Biology论文模板 – Directed Evolution of Ralstonia Eutropha Towards High Formic Acid Tolerance and Uptake


This review evaluates the directed evolution of Ralstonia eutropha towards formic acid tolerance and uptake. Ralstonia eutropha is a chemolithoautotrophic non-pathogenic prokaryote that has infinitely broad industrial applications including Biofuel Synthesis, Bioplastic production, nitrogenous waste degradation and pharmaceutical biosynthesis (Matsumoto, et Al, 2013; Jain, 1993 and Ventosa, 2004). The bacterium combines different evolved advanced genome architecture that encodes different enzymatic activity for high metabolic versatility. The substrate synthesised include Hydrogen gas, Oxygen gas molecule and Carbon (II) oxide (Matsumoto, et Al, 2013). However, these elementary sources require an advanced environmental condition that, other than being expensive, they are susceptible to explosions in industrial setup (Jain, 1993).

Formic acid offers a potential alternative substrate to the elementary substrates, including hydrogen and oxygen molecule, as the acid compound acts as both energy and carbon II oxide source for the synthetic cycle. Nonetheless, the toxicity nature of the small chain acid compound and the imbalanced substrate supply poses a great challenge to its exploitation. The economic value of Ralstonia eutropha’s products as well as their environmental sustainability, however, earns the organism a significant research interest. The Calvin-Benson-Bassham (CBB) cycle products are biodegradable and renewable and produces less toxic emission in the case of biofuel thus are environmentally sustainable.


Ralstonia eutropha or Cupriavidus necator is a chemolithoautotrophic non-pathogenic ubiquitous bacterium belonging to Burkholderiaceae family of Betaproteobacteria group (Matsumoto, et Al, 2013; Jain, 1993 and Ventosa, 2004). The organism has complex combination of survival mechanism that enhances its adaptation to versatile environmental conditions (Matsumoto, et Al, 2013). These adaptation strategies include high metabolic versatility [Fig.1] and the ability to store sufficient quantity of polyhydroxyalkanoates among others [Fig.1.2].

Ralstonia eutropha has multireplicon genome architecture consisting three genomes: 4052032bp Chromosome 1(Cr1), 2912490bp Chromosome 2 (Cr2) and 452156bp megaplasmid pHG1 that totals to 7416678bp (Hagins, & Silo-Suh, 2009; and TrčEk, Mira & Jarboe, 2015). Cr1 and Cr2 have equal gene coding regions as well as Guanine-Cytosine content. Megaplasmid, on the other hand, carries the genetic information for coding the lithoautotrophy and denitrification (TrčEk, Mira & Jarboe, 2015).

The expansion of the commercial application of Ralstonia eutropha is enhanced by the outstanding qualities its synthesised products such as environmental sustainability and biodegradability among others (Mobley, 1994; and Tsuge et Al, 2004). According to Mobley (1994), the environmental sustainable, biodegradable biopolymers, Polyhydroxyalkanoates (PHAs), are storage materials for carbon and energy in most prokaryote cells (including Ralstonia eutropha). Other than being readily disposed through biodegradation, the metabolic processes by which these materials are formed utilizes readily available precursors such as CO2 thereby reducing the environmental tensors.

Under anoxia condition with high CO2 concentration, Ralstonia eutropha piles up a significant quantity of PHAs through autotrophic Calvin-Benson-Bassham cycle using CO2 from the renewable catabolised sources such as carbohydrates, short chain carboxylic acids and lipid among others (Tsuge et Al, 2004). Polyhydroxyalkanoates are compounds of Hydrogen, Oxygen, and Carbon with similar chemical and physical properties to that of synthetic plastics. The product has a wide variety of subtypes that are quite promising for feature plastic development (Matsumoto et Al., 2013).

A study on the role of Ralstonia eutropha in biofuel production from palm oil has demonstrated that the engineered bacterium is a potential method for synthesis and accumulation of 3-hydroxybutyrate and 3-hydroxyhexanoate that are potentially important in the production of Polyhydroxyalkanoates (PHAs) (Matsumoto, 2013). The microarray study on the gene growth in palm oil revealed the active influence of acetoacetyl-CoA reductase (PhaB) activities on the amount of 3-hydroxyhexanoate production. Further, the Biosynthesis study by MüLler et Al (2011) unveiled three different enzyme catalysed reactions including acetoacetyl-CoA synthesis from acetyl-CoA molecules, acetoacetyl-CoA reduction to (R)-3-hydroxybutyryl-CoA and polymerisation of (R)-3-hydroxybutyryl-CoA monomers to form PHB [Fig.1.1]. These processes are encoded by three different gene namely phbA, phbB and phbC respectively.

Other than the microbial activities and the production medium, carbon source is also a significant factor that determines the type as well as the quality of Polyhydroxyalkanoates product (Mobley, 1994). Kinetic and stoichiometric study on Ralstonia eutropha organoautotrophic growth in formic acid medium (Meir, et Al, N.D.) demonstrate a relatively increasing Polyhydroxyalkanoates yield with substrate concentration. Besides, significant increase in density was also evident (Matsumoto et Al, 2013).

In this review, we investigate the genetic engineering application in directed evolution towards modifying the bacterium adaptation to formic acid medium. We evaluate the organism genome architect and their role in enhancing their adaptation strategy. The paper also reviews the successful experimental studies relating the genome’s sequence and formic acid tolerance and uptake. Finally, we derive the possible genetic control of the enzymatic activities to optimise Ralstonia eutropha’s growth and production in formic acid.

Fig.1.1: Carboxylation and oxygenation catalytic intermediate sequence (Ventosa, 2004),

Fig. 1.2: Schematic drawing illustrating the aggregation and accumulation of PBH in R. eutropha cell (Matsumoto, et Al, 2013),


3.1.           Ralstonia eutropha

Cupriavidus necator or Ralstonia eutropha formally known as Hydrogenomonas eutrophus is a ubiquitous gram negative bacterium capable of chemolithoautotrophic and organoautotrophic metabolism (Ventosa, 2004). In other words, Ralstonia eutropha has the ability of using the organic compounds such as carbohydrates and fatty acids as well as molecular substances including hydrogen and carbon (IV) oxide molecules as sources of energy. Cupriavidus necator is one of the bacterium species recorded as non-pathogenic. The organism has complex combination of survival mechanism including high metabolic versatility and the ability to accumulate polyhydroxyalkanoates sufficiently that enables its adaptation to a versatile environmental conditions (Matsumoto, et Al, 2013).

Cupriavidus necator belongs to Burkholderiaceae family of Betaproteobacteria group (Matsumoto, et Al, 2013; Jain, 1993 and Ventosa, 2004).  The microbe can proliferate in both aerobic and anaerobic environment due to its “knallgas” characteristics, for example, the influence of the two energy conserving hydrogenase. Cupriavidus necator have broad span of pathways that enables its survival under varying substrate environment. According to Ventosa (2004), Ralstonia eutropha depends on pentose phosphate pathway for carbon fixation in autotrophic conditions. It also survives and utilizes hydrogen molecules with carbon (IV) oxide for energy through denitrification pathway. Its ability to accumulate massive polyhydroxyalkanoate (PHA) and poly[R-(–)-3-hydroxybutyrate] (PHB) makes it preferred for bio-plastic synthesis.

Cupriavidus necator has flexible bioenergetics mode that enhances its survival strategies including heterotrophic and autotrophic growth mode (Serrano-Ruiz, 2015; and TrčEk, Mira & Jarboe, 2015). The bacterium undergoes Calvin-Benson-Bassham (CBB) cycle that enhances CO2 fixation and is capable of stockpiling organic hydrocarbons in the form of poly[R-(–)-3-hydroxybutyrate] (PHB). The piling of these compounds improves the microbe’s survival technique in fluctuating oxygen condition and growth-limiting nitrogen or phosphate complexes. R. eutropha can highly proliferate in diverse organic complexes such as fatty acids, sugar acids and tricarboxylic acid (TCA). Ralstonia eutropha has versatile biotechnological applications of significant importance including industrial production of biodegradable thermoplastics through lithoautotrophic fermentation, production of biofuel and the construction of H2-sensing device (Mittal, 2012; and Hu, 2004).

The genome of R. eutropha carries 59 transfer RNA (tRNA) genes: 51in Cr1, seven in Cr2 and one on megaplasmid pHG1 (Normi, et Al, 2005; and Oh & Bowien, 1999). Additionally, three ribosomal RNA (rRNA) operons are evident in their gene sequence on Cr1and two in Cr2. SIGI-HMM codon usage prediction demonstrates that Cr1 and Cr2 have universal value for Guanine-Cytosine content, ratio of laterally transferred alien DNA and coding sequence, which differs entirely from megaplasmid pHG1. Chr2 has stronger genome plasticity as compared with Chr1 as indicated by the number of genome copies per Megaplasmid SI content (Normi, et Al, 2005).

The origin Ralstonia eutropha genome replication units Chr1, Chr2 and pHG1 provides the basis of the principal bacterium identification (Falk, 2009; and Sudesh & Abe, 2010). The primary replication units’ cumulative GC skew of global minimum enhances the identification of the genome origins. DNA A-binding site as well as the dnaA gene of Chr1, and the repA with RepA-binding sites on the chromosome 2 and the plasmid coincide with the global minimum. RepA proteins are encoded on plasmids and enhance the replication of plasmid DNA while DnaA is located in direct propinquity to the genome origin indicating that plasmid genome unit the two chromosomes. The DnaA plays a significant role in the bacterium’s chromosomal replication while the plasmid partitioning is assisted by ParAB proteins (Meir, et Al (N.D.).

Meir and the colleagues (N.D.) have shown that the Ralstonia eutropha’s Chr2 can also be designated megaplasmid given that the corresponding GMI1000 chromosome replicon in Ralstonia solanacearum is a megaplasmid bacterium. In the study of Burkholderiaceae family’s genome organisation, Meir and the colleagues (N.D.) demonstrated a closes similarity between the Ralstonia eutropha H16’s DnaAChr1 (CAJ91153), RepAChr2 (CAJ94807) and Chr2 (CAD17152) of Rasltonia solanacearum. Meir and the colleagues (N.D.) are in line with Tanhaemami (2015) who demonstrated that RepApHG1 (AAP86121) had weak relation (31% identity) with RepA protein (S60672).

Going by the general genes distribution, there are two classifications of Ralstonia eutropha genome replicons including the Cr1 group, and the Chr2 and pHG1 category (Ayala-Del-Río, Héctor L., et Al., N.D; and Lawrence Berkeley National Laboratory, & United States, 2008). Cr1 genome replicon encodes the DNA replication, translation, and transcription as well as the synthesis of complex substrate’s building blocks (Ayala-Del-Río, Héctor L., et Al., N.D). The replicon encodes organic lipids, amino acids, and the nucleotides synthesis. On the other hand, chromosome 2 and plasmid group encodes the development of the bacterium adaptation qualities that enhance its sophisticated survival techniques (Falk, 2009; and Ayala-Del-Río, Héctor L., et Al., N.D). Cr2 replicon genes include 4-aminobutyrate aminotransferase (CAJ95772), argininosuccinate synthase (CAJ97313) and peptide chain release factor 3 (CAJ97353) (Ayala-Del-Río, Héctor L., et Al., N.D).

3.2.           Substrate Spectrum

High metabolic versatility and flexible nutritional modes characteristics of Cupriavidus necator are the potential survival mechanisms of the bacterium (Rehm et Al. 2002). According to Michigan Biotechnology Institute and the United States (1993), the ability of the organism to use both Hydrogen gas molecule and Hydrocarbons for aerobic metabolism (chemolitho-organoautotrophism) is an outstanding survival mechanism that provides energy for carbon dioxide assimilation. Additionally, Ralstonia eutropha has two energy-conserving hydrogenases: the reductant NiFe metalloproteinase that catalyses hydrogen gas molecule’s oxidation in addition to chemolitho-organoautotrophism that enhances its adaptation to both oxic and anoxic conditions (Buhrke et Al, 2005). Also, the bacterium has an alternative denitrification pathway for exploitation of electron acceptors including NO2 and NO3 in transient anoxia condition (Tsuge et Al, 2004).

The entire bacterium’s genome elucidation unveiled that, other than the chemoautotrophic encoding capability of Cr2 gene, as well as its ability to enhance anaerobic respiration, the replicon also encodes motility and chemolitho-organoautotrophism (Breitling, et Al, N.D.). According to Breitling, et Al, (N.D.), the primary lithoautotrophism gene components are located on Cr2 and plasmid replicons inthe Litho-Organoautotrophic Ralstonia eutropha. However, the fundamental characteristics of Ralstonia eutropha including litho-organoautotrophy and anoxia denitrification among others are exclusive role of the plasmid pHG1 replicon (Sundararajan, 2011).

Plasmid pHG1 contains hox and hyp genes that regulate the hydrogenases and auxiliary proteins activities while the autotrophic Calvin-Benson-Bassham cycle CO2 fixation gene is duplicated on both plasmid and CR2 replicons. CbbR (CAJ96185) is exclusive encoded on chromosome 2 (Lawrence Berkeley National Laboratory, & United States, 2008). The Cupriavidus necator’s substrate spectra include Facultative Anaerobiosis, Carbohydrates Digestion, and Aromatics Degradation.

Ralstonia eutropha’s has high concentration of denitrifying enzymes such as nitric oxide reductase and nitrate reductase on Cr2 and nitrous oxide reductase on Plasmid pHG1that facilitates anaerobic activities in anoxia condition. Plasmid pHG1 also carries ribonucleotide reductase (AAP85992) that is responsible for anaerobic class III. This procedure is in accordance with the Lawrence Berkeley National Laboratory and United States (2008). Entner-Doudoroff (2-keto-3-deoxy-6-phosphogluconate) pathway catalysing enzymes such as gluconate kinase that is located on Cr1 and glucokinase (CAJ97346) on Cr2, on the other hand, are indispensable for Ralstonia Eutropha’s proliferation in sugar medium such as glucosaminate and gluconate, 2-ketogluconate. The degeneration of these carbohydrates directly involves Entner-Doudoroff pathway that is exclusively regulated by the enzymes (Hagins, & Silo-Suh, 2009).

Substituted aromatic pollutants degeneration is one significant application of Ralstonia eutropha (Mittal, 2012; and Mobley, 1994). Cr2 replicon carries genetic encoding that is responsible for the degradation of a wide variety of aromatic compounds, including 3-hydroxylated benzoates, 4-hydroxylated benzoates4-cresol, and 4-biphenyl (Mobley, 1994). The replicon contains Protocatechuate of ketoadipate, which is complemented by Cr1 encoded Catechol, meta-cleavage and the gentisate pathway genes (Mittal, 2012).

Other than the degradation of aromatics and carbohydrates, as well as facultative anaerobiosis, Ralstonia eutropha’s exhibits motility in addition to secondary metabolism. The bacterium’s Cr2 encodes the biosynthesis of flagella and chemotaxis. The prokaryote is flagellated peritrichously (Falk 2009). Despite potentially toxic chromosome 2 fragmentary gene clusters, Ralstonia eutropha remains non-pathogenic. Putative insecticidal Tcc (CAJ96141-CAJ96142) and putative RTX (CAJ96468, CAJ96470-CAJ96472) encoded on Cr2 replicon are responsible for the formation of potential toxins (Falk 2009).

Comparative sequence analysis for investigating evolutionary origin unveils a possible indication that the Burkholderiaceae strains have one common ancestral origin (Falk 2009; and Meir, et Al, N.D.). This study, therefore, implies that the present Burkholderiaceae genomes content, size and order correspond to the single circular replicon of the primitive prototrophic soil or aquatic generation. Falk (2009) suggests the possibility of heterotrophic metabolism among the inferior bacterium base on the existence of CDS the primary components of TCA cycle, organic substrate and respiratory chain. According to Falk (2009), the need to adapt to the diverse ecological niche that is characterised by substrate demand facilitated evolution of the ancestral Burkholderiaceae bacterium family.

Falk’s (2009) theory is further supported by Hagins, & Silo-Suh’s (2009) findings who cites the size of the ancestral genome as an indication for the limited metabolism adaptation techniques with potential phenotypic traits developed as survival strategy towards the dynamic aquatic and soil environment. Also, Hagins, & Silo-Suh’s (2009) pointed to the continuous development and maintenance of megaplasmids encoded phenotypes as the separation point between the genera of Ralstonia and Burkholderia, which is analogous to MüLler’s et Al (2011) theory of multi-replicon genome architecture. The accumulation of the genetic materials responsible for encoding such traits is a potential explanation of megaplasmid development from plasmid.

According to Kivisaar (2011), the difference between the stable chromosome and other primitive variable encoded functions is the advanced genetic infrastructure that is developed as a result of the evolution. The elementary variation between the related Burkholderiaceae strains can appropriately be attributed development of megsplasmid encoded phenotype (Normi et Al, 2005). This gradual bacterium evolution is evidenced by the variation in the number of megaplasmid number. More particularly, the development of comparatively larger Cr2 replicon in Ralstonia eutropha, for example, is as aresult of genetic stabilization between selective trait adaptation strategy and the inert replicon essential genetic transfer.

Controlled metabolism of Hydrogen for industrial process is complicated and cost ineffective (Mobley, 1994). According to Mobley (1994), the process demands for critical gas ratio that would result into explosion if not considered. Besides, the complicated production system is comparatively expensive (Hu, 2004). Formic acid, therefore, comes as a potential alternative of the substrate as well as the energy source. Enzyme controlled oxidation of the acid releases the energy required for the process as well as the fuel in form of CO gas. The compound has an outstanding environmental sustainability and cost effective. None the less, formic acid is poisonous to the cell and inhibits prokaryote growth.

3.3.           Substrate Utilizing Pathways

Ralstonia eutropha’s substrate utilizing pathways are of significant importance in the directed industrial biotechnological production of material and chemical (Hagins, & Silo-Suh, 2009 and Kim & Zhang, 2015). According to Hagins, & Silo-Suh (2009), genetic manipulations of Ralstonia eutropha’s chromosomally pathway encoding genes is a potential method for defining the specification of the biosynthesised products. These pathways are substrate specific, and a simple alteration in the source would significantly change the end product. Examples of Ralstonia eutropha’s substrate utilizing channels include Calvin-Benson-Bassham (CBB) cycle, Tricarboxylic acid (TCA) cycle, Entner-Doudoroff (ED) pathway, Fatty acid β-oxidation, Glyoxylate cycle and Branched Chain Amino Acid Biosynthesis (Kim & Zhang, 2015).

The Ralstonia eutropha’s CBB cycle is carbon fixation pathway in which hexose sugar is produced from six turns of cyclic series of 3-phospho-D-glycerate. The molecules of 3-phospho-D-glycerate are generated from carboxylation of D-ribulose-1,5-bisphosphate (RuBP) molecules forming the molecules of the products (3-phospho-D-glycerate). The RuBP molecules act as the acceptors, and they are always regenerated at the end of the cycle. Strictly a single carbon (IV) molecule is added per single cycle and therefore, six such molecules are consumed by the end of the cycle. The entire cycle takes place in three stages including Fixation, Reduction, and Regeneration.

In the Fixation stage of Calvin-Benson-Bassham cycle, D-ribulose-1,5-bisphosphate molecules undergo reductive carboxylation forming two molecules of 3-phospho-D-glycerate in the presence of Ribulose Bisphosphate carboxylase (RubisCO) enzyme catalyst. The carboxylation phase is followed by phosphorylation of the 3-phospho-D-glycerate molecules from the fixation phase to form 1,3-diphosphateglycerate molecules in the Reductive stage. The 1,3-diphosphateglycerate molecules formed undergo reductive reversed dephosphorylation thus forming glyceraldehyde-3-phosphate.

In the Reductive phase, six cycles of phosphorylation and reductive dephosphorylation occur and produce 12 D-glyceraldehyde-3-phosphate molecules from six the simulated CO2 molecules. Ten of the twelve D-glyceraldehyde-3-phosphate molecules are passed to the Regeneration stage while the remaining two are converted D-fructose-6-phosphate and directed to biosynthetic pathways. In the third (Regeneration) stage, D-ribulose-1,5-bisphosphate restored from a series of D-fructose-6-phosphate enzymes controlled reactions of the ten D-glyceraldehyde-3-phosphate from the second phase.

A portion of D-glyceraldehyde-3-phosphate molecules converts to dihydroxyacetone phosphate molecules that are condensed with part of the remaining D-glyceraldehyde-3-phosphate molecules forming fructose-1,6-bisphosphate complex. This complex is dephosphorylated to form D-fructose-6-phosphate that forms a different compound upon combining with the remaining parts of D-glyceraldehyde-3-phosphate. The generated molecular compound separates into D-erythrose-4-phosphate and D-xylulose-5-phosphate molecules.

D-erythrose-4-phosphate molecule combines with dihydroxyacetone phosphate, the product of D-glyceraldehyde-3-phosphate molecules transformation process, and dephosphorylated thus forming D-sedoheptulose-7-phosphate compound. D-glyceraldehyde-3-phosphate molecule from the second phase of Calvin-Benson-Bassham cycle is added to D-sedoheptulose-7-phosphate complex thus forming a compound that is separates into D-erythrose-4-phosphate and D-xylulose-5-phosphate molecules. These compounds (D-erythrose-4-phosphate and D-xylulose-5-phosphate molecules) transforms to D-ribulose-5-phosphate that is phosphorylated to regenerate the D-ribulose-1,5-bisphosphate.

The Entner-Doudoroff pathway, on the other hand, is of vital importance to Ralstonia eutropha’s towards the uptake and tolerance to high formic acid. According to Hagins, & Silo-Suh (2009), the Entner-Doudoroff (ED) processes provide the pathway through which sugar acid from the microbes intercellular or extracellular environments including formic acid are metabolized. Entner-doudoroff is the joining cycle between glycolysis and pentose phosphate path way. The processes are controlled by a mass of inducible enzymes.

Other substrate utilizing pathways include tricarboxylic acid (TCA) cycle, which provide the pathway for biosynthesis of polyhydroxyalkanoate (PHA) mostly from carbon sources including formic acid, and fatty acid b-oxidation path way triacylglycerols uptake and utilization in production of biosynthesised products. Also, branched chain amino acid biosynthesis provides the pathway for branched carbon chain biosynthesis. Glyoxylate cycle is utilized in biomaterials and bioproducts synthesis that use acetate/acetyl-CoA as the raw products.

Fig.3.1: The schematic illustration of Calvin-Benson-Bassham (CBB) cycle (Kim & Zhang, 2015),

Fig.3.2: The growth of Ralstonia  eutropha in different substrate medium: Glu-5 is the glucose medium, Ace-5 is the Acetal acid and For5 and For-2 are formic acid medium (Matsumoto, et Al, 2013).

3.4.           Formic Acid

Ralstonia eutropha is capable of using an extensive variety of substrate including complex cane molasses (Hu, 2004), alkanes (Wu, 2014), lipid compounds (Mittal, 2012) and short chains carboxylic acid (Tanhaemami, 2015), which involves particular metabolic path way for monomer integration. For example, β-ketothiolase (PhaA) initiates the condensation of two acetyl-CoA moieties to form acetoacetyl-CoA. Also, PhaB reductase regulates NADPH activities during the production of 3-hydroxybutyryl-CoA (R)-isomers. Other substrates for Polyhydroxyalkanoates production using Ralstonia eutropha include lactic acid (Jain, 1993), alkanoates (Tsuge et Al, 2004), utilize carbon dioxide (Driggers, 2014), and 4-hydroxybutyric acid, 1, 4 – butanediol and γ-butyrolactone for an advanced integration of different Polyhydroxyalkanoates.

Several studies have demonstrated that short chain organic acids, including Formic acid (HCOOH or HCO2H), are potential substrate source for Ralstonia eutropha’s biosynthesis reactions (Sun et Al., 2012; Jain, 1993). According to Jain (1993), Valveric acid, Propionic acids, and Formic acids are excellent precursors for P(3HB-co-3HV) biosynthesis as well as PHA. Sun et Al. (2012) demonstrates in an experiment that up 70wt% of poly (3-hydroxybutyrate-co-3-hydroxy-4-pentenoate) can be obtained from sucrose, formic acid, and glucose. Sun et Al. (2012) has also shown that the use of organic acids, particularly formic acid has proven to more explosive safe compared with the autrophic Simulation of Hydrogen and Carbon (IV) oxide molecules and oxygen biomolecules [Fig 1.3].

These finding was further supported by Zeikus, et Al. (1993) who ascertained that up to 25g cell’s dry weight could be obtained using formic acid without safety risks associated with autotrophic growth. However, Zeikus, et Al. (1993) identified that Ralstonia eutropha had a reduced rate of growth in formic acid medium that continuously worsened with increase in concentration. According to Zeikus, et Al. (1993), formic acid molecules act as the discontinuity proton electrochemical gradient across the Ralstonia eutropha’s cell membrane thereby intoxicating the bacterium. Zeikus, et Al. (1993) ascertains that the substrate’s toxicity increases with increase in concentration of fall in pH value.

The toxicity of formic acid has been inhibition to its growth in high concentration of the acid thereby inhibiting the application of the bacterium for industrial biosynthesis. Referring to Jain (1993), Ralstonia eutropha can efficiently proliferate in formic acid of up to 2 g/L concentration and 6.5 pH value. Biosynthesis of the materials from formic acid as well fermentation production of the acid will be, therefore, strictly limited to these specifications hence the feeding and manufacturing process must remain concentration and pH controlled. In PHB production processes, the concentration and pH value are kept constant by continuous addition of new acid.

The biosynthesis of the Polyhydroxyalkanoates in prokaryotes is regulated by a series of operons clusters whose gene and enzyme constituent and arrangement, as well as their functionalities, varies from one bacterium to another (Hu, 2004; and Tsuge et Al, 2004). The production a particular type of PHA from a given substrate can be achieved by genetic engineering of the synthesis operons and the Polyhydroxyalkanoates synthases of Ralstonia eutropha (Sudesh & Abe, 2010). The mecanism has been observed in other bacteria such as E. coli.

Other than Polyhydroxyalkanoates, other biotechnical compounds that can be produced through direct evolution of Ralstonia eutropha’s Calvin–Benson–Bassham (CBB) cycle include biopetrols such as 3-methyl-butanol, isobutanol, and isopropanol (Fukuyama et Al, 2012). The bacterium converts CO into the biofuel using carbonic substrate such as carbohydrates and formic acid or inorganic hydrogen and oxygen atoms under controlled gas concentration. Formic acid a sustainable means as it is associated with minimal explosion thus does not require complex regulation of substrate composition.  

Formate dehydrogenase enzyme catalyses the oxidation of the carboxyl acid to nicotinamide adenine dinucleotide and CO that are recycled back to Calvin–Benson–Bassham proess (Oh & Bowien, 1999). Nonetheless, like all other short chain organic acid, formic acid is poisonous to Ralstonia eutropha’s cell. A study conducted on kinetic and stoichiometric rganoautotrophic growth of Ralstonia eutropha characterization (Matsumoto et Al. 2013) reveals that formic acid concentration lowers the industrial production of Biochemical [Fig. 4.1]. For optimum production of Polyhydroxyalkanoates from formic acid, there are several ways of controlling the toxicity of the substrate including pH controlled feeding (Matsumoto, 2013), chemoculture (Meir, et Al, N.D.) and directed evolution of the bacterium (Matsumoto, 2013).

5 NADH moles are required for every 3 CO moles assimilated through Calvin–Benson–Bassham cycle:

The disintegration of a single mole of formic acid through the cycles yields a single NADH mole thereby leaving a deficiency of 2 moles of NAD for three such decay processes [Fig.4.2]. As a result, bacteria generate low biomass under uncontrolled condition due to unbalanced substrate supply (Wang, Chen, & Quinn, 2012). Controlled feeding of the substrate can thus be used to optimise the production. Wang, Chen, & Quinn (2012) demonstrated low biomass production from Ralstonia eutropha growing on formic acid as compared to other substrates such as butyric acid, pyruvate, and fructose in a study investigating the factors influencing the lithoautotrophic growth of the bacterium in formic acid medium (Matsumoto, 2013). Oh and Bowien (1999) suggest that very high concentration of formic acid inhibits Ralstonia eutropha’s proliferation.

Fig.4.1: The growth of Ralstonia  eutropha in different substrate medium: Glu-5 is the glucose medium, Ace-5 is the Acetal acid and For5 and For-2 are formic acid medium (Matsumoto, et Al, 2013).

Fig.4.2: The schematic drawing for Formic Acid synthesis applied for production of IBT (Matsumoto, 2013).

3.5.           Directed Evolution

Genetic engineering technique of modifying the protein sequence of organism towards achieving a particular goal can be extensively applied in mutating Ralstonia eutropha towards the survival and uptake of formic acid substrates (Steven et Al., 2004). Steven et Al,  (2004) demonstrate that Nicotinamide adenine dinucleotide phosphate (NADPH) dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) Reductase can be genetically modified to enhance the Ralstonia eutropha’s kinetics for monomers synthesis.

AcAcCoA reductase catalyses the synthesis of the microbial of PHA, (R)-3-hydroxybutyryl (3HB)-CoA through the reduction process of 3-ketone group (Matsumoto, 2013). This enzyme is encoded by PhaB-encoding gene that is located together with PHA synthase (PhaC) and b-ketothiolase (PhaA) that synthesises P (3HB) from acetyl-CoA. The three genes are located on the phb operon and are responsible for efficient enzyme activities thus have been extensively exploited in bio-plastic production.

Additionally, Sudesh & Abe (2010) demonstrates an increased rate of Polyhydroxyalkanoates production through codon optimisation of   PhaB,  PhaB, and PhaA encoding genes. P(3HB) production has also been optimised through the increased dosage of the same encoding genes as well as codon optimisation. PhaB plays a significant role in Polyhydroxyalkanoates as it NADPH-dependent 3-ketoacyl-acyl-carrier proteins-(ACP)-like elementary structure that synthesise (R)-3-hydroxyacyl-ACP reductase (FabG) that plays an active part in the Bio-lipids synthesis. The directed codon evolution is achieved some approaches including stringent response, codon optimization engineering and design and optimisation of Synthetic ribosome binding site (Matsumoto, 2013).

Stringent response is the genetic engineering mechanism regulated by nucleotide guanosine tetraphosphate (ppGpp) that protects the microbes from the nutrient tensors (Dale, & Park, 2004). This method governs the rate of transcription in σ70 controlled genes and its homologs making RNA polymerase σ70 holoenzyme more stable. The inhibition of the transcription process stimulates other σ factors including σ54 apart from the σ70 that initiates and stimulates biosynthesised products such as PHB and PHA within Ralstonia eutropha systems. This process is further facilitated by guanosine tetraphosphate translation inhibition properties.

Stringent response can be enhanced in Ralstonia eutropha by eliminating guanosine tetraphosphate producing enzyme (Re1A) in the wild type to mitigate PHB synthesis while regulating branched chain amino acids pathway. The elimination of Re1A activities supresses PHB production while optimises biosynthesis of other organic materials, for example, IBT (Hungria, 2004). Hungria (2004) however points out that the method is not quite efficient and need improvements (Arnold & Georgiou, 2003).

According to Hungria (2004), efficiency optimisation of ribosome-binding site (RBS) sequences is a potential rationale to the regulation of branched-chain amino acid pathways enzymes limitations. This procedure involves the determination of the optimum ribosome-binding site sequence for the peak translation efficiency. RBS optimisation is there after followed by the design of a novel operon using synthetic RBSs to stimulate translation initiation rate (TIR) synthesis that catalyses the protein translation (Hungria, 2004).

The codon optimization engineering applies the principle of gene’s codon frequency dependency dependent characteristics (Hungria, 2004). According to Hungria (2004), ribosomes decoding speed as well as rate are functions of tRNA concentration. Therefore, specific codon and particular frequency can be used to define the gene sequence, and to perform target functions.

Other than the industrial performance of the bacterium, artificial genetic modification of Ralstonia eutropha’s protein sequence has extensively been used in alleviates growth inhibition in the formic acid medium (Sudesh & Abe, 2010). The evolution of Dehydrogenases (FDHs) to catalyse formic acid oxidation has earned a significant improvement has enhanced formic acid biosynthesis from atmospheric Carbon (IV) oxide (Das, 2014). According to Das (2014), FDH enzymes initiate electron transfer that enhances efficient industrial biocatalysts regeneration of NADH. Similar enzymatic activities were also observed naturally in Corynebacterium glutamicum that can be improved through directed genome evolution for industrial production of formic acid (Sudesh & Abe, 2010). The enzyme also plays the significant role of eliminating formic acid sense from the NADH products [Fig. 5.1].

Fig.5.1: Illustration of the R. eutropha’s central carbon and energy metabolism when exposed to formic acid,

Formic acid uptake has been observed to occur naturally in a wide variety of prokaryotes including Corynebacterium glutamicum and Escherichia coli. The enzymatic activities that initiate and catalyse formic acid consumptions in the two prokaryotes are encoded by two homologous genes. These FDH enzymes works particularly under anoxia condition using nitrate attached to the periplasmic side that provide electron acceptor terminals. FDHs include FdnGHI (FDH-N) and FdhF (FDHH) of Escherichia coli that incorporate Fe4S4 and molybdenum cofactor cluster catalyses electron transfer from formic acid molecule to hydrogenase and the fusion of the resulting hydrogen protons to form hydrogen gas molecules.

Similarly, Kim & Zhang (2015) propose the genetic engineering of the Ralstonia eutropha’s β-oxidation pathway towards degradation as well as synthesis of formic acid [Fig. 6]. The production of hydrogen based biofuel, bioplastics, and methyle ketons have been achieved through directed enzymatic reactions using genetic modification (Al-Adham et Al, 2012). This genetic engineering of Ralstonia eutropha’s β-oxidation pathway has been successfully applied in industrial blending of methyl ketones Biofuel from glucose substrate (Jain, 1993). This genetic mutation was also possible with Escherichia coli DH1 strain in which β-oxidation pathway engineered for excessive production of β-ketoacyl coenzymes A (β-ketoacyl-CoAs).

Fig. 5.2: The suggested glutathione biosynthesis pathway in R. eutropha to formic acid,


This study elucidates the directed evolution of Ralstonia eutropha towards the development of high formic acid tolerance. The unique genetic architect that encodes the standard survival mechanisms such as Facultative Anaerobiosis, Degradation of Carbohydrates, Degradation of Aromatics, Secondary Metabolism and Motility are discoursed. An extensive review of the experimental studies on the naturally derived adaptation mechanism among other families as well as the natural evolution among the prokaryotes is carried out to calculate the possible Ralstonia eutropha’s engineered genetic sequence to attain the formic resistance species. This study involves the gene replication, gene encodings, and enzymatic actions.

Directed evolution of Ralstonia eutropha is a potential biosynthesis technique that awaits exploitation. Biofuel production, bioplastic synthesis, and pharmaceutical development can significantly be improved through the genetic engineered microbe growth and activities. The products are environmentally sustainable, and application of formic acid makes the process cost effective. Nonetheless, the mechanical integrity of the products is poor due compared to the synthetic materials. Besides, the biofuel products have low calorific value. Therefore, more study is required to improve the shortcoming.


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