Turning waste into treasure: A new direction for low-cost production of lipid chemicals from Thraustochytrids.

Thraustochytrids are marine microorganisms known for their fast growth and ability to store lipids, making them useful for producing polyunsaturated fatty acids (PUFAs), biodiesel, squalene, and carotenoids. However, the high cost of production, mainly due to expensive fermentation components, limits their wider use. A significant challenge in this context is the need to balance production costs with the value of the end products. This review focuses on integrating the efficient utilization of waste with Thraustochytrids fermentation, including the economic substitution of carbon sources, nitrogen sources, and fermentation water. This approach aligns with the 3Rs principles (reduction, recycling, and reuse). Furthermore, it emphasizes the role of Thraustochytrids in converting waste into lipid chemicals and promoting sustainable circular production models. The aim of this review is to emphasize the value of Thraustochytrids in converting waste into treasure, providing precise cost reduction strategies for future commercial production.

Enhanced fatty acid storage combined with the multi-factor optimization of fermentation for high-level production of docosahexaenoic acid in Schizochytrium sp.

Schizochytrium sp. hasreceived much attention for itsability to synthesize and accumulate high-level docosahexaenoic acid (DHA), which can reach nearly 40 % of total fatty acids. In this study, the titer of DHA in Schizochytrium sp. was successfully improved by enhancing DHA storage through overexpressing the diacylglycerol acyltransferase (ScDGAT2C) gene, as well as optimizing the supply of precursors and cofactors required for DHA synthesis by response surface methodology. Notably, malic acid, citric acid, and biotin showed synergistic and time-dependent effects on DHA accumulation. The maximum lipid and DHA titers of the engineered Schizochytrium sp. strain reached 84.28 ± 1.02 g/L and 42.23 ± 0.69 g/L, respectively, with the optimal concentration combination (1.62 g/L malic acid + 0.37 g/L citric acid + 8.28 mg/L biotin) were added 48 h after inoculation. This study provides an effective strategy for improving lipid and DHA production in Schizochytrium sp.

Identification of lipid synthesis genes in Schizochytrium sp. and their application in improving eicosapentaenoic acid synthesis in Yarrowia lipolytica.

BACKGROUND: Eicosapentaenoic acid (EPA) is widely used in the functional food and nutraceutical industries due to its important benefits to human health. Oleaginous microorganisms are considered a promising alternative resource for the production of EPA lipids. However, the storage of EPA in triglyceride (TG) becomes a key factor limiting its level. RESULTS: This study aimed to incorporate more EPA into TG storage through metabolic engineering. Firstly, key enzymes for TG synthesis, the diacylglycerol acyltransferase (DGAT) and glycerol-3-phosphate acyltransferase (GPAT) genes from Schizochytrium sp. HX-308 were expressed in Yarrowia lipolytica to enhance lipid and EPA accumulation. In addition, engineering the enzyme activity of DGATs through protein engineering was found to be effective in enhancing lipid synthesis by replacing the conserved motifs "HFS" in ScDGAT2A and "FFG" in ScDGAT2B with the motif "YFP". Notably, combined with lipidomic analysis, the expression of ScDGAT2C and GPAT2 enhanced the storage of EPA in TG. Finally, the accumulation of lipid and EPA was further promoted by identifying and continuing to introduce the ScACC, ScACS, ScPDC, and ScG6PD genes from Schizochytrium sp., and the lipid and EPA titer of the final engineered strain reached 2.25 ± 0.03 g/L and 266.44 ± 5.74 mg/L, respectively, which increased by 174.39% (0.82 ± 0.02 g/L) and 282.27% (69.70 ± 0.80 mg/L) compared to the initial strain, respectively. CONCLUSION: This study shows that the expression of lipid synthesis genes from Schizochytrium sp. in Y. lipolytica effectively improves the synthesis of lipids and EPA, which provided a promising target for EPA-enriched microbial oil production.

Integration of genetic engineering and multi-factor fermentation optimization for co-production of carotenoid and DHA in Schizochytrium sp.

Schizochytrium sp., a microalga with high lipid content, holds the potential for co-producing docosahexaenoic acid (DHA) and carotenoids. In this study, the ability of Schizochytrium sp. to naturally produce carotenoids was systematically explored. Further, by enhancing the precursor supply of geranylgeranyl diphosphate, regulating carbon source through sugar limitation fermentation and employing a combination of response surface methodology and artificial neural networks to precisely optimize nitrogen sources, a new record of 43-fold increase in ß-carotene titer was achieved in the 5L bioreactor (653.2 mg/L). Meanwhile, a high DHA content was maintained (13.4 g/L). Furthermore, the use of corn stover hydrolysate has effectively lowered the production costs of carotenoid and DHA while sustaining elevated production levels (with total carotenoid titer and DHA titer reached 502.0 mg/L and 13.2 g/L, respectively). This study offers an efficient and cost-effective method for the co-production of carotenoid and DHA in Schizochytrium sp..

Enhancing the accumulation of lipid and docosahexaenoic acid in Schizochytrium sp. by co-overexpression of phosphopantetheinyl transferase and ω-3 fatty acid desaturase.

Docosahexaenoic acid (DHA) as one of ω-3 polyunsaturated fatty acids (PUFAs), plays a key role in brain development, and is widely used in food additives and the pharmaceutical industry. Schizochytrium sp. is often considered as a satisfactory strain for DHA industrialization. The aim of this study was to assess the feasibility of phosphopantetheinyl transferase (PPTase) and ω-3 fatty acid desaturase (FAD) for regulating DHA content in Schizochytrium sp. PPTase is essential to activate the polyketide-like synthase (PKS) pathway, which can transfer apo-acyl-carrier protein (apo-ACP) into holo-ACP, and plays a key role in DHA synthesis. Moreover, DHA and docosapentaenoic acid (DPA) are synthesized by the PKS pathway simultaneously, so high DPA synthesis limits the increase of DHA content. In addition, the detailed mechanisms of PKS pathway have not been fully elucidated, so it is difficult to improve DHA content by modifying PKS. However, ω-3 FAD can convert DPA into DHA, and it is the most direct and effective way to increase DHA content and reduce DPA content. Based on this, PPTase was overexpressed to enhance the synthesis of DHA by the PKS pathway, overexpressed ω-3 FAD to convert the co-product of the PKS pathway into DHA, and co-overexpressed PPTase and ω-3 FAD. With these strategies, compared with wild type, the final lipid, and DHA titer were 92.5 and 51.5 g L-1 , which increased by 46.4% and 78.1%, respectively. This study established an efficient DHA production strain, and provided some feasible strategies for industrial DHA production in Schizochytrium sp.

Metal cofactor regulation combined with rational genetic engineering of Schizochytrium sp. for high-yield production of squalene.

The increasing market demand for squalene requires novel biotechnological production platforms. Schizochytrium sp. is an industrial oleaginous host with a high potential for squalene production due to its abundant native acetyl-CoA pool. We first found that iron starvation led to the accumulation of 1.5 g/L of squalene by Schizochytrium sp., which was 40-fold higher than in the control. Subsequent transcriptomic and lipidomic analyses showed that the high squalene titer is due to the diversion of precursors from lipid biosynthesis and increased triglycerides (TAG) content for squalene storage. Furthermore, we constructed the engineered acetyl-CoA C-acetyltransferase (ACAT)-overexpressing strain 18S::ACAT, which produced 2.79 g/L of squalene, representing an 86% increase over the original strain. Finally, a nitrogen-rich feeding strategy was developed to further increase the squalene titer of the engineered strain, which reached 10.78 g/L in fed-batch fermentation, a remarkable 161-fold increase over the control. To our best knowledge, this is the highest squalene yield in thraustochytrids reported to date.

Progress of metabolic engineering for the production of eicosapentaenoic acid.

Eicosapentaenoic Acid (EPA) is an essential ω-3 polyunsaturated fatty acid for human health. Currently, high-quality EPA production is largely dependent on the extraction of fish oil, but this unsustainable approach cannot meet its rising market demand. Biotechnological approaches for EPA production from microorganisms have received increasing attention due to their suitability for large-scale production and independence of the seasonal or climate restrictions. This review summarizes recent research on different microorganisms capable of producing EPA, such as microalgae, bacteria, and fungi, and introduces the different EPA biosynthesis pathways. Notably, some novel engineering strategies have been applied to endow and improve the abilities of microorganisms to synthesize EPA, including the construction and optimization of the EPA biosynthesis pathway, an increase in the acetyl-CoA pool supply, the increase of NADPH and the inhibition of competing pathways. This review aims to provide an updated summary of EPA production.

Development of an Efficient Gene Editing Tool in Schizochytrium sp. and Improving Its Lipid and Terpenoid Biosynthesis.

Schizochytrium sp. HX-308 is a marine microalga with fast growth and high lipid content, which has potential as microbial cell factories for lipid compound biosynthesis. It is significant to develop efficient genetic editing tool and discover molecular target in Schizochytrium sp. HX-308 for lipid compound biosynthesis. In this study, we developed an efficient gene editing tool in HX-308 which was mediated by Agrobacterium tumefaciens AGL-1. Results showed that the random integration efficiency reached 100%, and the homologous recombination efficiency reached about 30%. Furthermore, the metabolic pathway of lipid and terpenoid biosynthesis were engineered. Firstly, the acetyl-CoA c-acetyltransferase was overexpressed in HX-308 with a strong constitutive promoter. With the overexpression of acetyl-CoA c-acetyltransferase, more acetyl-CoA was used to synthesize terpenoids, and the production of squalene, ß-carotene and astaxanthin was increased 5.4, 1.8, and 2.4 times, respectively. Interestingly, the production of saturated fatty acids and polyunsaturated fatty acids also changed. Moreover, three Acyl-CoA oxidase genes which catalyze the first step of ß-oxidation were knocked out using homologous recombination. Results showed that the production of lipids increased in the three knock-out strains. Our results demonstrated that the A. tumefaciens-mediated transformation method will be of great use for the study of function genes, as well as developing Schizochytrium sp. as a strong cell factory for producing high value products.

Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production.

Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PlacUV5), which is leakier and more active than wild-type lac promoter (PlacWT) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced PlacUV5 with other inducible promoters respectively, including arabinose promoter (ParaBAD), rhamnose promoter (PrhaBAD), tetracycline promoter (Ptet), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins.

Recent advances in the application of multiplex genome editing in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a widely used microorganism and a greatly popular cell factory for the production of various chemicals. In order to improve the yield of target chemicals, it is often necessary to increase the copy numbers of key genes or engineer the related metabolic pathways, which traditionally required time-consuming repetitive rounds of gene editing. With the development of gene-editing technologies such as meganucleases, TALENs, and the CRISPR/Cas system, multiplex genome editing has entered a period of rapid development to speed up cell factory optimization. Multi-copy insertion and removing bottlenecks in biosynthetic pathways can be achieved through gene integration and knockout, for which multiplexing can be accomplished by targeting repetitive sequences and multiple sites, respectively. Importantly, the development of the CRISPR/Cas system has greatly increased the speed and efficiency of multiplex editing. In this review, the various multiplex genome editing technologies in S. cerevisiae were summarized, and the principles, advantages, and the disadvantages were analyzed and discussed. Finally, the practical applications and future prospects of multiplex genome editing were discussed. KEY POINTS: • The development of multiplex genome editing in S. cerevisiae was summarized. • The pros and cons of various multiplex genome editing technologies are discussed. • Further prospects on the improvement of multiplex genome editing are proposed.

Developing GDi-CRISPR System for Multi-copy Integration in Saccharomyces cerevisiae.

In recent years, Saccharomyces cerevisiae has been widely used in the production of biofuels and value-added chemicals. To stably express the target products, it is necessary to integrate multiple target genes into the chromosome of S. cerevisiae. CRISPR multi-copy integration technology relying on delta sites has been developed, but it often requires the help of high-throughput screening or resistance markers, resulting in non-replicability and high cost. This study aims to develop a low-cost and easy-to-use multi-copy integration tool in S. cerevisiae. Firstly, twenty-one Cas proteins from different microorganisms were tested in S. cerevisiae to find the functional Cas proteins with optimal cleavage ability. Results showed that eight Cas proteins can complete gene editing. However, most of the transformants have low copy numbers, which may be caused by high cutting efficiency exceeding the repair rate. Therefore, the effect of donor translocation order was further investigated. Results showed that 4 copies were obtained by donor first translocation. Then, the gene drive delta site integration system by the CRISPR system (GDi-CRISPR) was developed by combining gene drive principle and CRISPR system. To be clear, the gRNA was put into donor fragments. Then, both of them were integrated into the genome, which can drive further cutting and repair due to increasing number of gRNA. Instead of high-throughput screening or resistance pressure, 6 copies were obtained in only 5-6 days using the GDi-CRISPR system. It is expected to further advance the development of S. cerevisiae multi-copy integration tools.

Biotechnological production of lipid and terpenoid from thraustochytrids.

As fungus-like protists, thraustochytrids have been increasingly studied for their faster growth rates and high lipid content. In the 1990s, thraustochytrids were used as docosahexaenoic acid (DHA) producers for the first time. Thraustochytrids genera, such as Thraustochytrium, Schizochytrium, and Aurantiochytrium have been developed and patented as industrial strains for DHA production. The high DHA yield is attributed to its unique and efficient polyketide-like synthase (PKS) pathway. Moreover, thraustochytrids possess a completed mevalonate (MVA) pathway, so it can be used as host for terpenoid production. In order to improve strain performance, the metabolic engineering strategies have been applied to promote or disrupt intracellular metabolic pathways, such as genetic engineering and addition of chemical activators. However, it is difficult to realize industrialization only by improving strain performance. Various operation strategies were developed to enlarge the production quantities from the laboratory-scale, including two-stage cultivation strategies, scale-up technologies and bioreactor design. Moreover, an economical and effective downstream process is also an important consideration for the industrial application of thraustochytrids. Downstream costs accounts for 20-60% of the overall process costs, which represents an attractive target for increasing the cost-competitiveness of thraustochytrids, including how to improve the efficiency of lipid extraction and the further application of biomass residues. This review aims to overview the whole lipid biotechnology of thraustochytrids to provide the background information for researchers.

Transposon-Associated CRISPR-Cas System: A Powerful DNA Insertion Tool.

The transposon-associated CRISPR-Cas system is emerging as a powerful tool for targeted genetic manipulation. Using this system, targeted genomic knockins and knockouts can be achieved, independently of the host-cell repair machinery, thereby significantly improving our ability to perform genome editing in a wider variety of cell types.

An emerging simple and effective approach to increase the productivity of thraustochytrids microbial lipids by regulating glycolysis process and triacylglycerols' decomposition.

BACKGROUND: The oleaginous microorganism Schizochytrium sp. is widely used in scientific research and commercial lipid production processes. However, low glucose-to-lipid conversion rate (GLCR) and low lipid productivity of Schizochytrium sp. restrict the feasibility of its use. RESULTS: Orlistat is a lipase inhibitor, which avoids triacylglycerols (TAGs) from hydrolysis by lipase. TAGs are the main storage forms of fatty acids in Schizochytrium sp. In this study, the usage of orlistat increased the GLCR by 21.88% in the middle stage of fermentation. Whereas the productivity of lipid increased 1.34 times reaching 0.73 g/L/h, the saturated fatty acid and polyunsaturated fatty acid yield increased from 21.2 and 39.1 to 34.9 and 48.5 g/L, respectively, indicating the advantages of using a lipase inhibitor in microbial lipids fermentation. Similarly, the system was also successful in Thraustochytrid Aurantiochytrium. The metabolic regulatory mechanisms stimulated by orlistat in Schizochytrium sp. were further investigated using transcriptomics and metabolomics. The results showed that orlistat redistributed carbon allocation and enhanced the energy supply when inhibiting the TAGs' degradation pathway. Therefore, lipase in Schizochytrium sp. prefers to hydrolyze saturated fatty acid TAGs into the ß-oxidation pathway. CONCLUSIONS: This study provides a simple and effective approach to improve lipid production, and makes us understand the mechanism of lipid accumulation and decomposition in Schizochytrium sp., offering new guidance for the exploitation of oleaginous microorganisms.

Thraustochytrid Cell Factories for Producing Lipid Compounds.

Thraustochytrids can accumulate over 150 g/l biomass, containing up to 55% lipids, without any genetic modification. Their broad substrate utilization capacity, several effective key metabolic pathways, and a well-developed suite of bioprocess engineering strategies all point toward great promise for the future development of these marine protists.