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.     

Engineering Native and Synthetic Pathways in Pseudomonas putida for the Production of Tailored Polyhydroxyalkanoates

Growing environmental concern sparks renewed interest in the sustainable production of (bio)materials that can replace oil-derived goods. Polyhydroxyalkanoates (PHAs) are isotactic polymers that play a critical role in the central metabolism of producer bacteria, as they act as dynamic reservoirs of carbon and reducing equivalents. PHAs continue to attract industrial attention as a starting point toward renewable, biodegradable, biocompatible, and versatile thermoplastic and elastomeric materials. Pseudomonas species have been known for long as efficient biopolymer producers, especially for medium-chain-length PHAs. The surge of synthetic biology and metabolic engineering approaches in recent years offers the possibility of exploiting the untapped potential of Pseudomonas cell factories for the production of tailored PHAs. In this article, an overview of the metabolic and regulatory circuits that rule PHA accumulation in Pseudomonas putida is provided, and approaches leading to the biosynthesis of novel polymers (e.g., PHAs including nonbiological chemical elements in their structures) are discussed. The potential of novel PHAs to disrupt existing and future market segments is closer to realization than ever before. The review is concluded by pinpointing challenges that currently hinder the wide adoption of bio-based PHAs, and strategies toward programmable polymer biosynthesis from alternative substrates in engineered P. putida strains are proposed.     

Bioproduction of polyhydroxyalkanoates from bacteria: a metabolic approach

The advent of molecular biological techniques and a developing environmental awareness initiated a renewed scientific interest in Polyhydroxyalkanoates (PHAs) and the biosynthetic machinery for PHA metabolism has been the area of research over the last two decades. PHAs are polyesters of hydroxyalkanoates synthesized by numerous bacterial species with atleast five different PHA biosynthetic pathways. These are accumulated as an intracellular carbon and energy storage material. This diversity, in combination with genetic and molecular engineering has opened up this area for development of optimum PHA producing organisms. Even though PHAs have been recognized as a good candidate for biodegradable plastics, their industrial application is limited owing to high production cost. The classical microbiology and modern molecular biology have been brought together to decipher the intricacies of PHA metabolism both for production purposes and for the unraveling of the natural role of PHA. This review provides an overview of the different PHA biosynthetic systems, the enzymes involved in PHA biosynthesis and there genetic background followed by a detailed summation of how this natural diversity is being used to develop commercially attractive recombinant process for large scale production of PHAs.     

Production of C2–C4 diols from renewable bioresources: new metabolic pathways and metabolic engineering strategies

C2–C4 diols classically derived from fossil resource are very important bulk chemicals which have been used in a wide range of areas, including solvents, fuels, polymers, cosmetics, and pharmaceuticals. Production of C2–C4 diols from renewable resources has received significant interest in consideration of the reducing fossil resource and the increasing environmental issues. While bioproduction of certain diols like 1,3-propanediol has been commercialized in recent years, biosynthesis of many other important C2–C4 diol isomers is highly challenging due to the lack of natural synthesis pathways. Recent advances in synthetic biology have enabled the de novo design of completely new pathways to non-natural molecules from renewable feedstocks. In this study, we review recent advances in bioproduction of C2–C4 diols, focusing on new metabolic pathways and metabolic engineering strategies being developed. We also discuss the challenges and future trends toward the development of economically competitive processes for bio-based diol production.     

Advance on the production of polyhydroxyalkanoates by mixed cultures

Polyhydroxyalkanoates (PHAs)are the polymers of hydroxyalkanoates that accumulate as carbon/energy or reducing-power storage material in various microorganisms.PHAs have attracted considerable attention as biodegradable substitutes for conventional polymers.Until now,however,industrial production of PHAs has encountered only limited success.The main barrier to the replacement of synthetic plastics by PHAs has been the higher cost.The use of mixed cultures and renewable sources obtained from waste organic carbon can substantially decrease the cost of PHA and increase their market potential.This work reviews two main methods of PHA production by mixed cultures,anaerobicaerobic processing and aerobic transient feeding processing,and analyzed the metabolic and effective factors.     

Advance on the production of polyhydroxyalkanoates by mixed cultures

Polyhydroxyalkanoates (PHAs) are the polymers of hydroxyalkanoates that accumulate as carbon/energy or reducing-power storage material in various microorganisms. PHAs have attracted considerable attention as biodegradable substitutes for conventional polymers. Until now, however, industrial production of PHAs has encountered only limited success. The main barrier to the replacement of synthetic plastics by PHAs has been the higher cost. The use of mixed cultures and renewable sources obtained from waste organic carbon can substantially decrease the cost of PHA and increase their market potential. This work reviews two main methods of PHA production by mixed cultures, anaerobic-aerobic processing and aerobic transient feeding processing, and analyzed the metabolic and effective factors.     

Biosynthesis of lactate-containing polyesters by metabolically engineered bacteria

Due to increasing concerns about environmental problems, climate change and limited fossil resources, bio-based production of chemicals and polymers is gaining attention as one of the solutions to these problems. Polyhydroxyalkanoates (PHAs) are polyesters that can be produced by microbial fermentation. PHAs are synthesized using monomer precursors provided from diverse metabolic pathways and are accumulated as distinct granules inside the cells. On the other hand, most so-called bio-based polymers including polybutylene succinate, polytrimethylene terephthalate, and polylactic acid (PLA) are synthesized by a chemical process using monomers produced by fermentation. PLA, an attractive biomass-derived plastic, is currently synthesized by heavy metal-catalyzed ring opening polymerization of L-lactide that is made from fermentation-derived L-lactic acid. Recently, a complete biological process for the production of PLA and PLA copolymers from renewable resources has been developed by direct fermentation of recombinant bacteria employing PHA biosynthetic pathways coupled with a novel metabolic pathway. This could be accomplished by establishing a pathway for generating lactyl-CoA and engineering PHA synthase to accept lactyl-CoA as a substrate combined with systems metabolic engineering. In this article, we review recent advances in the production of lactate-containing homo- and co-polyesters. Challenges remaining to efficiently produce PLA and its copolymers and strategies to overcome these challenges through metabolic engineering combined with enzyme engineering are discussed.     

Bacillus subtilis as potential producer for polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by microbes to overcome environmental stress. Commercial production of PHAs is limited by the high cost of production compared to conventional plastics. Another hindrance is the brittle nature and low strength of polyhydroxybutyrate (PHB), the most widely studied PHA. The needs are to produce PHAs, which have better elastomeric properties suitable for biomedical applications, preferably from inexpensive renewable sources to reduce cost. Certain unique properties of Bacillus subtilis such as lack of the toxic lipo-polysaccharides, expression of self-lysing genes on completion of PHA biosynthetic process – for easy and timely recovery, usage of biowastes as feed enable it to compete as potential candidate for commercial production of PHA.     

Synthesis of polyhydroxyalkanoate from palm oil and some new applications

Polyhydroxyalkanoate (PHA) is a potential substitute for some petrochemical-based plastics. This biodegradable plastic is derived from microbial fermentation using various carbon substrates. Since carbon source has been identified as one of the major cost-absorbing factors in PHA production, cheap and renewable substrates are currently being investigated as substitutes for existing sugar-based feedstock. Plant oils have been found to result in high-yield PHA production. Malaysia, being the world’s second largest producer of palm oil, is able to ensure continuous supply of palm oil products for sustainable PHA production. The biosynthesis and characterization of various types of PHA using palm oil products have been described in detail in this review. Besides, by-products and waste stream from palm oil industry have also demonstrated promising results as carbon sources for PHA biosynthesis. Some new applications in cosmetic and wastewater treatment show the diversity of PHA usage. With proper management practices and efficient milling processes, it may be possible to supply enough palm oil-based raw materials for human consumption and other biotechnological applications such as production of PHA in a sustainable manner.     

Rational engineering of natural polyhydroxyalkanoates producing microorganisms for improved synthesis and recovery

Microbial production of biopolymers derived from renewable substrates and waste streams reduces our heavy reliance on petrochemical plastics. One of the most important biodegradable polymers is the family of polyhydroxyalkanoates (PHAs), naturally occurring intracellular polyoxoesters produced for decades by bacterial fermentation of sugars and fatty acids at the industrial scale. Despite the advances, PHA production still suffers from heavy costs associated with carbon substrates and downstream processing to recover the intracellular product, thus restricting market positioning. In recent years, model-aided metabolic engineering and novel synthetic biology approaches have spurred our understanding of carbon flux partitioning through competing pathways and cellular resource allocation during PHA synthesis, enabling the rational design of superior biopolymer producers and programmable cellular lytic systems. This review describes these attempts to rationally engineering the cellular operation of several microbes to elevate PHA production on specific substrates and waste products. We also delve into genome reduction, morphology, and redox cofactor engineering to boost PHA biosynthesis. Besides, we critically evaluate engineered bacterial strains in various fermentation modes in terms of PHA productivity and the period required for product recovery.     

Valorization of rendering industry wastes and co-products for industrial chemicals,materials and energy: review

Over the past decades, strong global demand for industrial chemicals, raw materials and energy has been driven by rapid industrialization and population growth across the world. In this context, long-term environmental sustainability demands the development of sustainable strategies of resource utilization. The agricultural sector is a major source of underutilized or low-value streams that accompany the production of food and other biomass commodities. Animal agriculture in particular constitutes a substantial portion of the overall agricultural sector, with wastes being generated along the supply chain of slaughtering, handling, catering and rendering. The recent emergence of bovine spongiform encephalopathy (BSE) resulted in the elimination of most of the traditional uses of rendered animal meals such as blood meal, meat and bone meal (MBM) as animal feed with significant economic losses for the entire sector. The focus of this review is on the valorization progress achieved on converting protein feedstock into bio-based plastics, flocculants, surfactants and adhesives. The utilization of other rendering streams such as fat and ash rich biomass for the production of renewable fuels, solvents, drop-in chemicals, minerals and fertilizers is also critically reviewed.     

Microbial Cometabolism and Polyhydroxyalkanoate Co-polymers

Polyhydroxyalkanoate (PHAs) are natural, biodegradable biopolymers, which can be produced from renewable materials. PHAs have potential to replace petroleum derived plastics. Quite a few bacteria can produce PHA under nutritional stress. They generally produce homopolymers of butyrate i.e., polyhydroxybutyrate (PHB), as a storage material. The biochemical characteristics of PHB such as brittleness, low strength, low elasticity, etc. make these unsuitable for commercial applications. Co-polymers of PHA, have high commercial value as they overcome the limitations of PHBs. Co-polymers can be produced by supplementing the feed with volatile fatty acids or through hydrolysates of different biowastes. In this review, we have listed the potential bacterial candidates and the substrates, which can be co-metabolized to produce PHA co-polymers.     

Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic.

Poly(3-hydroxyalkanoates) (PHAs) are a class of microbially produced polyesters that have potential applications as conventional plastics, specifically thermoplastic elastomers. A wealth of biological diversity in PHA formation exists, with at least 100 different PHA constituents and at least five different dedicated PHA biosynthetic pathways. This diversity, in combination with classical microbial physiology and modern molecular biology, has now opened up this area for genetic and metabolic engineering to develop optimal PHA-producing organisms. Commercial processes for PHA production were initially developed by W. R. Grace in the 1960s and later developed by Imperial Chemical Industries, Ltd., in the United Kingdom in the 1970s and 1980s. Since the early 1990s, Metabolix Inc. and Monsanto have been the driving forces behind the commercial exploitation of PHA polymers in the United States. The gram-negative bacterium Ralstonia eutropha, formerly known as Alcaligenes eutrophus, has generally been used as the production organism of choice, and intracellular accumulation of PHA of over 90% of the cell dry weight have been reported. The advent of molecular biological techniques and a developing environmental awareness initiated a renewed scientific interest in PHAs, and the biosynthetic machinery for PHA metabolism has been studied in great detail over the last two decades. Because the structure and monomeric composition of PHAs determine the applications for each type of polymer, a variety of polymers have been synthesized by cofeeding of various substrates or by metabolic engineering of the production organism. Classical microbiology and modern molecular bacterial physiology have been brought together to decipher the intricacies of PHA metabolism both for production purposes and for the unraveling of the natural role of PHAs. This review provides an overview of the different PHA biosynthetic systems and their genetic background, followed by a detailed summation of how this natural diversity is being used to develop commercially attractive, recombinant processes for the large-scale production of PHAs.     

Bio- and chemocatalysis cascades as a bridge between biology and chemistry for green polymer synthesis

The development and integration of bio- and chemocatalytic processes to convert renewable or biomass feedstocks into polymers is a vibrant field of research with enormous potential for environmental protection and the mitigation of global warming. Here, we review the biotechnological and chemical synthetic strategies for producing platform monomers from bio-based sources and transforming them into eco-polymers. We also discuss their advanced bio-application using the example of polylactide (PLA), the most valuable green polymer on the market.     

The phylogenetic and global distribution of bacterial polyhydroxyalkanoate bioplastic-degrading genes

Polyhydroxyalkanoates (PHAs) are a family of microbially made polyesters commercialized as biodegradable plastics. PHA production rates are predicted to increase as concerns around environmental plastic contamination and limited fossil fuel resources have increased the importance of biodegradable and bio-based plastic alternatives. Microbially produced PHA depolymerases are the key enzymes mediating PHA biodegradation, but only a few PHA depolymerases have been well-characterized and screens employing metagenomic sequence data are lacking. Here, we used 3078 metagenomes to analyse the distribution of PHA depolymerases in microbial communities from diverse aquatic, terrestrial and waste management systems. We significantly expand the recognized diversity of this protein family by screening 1914 Gb of sequence data and identifying 13 869 putative PHA depolymerases in 1295 metagenomes. Our results indicate that PHA depolymerases are unevenly distributed across environments. We predicted the highest frequency of PHA depolymerases in wastewater systems and the lowest in marine and thermal springs. In tandem, we screened 5290 metagenome-assembled genomes to describe the phylogenetic distribution of PHA depolymerases, which is substantially broader compared with current cultured representatives. The Proteobacteria and Bacteroidota are key lineages encoding PHA depolymerases, but PHA depolymerases were predicted from members of the Bdellovibrionota, Methylomirabilota, Actinobacteriota, Firmicutes, Spirochaetota, Desulfobacterota, Myxococcota and Planctomycetota.     

Recent developments in bioreactor scale production of bacterial polyhydroxyalkanoates

Bioprocess and Biosystems Engineering - Polyhydroxyalkanoates (PHAs) are biological plastics that are sustainable alternative to synthetic ones. Numerous microorganisms have been identified as PHAs...     

Bacillus and biopolymer: Prospects and challenges

The microbially derived polyhydroxyalkanoates biopolymers could impact the global climate scenario by replacing the conventional non-degradable, petrochemical-based polymer. The biogenesis, characterization and properties of PHAs by Bacillus species using renewable substrates have been elaborated by many for their wide applications. On the other hand Bacillus species are advantageous over other bacteria due to their abundance even in extreme ecological conditions, higher growth rates even on cheap substrates, higher PHAs production ability, and the ease of extracting the PHAs. Bacillus species possess hydrolytic enzymes that can be exploited for economical PHAs production. This review summarizes the recent trends in both non-growth and growth associated PHAs production by Bacillus species which may provide direction leading to future research towards this growing quest for biodegradable plastics, one more critical step ahead towards sustainable development.     

Polymers of 3-hydroxyacids in plants

Polyhydroxyalkanoates (PHA) are polyesters of bacterial origin that have properties of biodegradable plastics and elastomers. Synthesis of PHA in crop plants would allow the large-scale production and use of these biodegradable and renewable polymers as substitutes for petroleum-derived plastics. Synthesis of a diversity of PHAs in plants, such as Arabidopsis thaliana, rapeseed, corn and cotton, has been demonstrated through the genetic engineering of metabolic pathways in the cytoplasm, plastid and peroxisome. PHA can also be used as a novel tool to study various aspects of plant metabolism, such as the regulation of carbon flux to the fatty acid biosynthetic and degradation pathways.     

Chemical recycling of polyhydroxyalkanoates as a method towards sustainable development

Chemical recycling of bio-based polymers polyhydroxyalkanoates (PHAs) by thermal degradation was investigated from the viewpoint of biorefinery. The thermal degradation resulted in successful transformation of PHAs into vinyl monomers using alkali earth compound (AEC) catalysts. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)s (PHBVs) were smoothly and selectively depolymerized into crotonic (CA) and 2-pentenoic (2-PA) acids at lower degradation temperatures in the presence of CaO and Mg(OH)₂ as catalysts. Obtained CA from 3-hydroxybutyrate sequences in PHBV was copolymerized with acrylic acid to produce useful water-soluble copolymers, poly(crotonic acid-co-acrylic acid) that have high glass-transition temperatures. The copolymerization of CA derived from PHA pyrolysis is an example of cascade utilization of PHAs, which meets the idea of sustainable development.     

Production of polyhydroxyalkanoates by mixed microbial cultures

Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics formed from renewable resources, like sugars, with similar characteristics of polypropylene. These bioplastics are industrially produced by pure cultures using expensive pure substrates. These factors lead to a much higher selling price of PHAs compared to petroleum-based plastics, like polypropylene. The use of mixed cultures and cheap substrates (waste materials) can reduce costs of PHA production by more than 50%. Storage of PHAs by mixed populations occurs under transient conditions mainly caused by discontinuous feeding and variation in the electron donor/acceptor presence. In the last years the mechanisms of storage, metabolism and kinetics of mixed cultures have been studied. The maximum capacity of PHA storage and production rate is dependent on the substrate and on the operating conditions used. In this paper an overview and discussion of various mechanisms and processes for PHA production by mixed cultures is presented.