Development of efficient colorimetric assay for sugar alcohol screening
Sugar alcohol-producing strains are usually screened and quantified by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and p-iodonitrotetrazolium violet (INT) methods [24–26]. However, these methods are time-consuming or suffer from high-cost and are limited for high throughput screening. Therefore, it is necessary to develop an efficient screening approach for sugar alcohol-producing microbes.
In our study, a colorimetric method previously applied in trace detection of polyols [27, 28] was developed and optimized for the high throughput assay of sugar alcohols (Additional file 1: Fig. S1). d-arabitol was selected as a standard for the method construction because it is the main sugar alcohol product of P. anomala. By optimizing the reaction system, the standardized assay demonstrated a linear detection range of d-arabitol from 0 to 12 g/L. Although the linear relation was noticeably altered at 20 g/L sugar alcohol, the colorimetric curve was positively related with the sugar alcohol concentration and could be applied in the preliminary screening (Fig. 1a, b). To analyze effects of the substrate and by-products on sugar alcohol screening, an interference experiment was performed at different concentrations of glucose and ethanol (2–30 g/L). The results showed that glucose and ethanol had no interference in the quantitative analysis of sugar alcohols by the colorimetric method (Fig. 1a), which indicated that the developed assay is highly efficient for the determination of the content of sugar alcohol in biological samples. To gain a further understanding of the accuracy, the reference HPLC and the proposed colorimetric methods were applied to analyze sugar alcohol at different concentration levels. The results showed that the data measured by the colorimetric procedure agree with those determined by the reference HPLC method, and a regression line with an R2 of 0.9673 was obtained (Fig. 1c; Additional file 1: Fig. S1).
In this study, a convenient, reliable and low-cost colorimetric assay was developed for efficient primary screening and selection of strains with high productivity. The method is highly specific for sugar alcohols and can be performed on crude, non-purified extracts. The method uses low hazard and inexpensive reagents and requires only commonly available equipment. Finally, the method is sensitive and highly reproducible. Compared with HPLC and TLC methods, the colorimetric method facilitated sugar alcohol detection and made the operation of screening of sugar alcohol-producing strains more convenient. Although INT is another efficient method for sugar alcohol detection by specific enzyme catalysis, it is not suitable for high throughput assay because of the complex process and the expensive substrate p-iodonitrotetrazolium violet [29]. Therefore, the proposed colorimetric assay has clear advantages over the other methods and can be applied to high throughput screening for different polyol-producing strains.
Development of a rapid hybrid cell selection procedure via FACS analysis
To achieve the efficient screening of hybrid cells without complementary genetic markers, FACS analysis based on fluorescent dyes was applied. In this process, hybrid cells are detected by carrying two dyes, and these cells can be analyzed and selected by FACS.
In this approach, parental protoplasts were prepared and then labeled with fluorescent dyes Nuclear Green and Nuclear Red, resulting in green and red fluorescence with laser excitation at 488 and 641 nm, respectively. After fusion, the hybrids were sorted by FCM, and the results are represented as dot plots (Fig. 2). As the control, strains without staining showed no fluorescence in the R4 gate (Fig. 2a). The parental strains showed single red and green fluorescence in different gates based on the staining with fluorescent dyes Red or Green (Fig. 2b, c). Overlap between the fluorescence regions of Green and Red was also observed, and possible compensation was performed. As shown in Fig. 2d, R3 is the sorting area showing cells that exhibit high intensity fluorescence with green and red and is identified as potential hybrid cells. In our study, some 2,500,000 protoplasts were rapidly sorted, and 15,300 potential hybrids were selected. Only approximately 1,000 colonies were found after incubation for regeneration; most protoplasts were not regenerated, probably because of damage during protoplast preparation, staining and laser sorting.
To facilitate screening and identification of the hybrid cells, different genetic markers were always necessary in previous studies, such as auxotroph [30] and drug resistance [31]. However, a genetic marker, such as auxotroph, affects the physiology and metabolism of the strain and leads to reduced performance in the production process. Furthermore, adding genetic markers to the parent strain is a difficult operation for some nonconventional strains. In this study the fluorescence-activated cell sorting was applied as a useful method for the hybrid cell selection of P. anomala without the need for genetic markers; in addition, this method is also available for the genome shuffling of other microbes. It may be possible to apply the technique for other native strains that are limited by unclear genetic backgrounds or unskilled genetic operations.
The construction of a mutant library for genome shuffling by random mutagenesis
In the genome shuffling process, the wild type strain is usually treated by the traditional physical and chemical mutation methods, and the strains with superior performance are collected to form the parental library for the next step of recursive protoplast fusion [31, 32]. In this study, a haploid of sugar alcohol-producing P. anomala TIB-x229 [5] was first isolated and identified as P. anomala HP. The mutant library was constructed by ultraviolet (UV) and atmospheric and room-temperature plasma (ARTP) mutagenesis methods to generate genetic diversity. After the mutagenesis processes, mutants with the maximum sugar alcohol production were selected from approximately 2,000 mutants by colorimetric screening and were then prepared for the next round of mutation and screening. Through five rounds of continuous mutagenesis, a parent library with approximately 10,000 mutants was constructed and analyzed by the aforementioned colorimetric method (Fig. 1d). The sugar alcohol yield of the positive mutants was further confirmed by an HPLC method, and the four mutants (U-7, U-9, A-4 and A-1) showed clear superiority for sugar alcohol production. Compared to the initial P. anomala HP, the mutants U-7 and U-9 treated by UV had 7.3 and 8.9% improvement of sugar alcohol production. The yields of mutants A-4 and A-1 treated by ARTP were increased by 12.3 and 12.9%, respectively (Fig. 3a). These results showed that there was a slight improvement in mutants after several rounds of traditional mutagenesis. However, the single traditional mutagenesis was still a time-consuming process for strain engineering because of the low mutation rate and less diversity.
Genome shuffling of P. anomala for improved sugar alcohol production
To further improve the performance of sugar alcohol productivity, the mutant strains (U-7, U-9, A-4 and A-1) with slightly improved performance were collected as the parental library for the next step of genome shuffling, which is a powerful means for rapid breeding of improved organisms without knowledge of the detailed genome information. To achieve the efficient screening for genome shuffling, the developed colorimetric assay of sugar alcohol and the FACS method were incorporated into the genome shuffling procedure for our nonconventional yeast P. anomala (Fig. 4).
The protoplasts were processed and fused by a chemical method induced by polyethylene glycol [33]. After the first protoplast fusion and screening by FACS, approximately 1,000 colonies with both red and green fluorescence were preliminarily cultivated and assayed for sugar alcohol production by colorimetric assay. The selected colonies exhibiting improved performance were further confirmed by HPLC. In the bioconversion process, d-arabitol and ribitol were produced from glucose by P. anomala. Compared to the parental strain P. anomala HP, three recombinants (GS1-1, GS1-2 and GS1-3) exhibited significantly improved productivity of total sugar alcohols by 19.5, 25.6 and 23.9%, respectively (Fig. 3a). The isolates GS1-2 and GS1-3 were used as the parent population for the following round of genome shuffling. Similarly, the resulting second-round isolates were further screened, and three isolates GS2-1, GS2-2 and GS2-3 were selected and evaluated and showed increased total sugar alcohol production of 46.1, 46.5 and 47.1 g/L, which was 29.5, 30.6, and 32.3% higher than that of parental strain P. anomala HP, respectively (Fig. 3a). We compared the relative DNA content between the parent strain and the shuffled strains by DAPI labeling and FCM (Additional file 1: Fig. S2). Compared to the parental strain P. anomala HP and the referenced haploid yeast Saccharomyces cerevisiae BY4741, the wild type TIB-x229, GS2-1, GS2-2 and GS2-3 strains had diploid DNA content. We assessed the performance and stability of shuffled strains through the bioconversion of sugar alcohols. For that purpose, bioconversion in sterile water containing 100 g/L glucose was used to compare the performance of the evolved strains, GS2-1, GS2-2 and GS2-3, with that of the original strain TIB-x229. Although the overall growth conditions were the same in all strains, the shuffled strains showed a slightly faster rate of glucose consumption (Fig. 3b, c). Likewise, the accumulation rate of d-arabitol and ribitol was higher in the shuffled strains. The yield of d-arabitol in the shuffled strains GS2-1, GS2-2 and GS2-3 was 0.29, 0.31 and 0.32 g/g, which was 11.5, 19.2, and 23.1% higher than that of the original strain P. anomala TIB-x229, respectively (Fig. 3d). The ribitol production in these shuffled strains was 8.46, 11.23 and 10.98 g/L (Fig. 3e), which was also slightly higher than that in the original strain (7.51 g/L). These results showed that the improvement of the shuffled strains in sugar alcohol production was due to the accumulation of d-arabitol and ribitol. In this study, two rounds of genome shuffling achieved efficient gains in sugar alcohol yield. The results further indicated that genome shuffling is a much more powerful means for breeding improved organisms, especially for those strains that have undergone classic strain improvement many times.
In recent years, there are also other different reports on sugar alcohols improvement including metabolic engineering [34], natural screening [5], fermentation optimization [35] and mutation breeding [36]. However, there was not any study reported about improving performance of sugar alcohol-producing strains by genome shuffling, because there were some obstacles in this process, such as a lack of efficient sugar alcohol-detection methods and available yeast selective markers. In our study, we developed the practicable genome shuffling for sugar alcohol-producing strains by combining the colorimetric assay and fluorescence-activated cell sorting, which provided a more efficient way for sugar alcohol-producing strain improvement.