With more than 130 clinical trials and 8 approved gene therapy products, adeno-associated virus (AAV) stands as one of the most popular vehicles to deliver therapeutic DNA in vivo. One critical quality attribute analyzed in AAV batches is the presence of residual DNA, as it could pose genotoxic risks or induce immune responses. Surprisingly, the presence of small cell-derived RNAs, such as microRNAs (miRNAs), has not been investigated previously. In this study, we examined the presence of miRNAs in purified AAV batches produced in mammalian or in insect cells. Our findings revealed that miRNAs were present in all batches, regardless of the production cell line or capsid serotype (2 and 8). Quantitative assays indicated that miRNAs were co-purified with the recombinant AAV particles in a proportion correlated with their abundance in the production cells. The level of residual miRNAs was reduced via an immunoaffinity chromatography purification process including a tangential flow filtration step or by RNase treatment, suggesting that most miRNA contaminants are likely non-encapsidated. In summary, we demonstrate, for the first time, that miRNAs are co-purified with AAV particles. Further investigations are required to determine whether these miRNAs could interfere with the safety or efficacy of AAV-mediated gene therapy.

Synthetic biology encompasses many kinds of ideas and techniques with the common theme of creating something novel. The industrially relevant microorganism, Ralstonia eutropha (also known as Cupriavidus necator), has long been a subject of metabolic engineering efforts to either enhance a product it naturally makes (polyhydroxyalkanoate) or produce novel bioproducts (e.g., biofuels and other small molecule compounds). Given the metabolic versatility of R. eutropha and the existence of multiple molecular genetic tools and techniques for the organism, development of a synthetic biology toolkit is underway. This toolkit will allow for novel, user-friendly design that can impart new capabilities to R. eutropha strains to be used for novel application. This article reviews the different synthetic biology techniques currently available for modifying and enhancing bioproduction in R. eutropha. KEY POINTS: • R. eutropha (C. necator) is a versatile organism that has been examined for many applications. • Synthetic biology is being used to design more powerful strains for bioproduction. • A diverse synthetic biology toolkit is being developed to enhance R. eutropha\'s capabilities.

The production of biologics in mammalian cells is hindered by some limitations including high production costs, prompting the exploration of other alternative expression systems that are cheaper and sustainable like microalgae. Successful productions of biologics such as monoclonal antibodies have already been demonstrated in the diatom Phaeodactylum tricornutum; however, limited production yields still remain compared to mammalian cells. Therefore, efforts are needed to make this microalga more competitive as a cell biofactory. Among the seventeen reported accessions of P. tricornutum, ten have been mainly studied so far. Among them, some have already been used to produce high-value-added molecules such as biologics. The use of \"omics\" is increasingly being described as useful for the improvement of both upstream and downstream steps in bioprocesses using mammalian cells. Therefore, in this context, we performed an RNA-Seq analysis of the ten most used P. tricornutum accessions (Pt1 to Pt10) and deciphered the differential gene expression in pathways that could affect bioproduction of biologics in P. tricornutum. Our results highlighted the benefits of certain accessions such as Pt9 or Pt4 for the production of biologics. Indeed, these accessions seem to be more advantageous. Moreover, these results contribute to a better understanding of the molecular and cellular biology of P. tricornutum.

Applying low-cost substrate is critical for sustainable bioproduction. Co-culture of phototrophic and heterotrophic microorganisms can be a promising solution as they can use CO2 and light as feedstock. This study aimed to create a light-driven consortium using a marine cyanobacterium Synechococcus sp. PCC 7002 and an industrial yeast Yarrowia lipolytica. First, the cyanobacterium was engineered to accumulate and secrete sucrose by regulating the expression of genes involved in sucrose biosynthesis and transport, resulting in 4.0 g/L of sucrose secretion. Then, Yarrowia lipolytica was engineered to efficiently use sucrose and produce β-caryophyllene that has various industrial applications. Then, co- and sequential-culture were optimized with different induction conditions and media compositions. A maximum β-caryophyllene yield of 14.1 mg/L was obtained from the co-culture. This study successfully established an artificial light-driven consortium based on a marine cyanobacterium and Y. lipolytica, and provides a foundation for sustainable bioproduction from CO2 and light through co-culture systems.

Xylose is an abundant, inexpensive and readily available carbohydrate common in minimally processed feedstocks such as seaweed and algae. While a wide variety of marine microbes have evolved to utilize seaweed and algae, only a few currently have the requisite characteristics and genetic engineering tools necessary to entertain the use of these underutilized feedstocks. The rapidly growing Gram-negative halophilic bacterium Vibrio natriegens is one such chassis. In this study, we engineered and tested xylose induction in V. natriegens as a tool for scalable bioproduction applications. First, we created a sensing construct based on the xylose operon from Escherichia coli MG1665 and measured its activity using a fluorescent reporter and identified that cellular import plays a key role in induction strength and that expression required the XylR transcription factor. Next, we identified that select deletions of the promoter region enhance gene expression, limiting the effect of carbohydrate repression when xylose is used as an inducer in the presence of industrially relevant carbon sources. Lastly, we used the optimized constructs to produce the biopolymer melanin using seawater mimetic media. One of these formulations utilized a nori-based seaweed extract as an inducer and demonstrated melanin yields comparable to previously optimized methods using a more traditional and costly inducer. Together, the results demonstrate that engineering xylose induction in V. natriegens can provide an effective and lower cost option for timed biosynthesis in scalable biomanufacturing applications using renewable feedstocks.

BACKGROUND: Limonene has a variety of applications in the foods, cosmetics, pharmaceuticals, biomaterials, and biofuels industries. In order to meet the growing demand for sustainable production of limonene at industry scale, it is essential to find an alternative production system to traditional plant extraction. A promising and eco-friendly alternative is the use of microbes as cell factories for the synthesis of limonene.
RESULTS: In this study, the oleaginous yeast Yarrowia lipolytica has been engineered to produce D- and L-limonene. Four target genes, l- or d-LS (limonene synthase), HMG (HMG-CoA reductase), ERG20 (geranyl diphosphate synthase), and NDPS1 (neryl diphosphate) were expressed individually or fused together to find the optimal combination for higher limonene production. The strain expressing HMGR and the fusion protein ERG20-LS was the best limonene producer and, therefore, selected for further improvement. By increasing the expression of target genes and optimizing initial OD, 29.4 mg/L of L-limonene and 24.8 mg/L of D-limonene were obtained. We also studied whether peroxisomal compartmentalization of the synthesis pathway was beneficial for limonene production. The introduction of D-LS and ERG20 within the peroxisome improved limonene titers over cytosolic expression. Then, the entire MVA pathway was targeted to the peroxisome to improve precursor supply, which increased D-limonene production to 47.8 mg/L. Finally, through the optimization of fermentation conditions, D-limonene production titer reached 69.3 mg/L.
CONCLUSIONS: In this work, Y. lipolytica was successfully engineered to produce limonene. Our results showed that higher production of limonene was achieved when the synthesis pathway was targeted to the peroxisome, which indicates that this organelle can favor the bioproduction of terpenes in yeasts. This study opens new avenues for the efficient synthesis of valuable monoterpenes in Y. lipolytica.

Lignocellulosic biomass is currently underutilized, but it offers promise as a resource for the generation of commercial end-products, such as biofuels, detergents, and other oleochemicals. Rhodococcus opacus PD630 is an oleaginous, Gram-positive bacterium with an exceptional ability to utilize recalcitrant aromatic lignin breakdown products to produce lipid molecules such as triacylglycerols (TAGs), which are an important biofuel precursor. Lipid carbon storage molecules accumulate only under growth-limiting low nitrogen conditions, representing a significant challenge toward using bacterial biorefineries for fuel precursor production. In this work, we screened overexpression of 27 native transcriptional regulators for their abilities to improve lipid accumulation under nitrogen-rich conditions, resulting in three strains that accumulate increased lipids, unconstrained by nitrogen availability when grown in phenol or glucose. Transcriptomic analyses revealed that the best strain (#13) enhanced FA production via activation of the β-ketoadipate pathway. Gene deletion experiments confirm that lipid accumulation in nitrogen-replete conditions requires reprogramming of phenylalanine metabolism. By generating mutants decoupling carbon storage from low nitrogen environments, we move closer toward optimizing R. opacus for efficient bioproduction on lignocellulosic biomass.

Bioproduction of diverse N-acetyl chitooligosaccharides from chitin is of great value. In the study, a novel GH family 18 bifunctional chitinase gene (PsChi82) from Paenibacillus shirakamiensis was identified, expressed and biochemically characterized. PsChi82 was most active at pH 5.0, and 55 °C, and displayed remarkable pH stability with the broad pH range of 3.0-12.0. It showed high chitosanase activity of 10.6 U mg-1 and diverse hydrolysis products of GlcNAc, (GlcNAc)2, GlcN-GlcNAc and (GlcN)2-GlcNAc, which may facilitate comprehensively understanding of structure-function relationships of N-acetyl COSs. Three engineered variants were then expressed and characterized. Among them, PsChi82-CBM26 possessed specific activity of 25.1 U mg-1 against colloidal chitin, which was 2.1 folds higher than that of PsChi82. The diverse N-acetyl COSs were subsequently produced by PsChi82-CBM26 with a sugar content of 23.2 g L-1. These excellent properties may make PsChi82-CBM26 potentially useful for N-acetyl COSs production in the food and chemical industries.

Cyanobacteria have been proposed as a potential alternative carbohydrate feedstock and multiple species have been successfully engineered to secrete fermentable sugars. To date, the most productive cyanobacterial strains are those designed to secrete sucrose, yet there exist considerable differences in reported productivities across different model species and laboratories. In this study, we investigate how cultivation conditions (specifically, irradiance, CO2, and cultivator type) affect the productivity of sucrose-secreting Synechococcus elongatus PCC 7942. We find that S. elongatus produces the highest sucrose yield in irradiances far greater than what is often experimentally utilized, and that high light intensities are tolerated by S. elongatus, especially under higher density cultivation where turbidity may attenuate the effective light experienced in the culture. By increasing light and inorganic carbon availability, S. elongatus cscB/sps produced a total of 3.8 g L-1 of sucrose and the highest productivity within that period being 47.8 mg L-1 h-1. This study provides quantitative description of the impact of culture conditions on cyanobacteria-derived sucrose that may assist to standardize cross-laboratory comparisons and demonstrates a significant capacity to improve productivity via optimizing cultivation conditions.

Microbes have inherent capacities for utilizing various carbon sources, however they often exhibit sub-par fitness due to low metabolic efficiency. To test whether a bacterial strain can optimally utilize multiple carbon sources, Escherichia coli was serially evolved in L-lactate and glycerol. This yielded two end-point strains that evolved first in L-lactate then in glycerol, and vice versa. The end-point strains displayed a universal growth advantage on single and a mixture of adaptive carbon sources, enabled by a concerted action of carbon source-specialists and generalist mutants. The combination of just four variants of glpK, ppsA, ydcI, and rph-pyrE, accounted for more than 80% of end-point strain fitness. In addition, machine learning analysis revealed a coordinated activity of transcriptional regulators imparting condition-specific regulation of gene expression. The effectiveness of the serial adaptive laboratory evolution (ALE) scheme in bioproduction applications was assessed under single and mixed-carbon culture conditions, in which serial ALE strain exhibited superior productivity of acetoin compared to ancestral strains. Together, systems-level analysis elucidated the molecular basis of serial evolution, which hold potential utility in bioproduction applications.