Synthetic biology (Oxford, England)最新文献

[This corrects the article DOI: 10.1093/synbio/ysab007.].

Cell-free extract and purified enzyme-based systems provide an attractive solution to study biosynthetic strategies towards a range of chemicals. 4-(4-hydroxyphenyl)-butan-2-one, also known as raspberry ketone, is the major fragrance component of raspberry fruit and is used as a natural additive in the food and sports industry. Current industrial processing of the natural form of raspberry ketone involves chemical extraction from a yield of ∼1-4 mg kg^-1 of fruit. Due to toxicity, microbial production provides only low yields of up to 5-100 mg L^-1. Herein, we report an efficient cell-free strategy to probe into a synthetic enzyme pathway that converts either L-tyrosine or the precursor, 4-(4-hydroxyphenyl)-buten-2-one, into raspberry ketone at up to 100% conversion. As part of this strategy, it is essential to recycle inexpensive cofactors. Specifically, the final enzyme step in the pathway is catalyzed by raspberry ketone/zingerone synthase (RZS1), an NADPH-dependent double bond reductase. To relax cofactor specificity towards NADH, the preferred cofactor for cell-free biosynthesis, we identify a variant (G191D) with strong activity with NADH. We implement the RZS1 G191D variant within a 'one-pot' cell-free reaction to produce raspberry ketone at high-yield (61 mg L^-1), which provides an alternative route to traditional microbial production. In conclusion, our cell-free strategy complements the growing interest in engineering synthetic enzyme cascades towards industrially relevant value-added chemicals.

Formate is an attractive feedstock for sustainable microbial production of fuels and chemicals, but its potential is limited by the lack of efficient assimilation pathways. The reduction of formate to formaldehyde would allow efficient downstream assimilation, but no efficient enzymes are known for this transformation. To develop a 2-step formate reduction pathway, we screened natural variants of acyl-CoA synthetase (ACS) and acylating aldehyde dehydrogenase (ACDH) for activity on one-carbon substrates and identified active and highly expressed homologs of both enzymes. We then performed directed evolution, increasing ACDH-specific activity by 2.5-fold and ACS lysate activity by 5-fold. To test for the in vivo activity of our pathway, we expressed it in a methylotroph which can natively assimilate formaldehyde. Although the enzymes were active in cell extracts, we could not detect formate assimilation into biomass, indicating that further improvement will be required for formatotrophy. Our work provides a foundation for further development of a versatile pathway for formate assimilation.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has become a standard tool in many genome engineering endeavors. The endonuclease-deficient version of Cas9 (dCas9) is also a powerful programmable tool for gene regulation. In this study, we made use of Saccharomyces cerevisiae transcription factor (TF) binding data to obtain a better understanding of the interplay between TF binding and binding of dCas9 fused to an activator domain, VPR. More specifically, we targeted dCas9-VPR toward binding sites of Gcr1-Gcr2 and Tye7 present in several promoters of genes encoding enzymes engaged in the central carbon metabolism. From our data, we observed an upregulation of gene expression when dCas9-VPR was targeted next to a TF binding motif, whereas a downregulation or no change was observed when dCas9 was bound on a TF motif. This suggests a steric competition between dCas9 and the specific TF. Integrating TF binding data, therefore, proved to be useful for designing guide RNAs for CRISPR interference or CRISPR activation applications.

Biofoundry is a place where biomanufacturing meets automation. The highly modular structure of a biofoundry helps accelerate the design-build-test-learn workflow to deliver products fast and in a streamlined fashion. In this perspective, we describe our efforts to build Biofoundry India, where we see the facility add a substantial value in supporting research, innovation and entrepreneurship. We describe three key areas of our focus, harnessing the potential of non-expressing parts of the sequenced genomes, using deep learning in pathway reconstruction and synthesising enzymes and metabolites. Toward the end, we describe specific challenges in building such facility in India and the path to mitigate some of those working with the other biofoundries worldwide.

Lutein is an industrially important carotenoid pigment, which is essential for photoprotection and photosynthesis in plants. Lutein is crucial for maintaining human health due to its protective ability from ocular diseases. However, its pathway engineering research has scarcely been performed for microbial production using heterologous hosts, such as Escherichia coli, since the engineering of multiple genes is required. These genes, which include tricky key carotenoid biosynthesis genes typically derived from plants, encode two sorts of cyclases (lycopene ε- and β-cyclase) and cytochrome P450 CYP97C. In this study, upstream genes effective for the increase in carotenoid amounts, such as isopentenyl diphosphate isomerase (IDI) gene, were integrated into the E. coli JM101 (DE3) genome. The most efficient set of the key genes (MpLCYe, MpLCYb and MpCYP97C) was selected from among the corresponding genes derived from various plant (or bacterial) species using E. coli that had accumulated carotenoid substrates. Furthermore, to optimize the production of lutein in E. coli, we introduced several sorts of plasmids that contained some of the multiple genes into the genome-inserted strain and compared lutein productivity. Finally, we achieved 11 mg/l as lutein yield using a mini jar. Here, the high-yield production of lutein was successfully performed using E. coli through approaches of pathway engineering. The findings obtained here should be a base reference for substantial lutein production with microorganisms in the future.

Assembly of minimal genomes revealed many genes encoding unknown functions. Three overlooked functional categories account for some of them. Cells are prone to make errors and age. As a first key function, discrimination between proper and changed entities is indispensable. Discrimination requires management of information, an authentic, yet abstract, currency of reality. For example proteins age, sometimes very fast. The cell must identify, then get rid of old proteins without destroying young ones. Implementing discrimination in cells leads to the second set of functions, usually ignored. Being abstract, information must nevertheless be embodied into material entities, with unavoidable idiosyncratic properties. This brings about novel unmet needs. Hence, the buildup of cells elicits specific but awkward material implementations, 'kludges' that become essential under particular settings, while difficult to identify. Finally, a third functional category characterizes the need for growth, with metabolic implementations allowing the cell to put together the growth of its cytoplasm, membranes, and genome, spanning different spatial dimensions. Solving this metabolic quandary, critical for engineering novel synthetic biology chassis, uncovered an unexpected role for CTP synthetase as the coordinator of nonhomothetic growth. Because a significant number of SynBio constructs aim at creating cell factories we expect that they will be attacked by viruses (it is not by chance that the function of the CRISPR system was identified in industrial settings). Substantiating the role of CTP, natural selection has dealt with this hurdle via synthesis of the antimetabolite 3'-deoxy-3',4'-didehydro-CTP, recruited for antiviral immunity in all domains of life.