(−)-Carvone is a member of monoterpenoid and key flavor compound of spearmint essential oil [1]. (−)-Carvone is utilized for spearmint flavor and fragrance in segments such as confectionery and oral care [2]. The annual production of (−)-carvone is approximately 3,800 tons per year, the majority of which (approximately 2,000 tons per year) is made by chemical synthesis from (+)-limonene [3]. Due to enhanced health- and environment-awareness in recent years, more consumers prefer natural flavors and fragrances for better perception [4]. Spearmint flavor is not an exception, so that the demand for natural spearmint flavor, or natural (−)-carvone is increasing. The spearmint essential oil is currently the only source of natural spearmint flavor, including natural (−)-carvone. There has been an issue that the demand for natural spearmint flavor exceeds the supply. To increase the supply is not simple, since current spearmint essential oil production method is water-intensive and requires improvement to be sustainable. Also, spearmint cultivation can be easily affected by the weather, such as drought, so that the spearmint essential oil supply volume and unit price is fluctuating [5]. Therefore, it is desirable to develop sustainable and stable natural (−)-carvone production method in order to accommodate the market demands.
One possible solution is (−)-carvone production by fermentation in a microbe. There are numerous attempts to produce flavor and fragrance compounds by biotechnology rather than extracting from its natural sources, because it has potential to be more sustainable and stable [6]. The regulatory circumstance is also supporting such attempts. For example, the flavor and fragrance compounds produced by biotechnology (regardless of the microbial or enzymatic process) can be labeled as “natural,” according to European regulation CE 1334/2008 [7]. Therefore, (−)-carvone produced by biotechnology method can be labeled as natural (−)-carvone, and sustainably replace natural (−)-carvone conventionally produced by extracting from spearmint. To our knowledge, there is no natural (−)-carvone produced by biotechnology in the market yet.
The purpose of our study is to develop the sustainable and cost-effective replacement of carvone production by using microbes. (−)-Carvone is synthesized from the precursor (−)-limonene in its native producer spearmint. Specifically, intracellular (−)-limonene is converted to (−)-trans-carveol by cytochrome P450 limonene-6-hydroxylase (along with cytochrome P450 reductase; CPR), while (−)-trans-carveol is converted to (−)-carvone by carveol dehydrogenase (CDH) (Fig. 1). The enzymology of (−)-carvone biosynthesis in spearmint has been studied in detail [8,9]. The cytochrome P450 limonene-6-hydroxylase and CDH have been functionally expressed in Escherichia coli [10,11]. However, when these three genes were expressed in a single E. coli cell, a very low level of (−)-carvone (up to 2 µM) can be obtained from whole-cell biocatalysis with (−)-limonene supplementation [12]. The reason for the low conversion rate was still not clear, however, one general issue among heterologous expression of cytochrome P450 of plant origin is the difficulty to express in a heterologous host such as E. coli [13]. In order to increase the target compound production by plant origin cytochrome P450 in E. coli, careful fine-tuning at protein level to balance the expression of P450 along with other pathway enzymes was necessary [14]. Indeed, heterologous expression of cytochrome P450 limonene-6-hydroxylase required intensive N-terminal modification [10], whereas CDH was expressed well in soluble form without any modification [11]. Kinetic parameter information of these two enzymes were limited, but it appeared that P450 reaction was rate-limiting in spearmint plant [9]. Based on these prior studies, we hypothesized that the expression levels and the enzymatic kinetics of P450 and CDH are quite different, leading imbalance in the carvone biosynthesis pathway in E. coli, and ultimately causing the low conversion rate from (−)-limonene to (−)-carvone. To investigate such hypothesis, it was desired to have a protein quantification method with high sensitivity to conduct comparative study among various strains by abundance ratio of pathway enzymes.
Proteome analysis is a powerful tool for quantification of proteins. Proteome analysis can be divided into two types, relative quantification and absolute quantification. The relative quantification method in proteome analysis can be conducted without the laborious preparation of standard proteins; however, different proteins cannot be compared for expression level. The absolute quantification method requires a synthetic, isotope-labeled standard peptide (AQUA) preparation [15]. AQUA peptide preparation is still relatively expensive so that it is hardly performed in strain construction for industrial biotechnology fields. Another method for quantification standard preparation is by individually purify each target proteins in an isotope-labeled form, called the PSAQ method [16]. Alternatively, the quantification concatemer (QconCAT) method enables the standard peptide preparation easier and cheaper [17]. In the QconCAT method, targeted peptides are concatenated into a QconCAT protein, which is spiked in as a standard in proteome analysis. For our purpose, QconCAT method appears to be an attractive choice. There are a few studies that used the QconCAT method for quantification of proteins in prokaryote [18–20]; however, to our knowledge, there is no reports that the QconCAT method was applied to genetic engineering for upgrading metabolic pathways. In recent years, DNA synthesis became extremely accessible, so that the QconCAT method has a potential to become more popular among the synthetic biology field.
In this study, we developed a QconCAT method to quantify the expression of carvone biosynthetic pathway enzymes, P450, CPR and CDH. Upon receiving our hypothesis of imbalanced P450 and CDH expression in E. coli, two strains independently expressing P450/CPR and CDH were mixed with different mixing ratio, and the QconCAT method was used to determine the optimum expression balance between the P450 and CDH. Based on this proteome analysis data, a single strain expressing both P450/CPR and CDH with superior expression ratio was constructed. This upgraded strain displayed 15-fold improvement of (−)-carvone production compared to our initial strain, and achieved increase of (−)-carvone titer approximately 150 times higher than the previous report. To our knowledge, this is the first report in genetic engineering for upgrading metabolic pathways using the QconCAT method.