Engineering

Design

Samples were collected from mangroves in Hainan, and several marine yeast strains were isolated in the laboratory. To screen for strains capable of growing on methanol, the isolated strains were first activated on YPD solid medium. Single colonies from the activated strains were then inoculated into 5 mL YNB liquid medium and cultured at 28 °C with shaking at 180 rpm for 12 h, followed by transfer into YPM methanol medium under the same conditions for 24 h. The obtained strains were subjected to morphological observation and sequencing analysis, confirming them as melanin-producing Aureobasidium melanogenum, among which one was designated as strain P16.

To systematically evaluate the metabolic potential of A. melanogenum under methanol conditions, this study first designed a comparative growth experiment involving multiple marine-derived A. melanogenum strains (TN3-1, P16, ZW03-4, DH177, and BZ). The experimental medium was YPM (2% peptone, 1% yeast extract, 2% methanol v/v), containing 20 g/L methanol as the sole carbon source. All strains were inoculated at the same initial cell density, and their methanol utilization capacity was assessed by measuring OD₆₀₀ after 48 h of cultivation.

In addition, each screened strain was subjected to self-comparison experiments, in which their growth in methanol-containing medium and methanol-free medium was compared, in order to determine whether the candidate strains had strong methanol utilization potential.

To ensure a relative evaluation of methanol metabolism levels, two types of control strains were included: a non-methylotrophic yeast, Saccharomyces cerevisiae, which lacks methanol metabolism; and a typical methylotrophic yeast, Komagataella pastoris X33, which is widely used in methanol metabolism research and industrial applications. By comparing the growth curves of A. melanogenum candidates with these control strains, the methanol adaptability and utilization efficiency of each strain could be revealed more intuitively, thus providing experimental evidence for the selection of chassis strains.

This experimental design not only aimed to screen out the candidate strain with the best growth performance but also, through multi-dimensional comparisons among different A. melanogenum strains, between A. melanogenum and non-methylotrophic yeasts, and between A. melanogenum and typical methylotrophic yeasts, laid the foundation for subsequent in-depth analysis of methanol metabolic pathways in A. melanogenum and future metabolic engineering modifications.

Build

To preliminarily verify the methanol utilization capacity of the strain obtained from mangrove sampling, designated as P16, it was inoculated into methanol-containing medium (YPM) and methanol-free medium (YP), with the same initial inoculum (OD₆₀₀ = 0.01). After 48 h of cultivation, P16 exhibited significantly higher growth in YPM than in YP, confirming its ability to metabolize and proliferate using methanol as a carbon source.

During the experimental stage, five marine-derived Aureobasidium melanogenum strains (TN3-1, P16, ZW03-4, DH177, and BZ) were comparatively cultured. Each strain was inoculated into YPM medium (2% peptone, 1% yeast extract, 20 g/L methanol, v/v 2% methanol) at an initial OD₆₀₀ of 0.01 and incubated at 28 °C and 200 rpm for 48 h. Growth was assessed by OD₆₀₀ measurement. The results showed significant differences in methanol-based growth performance among the five strains, with P16 exhibiting the highest biomass after 48 h. Thus, P16 was selected as the candidate chassis strain for subsequent studies.

To further benchmark its methanol utilization capacity, P16 was compared with two reference strains: the non-methylotrophic yeast Saccharomyces cerevisiae and the typical methylotrophic yeast Komagataella pastoris X33. The results revealed that while P16 exhibited weaker methanol utilization than K. pastoris X33, its performance was markedly superior to S. cerevisiae, supporting its potential as a promising chassis for methanol metabolism research.

Test

After identifying P16 as the candidate chassis strain, we further evaluated its tolerance to different methanol concentrations. A concentration gradient of 10, 20, 30, 40, and 50 g/L methanol was tested, with an initial inoculum of OD₆₀₀=0.01 under identical shake-flask cultivation conditions. Growth curves were monitored over 72 h. Results showed that P16 exhibited optimal growth at 20–30 g/L methanol, reaching average OD₆₀₀ values of 1.28 ± 0.05 and 1.31 ± 0.04 at 48 h, respectively. When the concentration increased to 40 g/L, the growth rate declined significantly, and at 50 g/L, cells nearly lost proliferative capacity. In summary, P16 can grow stably within a methanol range of 10–40 g/L, with 20–30 g/L being the optimal range, indicating its broad tolerance and potential as a chassis for methanol metabolism research.

Learn

Through comparative experiments and tolerance analysis, this study confirmed that the melanin-producing strain Aureobasidium melanogenum P16 is capable of using methanol as the sole carbon source for growth, with optimal adaptation observed at 20–30 g/L methanol. This demonstrates its strong metabolic plasticity and broad environmental tolerance. According to literature, A. melanogenum not only synthesizes high-value byproducts such as pullulan but also possesses natural melanin-producing ability and unique stress resistance as a marine-derived microorganism. These features confer dual advantages for its use as an engineering chassis: on the one hand, P16’s methanol utilization provides a metabolic foundation for constructing “methanol-driven” synthetic biology platforms; on the other, its byproduct synthesis potential and environmental resilience offer distinct benefits in marine biomanufacturing and stress-resistant production systems.

However, research on melanin-producing A. melanogenum remains limited. Its genetic background is not yet well characterized, and studies on its development as a microbial cell factory are still relatively weak, which partially restricts its industrial application potential. Taken together, P16 combines methanol metabolism capacity, secondary metabolite development potential, and ecological stress tolerance, making it an ideal candidate chassis strain for further engineering modification. Based on this, future work will focus on elucidating the methanol metabolic mechanisms of P16 and conducting targeted cell engineering to overcome current research limitations and further unlock its application potential.

Design

Based on whole-genome sequencing and annotation of Aureobasidium melanogenum P16, the methanol metabolism pathway of strain P16 was predicted using KEGG annotation. From the KEGG pathway comparison, this study focused on key methanol-metabolism enzymes: alcohol oxidase (AOX), dihydroxyacetone synthase (DAS), and dihydroxyacetone kinase (DAK). AOX is considered the primary rate-limiting enzyme in methanol metabolism, catalyzing the oxidation of methanol to formaldehyde while producing hydrogen peroxide. The subcellular localization of AOX therefore determines the spatial locus of methanol oxidation reactions; clarifying AOX localization is thus critical for elucidating the methanol metabolic pathway in P16.

Build

To validate AOX subcellular localization, a fluorescence-labeling strategy was employed to construct localization verification plasmids. The AOX gene was fused to green fluorescent protein (GFP) and co-transformed into P16 together with mCherry-SKL, a red fluorescent peroxisomal marker carrying a canonical peroxisomal targeting signal. These plasmid constructions and transformations enabled co-expression of GFP–AOX and mCherry–SKL in cells, providing the basis for subsequent fluorescence microscopy.

Test

After transforming P16 with the co-expression plasmids (GFP–AOX and mCherry–SKL), cells were imaged by fluorescence microscopy. The green GFP signal and the red peroxisomal marker signal showed clear intracellular colocalization and appeared as yellow–orange spots in the merged channel, indicating that AOX is primarily localized to peroxisomes. This result directly verifies the subcellular localization of AOX.

Learn

The experimental results confirm that AOX functions within peroxisomes in A. melanogenum P16, indicating that the methanol-oxidation step occurs mainly in peroxisomes. This finding is consistent with methanol metabolism patterns observed in model methylotrophic yeasts and further supports P16’s potential as a chassis for methanol metabolism studies. Considering that DAS and DAK do not display canonical peroxisomal targeting signals in KEGG annotations, their localization mechanisms may differ from AOX; therefore, follow-up experiments are required to validate the subcellular localization of DAS and DAK.

Design

The sequences of DAK and DAS were analyzed using UniProt for subcellular localization prediction, which suggested dual distribution in peroxisomes and cytoplasm. Literature review indicated that DAS and DAK may rely on a ribosomal stop-codon readthrough mechanism, enabling translation beyond the first conventional stop codon to reveal a latent peroxisomal targeting signal (PTS) located downstream. If true, the subcellular localization of DAS and DAK would depend on translational readthrough, thereby affecting the spatial organization of key methanol-metabolism reactions. To test this hypothesis, we designed a simulated readthrough experiment by constructing plasmid variants to verify whether readthrough regulates the localization of DAS and DAK.

Build

To validate the hypothesis, multiple GFP-fusion expression constructs were generated, including Das, Das cyt, Das pex, Dak, Dak cyt, and Dak pex. Das: contained the coding sequence up to the second stop codon. If readthrough occurs, it should include the complete PTS and localize to peroxisomes. Das cyt: truncated at the first stop codon, lacking the downstream PTS region; used as a negative control, expected to localize exclusively to the cytoplasm. Das pex: a simulated readthrough mutant, where the first stop codon was altered to enforce readthrough, allowing validation of whether the downstream latent PTS is functional.

Similarly, Dak, Dak cyt, and Dak pex constructs were built to examine the localization properties of DAK. All constructs were co-transformed with the peroxisomal marker protein mCherry-SKL into P16 for fluorescence co-localization analysis.

Test

After transformation into P16, fluorescence microscopy revealed that GFP–Das and GFP–Das cyt both displayed diffuse cytoplasmic distribution with little overlap with the peroxisomal marker. GFP–Das pex also failed to show punctate fluorescence, indicating that DAS localization did not exhibit the expected readthrough dependence.

In contrast, GFP–Dak showed both diffuse cytoplasmic fluorescence and partial peroxisomal co-localization. When the downstream latent PTS was removed (Dak cyt), GFP fluorescence was completely cytoplasmic, with no peroxisomal overlap. Meanwhile, the Dak pex mutant exhibited clear punctate green fluorescence strongly colocalized with the peroxisomal marker, demonstrating that DAK’s peroxisomal localization depends on the release of a functional downstream PTS via stop-codon readthrough.

Learn

The results indicate that in A. melanogenum P16, DAK localization to peroxisomes is mediated by a functional PTS revealed through stop-codon readthrough, while DAS did not display the expected localization pattern, leaving its mechanism uncertain. Based on these findings, the next step will involve heterologous overexpression of the Pichia pastoris DAS gene to overcome potential limitations of DAS localization in P16 and further enhance methanol metabolism efficiency.

Design

Given that the endogenous DAS in A. melanogenum P16 did not exhibit the expected peroxisomal localization—possibly due to low readthrough efficiency or insufficient functionality of its latent PTS—this study proposed a heterologous optimization strategy. Specifically, the DAS gene from the model methylotrophic yeast Pichia pastoris was selected for overexpression. Since P. pastoris DAS has evolved efficient catalytic activity and a robust peroxisomal localization mechanism, its heterologous expression in P16 is expected to enhance methanol metabolic flux and improve growth performance under methanol-based cultivation.

Build

To achieve heterologous expression, an expression plasmid harboring the P. pastoris Das gene was constructed under the control of a strong promoter and fused with a GFP tag for verification. The plasmid was introduced into P16 via electroporation, and positive transformants were screened on selective media. PCR and sequencing confirmed correct integration and stable expression, providing a reliable genetic background for subsequent metabolic performance assays.

Test

Wild-type P16 and the P. pastoris Das-overexpressing strain were inoculated into YPM medium containing 20 g/L methanol with an initial OD₆₀₀ of 0.01 and cultured under identical shaking conditions for 48 h. Results showed that wild-type P16 reached an average OD₆₀₀ of 1.29 ± 0.03, whereas the overexpression strain achieved 1.46 ± 0.01. This represents an approximately 12% increase in biomass, indicating enhanced methanol adaptation and growth.

Learn

In this study, through the heterologous introduction of the Das gene from Pichia pastoris, it was verified that the methanol metabolic capacity of strain P16 could be significantly enhanced via the exogenous optimization of key enzymes. This result indicates that the DAS from P. pastoris not only exhibits advantages in methanol metabolism efficiency but also, with its clear peroxisomal targeting signal, effectively overcomes the bottleneck caused by the uncertain localization of endogenous DAS in the P16 strain. Thus, the introduction of heterologous DAS is a feasible strategy to improve the methanol metabolism efficiency of P16, and it also provides a strong basis for the subsequent construction of high-efficiency methanol-utilizing chassis strains through multi-gene synergistic optimization.

Design

To enhance the glucose production capacity of Aureobasidium melanogenum P16 under methanol conditions, this study proposed a metabolic engineering strategy of “enhancing the synthetic pathway.” Metabolic pathway analysis indicated that the intermediate metabolite glucose-1-phosphate (G1P) generated from methanol assimilation tends to be diverted into glycogen synthesis or UDP-glucose synthesis, causing carbon flux dispersion. To channel the carbon flux directly toward free glucose accumulation, the YihX gene encoding a haloacid dehalogenase-like phosphatase from E. coli was introduced. This enzyme can catalyze the dephosphorylation of G1P to generate free glucose, thereby potentially increasing intracellular glucose accumulation.

Build

Based on this design, a strong-promoter-driven YihX overexpression plasmid was constructed and introduced into A. melanogenum P16. Stable transformants were obtained via electroporation and designated as Yihx. Positive clones were verified by PCR and sequencing to confirm correct integration and expression of the YihX gene. Subsequently, the Yihx recombinant strain and wild-type P16 were cultured in parallel to evaluate their glucose production performance.

Test

Wild-type P16 and the Yihx recombinant strain were cultured in YPM medium containing 20 g/L methanol, with an initial inoculum of OD₆₀₀ = 0.01, under identical conditions for 48 h. Glucose production was measured at the end of cultivation. The results showed that the Yihx strain had an average glucose yield of 0.042 g/L, which was not significantly different from that of wild-type P16 (0.040 g/L, P > 0.05). This indicates that the sole introduction of YihX did not significantly enhance glucose production.

Learn

The experimental results indicate that although the YihX gene can be functionally expressed in P16, its effect on enhancing glucose synthesis is limited. This is likely because P16 possesses strong glucose consumption pathways, causing the newly synthesized glucose to be rapidly funneled into downstream metabolic processes such as glycolysis, thereby offsetting the contribution of YihX-catalyzed conversion. Therefore, merely “enhancing the synthetic pathway” is insufficient to improve glucose production efficiency. Future strategies should combine pathway enhancement with reduction of glucose-consuming pathways to effectively redirect carbon flux toward the accumulation of free glucose.

Design

In previous experiments, the sole introduction of the Escherichia coli haloacid dehalogenase-like phosphatase 4 (YihX) did not significantly increase glucose production in P16, suggesting that intracellular glucose synthesis was limited by strong consumption pathways. To overcome this bottleneck, this study proposes a combined metabolic engineering strategy of “reducing consumption pathways + enhancing synthesis pathways”. Specifically, based on the existing YihX recombinant strain, the CRISPR-Cas9-Am system was employed to knock out the rate-limiting glycolytic enzyme gene pfk (phosphofructokinase), thereby blocking further glucose consumption through glycolysis and constructing the dual-modified strain YihX-Δpfk. This strategy is expected to effectively accumulate glucose and achieve a significant increase in yield.

Build

First, sgRNAs targeting the pfk gene were designed based on the CRISPR-Cas9-Am system, and the corresponding gene knockout vector was constructed. The vector was then introduced into the YihX recombinant strain via protoplast transformation to obtain resistant clones. PCR detection and sequencing confirmed the successful deletion of pfk, resulting in the dual-modified strain YihX-Δpfk. This strain carries both the heterologous overexpressed YihX gene and the pfk deletion, providing a reliable chassis for subsequent fermentation performance evaluation.

Test

Wild-type P16 and the YihX-Δpfk recombinant strain were cultured in YPM medium containing 20 g/L methanol, with fermentation for 48 h, followed by measurement of extracellular glucose. The results showed that the average glucose production of wild-type P16 was 0.040 g/L, whereas the YihX-Δpfk strain reached 0.844 g/L, representing over a 20-fold increase with statistical significance (P < 0.01).

Learn

This experiment validated the effectiveness of the “reduce consumption + enhance synthesis” combined strategy in P16. By simultaneously introducing YihX and knocking out pfk, glucose accumulation was significantly promoted, overcoming the limitation of the single-pathway enhancement strategy. The results indicate that blocking glucose consumption pathways is a key step for achieving efficient glucose production, and the coordinated regulation of synthesis and consumption pathways can effectively redirect carbon flux toward the target product. This strategy not only provides an effective example for constructing a methanol-based glucose-producing cell factory but also lays the foundation for producing other high-value products in the future.