Recent advances in microbial production of diamines, aminocarboxylic acids, and diacids as potential platform chemicals and bio-based polyamides monomers.

Recently, bio-based manufacturing processes of value-added platform chemicals and polymers in biorefineries using renewable resources have extensively been developed for sustainable and carbon dioxide (CO2) neutral-based industry. Among them, bio-based diamines, aminocarboxylic acids, and diacids have been used as monomers for the synthesis of polyamides having different carbon numbers and ubiquitous and versatile industrial polymers and also as precursors for further chemical and biological processes to afford valuable chemicals. Until now, these platform bio-chemicals have successfully been produced by biorefinery processes employing enzymes and/or microbial host strains as main catalysts. In this review, we discuss recent advances in bio-based production of diamines, aminocarboxylic acids, and diacids, which has been developed and improved by systems metabolic engineering strategies of microbial consortia and optimization of microbial conversion processes including whole cell bioconversion and direct fermentative production.

Production of γ-Aminobutyrate (GABA) in Recombinant Corynebacterium glutamicum by Expression of Glutamate Decarboxylase Active at Neutral pH.

γ-Aminobutyrate (GABA) is an important chemical by itself and can be further used for the production of monomer used for the synthesis of biodegradable polyamides. Until now, GABA production usingCorynebacterium glutamicum harboring glutamate decarboxylases (GADs) has been limited due to the discrepancy between optimal pH for GAD activity (pH 4.0) and cell growth (pH 7.0). In this study, we developed recombinant C. glutamicum strains expressing mutated GAD from Escherichia coli (EcGADmut) and GADs from Lactococcus lactis CICC20209 (LlGAD) and Lactobacillus senmaizukei (LsGAD), all of which showed enhanced pH stability and adaptability at a pH of approximately 7.0. In shake flask cultivations, the GABA productions of C. glutamicum H36EcGADmut, C. glutamicum H36LsGAD, and C. glutamicum H36LlGAD were examined at pH 5.0, 6.0, and 7.0, respectively. Finally, C. glutamicum H36EcGADmut (40.3 and 39.3 g L-1), H36LlGAD (42.5 and 41.1 g L-1), and H36LsGAD (41.6 and 40.2 g L-1) produced improved GABA titers and yields in batch fermentation at pH 6.0 and pH 7.0, respectively, from 100 g L-1 glucose. The recombinant strains developed in this study could be used for the establishment of sustainable direct fermentative GABA production from renewable resources under mild culture conditions, thus increasing the availability of various GADs.

Valorization of lignocellulosic biomass for polyhydroxyalkanoate production: Status and perspectives.

With the increasing concerns regarding climate, energy, and plastic crises, bio-based production of biodegradable polymers has become a dire necessity. Significant progress has been made in biotechnology for the production of biodegradable polymers from renewable resources to achieve the goal of zero plastic waste and a net-zero carbon bioeconomy. In this review, an overview of polyhydroxyalkanoate (PHA) production from lignocellulosic biomass (LCB) was presented. Having established LCB-based biorefinery with proper pretreatment techniques, various PHAs could be produced from LCB-derived sugars, hydrolysates, and/or aromatic mixtures employing microorganisms. This provides a clue for addressing the current environmental crises because "biodegradable polymers" could be produced from one of the most abundant resources that are renewable and sustainable in a "carbon-neutral process". Furthermore, the potential future of LCB-to-non-natural PHA production was discussed with particular reference to non-natural PHA biosynthesis methods and LCB-derived aromatic mixture biofunnelling systems.

Recent advances in the microbial production of C4 alcohols by metabolically engineered microorganisms.

BACKGROUND: The heavy global dependence on petroleum-based industries has led to serious environmental problems, including climate change and global warming. As a result, there have been calls for a paradigm shift towards the use of biorefineries, which employ natural and engineered microorganisms that can utilize various carbon sources from renewable resources as host strains for the carbon-neutral production of target products. PURPOSE AND SCOPE: C4 alcohols are versatile chemicals that can be used directly as biofuels and bulk chemicals and in the production of value-added materials such as plastics, cosmetics, and pharmaceuticals. C4 alcohols can be effectively produced by microorganisms using DCEO biotechnology (tools to design, construct, evaluate, and optimize) and metabolic engineering strategies. SUMMARY OF NEW SYNTHESIS AND CONCLUSIONS: In this review, we summarize the production strategies and various synthetic tools available for the production of C4 alcohols and discuss the potential development of microbial cell factories, including the optimization of fermentation processes, that offer cost competitiveness and potential industrial commercialization.

A shortcut to carbon-neutral bioplastic production: Recent advances in microbial production of polyhydroxyalkanoates from C1 resources.

Since the 20th century, plastics that are widely being used in general life and industries are causing enormous plastic waste problems since improperly discarded plastics barely degrade and decompose. Thus, the demand for polyhydroxyalkanoates (PHAs), biodegradable polymers with material properties similar to conventional petroleum-based plastics, has been increased so far. The microbial production of PHAs is an environment-friendly solution for the current plastic crisis, however, the carbon sources for the microbial PHA production is a crucial factor to be considered in terms of carbon-neutrality. One­carbon (C1) resources, such as methane, carbon monoxide, and carbon dioxide, are greenhouse gases and are abundantly found in nature and industry. C1 resources as the carbon sources for PHA production have a completely closed carbon loop with much advances; i) fast carbon circulation with direct bioconversion process and ii) simple fermentation procedure without sterilization as non-preferable nutrients. This review discusses the biosynthesis of PHAs based on C1 resource utilization by wild-type and metabolically engineered microbial host strains via biorefinery processes.

Chemoautotroph Cupriavidus necator as a potential game-changer for global warming and plastic waste problem: A review.

Cupriavidus necator, a versatile microorganism found in both soil and water, can have both heterotrophic and lithoautotrophic metabolisms depending on environmental conditions. C. necator has been extensively examined for producing Polyhydroxyalkanoates (PHAs), the promising polyester alternatives to petroleum-based synthetic polymers because it has a superior ability for accumulating a considerable amount of PHAs from renewable resources. The development of metabolically engineered C. necator strains has led to their application for synthesizing biopolymers, biofuels and biochemicals such as ethanol, isobutanol and higher alcohols. Bio-based processes of recombinant C. necator have made much progress in production of these high-value products from biomass wastes, plastic wastes and even waste gases. In this review, we discuss the potential of C. necator as promising platform host strains that provide a great opportunity for developing a waste-based circular bioeconomy.

Rapid analysis of polyhydroxyalkanoate contents and its monomer compositions by pyrolysis-gas chromatography combined with mass spectrometry (Py-GC/MS).

Here, we report an analysis method for determining PHA (polyhydroxyalkanoates) contents and their monomer composition in microbial cells based on pyrolysis gas chromatography combined with mass spectrometry (Py-GC/MS). Various kinds of microbial cells accumulating different PHA contents and monomer compositions were prepared through the cultivation of Ralstonia eutropha and recombinant Escherichia coli. Py-GC/MS could analyse these samples in a short time without complicated pretreatment steps. Characteristic peaks such as 2-butenoic acid, 2-pentenoic acid, and hexadecanoic acid regarding PHA compositions and cell components were identified. Considering constituents of cells and ratios of peak areas of dehydrated monomers to hexadecanoic acid, a simple equation for estimation of PHA contents in microbial cells was derived. Also, monomer compositions of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in R. eutropha could be successfully determined based on peak area of 2-butenoic acid and 2-pentenoic acid of Py-GC/MS, which are the corresponding species of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) in PHBV. Correlation of results between GC-FID and Py-GC/MS could be fitted very well. This method shows similar results for the samples obtained from same experimental conditions, allowing rapid and reliable analysis. Py-GC/MS can be a promising tool to rapidly screen PHA-positive strains based on polymer contents along with monomer compositions.

Recent Advances in Sustainable Plastic Upcycling and Biopolymers.

Advances in scientific technology in the early twentieth century have facilitated the development of synthetic plastics that are lightweight, rigid, and can be easily molded into a desirable shape without changing their material properties. Thus, plastics become ubiquitous and indispensable materials that are used in various manufacturing sectors, including clothing, automotive, medical, and electronic industries. However, strong physical durability and chemical stability of synthetic plastics, most of which are produced from fossil fuels, hinder their complete degradation when they are improperly discarded after use. In addition, accumulated plastic wastes without degradation have caused severe environmental problems, such as microplastics pollution and plastic islands. Thus, the usage and production of plastics is not free from environmental pollution or resource depletion. In order to lessen the impact of climate change and reduce plastic pollution, it is necessary to understand and address the current plastic life cycles. In this review, "sustainable biopolymers" are suggested as a promising solution to the current plastic crisis. The desired properties of sustainable biopolymers and bio-based and bio/chemical hybrid technologies for the development of sustainable biopolymers are mainly discussed.

Effect of DR1558, a Deinococcus radiodurans response regulator, on the production of GABA in the recombinant Escherichia coli under low pH conditions.

BACKGROUND: Gamma aminobutyric acid (GABA) is an important platform chemical, which has been used as a food additive and drug. Additionally, GABA is a precursor of 2-pyrrolidone, which is used in nylon synthesis. GABA is usually synthesized from glutamate in a reaction catalyzed by glutamate decarboxylase (GAD). Currently, there are several reports on GABA production from monosodium glutamate (MSG) or glucose using engineered microbes. However, the optimal pH for GAD activity is 4, which is the limiting factor for the efficient microbial fermentative production of GABA as fermentations are performed at pH 7. Recently, DR1558, a response regulator in the two-component signal transduction system was identified in Deinococcus radiodurans. DR1558 is reported to confer cellular robustness to cells by binding the promoter regions of genes via DNA-binding domains or by binding to the effector molecules, which enable the microorganisms to survive in various environmental stress conditions, such as oxidative stress, high osmotic shock, and low pH. RESULTS: In this study, the effect of DR1558 in enhancing GABA production was examined using two different strategies: whole-cell bioconversion of GABA from MSG and direct fermentative production of GABA from glucose under acidic culture conditions. In the whole-cell bioconversion, GABA produced by E. coli expressing GadBC and DR1558 (6.52 g/L GABA from 13 g/L MSG·H2O) in shake flask culture at pH 4.5 was 2.2-fold higher than that by E. coli expressing only GadBC (2.97 g/L of GABA from 13 g/L MSG·H2O). In direct fermentative production of GABA from glucose, E. coli ∆gabT expressing isocitrate dehydrogenase (IcdA), glutamate dehydrogenase (GdhA), GadBC, and DR1558 produced 1.7-fold higher GABA (2.8 g/L of GABA from 30 g/L glucose) than E. coli ∆gabT expressing IcdA, GdhA, and GadBC (1.6 g/L of GABA from 30 g/L glucose) in shake flask culture at an initial pH 7.0. The transcriptional analysis of E. coli revealed that DR1558 conferred acid resistance to E. coli during GABA production. The fed-batch fermentation of E. coli expressing IcdA, GdhA, GadBC, and DR1558 performed at pH 5.0 resulted in the final GABA titer of 6.16 g/L by consuming 116.82 g/L of glucose in 38 h. CONCLUSION: This is the first report to demonstrate GABA production by acidic fermentation and to provide an engineering strategy for conferring acid resistance to the recombinant E. coli for GABA production.

Biosynthesis of polyhydroxyalkanoates from sucrose by metabolically engineered Escherichia coli strains.

Sucrose utilization has been established in Escherichia coli strains by expression of Mannheimia succiniciproducens ß-fructofuranosidase (SacC), which hydrolyzes sucrose into glucose and fructose. Recombinant E. coli strains that can utilize sucrose were examined for their abilities to produce poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] from sucrose. When recombinant E. coli strains expressing Ralstonia eutropha PhaCAB and SacC were cultured in MR medium containing 20 g/L of sucrose, all recombinant E. coli strains could produce P(3HB) from sucrose. Also, recombinant E. coli strains expressing Pseudomonas sp. MBEL 6-19 PhaC1437, Clostridium propionicum Pct540, R. eutropha PhaAB enzymes along with SacC could produce P(3HB-co-LA) from sucrose. Among the examined E. coli strains, recombinant E. coli XL1-Blue produced the highest contents of P(3HB) (53.60 ± 2.55 wt%) and P(3HB-co-LA) (29.44 ± 0.39 wt%). In the batch fermentations, recombinant E. coli XL1-Blue strains completely consumed 20 g/L of sucrose as the sole carbon source and supported the production of 3.76 g/L of P(3HB) and 1.82 g/L of P(3HB-co-LA) with 38.21 wt% P(3HB) and 20.88 wt% P(3HB-co-LA) contents, respectively. Recombinant E. coli strains developed in this study can be used to establish a cost-efficient biorefinery for the production of polyhydroxyalkanoates (PHAs) from sucrose, which is an abundant and inexpensive carbon source.

Metabolic engineering for the synthesis of polyesters: A 100-year journey from polyhydroxyalkanoates to non-natural microbial polyesters.

As concerns increase regarding sustainable industries and environmental pollutions caused by the accumulation of non-degradable plastic wastes, bio-based polymers, particularly biodegradable plastics, have attracted considerable attention as potential candidates for solving these problems by substituting petroleum-based plastics. Among these candidates, polyhydroxyalkanoates (PHAs), natural polyesters that are synthesized and accumulated in a range of microorganisms, are considered as promising biopolymers since they have biocompatibility, biodegradability, and material properties similar to those of commodity plastics. Accordingly, substantial efforts have been made to gain a better understanding of mechanisms related to the biosynthesis and properties of PHAs and to develop natural and recombinant microorganisms that can efficiently produce PHAs comprising desired monomers with high titer and productivity for industrial applications. Recent advances in biotechnology, including those related to evolutionary engineering, synthetic biology, and systems biology, can provide efficient and effective tools and strategies that reduce time, labor, and costs to develop microbial platform strains that produce desired chemicals and materials. Adopting these technologies in a systematic manner has enabled microbial fermentative production of non-natural polyesters such as poly(lactate) [PLA], poly(lactate-co-glycolate) [PLGA], and even polyesters consisting of aromatic monomers from renewable biomass-derived carbohydrates, which can be widely used in current chemical industries. In this review, we present an overview of strain development for the production of various important natural PHAs, which will give the reader an insight into the recent advances and provide indicators for the future direction of engineering microorganisms as plastic cell factories. On the basis of our current understanding of PHA biosynthesis systems, we discuss recent advances in the approaches adopted for strain development in the production of non-natural polyesters, notably 2-hydroxycarboxylic acid-containing polymers, with particular reference to systems metabolic engineering strategies.