Platform Microorganism Research Articles

Heterologous production of 3-hydroxypropionic acid in Methylorubrum extorquens by introducing the mcr gene via a multi-round chromosomal integration system based on cre-lox71/lox66 and transposon

Background and aimReprogramming microorganisms to enhance the production of metabolites is a part of contemporary synthetic biology, which relies on the availability of genetic tools to successfully manipulate the bacteria. Methylorubrum extorquens AM1 is a platform microorganism used to convert C1 compounds into various value-added products. However, the repertoire of available plasmids to conveniently and quickly fine-tune the expression of multiple genes in this strain is extremely limited compared with other model microorganisms such as Escherichia coli. Thus, this study aimed to integrate existing technologies, such as transposon-mediated chromosomal integration and cre-lox-mediated recombination, to achieve the diversified expression of target genes through multiple chromosomal insertions in M. extorquens AM1.ResultsA single plasmid toolkit, pSL-TP-cre-km, containing a miniHimar1 transposon and an inducible cre-lox71/lox66 system, was constructed and characterized for its multiple chromosomal integration capacity. A co-transcribed mcr-egfp cassette [for the production of 3-hydroxypropionic acid (3-HP) and a reporting green fluorescent protein] was added to construct pTP-cre-mcr-egfp for evaluating its utility in mediating the expression of heterologous genes, resulting in the production of 3-HP with a titer of 34.7–55.2 mg/L by two chromosomal integration copies. Furthermore, in association with the expression of plasmid-based mcr, 3-HP production increased to 65.5–92.4 mg/L.ConclusionsThis study used a multi-round chromosomal integration system based on cre-lox71/lox66 and a transposon to construct a single constructed vector. A heterologous mcr gene was introduced through this vector, and high expression of 3-hydroxypropionic acid was achieved in M. extorquens. This study provided an efficient genetic tool for manipulating M. extorquens, which not only help increase the expression of heterologous genes in M. extorquens but also provide a reference for strains lacking genetic manipulation vectors.

Development of the Cre-Cas 2.0 system for evaluating acetoin production in Bacillus subtilis

BackgroundAcetoin is a vital platform compound widely used in the food, pharmaceutical, and chemical industries. Since 2004, the United States Department of Energy has listed it as one of the 30 platform compounds for priority development. Bacillus subtilis, a generally recognized safe microorganism, is an ideal platform microorganism for the engineered production of valuable bio-based chemicals. MethodsThe Cre-Cas 2.0 system derived from the Cre-Cas system was applied to inactive, enhance, or recover native metabolic genes. This system was designed to evaluate which native metabolic genes affect acetoin production in B. subtilis. Significant findingsUtilizing the Cre-Cas 2.0 system, acetoin reductase (bdhA), l-lactate dehydrogenase (ldh), and acetoin dehydrogenase (acoA) were inactivated, while the native acetoin synthesis genes, alsSD operon and alsR, were enhanced. This approach resulted in the creation of sixteen recombinant strains for the assessment of acetoin production. Finally, the acetoin production of strain ACN11 was 55 % higher than the original strain B. subtilis DB428. Moreover, employing the Cre-Cas 2.0 concept for editing the chromosomal genes of B. subtilis to enhance acetoin and other biochemical production could offer flexibility and applicability.

From Saccharomyces cerevisiae to Ethanol: Unlocking the Power of Evolutionary Engineering in Metabolic Engineering Applications.

Increased human population and the rapid decline of fossil fuels resulted in a global tendency to look for alternative fuel sources. Environmental concerns about fossil fuel combustion led to a sharp move towards renewable and environmentally friendly biofuels. Ethanol has been the primary fossil fuel alternative due to its low carbon emission rates, high octane content and comparatively facile microbial production processes. In parallel to the increased use of bioethanol in various fields such as transportation, heating and power generation, improvements in ethanol production processes turned out to be a global hot topic. Ethanol is by far the leading yeast output amongst a broad spectrum of bio-based industries. Thus, as a well-known platform microorganism and native ethanol producer, baker's yeast Saccharomyces cerevisiae has been the primary subject of interest for both academic and industrial perspectives in terms of enhanced ethanol production processes. Metabolic engineering strategies have been primarily adopted for direct manipulation of genes of interest responsible in mainstreams of ethanol metabolism. To overcome limitations of rational metabolic engineering, an alternative bottom-up strategy called inverse metabolic engineering has been widely used. In this context, evolutionary engineering, also known as adaptive laboratory evolution (ALE), which is based on random mutagenesis and systematic selection, is a powerful strategy to improve bioethanol production of S. cerevisiae. In this review, we focus on key examples of metabolic and evolutionary engineering for improved first- and second-generation S. cerevisiae bioethanol production processes. We delve into the current state of the field and show that metabolic and evolutionary engineering strategies are intertwined and many metabolically engineered strains for bioethanol production can be further improved by powerful evolutionary engineering strategies. We also discuss potential future directions that involve recent advancements in directed genome evolution, including CRISPR-Cas9 technology.

Fermentative conversion of unpretreated plant biomass: A thermophilic threshold for indigenous microbial growth

Naturally occurring, microbial contaminants were found in plant biomasses from common bioenergy crops and agricultural wastes. Unexpectedly, indigenous thermophilic microbes were abundant, raising the question of whether they impact thermophilic consolidated bioprocessing fermentations that convert biomass directly into useful bioproducts. Candidate microbial platforms for biomass conversion, Acetivibrio thermocellus (basionym Clostridium thermocellum; Topt 60 °C) and Caldicellulosiruptor bescii (Topt 78 °C), each degraded a wide variety of plant biomasses, but only A. thermocellus was significantly affected by the presence of indigenous microbial populations harbored by the biomass. Indigenous microbial growth was eliminated at ≥75 °C, conditions where C. bescii thrives, but where A. thermocellus cannot survive. Therefore, 75 °C is the thermophilic threshold to avoid sterilizing pre-treatments on the biomass that prevents native microbes from competing with engineered microbes and forming undesirable by-products. Thermophiles that naturally grow at and above 75 °C offer specific advantages as platform microorganisms for biomass conversion into fuels and chemicals.

Evaluation of Saccharomyces cerevisiae modified via CRISPR/Cas9 as a cellulosic platform microorganism in simultaneously saccharification and fermentation processes.

The nonrenewable character and deleterious effects of fossil fuels foster the need for cleaner and more inexhaustible energy sources, such as bioethanol. Especially from lignocellulosic biomasses. However, the economic viability of this product in the market depends on process optimization and cost reduction. This research applied a sequential experimental project to investigate the process of enzymatic saccharification and simultaneous fermentation to produce ethanol with sugarcane bagasse. The differential of the work was the application of the strain of Saccharomyces cerevisiae AGY001 which was improved by evolutionary engineering to become thermotolerant and by a heterologous expression based on genomic integration by CRISPR/Cas9 to produce endoglucanase and β-glucosidase (AsENDO-AsBGL). The maximum ethanol yield found was 89% of the maximum theoretical yield (released sugars), obtained at temperature concentrations, sugarcane bagasse and inoculum at 40°C, 16.5%, and 4.0g/L, respectively (12.5 FPU/g bagasse). The mathematical model obtained can predict approximately 83% of the data set with 95% confidence. Therefore, these findings demonstrated the potential of sugarcane bagasse and S. cerevisiae AGY001 strain (CRISPR/Cas9 modified) in bioethanol production without the need for impractical selection media on an industrial scale, in addition to providing useful insights for the development of SSF processes.

The soil bacterium, Corynebacterium glutamicum, from biosynthesis of value-added products to bioremediation: A master of many trades

Ever since its discovery in 1957, Corynebacterium glutamicum has become a well-established industrial strain and is known for its massive capability of producing various amino acids (like L-lysine and L-glutamate) and other value-added chemicals. With the rising demand for these bio-based products, the revelation of the whole genome sequences of the wild type strains, and the astounding advancements made in the fields of metabolic engineering and systems biology, our perspective of C. glutamicum has been revolutionized and has expanded our understanding of its strain development. With these advancements, a new era for C. glutamicum supremacy in the field of industrial biotechnology began. This led to remarkable progress in the enhancement of tailor-made over-producing strains and further development of the substrate spectrum of the bacterium, to easily accessible, economical, and renewable resources. C. glutamicum has also been metabolically engineered and used in the degradation/assimilation of highly toxic and ubiquitous environmental contaminant, arsenic, present in water or soil. Here, we review the history, current knowledge, progress, achievements, and future trends relating to the versatile metabolic factory, C. glutamicum. This review paper is devoted to C. glutamicum which is one of the leading industrial microbes, and one of the most promising and versatile candidates to be developed. It can be used not only as a platform microorganism to produce different value-added chemicals and recombinant proteins, but also as a tool for bioremediation, allowing to enhance specific properties, for example in situ bioremediation.

Developing Synthetic Methylotrophs by Metabolic Engineering-Guided Adaptive Laboratory Evolution.

Methanol is a promising alternative feedstock for biomanufacturing. However, natural platform industrial microorganisms such as Escherichia coli and Corynebacterium glutamicum cannot assimilate methanol. Although some methanol assimilation pathways differ from the typical sugar metabolism by only a few enzymes, engineering platform microorganisms to efficiently utilize methanol is very challenging. Recent advances in creating the first methylotrophic E. coli growing solely on methanol have underscored the value of metabolic engineering-guided adaptive laboratory evolution (ME-ALE) in developing non-natural methanol utilizers. This chapter reviews the recent progress in engineering platform microorganisms to utilize methanol by ME-ALE and discusses the future challenges in developing superior synthetic methylotrophs for methanol-based biomanufacturing.

Methanol Dehydrogenases as a Key Biocatalysts for Synthetic Methylotrophy.

One-carbon (C1) chemicals are potential building blocks for cheap and sustainable re-sources such as methane, methanol, formaldehyde, formate, carbon monoxide, and more. These resources have the potential to be made into raw materials for various products used in our daily life or precursors for pharmaceuticals through biological and chemical processes. Among the soluble C1 substrates, methanol is regarded as a biorenewable platform feedstock because nearly all bioresources can be converted into methanol through syngas. Synthetic methylotrophy can be exploited to produce fuels and chemicals using methanol as a feedstock that integrates natural or artificial methanol assimilation pathways in platform microorganisms. In the methanol utilization in methylotrophy, methanol dehydrogenase (Mdh) is a primary enzyme that converts methanol to formaldehyde. The discovery of new Mdhs and engineering of present Mdhs have been attempted to develop synthetic methylotrophic bacteria. In this review, we describe Mdhs, including in terms of their enzyme properties and engineering for desired activity. In addition, we specifically focus on the application of various Mdhs for synthetic methylotrophy.

Constructing a methanol-dependent Bacillus subtilis by engineering the methanol metabolism

Methanol is a promising green feedstock for producing fuels and chemicals because it is inexpensive, clean, environmentally friendly, and easily prepared. Thus, many studies have been devoted to engineering non-native methylotrophic platform microorganisms to utilize methanol. This study adopted a series of strategies to develop a synthetic methylotrophic Bacillus subtilis that can use methanol as the carbon source, including the heterologous expression of methanol dehydrogenase (Mdh), enhancement of the expressions of 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi), regulation of the expressions of key enzymes at both the translational and transcriptional levels, stabilization of the key enzyme expression through a dual-system for expressing the target genes on both the plasmid and genome, and improvement of the catalytic activity of Mdh with a recycling strategy for NAD+. As a result, the methanol consumption of the synthetic methylotrophic B. subtilis reached 4.09 g/L, with the maximum OD600 showing a 2.21-fold increase compared with the wild-type B. subtilis, which cannot use methanol. We further deleted the phosphoglucose isomerase (Pgi) and added co-substrates to increase the supply of ribulose-5-phosphate (Ru-5-P), and the specific methanol consumption rate increased by an additional 27.54%. Finally, we successfully constructed two strains that cannot grow in M9 medium with xylose or ribose unless methanol is utilized. The strategies used in this study are generally applicable to other studies on synthetic methylotrophy.

Recombinant and endogenous ways to produce methylated phospholipids in Escherichia coli

Escherichia coli is the daily workhorse in molecular biology research labs and an important platform microorganism in white biotechnology. Its cytoplasmic membrane is primarily composed of the phospholipids phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL). As in most other bacteria, the typical eukaryotic phosphatidylcholine (PC) is not a regular component of the E. coli membrane. PC is known to act as a substrate in various metabolic or catabolic reactions, to affect protein folding and membrane insertion, and to activate proteins that originate from eukaryotic environments. Options to manipulate the E. coli membrane to include non-native lipids such as PC might make it an even more powerful and versatile tool for biotechnology and protein biochemistry. This article outlines different strategies how E. coli can be engineered to produce PC and other methylated PE derivatives. Several of these approaches rely on the ectopic expression of genes from natural PC-producing organisms. These include PC synthases, lysolipid acyltransferases, and several phospholipid N-methyltransferases with diverse substrate and product preferences. In addition, we show that E. coli has the capacity to produce PC by its own enzyme repertoire provided that appropriate precursors are supplied. Screening of the E. coli Keio knockout collection revealed the lysophospholipid transporter LplT to be responsible for the uptake of lyso-PC, which is then further acylated to PC by the acyltransferase-acyl carrier protein synthetase Aas. Overall, our study shows that the membrane composition of the most routinely used model bacterium can readily be tailored on demand.Key points• Escherichia coli can be engineered to produce non-native methylated PE derivatives.• These lipids can be produced by foreign and endogenous proteins.• Modification of E. coli membrane offers potential for biotechnology and research.Graphical abstract

Metabolic engineering design to enhance (R,R)-2,3-butanediol production from glycerol in Bacillus subtilis based on flux balance analysis

BackgroundGlycerol is a desirable alternative substrate for 2,3-butanediol (2,3-BD) production for sustainable development in biotechnological industries and non-food competitive feedstock. B. subtilis, a “generally recognized as safe” organism that is highly tolerant to fermentation products, is an ideal platform microorganism to engineer the pathways for the production of valuable bio-based chemicals, but it has never been engineered to improve 2,3-BD production from glycerol. In this study, we aimed to enhance 2,3-BD production from glycerol in B. subtilis through in silico analysis. Genome-scale metabolic model (GSM) simulations was used to design and develop the metabolic pathways of B. subtilis. Flux balance analysis (FBA) simulation was used to evaluate the effects of step-by-step gene knockouts to improve 2,3-BD production from glycerol in B. subtilis.ResultsB. subtilis was bioengineered to enhance 2,3-BD production from glycerol using FBA in a published GSM model of B. subtilis, iYO844. Four genes, ackA, pta, lctE, and mmgA, were knocked out step by step, and the effects thereof on 2,3-BD production were evaluated. While knockout of ackA and pta had no effect on 2,3-BD production, lctE knockout led to a substantial increase in 2,3-BD production. Moreover, 2,3-BD production was improved by mmgA knockout, which had never been investigated. In addition, comparisons between in silico simulations and fermentation profiles of all B. subtilis strains are presented in this study.ConclusionsThe strategy developed in this study, using in silico FBA combined with experimental validation, can be used to optimize metabolic pathways for enhanced 2,3-BD production from glycerol. It is expected to provide a novel platform for the bioengineering of strains to enhance the bioconversion of glycerol into other highly valuable chemical products.

Metabolic engineering for the utilization of carbohydrate portions of lignocellulosic biomass

The petrochemical industry has grown to meet the need for massive production of energy and commodities along with an explosive population growth; however, serious side effects such as greenhouse gas emissions and global warming have negatively impacted the environment. Lignocellulosic biomass with myriad quantities on Earth is an attractive resource for the production of carbon-neutral fuels and chemicals through environmentally friendly processes of microbial fermentation. This review discusses metabolic engineering efforts to achieve economically feasible industrial production of fuels and chemicals from microbial cell factories using the carbohydrate portion of lignocellulosic biomass as substrates. The combined knowledge of systems biology and metabolic engineering has been applied to construct robust platform microorganisms with maximum conversion of monomeric sugars, such as glucose and xylose, derived from lignocellulosic biomass. By comprehensively revisiting carbon conversion pathways, we provide a rationale for engineering strategies, as well as their features, feasibility, and recent representative studies. In addition, we briefly discuss how tools in systems biology can be applied in the field of metabolic engineering to accelerate the development of microbial cell factories that convert lignocellulosic biomass into carbon-neutral fuels and chemicals with economic feasibility.

Integrating transcriptomic and metabolomic analysis of the oleaginous yeast Rhodosporidium toruloides IFO0880 during growth under different carbon sources.

Rhodosporidium toruloides is an oleaginous yeast capable of producing a variety of biofuels and bioproducts from diverse carbon sources. Despite numerous studies showing its promise as a platform microorganism, little is known about its metabolism and physiology. In this work, we investigated the central carbon metabolism in R. toruloides IFO0880 using transcriptomics and metabolomics during growth on glucose, xylose, acetate, or soybean oil. These substrates were chosen because they can be derived from plants. Significant changes in gene expression and metabolite concentrations were observed during growth on these four substrates. We mapped these changes onto the governing metabolic pathways to better understand how R. toruloides reprograms its metabolism to enable growth on these substrates. One notable finding concerns xylose metabolism, where poor expression of xylulokinase induces a bypass leading to arabitol production. Collectively, these results further our understanding of central carbon metabolism in R. toruloides during growth on different substrates. They may also help guide the metabolic engineering and development of better models of metabolism for R. toruloides.Key points• Gene expression and metabolite concentrations were significantly changed.• Reduced expression of xylulokinase induces a bypass leading to arabitol production.• R. toruloides reprograms its metabolism to allow growth on different substrates.

Transcriptome analysis reveals the roles of nitrogen metabolism and sedoheptulose bisphosphatase pathway in methanol-dependent growth of Corynebacterium glutamicum.

SummaryMethanol is a promising feedstock for biomanufacturing of fuels and chemicals. Although efforts have been made to engineer platform microorganisms for methanol bioconversion, the substrate uptake and cell growth rates on methanol are still unsatisfactory, suggesting certain limiting factors remain unsolved. Herein, we analysed the global metabolic regulation changes between an evolved methanol‐dependent Corynebacterium glutamicum mutant and its ancestral strain by transcriptome analysis. Many genes involved in central metabolism including glycolysis, amino acid biosynthesis and energy generation were regulated, implying the adaptive laboratory evolution reprogrammed the cellular metabolism for methanol utilization. We then demonstrated that nitrate could serve as a complementary electron acceptor for aerobic methanol metabolism, and the biosynthesis of several amino acids limited methylotrophic growth. Finally, the sedoheptulose bisphosphatase pathway for generating methanol assimilation acceptor was found effective in C. glutamicum. This study identifies limiting factors of methanol metabolism and provides engineering targets for developing superior synthetic methylotrophs.

Adaptive laboratory evolution enhances methanol tolerance and conversion in engineered Corynebacterium glutamicum

Synthetic methylotrophy has recently been intensively studied to achieve methanol-based biomanufacturing of fuels and chemicals. However, attempts to engineer platform microorganisms to utilize methanol mainly focus on enzyme and pathway engineering. Herein, we enhanced methanol bioconversion of synthetic methylotrophs by improving cellular tolerance to methanol. A previously engineered methanol-dependent Corynebacterium glutamicum is subjected to adaptive laboratory evolution with elevated methanol content. Unexpectedly, the evolved strain not only tolerates higher concentrations of methanol but also shows improved growth and methanol utilization. Transcriptome analysis suggests increased methanol concentrations rebalance methylotrophic metabolism by down-regulating glycolysis and up-regulating amino acid biosynthesis, oxidative phosphorylation, ribosome biosynthesis, and parts of TCA cycle. Mutations in the O-acetyl-l-homoserine sulfhydrylase Cgl0653 catalyzing formation of l-methionine analog from methanol and methanol-induced membrane-bound transporter Cgl0833 are proven crucial for methanol tolerance. This study demonstrates the importance of tolerance engineering in developing superior synthetic methylotrophs.

Simultaneous depolymerization and fermentation of lignin into value-added products by the marine protist, Thraustochytrium striatum

Biodegradation of lignin has been focused on bacteria and fungi, but little information is available on lignin biotransformation with marine protists. In this paper, kraft pine lignin was used as a carbon source for a newly recognized and patented lignin degrader, Thraustochytrium striatum HB. The fermentation medium was optimized in terms of nitrogen source and trace elements. The results showed NH4Cl was the best nitrogen source to enhance lignin consumption from 3 (for yeast extract or peptone) to 17%, while the utilization of yeast extract or peptone was found to inhibit lignin consumption by T. striatum HB. The presence of trace elements and vitamins improved lignin utilization from 17 to 25% by inducing the production of lignolytic enzymes. Different lignin depolymerization methods, including laccase, fungal secretome and Fenton reagent were used to enhance lignin fermentation. In the presence of laccase, both cell growth and lignin consumption were improved, and it was found that different laccases had different working mechanisms. Enzymatic depolymerization resulted in 25–30% lignin degradation, slightly higher than that (22%) from Fenton reagent. NMR analysis indicated that the fungus-derived laccase primarily worked on the breakdown of inter-unit linkages in lignin molecules, while plant-derived laccase possibly attacked the aromatic ring structures. With the function of laccase, more low-molecular-weight lignin fragments can be generated, and laccase-catalyzing reaction would direct the equilibrium toward depolymerization by preventing repolymerization of small fragments. On the contrary, lignin peroxidase-dominant fungal secretome and Fenton reagent facilitated lignin degradation but not cell growth, possibly due to the generation of inhibitory compounds from lignin depolymerization by fungal secretome and Fenton reagent. The results from this research demonstrate that marine protists, such as T. striatum HB are a class of promising platform microorganisms for lignin valorization into valuable bioproducts.

Metabolic Engineering Strategies for Sustainable Terpenoid Flavor and Fragrance Synthesis.

Terpenoids derived from plant material are widely applied in the flavor and fragrance industry. Traditional extraction methods are unsustainable, but microbial synthesis offers a promising solution to attain efficient production of natural-identical terpenoids. Overproduction of terpenoids in microbes requires careful balancing of the synthesis pathway constituents within the constraints of host cell metabolism. Advances in metabolic engineering have greatly facilitated overcoming the challenges of achieving high titers, rates, and yields (TRYs). The review summarizes recent development in the molecular biology toolbox to achieve high TRYs for terpenoid biosynthesis, mainly in the two industrial platform microorganisms: Escherichia coli and Saccharomyces cerevisiae. The biosynthetic pathways, including alternative pathway designs, are briefly introduced, followed by recently developed methodologies used for pathway, genome, and strain optimization. Integrated applications of these tools are important to achieve high "TRYs" of terpenoid production and pave the way for translating laboratory research into successful commercial manufacturing.

Bicistronic Design-Based Continuous and High-Level Membrane Protein Production in Escherichia coli.

Escherichia coli has been widely used as a platform microorganism for both membrane protein production and cell factory engineering. The current methods to produce membrane proteins in this organism require the induction of target gene expression and often result in unstable, low yields. Here, we present a method combining a constitutive promoter with a library of bicistronic design (BCD) elements, which enables inducer-free, tuned translation initiation for optimal protein production. Our system mediates stable, constitutive production of bacterial membrane proteins at yields that outperform those obtained with E. coli Lemo21(DE3), the current gold standard for bacterial membrane protein production. We envisage that the continuous, fine-tunable, and high-level production of membrane proteins by our method will greatly facilitate their study and their utilization in engineering cell factories.

A Synthetic Plasmid Toolkit for Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is a platform microorganism for understanding extracellular electron transfer (EET) with a fully sequenced and annotated genome. In comparison to other model microorganisms such as Escherichia coli, the available plasmid parts (such as promoters and replicons) are not sufficient to conveniently and quickly fine-tune the expression of multiple genes in S. oneidensis MR-1. Here, we constructed and characterized a plasmid toolkit that contains a set of expression vectors with a combination of promoters, replicons, antibiotic resistance genes, and an RK2 origin of transfer (oriT) cassette, in which each element can be easily changed by fixed restriction enzyme sites. The expression cassette is also compatible with BioBrick synthetic biology standards. Using green fluorescent protein (GFP) as a reporter, we tested and quantified the strength of promoters. The copy number of different replicons was also measured by real-time quantitative PCR. We further transformed two compatible plasmids with different antibiotic resistance genes into the recombinant S. oneidensis MR-1, enabling control over the expression of two different fluorescent proteins. This plasmid toolkit was further used for overexpression of the MtrCAB porin-c-type cytochrome complex in the S. oneidensis ΔmtrA strain. Tungsten trioxide (WO3) reduction and microbial fuel cell (MFC) assays revealed that the EET efficiency was improved most significantly when MtrCAB was expressed at a moderate level, thus demonstrating the utility of the plasmid toolkit in the EET regulation in S. oneidensis. The plasmid toolkit developed in this study is useful for rapid and convenient fine-tuning of gene expression and enhances the ability to genetically manipulate S. oneidensis MR-1.

Recent Advances in the Metabolic Engineering of Klebsiella pneumoniae: A Potential Platform Microorganism for Biorefineries

The production of industrial chemicals from renewable biomass resources is a promising solution to overcome the society’s dependence on petroleum and to mitigate the pollution resulting from petroleum processing. Klebsiella pneumoniae is a nutritionally versatile bacterium with numerous native pathways for the production of well-known and industrially important platform chemicals derived from various sugars. Genomic sequence analyses have shown that the K. pneumoniae genome has a high similarity with that of Escherichia coli, the most studied organism, which is used in industrial biotechnology processes for fuel and chemical production. Hence, K. pneumoniae can be considered as a promising platform microorganism that can be metabolically engineered for the high-level production of bio-based chemicals. This review highlights the substrate metabolism and the metabolic engineering strategies developed in K. pneumoniae for the production of bio-based chemicals.