Systems biology and synthetic biology are increasingly used to examine and modulate complex biological systems. As such, many issues arising during scaling-up microbial production processes can be addressed using these approaches. We review differences between laboratory-scale cultures and larger-scale processes to provide a perspective on those strain characteristics that are especially important during scaling. Systems biology has been used to examine a range of microbial systems for their response in bioreactors to fluctuations in nutrients, dissolved gases, and other stresses. Synthetic biology has been used both to assess and modulate strain response, and to engineer strains to improve production. We discuss these approaches and tools in the context of their use in engineering robust microbes for applications in largescale production. Challenges during Industrial Scale-up Highlights Strain engineering in the laboratory often does not consider process requirements in larger-scale bioreactors. Systems and synthetic biology can be applied to design microbial strains that allow reliable and robust production on a large scale. Commercial microbial platforms should be selected and developed based on their relevance to final process goals.

Decarbonizing the air transportation sector remains one of the most challenging hurdles to mitigating climate change.

Concerns over climate change have necessitated a rethinking of our transportation infrastructure. One possible alternative to carbon-polluting fossil fuels are biofuels produced from a renewable carbon source using engineered microorganisms. Two biofuels, ethanol and biodiesel, have been made inroads to displacing petroleum-based fuels, but their penetration has been limited by the amounts that can be used in conventional engines and by cost. Advanced biofuels that mimic petroleum-based fuels are not limited by the amounts that can be used in existing transportation infrastructure, but have had limited penetration due to costs. In this review, we will discuss the advances in engineering microbial metabolism to produce advanced biofuels and prospects for reducing their costs.

Naturally occurring plasmids constitute a major category of mobile genetic elements responsible for harboring and transferring genes important in survival and fitness. A targeted evaluation of plasmidomes can reveal unique adaptations required by microbial communities. We developed a model system to optimize plasmid DNA isolation procedures targeted to groundwater samples which are typically characterized by low cell density (and likely variations in the plasmid size and copy numbers). The optimized method resulted in successful identification of several hundred circular plasmids, including some large plasmids (11 plasmids more than 50 kb in size, with the largest being 1.7 Mb in size). Several interesting observations were made from the analysis of plasmid DNA isolated in this study. The plasmid pool (plasmidome) was more conserved than the corresponding microbiome distribution (16S rRNA based). The circular plasmids were diverse as represented by the presence of seven plasmid incompatibility groups. The genes carried on these groundwater plasmids were highly enriched in metal resistance. Results from this study confirmed that traits such as metal, antibiotic, and phage resistance along with toxin-antitoxin systems are encoded on abundant circular plasmids, all of which could confer novel and advantageous traits to their hosts. This study confirms the ecological role of the plasmidome in maintaining the latent capacity of a microbiome, enabling rapid adaptation to environmental stresses. IMPORTANCE Plasmidomes have been typically studied in environments abundant in bacteria, and this is the first study to explore plasmids from an environment characterized by low cell density. We specifically target groundwater, a significant source of water for human/agriculture use. We used samples from a well-studied site and identified hundreds of circular plasmids, including one of the largest sizes reported in plasmidome studies. The striking similarity of the plasmid-borne ORFs in terms of taxonomical and functional classifications across several samples suggests a conserved plasmid pool, in contrast to the observed variability in the 16S rRNA-based microbiome distribution. Additionally, the stress response to environmental factors has stronger conservation via plasmid-borne genes as marked by abundance of metal resistance genes. Last, identification of novel and diverse plasmids enriches the existing plasmid database(s) and serves as a paradigm to increase the repertoire of biological parts that are available for modifying novel environmental strains.

Background: Many microbes used for the rapid discovery and development of metabolic pathways have sensitivities to final products and process reagents. Isopentenol (3-methyl-3-buten-1-ol), a biogasoline candidate, has an established heterologous gene pathway but is toxic to several microbial hosts. Reagents used in the pretreatment of plant biomass, such as ionic liquids, also inhibit growth of many host strains. We explored the use of Corynebacterium glutamicum as an alternative host to address these constraints. Results: We found C. glutamicum ATCC 13032 to be tolerant to both the final product, isopentenol, as well to three classes of ionic liquids. A heterologous mevalonate-based isopentenol pathway was engineered in C. glutamicum. Targeted proteomics for the heterologous pathway proteins indicated that the 3-hydroxy-3-methylglutaryl-coenzyme A reductase protein, HmgR, is a potential rate-limiting enzyme in this synthetic pathway. Isopentenol titers were improved from undetectable to 1.25 g/L by combining three approaches: media optimization; substitution of an NADH-dependent HmgR homolog from Silicibacter pomeroyi; and development of a C. glutamicum ∆poxB ∆ldhA host chassis. Conclusions: We describe the successful expression of a heterologous mevalonate-based pathway in the Grampositive industrial microorganism, C. glutamicum, for the production of the biogasoline candidate, isopentenol. We identified critical genetic factors to harness the isopentenol pathway in C. glutamicum. Further media and cultivation optimization enabled isopentenol production from sorghum biomass hydrolysates.

Synthetic biology enables microbial hosts to produce complex molecules that are otherwise produced by organisms that are rare or di cult to cultivate, but the structures of these molecules are limited to those formed by chemical reactions catalyzed by natural enzymes. The integration of arti cial metalloenzymes (ArMs) that catalyze unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically by microorganisms. We report an engineered microbial cell expressing a heterologous biosynthetic pathway, which contains both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. To create this hybrid biosynthetic organism, we engineered Escherichia coli (E. coli) with a heterologous terpene biosynthetic pathway and an ArM containing an iridium-porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titer of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, together with natural and arti cial enzymes in whole cells, molecules that were previously inaccessible to nature.

Near-term decarbonization of aviation requires energy-dense, renewable liquid fuels. Biomass-derived 1,4-dimethylcyclooctane (DMCO), a cyclic alkane with a volumetric net heat of combustion up to 9.2% higher than Jet A, has the potential to serve as a low-carbon, high-performance jet fuel blendstock that may enable paraffinic bio-jet fuels to operate without aromatic compounds. DMCO can be produced from bio-derived isoprenol (3-methyl-3buten-1-ol) through a multistep upgrading process. This study presents detailed process configurations for DMCO production to estimate the minimum selling price and life-cycle greenhouse gas (GHG) footprint considering three different hydrogenation catalysts and two bioconversion pathways. The platinum-based catalyst offers the lowest production cost and GHG footprint of $9.0/L-Jet-A eq and 61.4 gCO 2e /MJ, given the current state of technology. However, when the supply chain and process are optimized, hydrogenation with a Raney nickel catalyst is preferable, resulting in a $1.5/L-Jet-A eq cost and 18.3 gCO 2e /MJ GHG footprint if biomass sorghum is the feedstock. This price point requires dramatic improvements, including 28 metric-ton/ha sorghum yield and 95−98% of the theoretical maximum conversion of biomass-to-sugars, sugars-to-isoprenol, isoprenol-to-isoprene, and isoprene-to-DMCO. Because increased gravimetric energy density of jet fuels translates to reduced aircraft weight, DMCO also has the potential to improve aircraft efficiency, particularly on long-haul flights.

While Pseudomonas putida KT2440 has great potential for biomass-converting processes, its inability to utilize the biomass abundant sugars xylose and galactose has limited its applications. In this study, we utilized Adaptive Laboratory Evolution (ALE) to optimize engineered KT2440 with heterologous expression of xylD encoding xylonate dehydratase from Caulobacter crescentus and galETKM encoding UDP-glucose 4-epimerase, galactose-1-phosphate uridylyltransferase, galactokinase, and galactose-1-epimerase from Escherichia coli K-12 MG1655. Poor starting strain growth (<0.1 h −1 or none) was evolutionarily optimized to rates of up to 0.25 h −1 on xylose and 0.52 h −1 on galactose. Wholegenome sequencing, transcriptomic analysis, and growth screens revealed significant roles of kguT encoding a 2-ketogluconate operon repressor and 2-ketogluconate transporter, and gtsABCD encoding an ATP-binding cassette (ABC) sugar transporting system in xylose and galactose growth conditions, respectively. Finally, we expressed the heterologous indigoidine production pathway in the evolved and unevolved engineered strains and successfully produced 3.2 g/L and 2.2 g/L from 10 g/L of either xylose or galactose in the evolved strains whereas the unevolved strains did not produce any detectable product. Thus, the generated KT2440 strains have the potential for broad application as optimized platform chassis to develop efficient microorganism-based biomass-utilizing bioprocesses.