Hydroxycinnamic acids are a class of polyphenols having a C6-C3 skeleton. These compounds are hydroxy derivative of cinnamic acid. Hydroxycinnamic acids are phenolic phytochemicals found in a wide range of plants and fungi and are found abundantly in nature. For example, hydroxycinnamic acid such as caffeic acid and ferulic acid (4-hydroxy-3-methoxy-trans-cinnamic acid) can constitute about 3% of the dry weight of graminaceous plants (1) such as flax seed with the highest concentrations found in seeds and the bran of graminaceous plants.
Ferulic acid links arabinoxylans to lignin, the lignin-ferulate-xylan complex (FIG. 3) can contribute to the recalcitrance of plant tissues, thereby lowering the efficiency of the biomass conversion in biofuel production (2). As such, to reduce substrate complexity and increasing cellulose accessibility, biomass can be pretreated with hemicellulases such as xylanase and feruloyl esterase (3-5), with ferulic acid released as a major by-product (6). With the espousal of using switchgrass (a rich source of ferulic acid) (7) as the renewable source for biofuel production, the by-product, ferulic acid is currently a waste product.
Another type of hydroxycinnamic acid found in graminaceous plants is p-coumaric acid (4-hydroxybenzaldehyde). Oxidizing 4-hydroxybenzaldehyde can lead to the production of p-hydroxybenzoate which is used as a monomer for synthesizing liquid crystal polymers. The aromatic compound p-hydroxybenzoate is a building block of liquid crystal polymers, high performance plastics that are employed in electronic devices such as mobile phones electronic devices for telecommunication and aerospace applications. p-hydroxybenzoate is also used in chemical and food packaging applications. The cascade bio-synthesis pathway of p-coumaric acid to p-hydroxybenzoate is realized through the biological pathway of 4-hydroxybenzaldehyde formation by feruloyl-CoA synthetase (Fcs), followed by oxidation to p-hydroxybenzoate by enoyl-CoA hydrotase (Ech).
Hydroxycinnamic acid can provide a substrate for the production of high value chemicals. Another such economic interest is the production of vanillin (4-hydroxy-3-methoxybenzaldehyde), the key flavor component of vanilla from hydroxycinnamic acids such as ferulic acid. Vanillin is used extensively in food, cosmetic, and pharmaceutical industries (8). Due to popular demand, vanillin derived from natural sources such as vanilla pod (Vanilla planifolia) can reach as high as US$4000/kg (9). The bio-synthesis of vanillin from ferulic acid can be realized through the established biological pathway of feruloyl-CoA thioester formation by feruloyl-CoA synthetase (Fcs), followed by hydration to β-hydroxy derivative and then cleavage to give vanillin and acetyl-CoA by enoyl-CoA hydrotase (Ech) (10) (See FIG. 4). Many studies had reported the use of these enzymes from various microorganisms for the bio-production of vanillin (11-16). Microorganisms such as Pseudomonas fluorescens BF13 (11), Pseudomonas sp. HR199 (12), Amycolatopsis sp. HR167(13), Pseudomonas putida (14), Bacillus subtilis (15), Delia acidovorans (16), and Streptomyces setonii (17) had been proposed as candidates for the bioconversion of ferulic acid to vanillin. However, these organisms produce natural ferulic acid degraders that are capable of using vanillin as a source of carbon and energy. Thus, to avoid any reduction in the yield, downstream genes such as vanillin dehydrogenase (Vdh) which converts vanillin to vanillic acid may have to be knocked out (12).
An alternative approach has been developed to use a non-native vanillin producer such as Escherichia coli (14, 18-21) to host the genes responsible for converting ferulic acid to vanillin. Among bacteria, recombinant E. coli has been touted as the most efficient biocatalyst for vanillin production (11).
However, in many of the studies where E. coli was used as the host for bio-vanillin production, artificial induction with common inducers such as isopropyl-b-D-thiogalactoside (IPTG) and arabinose were performed. Although generally, artificial induction offers the control over protein expression, artificial induction in many cases is less favorable due to high economic cost of inducers, inducer toxicity, incompatibilities with industrial scale-up and detrimental growth conditions (22). Thus, these issues often limit the usage of the artificial inducible systems in industrial scale protein production. Using recombinant E. coli as a biocatalyst for vanillin production with and without the inhibiter IPTG Lee at al. (19) demonstrated that the addition of IPTG only further decreased production of vanillin.
A substitute to the artificial inducible systems is the use of constitutive promoters which can initiate protein expression in the absence of the inducers. Though constitutive promoters may offer comparative economic advantage, strong constitutive expression of recombinant proteins may divert the cellular resources away from essential metabolic activities to overproduction of unnecessary RNAs, and proteins. This may subsequently leads to growth retardation or adaptive responses from the host cells that may reduce yield and productivity (23). To overcome such problem, previous work by Barghini et al. (2007) (20) had tried to use low-copy number vector in vanillin synthesis. Nonetheless, the instability issue brought by strong constitutive promoter still remains.
One strategy for that has been seen in recent studies for avoiding instability is to use the Vibrio fischeri's quorum sensing system for cell density regulated protein production (22), fatty acid bio-sensor for biodiesel production (23) and pathogen detection for anti-microbial peptide production (24). This strategy uses the lux regulon. It is unclear if the lux regulon would work in the complex control of hydroxycinnamic acid catabolism.
Gamma-aminobutyrate (GAB) expression is activated under carbon or nitrogen deficient cell stress. Expression of GAB is activated by the sigma factor RpoS a stress induced transcription factor often transcribed in the stationary phase of bacterial cell growth. There are about 70 stress genes having RpoS dependent expression.