healthmedicinet

Transcriptional analysis of the RIB genes in A. gossypii

As previously described, several reports have established a relationship between the
transcriptional regulation of some of the RIB genes and the increase of riboflavin production 11]–14]. In addition, the analysis of metabolic flux changes related to the production of
riboflavin using the iRL766 model predicted the up-regulation of most of the RIB genes during the production phase in A. gossypii4]. Accordingly, we were prompted to determine the RNA levels of all the six RIB genes encoded in the genome of A. gossypii.

The transcriptional analysis of the RIB genes was carried out by qRT-PCR. Total RNA from A. gossypii MA2 cultures in both the trophic phase (24 h of culture) and the production phase
(120 h of culture) were obtained. Quantitative analysis of the RIB genes in cDNA samples was carried out using the housekeeping gene ACT1 as a reference 6]. With the exception of RIB4, the level of RNA for all the RIB genes was very low in both the trophic and production phases; ranging from 1–5  (for
RIB2, RIB5 and RIB7) to 10–15 % (for RIB1 and RIB3) of the transcript level of the moderately transcribed gene ACT1 (Fig. 2). RIB4 showed a high transcriptional level, similar to that of the GPD gene (Fig. 2), whose promoter is widely used for gene overexpression in A. gossypii9]. In contrast to the prediction of the iRL766 model, the transcription rate of the
RIB genes showed no or only minor increments during the production phase (Fig. 2).

Fig. 2. Quantitative-real time PCR of the RIB genes in A. gossypii. Relative transcription levels of the RIB genes in our wild-type strain of A. gossypii during both the trophic phase (24 h, exponential growth) and the production phase
(120 h, stationary growth). The GPD1 gene was included as a control. Transcription levels were normalized using the A. gossypii ACT1 gene as a reference. The results are the means of two independent experiments performed
in duplicate and are expressed as a ratio of the cDNA abundances of the target genes
with respect to the ACT1 mRNA levels

Single gene overexpression of the RIB genes in A. gossypii

The results reported above revealed that the transcription of most of the RIB genes remains low throughout the trophic and production phases of A. gossypii culture. Accordingly we wished to determine the effect of the overexpression of each
RIB gene on riboflavin production in A. gossypii. The RIB4 was excluded from the overexpressions experiments, since this gene already showed
a high transcriptional level in the wild-type.

Gene overexpressions were carried out using an integrative cassette harboring a geneticin-resistance
selection marker and the strong promoter P _AgGPD
. The cassette was flanked by recombinogenic sequences for each RIB gene and was integrated by homologous recombination to replace the native promoter
region of each gene. Overexpression of the RIB genes was verified by qRT-PCR analysis (Additional file 1: Fig. S1a) and the riboflavin production of the mutant strains was determined. As
shown in the Fig. 3, the overexpression of RIB1, RIB2 or RIB3 triggered an increase in riboflavin production. The overexpression of either RIB1 or RIB3, which code for the first enzymes of each branch in the riboflavin pathway, induced
the highest increases in riboflavin production. In contrast, the overexpression of
either RIB5 or RIB7 did not cause any significant change in the production of riboflavin (Fig. 3).

Fig. 3. Riboflavin production after the overexpression of RIB genes in A. gossypii. Quatification of riboflavin production in the A. gossypii strains that overexpress one of the RIB genes. The data are the means of three independent experiments performed in duplicate

Global overexpression of the RIB genes in A. gossypii

We observed that overexpression of some of the RIB genes induced an increase in riboflavin production in A. gossypii. Thus, the low transcription rate of the RIB genes may be a limiting-rate step for riboflavin overproduction and, consequently,
a maximal yield of riboflavin might be obtained with the overexpression of all the
RIB genes. Accordingly, it was decided to overexpress all the RIB genes to quantify the riboflavin production in a quintuple overexpressing strain.
A loxP–KanMX–loxP selection marker was used for gene overexpression. The loxP repeated inverted sequences enabled the selection marker to be eliminated, and subsequently
reused, by expressing a Cre recombinase after each round of transformation, as described
elsewhere 6]. The gene overexpression pipeline is depicted in Fig. 4a. The A329 strain, which overexpresses the RIB1, RIB2, RIB3, RIB5 and RIB7 genes, was obtained after 10 transformations either to integrate the P _AgGPD
into the target loci or to remove the KanMX selection marker (Fig. 4a). The overexpression of the RIB genes in the new mutant strains was verified by qRT-PCR (Additional file 1: Fig. S1b).

Fig. 4. Overexpression of the RIB genes in a single strain of A. gossypii. a Pipeline of the construction of the A329 strain and b quatification of riboflavin production in the A. gossypii strains. The data are the means of three independent experiments performed in duplicate

Riboflavin production was measured in the quintuple overexpressing strain and also
in all the intermediate strains (Fig. 4b). As shown above, the overexpression of RIB3 alone triggered a remarkable increase in riboflavin production (46 % more riboflavin
than the WT strain). In addition, the combination of the overexpression of both RIB1 and RIB3 (strain A273) increased the riboflavin yield by more than 2.5-fold in comparison
with the WT. Moreover, the overexpression of RIB5, RIB2 and RIB7 caused an additional increase in riboflavin production with respect to the A273 strain.
Overall, the A329 strain, which simultaneously overexpressed the RIB1, RIB2, RIB3, RIB5 and RIB7 genes, showed the highest level of riboflavin production (326.6 mg/L), representing
a 3.1-fold increase over the WT production (Fig. 4b).

Flux balance analysis of the purine pathway for riboflavin production

In the absence of enzymatic regulation constrains, the strain containing the overexpressed,
deregulated riboflavin biosynthetic pathway should not be limited in the riboflavin
pathway itself, but rather it should be able to respond to increases in the pool of
the GTP substrate by efficiently increasing the rate of riboflavin product formation.
To assess this hypothesis we decided to increase the GTP supply available for riboflavin
synthesis and determine the effect on riboflavin production in both the WT and RIB-overexpressing
(A329) strains.

The purine pathway provides GTP, which is one of the main precursors of riboflavin.
Since our previous results demonstrated that the deregulation of the purine pathway
induces a significant increase of riboflavin overproduction in A. gossypii7]–9], an increase in the intracellular pool of GTP could be correlated with a higher riboflavin
yield in A. gossypii. IMP is the central metabolite of the purine pathway, which can subsequently be converted
either to AMP or GMP via a two-branch pathway (Fig. 1). Thus, IMP metabolic flux towards GMP/GTP production compete with the AMP branch,
which consists of two enzymatic reactions controlled by adenylosuccinate synthase
(ADE12) and adenylosuccinate lyase (ADE13). Indeed, flux balance analysis of riboflavin production using the A. gossypii iRL766 model predicted that the AMP branch of the purine pathway is down-regulated
during the production phase, while the GMP branch, leading to GTP and thereafter riboflavin,
is not transcriptionally regulated. These results suggest that a control over the
IMP hub affects riboflavin production.

Accordingly, we used the A. gossypii iRL766 model to simulate a reduction in metabolic flux towards the AMP branch by
decreasing the activity of the Ade12 enzyme (Fig. 1). The in silico simulation comprised 316 metabolic reactions, including enzymatic
cofactors that could affect metabolic flux exchange in both the purine and riboflavin
pathways. The output of the model predicted a linear increase in riboflavin production
that was directly correlated with the reduction in Ade12 activity (Additional file
2: Fig. S2). Thus, in order to increase the supply of GTP available for riboflavin
synthesis in A. gossypii, the ADE12 gene can be considered a solid candidate for metabolic engineering through deletion.
In addition, engineering the IMP metabolic hub can serve to analyze the performance
of a deregulated, overexpressed riboflavin pathway in response to a greater availability
of guanine precursors.

Metabolic engineering of the IMP hub gene in A. gossypii

To analyze the effect of the ADE12 gene-deletion on riboflavin production in A. gossypii a gene knockout strain was constructed using a loxP–KanMX–loxP selection marker flanked by ADE12-flanking recombinogenic sequences, as described above (see “Methods” section). As
expected, the ade12? strain was viable but showed adenine auxotrophy. Analysis of riboflavin production
revealed that the ade12? strain was able to produce up to 246 mg/L of riboflavin, which represents a 2.5-fold
increase in riboflavin production compared to the wild-type strain. In this sense,
our results confirm the idea of GTP availability as a limiting step for riboflavin
overproduction.

Although the deletion of ADE12 triggers the production of riboflavin, the presence of auxotrophic mutations in the
strains is highly undesirable for large-scale industrial fermentations. Therefore,
we decided to analyze the effect of ADE12 gene underexpression on riboflavin production. As shown above, the RIB7 gene has very low levels of mRNA in the WT strain. Indeed, a qRT-PCR analysis revealed
that the transcription rate of RIB7 was approximately 62-fold lower than that of the ADE12 gene. Accordingly, the promoter sequence of the RIB7 gene (P _RIB7
) was used for ADE12 gene underexpression. Thus, the ADE12 native promoter was replaced by P _RIB7
using a loxP–KanMX–loxP selection marker, which was subsequently eliminated as described above (Fig. 5a). The promoter replacement was confirmed both by DNA sequencing of the genomic amplicon
(data not shown) and by qRT-PCR analysis. As expected, the mRNA levels of ADE12 were extremely reduced in the P _RIB7
–ADE12 strain (approximately 70-fold lower than those in the wild-type strain). However,
this transcription level was sufficient to sustain the growth of the mutant in MA2
media without adenine supplementation. Moreover, the underexpression of ADE12 did not significantly affect biomass formation and riboflavin production was similar
to that of the ade12? strain (Fig. 5b).

Fig. 5. Underexpression of the ADE12 gene in A. gossypii. a Diagram of the promoter replacement of the ADE12 gene and b biomass and riboflavin production analyses after the underexpression of the ADE12 gene in A. gossypii. The data are the means of three independent experiments performed in duplicate

Engineering the metabolism of purines and overexpression of the RIB genes trigger riboflavin overproduction

As shown above, the supply of guanine precursors and the transcription of the RIB genes largely determine the riboflavin yield in A. gossypii. Accordingly, in order to analyze the a riboflavin overproducer strain, we decided
to combine both the overexpression of the RIB genes and the underexpression of the ADE12 gene.

The quintuple RIB-engineered strain A329, which overexpresses the RIB1, RIB2, RIB3, RIB5 and RIB7 genes, was transformed with either the ade12? deletion cassette or the P _RIB7
cassette to replace the ADE12 native promoter. The genomic integrations of each module were confirmed both by analytical
PCR and qRT-PCR to test ADE12 mRNA (data not shown). Other parameters, such as the growth rate, biomass production
and sporulation ability, were analyzed but no significant differences were found between
these new strains and the wild-type strain (data not shown). Finally, a marked increase
in riboflavin production was found when these two approaches were conjugated in a
single strain (Fig. 6). A total yield of 523 mg/L of riboflavin was obtained when the overexpression of
RIB genes and the underexpression of ADE12 were combined. This riboflavin level in our mutant strain represents an increase
of 5.4-fold with respect to production by the wild-type strain (Fig. 6).

Fig. 6. Riboflavin overproduction in the engineered A. gossypii. a quatification of riboflavin production in A. gossypii strains. The data are the means of three independent experiments performed in duplicate.
b solid MA2 plates of the strains A330, A324 and wild-type of A. gossypii. The strain A330 that was modified both for the underexpression of the ADE12 gene and for the overexpression of five RIB genes afforded the highest riboflavin yield (in yellow)