Biology subjects

Efimova, E.

Publications and source records attributed to Efimova, E..

9 recordsLinked to original sources

bioRxiv2025

Metabolic engineering of Acinetobacter baylyi ADP1 for efficient utilization of pentose sugars and production of glutamic acid

Efficient utilization of pentose sugars is critical for advancing sustainable biomanufacturing using lignocellulosic biomass. However, many potential host strains capable of consuming glucose and lignin-derived monomers are unable to utilize pentose sugars. Here, we engineered Acinetobacter baylyi ADP1 for the utilization of D-xylose and L-arabinose. We first modelled different pentose utilization pathways using flux balance analysis to choose the most optimal pathway. A novel marker-free approach facilitated the seamless integration of the pentose catabolic gene clusters of the selected Weimberg pathway into the A. baylyi genome, generating strains capable of efficiently utilizing both D-xylose and L-arabinose as sole carbon sources without any additional engineering or adaptation. For D-xylose, the cells achieved the highest growth rate ({micro}=0.73 h-1) reported to date for non-native hosts engineered for pentose utilization. For L-arabinose, a growth rate of {micro}=0.35 h-1 was achieved, which also surpassed the growth rate on a native substrate of A. baylyi, glucose ({micro}=0.31 h-1). Importantly, pentose utilization occurred simultaneously with glucose utilization. We then applied metabolic flux analysis using 13C labeled xylose to reveal D-xylose metabolism in the engineered strain. To demonstrate the potential for value-added bioproduct synthesis, L-glutamate was selected as a target compound. Deletion of sucAB and gabT, and the overexpression of gdhA enabled the production of L-glutamate. With the engineered strain, a carbon yield of 34% during co-utilization with succinate and 70% via whole-cell catalysis using resting cells was achieved. This study establishes a robust platform for pentose utilization and value-added bioproduct synthesis in A. baylyi ADP1 and highlights the potential for further metabolic optimization.

bioengineering↗

bioRxiv2024

Metabolic engineering of Acinetobacter baylyi ADP1 for naringenin production

Naringenin, a flavanone and a precursor for a variety of flavonoids, has potential applications in the health and pharmaceutical sectors. The biological production of naringenin using genetically engineered microbes is considered as a promising strategy. The naringenin synthesis pathway involving chalcone synthase (CHS) and chalcone isomerase (CHI) relies on the efficient supply of key substrates, malonyl-CoA and coumaroyl-CoA. In this research, we utilized a soil bacterium, Acinetobacter baylyi ADP1, which exhibits several characteristics that make it a suitable candidate for naringenin biosynthesis; the strain naturally tolerates and can uptake and metabolize coumarate, a primary compound in alkaline-pretreated lignin and a precursor for naringenin production. A. baylyi ADP1 also produces intracellular lipids, such as wax esters, thereby being able to provide an excess of malonyl-CoA for naringenin biosynthesis. Moreover, the genomic engineering of this strain is notably straightforward. In the course of the construction of a naringenin-producing strain, the coumarate catabolism was eliminated by a single gene knockout ({Delta}hcaA) and various combinations of plant-derived CHS and CHI were evaluated. The best performance was obtained by a novel combination of genes encoding for a CHS from Hypericum androsaemum and a CHI from Medicago sativa, that enabled the production of 18 mg/L naringenin in batch cultivations from coumarate. Furthermore, the implementation of a fed-batch system led to a significant 3.7-fold increase (66 mg/L) in naringenin production. These findings underscore the potential of A. baylyi ADP1 as a host for naringenin biosynthesis as well as advancement of lignin-based bioproduction.

bioengineering↗

bioRxiv2024

Novel approach for carbon-wise utilization of lignin-related compounds by synergistically employing anaerobic and aerobic bacteria

Lignin is a highly abundant but strongly underutilized natural resource that could serve as a sustainable feedstock to produce chemicals by microbial cell factories. However, the production from lignin-related aromatics is hindered by limited substrate range and inefficient catabolism of the production hosts. Particularly, the aerobic demethylation reactions are energy-limited and cause growth inhibition and loss of CO2. Here, we present a novel approach for carbon-wise utilization of lignin-related aromatics by the integration of anaerobic and aerobic metabolisms. In practice, we employed an acetogenic bacterium Acetobacterium woodii for anaerobic O-demethylation of aromatic compounds, which distinctively differs from the aerobic demethylation; in the process, the carbon from the methoxyl groups is fixated together with CO2 to form acetate while the aromatic ring remains unchanged. These accessible end-metabolites were then utilized by an aerobic bacterium Acinetobacter baylyi ADP1. Finally, we demonstrated the production of muconic acid from guaiacol, an abundant but inaccessible substrate to most microbes, with a nearly equimolar yield with only a minor genetic engineering and without the need for additional organic carbon source. This study highlights the power of synergistic integration of distinctive metabolic features of bacteria, thus unlocking new opportunities for harnessing microbial cocultures in upgrading challenging feedstocks.

bioengineering↗

bioRxiv2022

Engineering cell morphology by CRISPR interference in Acinetobacter baylyi ADP1

Microbial production of intracellular compounds can be engineered by, for example, redirecting the carbon flux towards products and increasing the cell size. Potential engineering strategies include exploiting clustered regularly interspaced short palindromic repeats interference (CRISPRi)-based tools for controlling gene expression. Here, we applied CRISPRi for engineering Acinetobacter baylyi ADP1, a model bacterium for synthesizing intracellular storage lipids, namely wax esters. We firstly established an inducible CRISPRi system for strain ADP1, which enables tightly controlled repression of target genes. We then targeted the glyoxylate shunt to redirect carbon flow towards wax esters. Secondly, we successfully employed CRISPRi for modifying cell morphology by repressing ftsZ, an essential gene required for cell division, in combination with targeted knock-outs to generate significantly enlarged filamentous or spherical cells, respectively. The engineered cells sustained increased wax ester production metrics, demonstrating the potential of cell morphology engineering in the production of intracellular lipids.

bioengineering↗

bioRxiv2019

Wax ester production in nitrogen-rich conditions by metabolically engineered Acinetobacter baylyi ADP1

Metabolic engineering can be used as a powerful tool to redirect cell resources towards product synthesis, also in conditions that are not optimal. An example of a synthesis pathway strongly dependent on external conditions is the production of storage lipids, which typically requires high carbon/nitrogen ratio. Acinetobacter baylyi ADP1 is known for its ability to produce industrially interesting storage lipids, namely wax esters (WEs). Here, we engineered the central carbon metabolism of A. baylyi ADP1 by deletion of the gene aceA encoding for isocitrate lyase in order to allow redirection of carbon towards WEs. The production was further enhanced by overexpression of fatty acyl-CoA reductase Acr1 in the wax ester production pathway. This strategy led to 3-fold improvement in yield (0.075 g/g glucose) and 3.15-fold improvement in titer (1.82 g/L) and productivity (0.038 g/L/h) by a simple one-stage batch cultivation with glucose as carbon source. The engineered strain accumulated up to 27% WEs of cell dry weight. The titer and cellular WE content are the highest reported to date among microbes. We further showed that the engineering strategy alleviated the inherent requirement for high carbon/nitrogen ratio and demonstrated the production of wax esters using nitrogen-rich substrates including casamino acids, yeast extract and bakers yeast hydrolysate, which support biomass production but not WE production in wild-type cells. The study demonstrates the power of metabolic engineering in overcoming natural limitations in the production of storage lipids.

bioengineering↗

bioRxiv2018

Alkane and wax ester production from lignin derived molecules

Lignin has potential as a sustainable feedstock for microbial production of industrially relevant molecules. However, the required lignin depolymerization yields a heterogenic mixture of aromatic monomers that are challenging substrates for the microorganisms commonly used in industry. Here, we investigated the properties of lignin-derived molecules (LDMs), namely coumarate, ferulate, and caffeate, in the synthesis of biomass and products in a LDM-utilizing bacterial host Acinetobacter baylyi ADP1. The biosynthesis products, wax esters and alkanes, are relevant compounds for the chemical and fuel industries. In A. baylyi ADP1, wax esters are produced by a native pathway, whereas alkanes are produced by a synthetic pathway introduced to the host. Using individual LDMs as substrates, the growth, product formation, and toxicity to cells were monitored with internal biosensors. Of the tested LDMs, coumarate was the most propitious in terms of product synthesis. Wax esters were produced from coumarate with a yield and titer of 40 mg /gcoumarate and 221 mg/L, whereas alkanes were produced with a yield of 62.3 g /gcoumarate and titer of 152 g/L. This study demonstrates the microbial preference for certain LDMs, and highlights the potential of A. baylyi ADP1 as a convenient host for LDM upgrading to value-added products.

bioengineering↗

bioRxiv2018

Synthetic Metabolic Pathway for the Production of 1-Alkenes from Lignin-derived Molecules

Integration of synthetic metabolic pathways to catabolically diverse chassis provides new opportunities for sustainable production. One attractive scenario is the use of abundant waste material to produce readily collectable product, minimizing production costs. Towards that end, we established the production of semivolatile medium-chain -olefins from lignin-derived monomers: we constructed 1-undecene synthesis pathway in Acinetobacter baylyi ADP1 using ferulate as the sole carbon source. In order to overcome the toxicity of ferulate, we first applied adaptive laboratory evolution, resulting in a highly ferulate-tolerant strain. Next, we demonstrated the 1-undecene production from glucose by heterologously expressing a fatty acid decarboxylase UndA and a thioesterase TesA in the wild type strain. Finally, we constructed the alkene synthesis pathway in the ferulate-tolerant strain. We were able to produce 1-undecene from ferulate and collect the product from the culture headspace without downstream processing. This study demonstrates the potential of bacterial lignin upgradation into value-added products.

bioengineering↗

bioRxiv2018

Dynamic decoupling of biomass and lipid biosynthesis by autonomously regulated switch

For improving the microbial production of fuels and chemicals, gene knock-outs and overexpression are routinely applied to intensify the carbon flow from substrate to product. However, their possibilities in dynamic control of the flux between the biomass and product synthesis are limited, whereas dynamic metabolic switches can be used for optimizing the distribution of carbon and resources. The production of single cell oils is especially challenging, as the synthesis is strongly regulated, competes directly with biomass, and requires defined conditions, such as nitrogen limitation. Here, we engineered a metabolic switch for redirecting carbon flow from biomass to wax ester production in Acinetobacter baylyi ADP1 using acetate as a carbon source. Isocitrate lyase, an essential enzyme for growth on acetate, was expressed under an arabinose inducible promoter. The autonomous downregulation of the expression is based on the gradual oxidation of the arabinose inducer by a glucose dehydrogenase gcd. The depletion of the inducer, occurring simultaneously to acetate consumption, switches the cells from a biomass mode to a lipid synthesis mode, enabling the efficient channelling of carbon to wax esters in a simple batch culture. In the engineered strain, the yield and titer of wax esters were improved by 3.8 and 3.1 folds, respectively, over the control strain. In addition, the engineered strain accumulated wax esters 19% of cell dry weight, being the highest reported among microbes. The study provides important insights into the dynamic engineering of the biomass-dependent synthesis pathways for the improved production of biocompounds from low-cost, sustainable substrates.\n\nSignificance statementIn the biological production, one of the greatest challenges is to find ways for optimal distribution of resources between cell growth, maintenance, and product synthesis. Robust and reliable circuits are required to allow autonomous switching of cells from biomass mode to lipid synthesis mode. Dynamic production of single cell oils such as triacylglycerols and wax esters is especially challenging due to the strong regulation. We present a dynamic genetic circuit based on conditional knockdown of a glyoxylate shunt enzyme, which is essential for cell growth. By gradual repression of the gene, the cells autonomously switch from biomass mode to product synthesis mode. We demonstrate the functionality of the circuit by using bacterium Acinetobacter baylyi ADP1 for the production of long chain alkyl esters, namely wax esters, with titer and yield improved by over 3-fold using acetate as the carbon source.

synthetic biology↗

bioRxiv2018

Metabolic pairing of aerobic and anaerobic production in a one-pot batch cultivation

BackgroundThe versatility of microbial metabolic pathways enables their utilization in vast number of applications. However, the electron and carbon recovery rates, essentially constrained by limitations of cell energetics, are often too low in terms of process feasibility. Cocultivation of divergent microbial species in a single process broadens the metabolic landscape and thus, the possibilities for more complete carbon and energy utilization.\n\nResultsIn this study, we integrated the metabolisms of two bacteria, an obligate anaerobe Clostridium butyricum and an obligate aerobe Acinetobacter baylyi ADP1. In the process, a glucose-negative mutant of A. baylyi ADP1 first deoxidized the culture allowing C. butyricum to grow and produce hydrogen from glucose. In the next phase, ADP1 produced long chain alkyl esters utilizing the by-products of C. butyricum, namely acetate and butyrate.\n\nConclusionsThe cocultivation of strictly anaerobic and aerobic bacteria allowed the production of both hydrogen gas and long-chain alkyl esters in a simple one-pot batch process. The study demonstrates the potential of metabolic pairing using designed microbial consortia for optimal electron and carbon recovery.

bioengineering↗