Sole Carbon Source Research Articles

Microalgae-bacterial consortiums for enhanced degradation of nonylphenol: Biodegradability and kinetic analysis

The widespread use of non-ionic surfactant nonylphenol (NP) has led to significant water pollution, posing a threat to both ecological stability and human health. However, the efficient biodegradation method and system of NP-biodegradation remain complex scientific challenges. In this study, we isolated and characterized three Pseudomonas sp. strains SW-1 (Scenedesmus quadricauda-associated), ZL-2 (Ankistrodesmus acicularis-associated), XQ-3 (Chlorella vulgaris-associated), and one NP-degrading Cupriavidus sp. strain EB-4, which exhibited the ability to utilize NP as the sole carbon source. Furthermore, four consortiums of microalgae-bacterial, S. quadricauda and SW-1 (S-SW), A. acicularis and ZL-2 (A-ZL), C. vulgaris and XQ-3 (C-XQ), S. quadricauda and EB-4 (S-EB), were constructed to investigate their biodegradability and kinetic characteristics of NP degradation from water. The consortiums showed higher degradation efficiency compared to individual microalgae or bacteria. The C-XQ consortium exhibited the highest degradation rate, removing over 94% of NP within just seven days. The first-order model with the following order of degradation rate by consortiums was C-XQ (0.3960 d−1) > S-SW (0.3506 d−1) > A-ZL (0.1968 d−1) > S-EB (0.1776 d−1). Compared with the results of our previous study, the interaction between microalgae and bacteria is not a simple additive relationship. Our findings highlight the potential of an algal-bacterial consortium for the remediation of NP-contaminated environments.

Metabolic engineering of Escherichia coli for the utilization of methylsuccinate, the product of methane activation via fumarate addition.

Methylsuccinate is a branched-chain, 5-carbon (C5) dicarboxylate that can be generated from the O2-independent activation of methane via fumarate addition. However, no established metabolic pathway enables growth and product synthesis from methylsuccinate. Here, we report a synthetic pathway that converts methylsuccinate into two precursor metabolites: pyruvate and acetyl-CoA. The pathway was constructed through rational design and validated both in vitro and in vivo using E. coli as the host. Subsequently, growth on methylsuccinate as the sole carbon source was achieved using two parallel strategies: adaptive laboratory evolution and enzyme mining. Through the latter approach, we identified a heterologous electron transfer pathway mediated by previously uncharacterized enzymes and integrated into E. coli enabling the conversion of methylsuccinyl-CoA to mesaconyl-C4-CoA. The engineered strain demonstrated efficient growth on various C5 dicarboxylates including methylsuccinate, mesaconate, and itaconate, with a specific growth rate of 0.11 h-1 on methylsuccinate. This study represents an important step toward achieving synthetic methanotrophy, as the engineered strain can serve as a platform for screening potential methane activation enzymes and ultimately as a production chassis for the bioconversion of methane into various value-added products.

Draft genome of hydrocarbon-degrader Burkholderia cepacia BCTT, isolated from soil chronically polluted with crude oil in Trinidad.

Burkholderia cepacia BCTT, isolated from chronically polluted soil in Trinidad, shows a capacity to survive in crude oil as a sole carbon source. Here, we report its high-quality draft genome sequence and highlight those pathways and genes involved in xenobiotic degradation. These data give a clearer insight into this organism's biotechnological potential.

Lignin valorization to bioplastics with an aromatic hub metabolite-based autoregulation system

Exploring microorganisms with downstream synthetic advantages in lignin valorization is an effective strategy to increase target product diversity and yield. This study ingeniously engineers the non-lignin-degrading bacterium Ralstonia eutropha H16 (also known as Cupriavidus necator H16) to convert lignin, a typically underutilized by-product of biorefinery, into valuable bioplastic polyhydroxybutyrate (PHB). The aromatic metabolism capacities of R. eutropha H16 for different lignin-derived aromatics (LDAs) are systematically characterized and complemented by integrating robust functional modules including O-demethylation, aromatic aldehyde metabolism and the mitigation of by-product inhibition. A pivotal discovery is the regulatory element PcaQ, which is highly responsive to the aromatic hub metabolite protocatechuic acid during lignin degradation. Based on the computer-aided design of PcaQ, we develop a hub metabolite-based autoregulation (HMA) system. This system can control the functional genes expression in response to heterologous LDAs and enhance metabolism efficiency. Multi-module genome integration and directed evolution further fortify the strain’s stability and lignin conversion capacities, leading to PHB production titer of 2.38 g/L using heterologous LDAs as sole carbon source. This work not only marks a leap in bioplastic production from lignin components but also provides a strategy to redesign the non-LDAs-degrading microbes for efficient lignin valorization.

Evaluation of hydrocarbon degrading potentials of cellulolytic edaphic borne fungi

This study assessed the hydrocarbon degradative potentials of cellulolytic fungi cultured from top soil samples within Benin City, Edo State. Soil samples were collected from 3 sites; a farmland, a fallow plant covered land, and municipal dumpsite. Physicochemical parameters of the soil samples were based on standard methods. Standard cultural and molecular techniques were used to isolate and identify fungi present in samples. Cellulolytic activity of fungal isolates was determined with Czapek-dox agar supplemented with 0.1% sodium carboxymethyl cellulose (CMC). Cellulolytic fungi were subsequently screened for the ability to utilize blended petroleum as sole carbon source. Further, two isolates having the highest optical density were used for growth profile study as axenic culture and in consortium. The growth profile study assessed pH, optical density, total petroleum hydrocarbon content and fungal dry mass. Results showed that the organic carbon value of the top soils ranged from 4.14 g/kg ± 0.55 to 8.56 g/kg ± 0.05. All collected soil samples were sandy, while total hydrocarbon content ranged from 0.38 g/kg ± 0.04 to 1.13 g/kg ± 0.01. The mean fungal counts were highest in dumpsite soils and ranged from 4.0 × 103 cfu/g ± 1414 to 7.0 × 103 cfu/g ± 1003. Eight fungal isolates with cellulolytic properties were identified from the soil samples. Trichosporon asahii and Curvularia bothriochloae had highest (p < 0.05) OD when grown on blended petroleum and were used for growth profile studies. OD was highest (p < 0.05) in C. bothriochloae (0.372 ± 0.0005) relative to T. asahii and the consortium at 15 d. THC reduced significantly (p < 0.05) in both axenic cultures and the consortium after 15 days, while fungal biomass was highest (p < 0.05) in consortium relative to axenic cultures after 15 d. This study showed hydrocarbon degrading capabilities of cellulolytic fungi from different top soils.

Draft genome sequence of Methyloversatilis sp. strain NSM2, isolated from a wastewater treatment plant.

Here, we report the draft genome sequence of Methyloversatilis sp. NSM2, isolated from a wastewater treatment plant. This strain was enriched using methane as the sole carbon source. The 4.5-Mb genome, with a GC content of 66.5%, provides valuable insights into the strain's ecological role and metabolic capabilities.

Isolation of Sphingopyxis kveilinensis sp. nov., a Potential Antibiotic-Degrading Bacterium, from a Karst Wetland.

A Gram-stain-negative, aerobic, mesophilic, motile, rod-shaped bacterium, designated strain TUF1T, was isolated from a karst wetland in south-west China. It was demonstrated to be capable of growing on plates containing oxytetracycline, streptomycin, or ofloxacin as the sole carbon source. Phylogenetic analysis of the 16S rRNA gene sequence revealed that this organism belongs to the genus Sphingopyxis and is closely related to S. chilensis S37T (99.17%) and S. alaskensis RB2256T (99.12%). The orthologous average nucleotide identity values (OrthoANIu, 84.42% and 87.53%) and digital DNA-DNA hybridization values (dDDH, 41.7% and 48.9%) between strain TUF1T and its close relatives were all below the standard recommended threshold values for species discrimination. The genomic DNA G + C content was determined to be 64.7%. The predominant cellular fatty acids were identified as summed feature 8 (C18:1ω7c and/or C18:1ω6c) and summed feature 3 (C16:1ω7c and/or C16:1ω6c). The major polar lipids found to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, and sphingoglycolipid. The sole respiratory quinone present was ubiquinone Q10. Based on the phylogenetic, biochemical, physiological, and chemotaxonomic analyses, strain TUF1T represents a novel species of the genus Sphingopyxis. The designation "Sphingopyxis kveilinensis sp. nov." is proposed as the name for this novel species, and the strain TUF1T (= CGMCC1.62043T = JCM36394T) is designated as the type strain.

Biodegradation of dimethachlon by Arthrobacter sp. K5: Mechanistic insights and ecological implications

Dimethachlon, an agricultural fungicide, poses a potential risk to both the ecosystem and human health. Microbial degradation provides a safe, efficient, and environmentally friendly method for eliminating pesticide residues from the environment. In this study, a dimethachlon-degrading bacterium, Arthrobacter sp. K5, was isolated and characterized by the enrichment technique. Strain K5 was observed to initially convert dimethachlon into 4-(3,5-dichloroanilino)-4-oxobutanoic acid, which was subsequently transformed into 3,5-dichloroaniline and succinic acid. Strain K5 effectively utilized dimethachlon as the sole carbon source, converting 100 mg L−1 dimethachlon to 3,5-dichloroaniline within 36 h at 30°C. The optimum degradation conditions were pH 7.0, a temperature of 30°C, and a NaCl concentration range of 0–0.1 % (w/v). In addition, 1 mM Cd2+, Cu2+, and Zn2+ could considerably decrease the dimethachlon degradation activity. Enzyme activity analysis revealed that strain K5 contains intracellular enzymes that can metabolize dimethachlon into 3,5-dichloroaniline. The strain K5 genome was sequenced, and four putative dimethachlon-degrading enzymes were hypothesized in strain K5. In addition, the genus Arthrobacter is extensively dispersed in metagenomes from various soil environments. This study enhances our understanding of the role of the genus Arthrobacter in dimethachlon degradation.

Decomposition of Polythene by Bacteria Isolated from Waste Disposal Sites in Gusau City

The accumulation of polythene can be hazardous and can cause various environmental problems. This work was aimed at studying the biodegradation of polythene by bacteria isolated from waste disposal areas in North West, (Zamfara state) Nigeria. A total of twenty soil samples were collected from four different locations, such as waste disposal areas, hospitals, roadsides, and petrol pumps station. Spread plate method was carried out on Nutrient agar for the growth of bacteria. A total of 13 bacteria were isolated from different areas. The total bacteria count in hospital ranged from 80 × 10-4 - 248 × 10-4 cfu/mL, roadsides ranged from 116 × 10-4 - 208 × 10-4 cfu/mL, petrol pump ranged from 88 × 10-4 - 184 × 10-4 cfu/mL and waste disposal areas ranged from 116 × 10-4 - 264 × 10-4 cfu/mL. The microbial species associated with the polythene materials were identified as Pseudomonas sp, Bacillus sp, Staphylococcus aureus, Providencia stuartii, Clostridium perfringes, Corynebacterium diphtheria, Listeria monocytogen. The bacteria isolates were screened for polyethylene degradation using mineral salt medium containing polyethylene as sole source of carbon and nitrogen for their ability to degrade polyethylene for the period of 2 months. The efficacy of the microbes in the degradation of polythene were analyzed using liquid culture method, among the bacteria Pseudomonas aeruginosa degraded 75% of polythene, Bacillus subtilis 71.4% in a period of 2 months.

Draft genome of the caprolactam-degrading Paenarthrobacter sp. CAP02 isolated from a landfill.

Here, we report the draft genome sequence of Paenarthrobacter sp. CAP02, which metabolizes caprolactam as a sole source of carbon and nitrogen. This bacterium was isolated from Dane County Landfill Site No. 2 in Madison, WI, USA.

Characterizing and Engineering Rhamnose-Inducible Regulatory Systems for Dynamic Control of Metabolic Pathways in Streptomyces.

Fine-tuning gene expression is of great interest for synthetic biotechnological applications. This is particularly true for the genus Streptomyces, which is well-known as a prolific producer of diverse natural products. Currently, there is an increasing demand to develop effective gene induction systems. In this study, bioinformatic analysis revealed a putative rhamnose catabolic pathway in multiple Streptomyces species, and the removal of the pathway in the model organism Streptomyces coelicolor impaired its growth on minimal media with rhamnose as the sole carbon source. To unravel the regulatory mechanism of RhaR, a LacI family transcriptional regulator of the catabolic pathway, electrophoretic mobility shift assays (EMSAs) were performed to identify potential target promoters. Multiple sequence alignments retrieved a consensus sequence of the RhaR operator (rhaO). A synthetic biology-based strategy was then deployed to build rhamnose-inducible regulatory systems, referred to as rhaRS1 and rhaRS2, by assembling the repressor/operator pair RhaR/rhaO with the well-defined constitutive kasO* promoter. Both rhaRS1 and rhaRS2 exhibited a high level of induced reporter activity, with no leaky expression. rhaRS2 has been proven successful for the programmable production of actinorhodin and violacein in Streptomyces. Our study expanded the toolkit of inducible regulatory systems that will be broadly applicable to many other Streptomyces species.

Synthesis of β-ionone from xylose and lignocellulosic hydrolysate in genetically engineered oleaginous yeast Yarrowia lipolytica.

β-ionone, an apocarotenoid derived from a C40 terpenoid has an intense, woody smell and a low odor threshold that has been widely used in as an ingredient in food and cosmetics. Yarrowia lipolytica is a promising host for β-ionone production because of its oleaginous nature, its ability to produce high levels of acetyl-CoA (an important precursor for terpenoids), and the availability of synthetic biology tools to engineer the organism. In this study, β-carotene-producing Y. lipolytica strain XK17 was employed for β-ionone biosynthesis. First, we explored the effect of different sources of carotenoid cleavage dioxygenase (CCD) genes on β-ionone production. A high-yielding strain rUinO-D14 with 122mg/L of β-ionone was obtained by screening promoters combined with rDNA mediated multi-round iterative transformations to optimize the expression of the CCD gene of Osmanthus fragrans. Second, to further develop a high-level production strain for β-ionone, we optimized key genes in the mevalonate pathway by multi-round iterative transformations mediated by non-homologous end joining, combined with a protein tagging strategy. Finally, the introduction of a heterologous oxidoreductase pathway enabled the engineered Y. lipolytica strain to use xylose as a sole carbon source and produce β-ionone. In addition, the potential for use of lignocellulosic hydrolysate as the carbon source for β-ionone production showed that the NHA-A31 strain had a high β-ionone productivity level. This study demonstrates that engineered Y. lipolytica can be used for the efficient, green and sustainable production of β-ionone.

Producción de esterasa por Fusarium culmorum crecido en presencia de diferentes concentraciones di(2-etilhexil) ftalato y de ion calcio en fermentación sumergida

Phthalates are plastic additives used as plasticizers in the manufacture of flexible plastic materials. These compounds, such as di(2-ethylhexyl) phthalate (DEHP) are toxic substances to the environment and human health, and have been detected as environmental pollutants. In this study, the activity of esterase produced by Fusarium culmorum grown in media supplemented with different concentrations of DEHP (1.5, 3 and 4 g/L) and calcium ion (0.1 and 0.5 g/L) in submerged fermentation was characterized by biochemical tests and polyacrylamide gel electrophoresis. The greatest esterase activity was observed in cultures grown in media added with 3 g of DEHP/L and 0.5 g of calcium ion/L, followed by those cultures grown in media containing 1.5 g of DEHP/L. Whereas cultures grown on 4 g of DEHP/L had the lowest esterase activity. In general, cultures added with 0.5 g of calcium ion/L had the maximum enzymatic activity and the highest enzymatic productivity in the media tested. High concentration of DEHP were used as the sole source of carbon and energy by F. culmorum. It is suggested that the calcium ion favored the esterase production during the DEHP biodegradation. However, further studies are needed to understand the relationship between calcium ion, fungal growth, and enzyme production to provide the appropriate concentration of this ion in the culture medium.

Microalgal Metabolomes and Recent Biotechnological Advances for Their Industrial Application

In recent decades, microalgae have emerged as new sustainable resources for the production of various bioactive and biochemical compounds. These organisms are photosynthetic, use CO2 as their sole carbon source, and grow rapidly under different environmental conditions. This makes microalgae a promising source of feedstock for many bio-commodities. However, the economic viability for large-scale production through microalgal cells is limited to certain organisms. Recent advances in omics technologies, along with high-throughput approaches, have provided new insights for understanding microalgal metabolites. With the improvement in metabolomic technology, sensitivity for the detection of cellular metabolites has increased, and it has directly enhanced our understanding of cellular metabolism and the corresponding metabolic pathways. Moreover, these metabolic techniques, along with other omics technologies, help us to characterize the changes in the cellular functioning of the different cells under environmental conditions. Metabolomic research on microalgae explores new metabolites and the understanding of their biosynthesis at the metabolic level. In this review, we discuss how these metabolomics techniques are applied to microalgae to study their metabolic networks. Additionally, we also discuss how integrating other tools of systems biology can improve the productivity of microalgal cells, especially for industrially important commodities.

Carbohydrate Derived Value-added Products from Lignocelluloses

Abstract: Chemistry is confronted with the pressing issues of depleting non-renewable fossil resources and the imperative to combat environmental pollution, which is crucial for a sustainable future. Biomass stands out as the sole organic carbon source in nature among the array of sustainable resources available, positioning it as a prime substitute for fossil-derived chemicals and fuels. Extensive research has been conducted on the abundant lignocelluloses as a potential source for biofuels, bioenergy, and various valuable products, wherein, the incorporation of various processes in biomass fractionation to separate biopolymers (such as lignin, cellulose, and hemicellulose) has the potential to enhance the overall value of the process. However, industrial demonstration of biomass utilization for commercial products has been limited due to the challenges posed by the recalcitrance and complexity of biomass. Therefore, there is a need for efficient reaction processes to enable the production of bio-chemicals and fuels from renewable lignocellulose. This review focuses on the latest chemical methods developed for producing value-added chemicals from biomass-derived cellulose as a renewable feedstock.

Biodegradation of phthalic acid esters by a novel marine bacterial strain RL-BY03: Characterization, metabolic pathway, bioaugmentation and genome analysis

Biodegradation is recognized as the main route for the decomposition of phthalic acid esters (PAEs) in nature, but the fate of PAEs in marine ecosystems is not well understood. Herein, a novel marine bacterium, Gordonia sihwaniensis RL-BY03, was identified and analyzed for its ability to degrade PAEs. Furthermore, the metabolic mechanism of di-(2-ethylhexyl) phthalate (DEHP) was examined through UPLC-MS/MS and genomic analysis. RL-BY03 could rely solely on several types of PAEs as its sole carbon source. Initial pH and temperature for DEHP degradation were optimized as 8.0 and 30°C, respectively. Surprisingly, RL-BY03 could simultaneously degrade ethyl acetate and DEHP and they could increase the cell surface hydrophobicity. DEHP degradation kinetics fitted well with the first-order decay model. The metabolic pathway of DEHP was deduced following the detection of five metabolic intermediates. Further, genes that are related to DEHP degradation were identified through genomic analysis and their expression levels were validated through RT-qPCR. A co-related metabolic pathway at biochemical and molecular level indicated that DEHP was turned into DBP and DEP by β-oxidation, which was further hydrolyzed into phthalic acid. Phthalic acid was utilized through catechol branch of β-ketoadipate pathway. Additionally, RL-BY03 exhibited excellent bioremediation potential for DEHP-contaminated marine samples. In general, these findings have the potential to enhance our understanding of the fate of PAEs in marine ecosystems.

Multi-omic profiling of a novel Myrothecium species reveals its potential mechanism of lignin degradation.

Lignin utilization is one of the key challenges in the valorziation of lignocellulose. Filamentous fungi are promising candidates for lignin degradation and mineralization. However, novel lignin-degrading species are underexplored and the mechanism of lignin degradation is not fully understood. Here we isolated and characterized a novel species, Myrothecium wuxin, capable of utilizing lignosulfonate as the sole carbon source. To understand the mechanism of lignin degradation, genomic, transcriptomic and metabolic analyses were performed. The genome was sequenced, and assembled to a size of 48.55 Mb, with a contig N50 size of 5.67Mb. A total of 14,221 protein-coding genes were predicted, including a high number of potential ligninolytic enzymes. Transcriptomic analysis revealed a pronounced effect of lignosulfonate on gene expression profiles. More than twenty intermediate aromatic metabolites were identified during lignosulfonate utilization. Through genomic annotation, the genes potentially involved in lignin degradation were identified, and more than nine metabolic pathways of lignin-derived aromatic intermediates were predicted, including the homogentisate pathway, benzoic acid pathway, as well as the tree-branched β-ketoadipate pathway. The genomic information will provide a valuable resource for lignin degradation, while the elucidated catabolic pathways and associated enzymes provide exciting biotechnological opportunities for lignin valorization and production of valuable chemicals.

Fungal degradation of phenylacetate focusing on CRISPR/Cas9-assisted characterization of two oxidative enzyme genes of Akanthomyces muscarius AM1091

The degradation of phenylacetate (PA) was investigated as a model to explore aromatic compound breakdown in the fungal system. Fungal strains capable of utilizing PA as their sole carbon source were isolated using a minimal solid medium supplemented with 0.5 % PA. Subsequent cultivation in minimum liquid medium revealed that selected fungal strains, including Trametes versicolor TV0876 and TV3295, Paecilomyces hepiali PH4477, and Akanthomyces muscarius AM1091, efficiently removed PA within 24 h. HPLC analysis of culture supernatants from various fungal strains revealed a time-dependent accumulation of 2-hydroxyphenylacetate (2-HPA) and 4-hydroxyphenylacetate (4-HPA), two key major metabolic products primarily found in ascomycetes and basidiomycetes, respectively. This suggests that the first hydroxylation of PA is catalyzed by two distinct hydroxylases, one for each fungal group. Furthermore, fungal species that make 4-HPA also produce phenylethanol (PE), indicating a distinct catabolic mechanism to remove PA by direct reduction of PA to PE. A. muscarius AM1091, identified as the most efficient PA degrader in this study, was studied further to determine the biochemical pathway of PA degradation. RNA-Seq and RT-PCR analyses of AM1091 revealed two oxidative enzyme genes, CYP1 and DIO4, upregulated in the presence of PA. Targeted disruption utilizing preassembled Cas9-gRNA ribonucleoprotein complexes and homologous DNAs harboring the URA3 gene as an auxotrophic marker resulted in the cyp1 and dio4 mutant strains. The cyp1 mutant was incapable of converting PA to 2-HPA, indicating its involvement in the C2 hydroxylation, whereas the dio4 mutant was unable to degrade 2,5-dihydroxyphenylacetate (2,5-DHPA), resulting in the accumulation of 2,5-DHPA. Our findings indicate that A. muscarius AM1091 degrades PA through the activities of CYP1 and DIO4 for the C2 hydroxylation and subsequent ring-opening reactions, respectively.

Extracytoplasmic polysaccharides control cellulosomal and non-cellulosomal systems in Herbivorax saccincola A7

Herbivorax saccincola A7 is an anaerobic alkali-thermophilic lignocellulolytic bacterium that possesses a cellulosome and high xylan degradation ability. To understand the expression profile of extracellular enzymes by carbon sources, quantitative real-time PCR was performed on all cellulosomal and non-cellulosomal enzyme genes of H. saccincola A7 using cellulose and xylan as carbon sources. The results confirmed that the scaffolding proteins of H. saccincola A7 were expressed. In general, the cellulosomal genes belonging to the glycoside hydrolase families 9, 10, 11, and 48 were repressed when xylan was the sole carbon source, but these genes were significantly induced in the presence of cellulose. These results indicate that cellulose, not xylan, is a key inducer of cellulosomal genes in H. saccincola A7. The RsgI-like proteins, which regulate a carbohydrate-sensing mechanism in Clostridium thermocellum, were also found to be encoded in the H. saccincola A7 genome. To confirm the regulation by RsgI-like proteins, the relative expression of σI1–σI4 factors was analyzed on both carbon sources. The expression of alternative σI1 and σI2 factors was enhanced by the presence of cellulose. By contrast, the expression of σI3 and σI4 factors was activated by both cellulose and xylan. Taken together, the results reveal that the cellulosomal and non-cellulosomal genes of H. saccincola A7 are regulated through a carbohydrate-sensing mechanism involving anti-σ regulator RsgI-like proteins.Key points• qRT-PCR performed on cellulosomal and non-cellulosomal genes of H. saccincola A7• Cellulose is a key inducer of the cellulosome of H. saccincola A7• H. saccincola A7 possesses a similar system of anti-σ regulator RsgI-like proteins

Production of L-lactic acid from methanol by engineered yeast Pichia pastoris.

Lactic acid (LA) serves as a widely used platform compound and has received significant attention as a raw material for synthesis of biodegradable polylactic acid. Currently, LA is mainly produced through microbial fermentation, but its high costs undermine its competitive advantage against other materials, necessitating the development of novel production routes. Methanol bioconversion represents an emerging low-carbon circular economy, where LA could become an outstanding representative product. This study successfully established an efficient methanol-based LA synthesis route in Pichia pastoris. Through systematic metabolic engineering strategies, including screening lactate dehydrogenase, modification of cofactor preference, blocking LA consumption pathway, and mitochondrial LA synthesis compartmentalization, 4.2 g/L L-LA was produced in fed-batch fermentation by using methanol as the sole carbon source. Through multi-dimensional and spatial engineering of enzyme, a cell factory was developed for efficient synthesis of L-LA, highlights the significant potential of the low-carbon synthesis route for L-LA via methanol bioconversion.