays a key role in fermentative metabolism

Wild-type Bacillus subtilis ferments 20 g/liter glucose in 48 h, producing lactate and butanediol, but not ethanol or acetate. To construct an ethanologenic B. subtilis strain, homologous recombination was used to disrupt the native lactate dehydrogenase (LDH) gene (ldh) by chromosomal insertion of the Zymomonas mobilis pyruvate decarboxylase gene (pdc) and alcohol dehydrogenase II gene (adhB) under the control of the ldh native promoter. The values of the intracellular PDC and ADHII enzymatic activities of the engineered B. subtilis BS35 strain were similar to those found in an ethanologenic Escherichia coli strain. BS35 produced ethanol and butanediol; however, the cell growth and glucose consumption rates were reduced by 70 and 65%, respectively, in comparison to those in the progenitor strain. To eliminate butanediol production, the acetolactate synthase gene (alsS) was inactivated. In the BS36 strain (BS35 delta alsS), ethanol production was enhanced, with a high yield (89% of the theoretical); however, the cell growth and glucose consumption rates remained low. Interestingly, kinetic characterization of LDH from B. subtilis showed that it is able to oxidize NADH and NADPH. The expression of the transhydrogenase encoded by udhA from E. coli allowed a partial recovery of the cell growth rate and an early onset of ethanol production. Beyond pyruvate-to-lactate conversion and NADH oxidation, an additional key physiological role of LDH for glucose consumption under fermentative conditions is suggested. Long-term cultivation showed that 8.9 g/liter of ethanol can be obtained using strain BS37 (BS35 delta alsS udhA+). As far as we know, this is the highest ethanol titer and yield reported with a B. subtilis strain.     

Tailoring structure-function properties of L-asparaginase: engineering resistance to trypsin cleavage

Bacterial L-ASNases (L-asparaginases) catalyse the conversion of L-asparagine into L-aspartate and ammonia, and are widely used for the treatment of ALL (acute lymphoblastic leukaemia). In the present paper, we describe an efficient approach, based on protein chemistry and protein engineering studies, for the construction of trypsin-resistant PEGylated L-ASNase from Erwinia carotovora (EcaL-ASNase). Limited proteolysis of EcaL-ASNase with trypsin was found to be associated with a first cleavage of the peptide bond between Lys53 and Gly54, and then a second cleavage at Arg206-Ser207 of the C-terminal fragment, peptide 54-327, showing that the initial recognition sites for trypsin are Lys53 and Arg206. Site-directed mutagenesis of Arg206 to histidine followed by covalent coupling of mPEG-SNHS [methoxypoly(ethylene glycol) succinate N-hydroxysuccinimide ester] to the mutant enzyme resulted in an improved modified form of EcaL-ASNase that retains 82% of the original catalytic activity, exhibits enhanced resistance to trypsin degradation, and has higher thermal stability compared with the wild-type enzyme.     

From zero to hero--design-based systems metabolic engineering of Corynebacterium glutamicum for L-lysine production

Here, we describe the development of a genetically defined strain of l-lysine hyperproducing Corynebacterium glutamicum by systems metabolic engineering of the wild type. Implementation of only 12 defined genome-based changes in genes encoding central metabolic enzymes redirected major carbon fluxes as desired towards the optimal pathway usage predicted by in silico modeling. The final engineered C. glutamicum strain was able to produce lysine with a high yield of 0.55 g per gram of glucose, a titer of 120 g L(-1) lysine and a productivity of 4.0 g L(-1) h(-1) in fed-batch culture. The specific glucose uptake rate of the wild type could be completely maintained during the engineering process, providing a highly viable producer. For these key criteria, the genetically defined strain created in this study lies at the maximum limit of classically derived producers developed over the last fifty years. This is the first report of a rationally derived lysine production strain that may be competitive with industrial applications. The design-based strategy for metabolic engineering reported here could serve as general concept for the rational development of microorganisms as efficient cellular factories for bio-production.     

Beyond the semi-synthetic artemisinin: metabolic engineering of plant-derived anti-cancer drugs

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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.