Metabolic engineering最新文献

Methanol, a rich one-carbon feedstock, can be massively produced from CO₂ by the liquid sunshine route, which is helpful to realize carbon neutrality. β-Farnesene is widely used in the production of polymers, surfactants, lubricants, and also serves as a suitable substitute for jet fuel. Constructing an efficient cell factory is a feasible approach for β-farnesene production through methanol biotransformation. Here, we extensively engineered the methylotrophic yeast Ogataea polymorpha for the efficient bio-production of β-farnesene using methanol as the sole carbon source. Our study demonstrated that sufficient supply of precursor acetyl-CoA and cofactor NADPH in an excellent yeast chassis had a 1.3-fold higher β-farnesene production than that of wild-type background strain. Further optimization of the mevalonate pathway and enhancement of acetyl-CoA supply led to a 7-fold increase in β-farnesene accumulation, achieving the highest reported sesquiterpenoids production (14.7 g/L with a yield of 46 mg/g methanol) from one-carbon feedstock under fed-batch fermentation in bioreactor. This study demonstrates the great potential of engineering O. polymorpha for high-level terpenoid production from methanol.

Tanshinones and phenolic acids are the two main chemical constituents in Salvia miltiorrhiza, which are used clinically for the treatment of hypertension, coronary heart disease, atherosclerosis, and many other diseases, and have broad medicinal value. The efficient synthesis of the target products of these two metabolites in isolated plant tissues cannot be achieved without the regulation and optimization of metabolic pathways, and transcription factors play an important role as common regulatory elements in plant tissue metabolic engineering. However, most of the regulatory effects are specific to one class of metabolites, or an opposing regulation of two classes of metabolites exists. In this study, an artificially modified transcription factor, SmMYB36-VP16, was constructed to enhance tanshinones and phenolic acids in Salvia miltiorrhiza hair roots simultaneously. Further in combination with the elicitors dual-screening technique, by applying the optimal elicitors screened, the tanshinones content in the transgenic hairy roots of Salvia miltiorrhiza reached 6.44 mg/g DW, which was theoretically 6.08-fold that of the controls without any treatment, and the content of phenolic acids reached 141.03 mg/g DW, which was theoretically 5.05-fold that of the controls without any treatment. The combination of artificially modified transcriptional regulatory and elicitors dual-screening techniques has facilitated the ability of plant isolated tissue cell factories to produce targeted medicinal metabolites. This strategy could be applied to other species, laying the foundation for the production of potential natural products for the medicinal industry.

Advancing the formation of artificial membraneless compartments with organizational complexity and diverse functionality remains a challenge. Typically, synthetic compartments or membraneless organelles are made up of intrinsically disordered proteins featuring low-complexity sequences or polypeptides with repeated distinctive short linear motifs. In order to expand the repertoire of tools available for the formation of synthetic membraneless compartments, here, a range of DIshevelled and aXin (DIX) or DIX-like domains undergoing head-to-tail polymerization were demonstrated to self-assemble into aggregates and generate synthetic compartments within E. coli cells. Then, synthetic complex compartments with diverse intracellular morphologies were generated by coexpressing different DIX domains. Further, we genetically incorporated a pair of interacting motifs, comprising a homo-dimeric domain and its anchoring peptide, into the DIX domain and cargo proteins, respectively, resulting in the alteration of both material properties and client recruitment of synthetic compartments. As a proof-of-concept, several human milk oligosaccharide biosynthesis pathways were chosen as model systems. The findings indicated that the recruitment of pathway sequential enzymes into synthetic compartments formed by DIX–DIX heterotypic interactions or by DIX domains embedded with specific interacting motifs efficiently boosted metabolic pathway flux and improved the production of desired chemicals. We propose that these synthetic compartment systems present a potent and adaptable toolkit for controlling metabolic flux and facilitating cellular engineering.

Despite being present in trace amounts, ethyl esters play a crucial role as flavour compounds in lager beer. In yeast, ethyl hexanoate, ethyl octanoate and ethyl decanoate, responsible for fruity and floral taste tones, are synthesized from the toxic medium chain acyl-CoA intermediates released by the fatty acid synthase complex during the fatty acid biosynthesis, as a protective mechanism. The aim of this study was to enhance the production of ethyl esters in the hybrid lager brewing yeast Saccharomyces pastorianus by improving the medium chain acyl-CoA precursor supply. Through CRISPR-Cas9-based genetic engineering, specific FAS1 and FAS2 genes harbouring mutations in domains of the fatty acid synthesis complex were overexpressed in a single and combinatorial approach. These mutations in the ScFAS genes led to specific overproduction of the respective ethyl esters: overexpression of ScFAS1^I306A and ScFAS2^G1250S significantly improved ethyl hexanoate production and ScFAS1^R1834K boosted the ethyl octanoate production. Combinations of ScFAS1 mutant genes with ScFAS2^G1250S greatly enhanced predictably the final ethyl ester concentrations in cultures grown on full malt wort, but also resulted in increased levels of free medium chain fatty acids causing alterations in flavour profiles. Finally, the elevated medium chain fatty acid pool was directed towards the ethyl esters by overexpressing the esterase ScEEB1. The genetically modified S. pastorianus strains were utilized in lager beer production, and the resulting beverage exhibited significantly altered flavour profiles, thereby greatly expanding the possibilities of the flavour palette of lager beers.

Advanced microbiome therapeutics have emerged as a powerful approach for the treatment of numerous diseases. While the genetic instability of genetically engineered microorganisms is a well-known challenge in the scale-up of biomanufacturing processes, it has not yet been investigated for advanced microbiome therapeutics. Here, the evolution of engineered Escherichia coli Nissle 1917 strains producing Interleukin 2 and Aldafermin were investigated in two strain backgrounds with and without the three error-prone DNA polymerases polB, dinB, and umuDC, which contribute to the mutation rate of the host strain. Whole genome short-read sequencing revealed the genetic instability of the pMUT-based production plasmid after serial passaging for approximately 150 generations using an automated platform for high-throughput microbial evolution in five independent lineages for six distinct strains. While a reduction of the number of mutations of 12%–43% could be observed after the deletion of the error-prone DNA polymerases, the interruption of production-relevant genes could not be prevented, highlighting the need for additional strategies to improve the stability of advanced microbiome therapeutics.

A bio-based production of chemical building blocks from renewable, sustainable and non-food substrates is one key element to fight climate crisis. Lactic acid, one such chemical building block is currently produced from first generation feedstocks such as glucose and sucrose, both requiring land and water resources. In this study we aimed for lactic acid production from methanol by utilizing Komagataella phaffii as a production platform. Methanol, a single carbon source has potential as a sustainable substrate as technology allows (electro)chemical hydrogenation of CO₂ for methanol production. Here we show that expression of the Lactiplantibacillus plantarum derived lactate dehydrogenase leads to L-lactic acid production in Komagataella phaffii, however, production resulted in low titers and cells subsequently consumed lactic acid again. Gene expression analysis of the methanol-utilizing genes AOX1, FDH1 and DAS2 showed that the presence of lactic acid downregulates transcription of the aforementioned genes, thereby repressing the methanol-utilizing pathway. For activation of the methanol-utilizing pathway in the presence of lactic acid, we constructed strains deficient in transcriptional repressors Nrg1, Mig1-1, and Mig1-2 as well as strains with overrepresentation of transcriptional activators Mxr1 and Mit1. While loss of transcriptional repressors had no significant impact on lactic acid production, overexpression of both transcriptional activators, MXR1 and MIT1, increased lactic acid titers from 4 g L⁻¹ to 17 g L⁻¹ in bioreactor cultivations.

Nanobodies are single-domain antibody fragments that have garnered considerable use as diagnostic and therapeutic agents as well as research tools. However, obtaining pure VHHs, like many proteins, can be laborious and inconsistent. High level cytoplasmic expression in E. coli can be challenging due to improper folding and insoluble aggregation caused by reduction of the conserved disulfide bond. We report a systems engineering approach leveraging engineered strains of E. coli, in combination with a two-stage process and simplified downstream purification, enabling improved, robust, soluble cytoplasmic nanobody expression, as well as rapid cell autolysis and purification. This approach relies on the dynamic control over the reduction potential of the cytoplasm, incorporates lysis enzymes for purification, and can also integrate dynamic expression of protein folding catalysts. Collectively, the engineered system results in more robust growth and protein expression, enabling efficient scalable nanobody production, and purification from high throughput microtiter plates, to routine shake flask cultures and larger instrumented bioreactors. We expect this system will expedite VHH development.

Characterizing the phenotypic diversity and metabolic capabilities of industrially relevant manufacturing cell lines is critical to bioprocess optimization and cell line development. Metabolic capabilities of production hosts limit nutrient and resource channeling into desired cellular processes and can have a profound impact on productivity. These limitations cannot be directly inferred from measured data such as spent media concentrations or transcriptomics. Here, we present an integrated multi-omic analysis pipeline combining exo-metabolomics, transcriptomics, and genome-scale metabolic network analysis and apply it to three antibody-producing Chinese Hamster Ovary cell lines to identify reprogramming features associated with high-producing clones and metabolic bottlenecks limiting product formation in an industrial bioprocess. Analysis of individual datatypes revealed a decreased nitrogenous byproduct secretion in high-producing clones and the topological changes in peripheral metabolic pathway expression associated with phase shifts. An integrated omics analysis in the context of the genome-scale metabolic model elucidated the differences in central metabolism and identified amino acid utilization bottlenecks limiting cell growth and antibody production that were not evident from exo-metabolomics or transcriptomics alone. Thus, we demonstrate the utility of a multi-omics characterization in providing an in-depth understanding of cellular metabolism, which is critical to efforts in cell engineering and bioprocess optimization.

Subcellular compartmentalization is a crucial evolution characteristic of eukaryotic cells, providing inherent advantages for the construction of artificial biological systems to efficiently produce natural products. The establishment of an artificial protein transport system represents a pivotal initial step towards developing efficient artificial biological systems. Peroxisome has been demonstrated as a suitable subcellular compartment for the biosynthesis of terpenes in yeast. In this study, an artificial protein transporter ScPEX5* was firstly constructed by fusing the N-terminal sequence of PEX5 from S. cerevisiae and the C-terminal sequence of PEX5. Subsequently, an artificial protein transport system including the artificial signaling peptide YQSYY and its enhancing upstream 9 amino acid (9AA) residues along with ScPEX5* was demonstrated to exhibit orthogonality to the internal transport system of peroxisomes in S. cerevisiae. Furthermore, a library of 9AA residues was constructed and selected using high throughput pigment screening system to obtain an optimized signaling peptide (oPTS1*). Finally, the ScPEX5*-oPTS1* system was employed to construct yeast cell factories capable of producing the sesquiterpene α-humulene, resulting in an impressive α-humulene titer of 17.33 g/L and a productivity of 0.22 g/L/h achieved through fed-batch fermentation in a 5 L bioreactor. This research presents a valuable tool for the construction of artificial peroxisome cell factories and effective strategies for synthesizing other natural products in yeast.