Biology subjects

Santala, V.

Publications and source records attributed to Santala, V..

15 recordsLinked to original sources

bioRxiv2026

Metabolic adaptation to TCA cycle deficiency in a strictly aerobic bacterium

The tricarboxylic acid (TCA) cycle is central to cellular metabolism, yet its disruption offers an attractive opportunity to redirect carbon toward biotechnological products. A key challenge is that severe TCA cycle interruption often results in auxotrophic phenotypes, limiting their practical application. Here, we used adaptive laboratory evolution to restore growth of the strictly aerobic bacterium Acinetobacter baylyi ADP1 following major disruption of the TCA cycle during growth on glucose minimal medium. Evolved strains recovered rapid growth through extensive reorganization of central metabolism, including reduced flux through the oxidative branch of the TCA cycle, enhanced anaplerotic cycling, increased acetate overflow, and remodeling of oxidative phosphorylation. Adaptation involved coordinated changes in quinone-linked electron transport and intracellular redox balancing, enabling continued growth despite reduced respiratory efficiency. These results demonstrate that adaptive evolution can overcome fundamental physiological constraints imposed by TCA cycle disruption and establish a framework for engineering carbon-partitioning phenotypes in aerobic production hosts.

bioengineering↗

bioRxiv2026

3D-Printable and Cytocompatible Hydrogel from Acinetobacter baylyi ADP1 Extracellular Matrix

Tissue engineering has advanced significantly, yet multicomponent hydrogels inspired by the compositional complexity of natural extracellular matrices (ECMs) are still underexplored. Most current hydrogels are based on single-component formulations, which can limit their biochemical and mechanical versatility. Developing synthetic multicomponent hydrogels remains challenging because it requires the controlled integration of multiple functional groups within a single material platform. Here, a biologically driven strategy is introduced by leveraging Acinetobacter baylyi ADP1, a bacterium that naturally produces extracellular polymeric substances (EPS) composed of a multicomponent matrix of polysaccharides and proteins. Through three-day cultivation and a simple extraction method, a hydrogel is obtained that can be methacrylated and photocrosslinked using red or blue light. This hydrogel is porous, cytocompatible, 3D-bioprintable, injectable, and undergoes rapid gelation for in situ crosslinking. This work highlights the potential of using bacterial-derived multicomponent hydrogels for biofabrication.

bioengineering↗

bioRxiv2026

Dual-Chassis Strategy for Bridging Adaptive Evolution and Rational Design for Synthetic Biology

Genome streamlining and pathway refactoring are powerful strategies for constructing controllable microbial chassis for both fundamental studies and applications. While rational design benefits from reduced genetic complexity, adaptive laboratory evolution (ALE) thrives on metabolic redundancy, creating a mismatch between optimal hosts for design and evolution. Here, we introduce a dual chassis framework (DUET) in which rational pathway construction and adaptive evolution are first carried out in an evolution-competent host, and the resulting optimized designs are subsequently transferred into a genetically stable chassis for deployment. Using the naturally evolvable bacterium Acinetobacter baylyi ADP1 and its genome-stabilized derivative (ISx), we applied this framework to the {beta}-ketoadipate pathway, a central hub for aromatic compound catabolism. We first streamlined the native network by deleting individual pathway branches and then engineered a minimal synthetic route that merges protocatechuate and catechol metabolism. Subsequent ALE enabled efficient growth through this synthetic pathway, and reverse-engineering identified key adaptive mutations underlying functional recovery. Both the synthetic pathway and the mutations were transferred unchanged into ISx, where robust growth was maintained without further adaptation. These results demonstrate that DUET enables portable, host-independent deployment of rational metabolic streamlining combined with evolution, providing a generalizable strategy for building reduced yet robust microbial platforms. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/729570v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@19ba68forg.highwire.dtl.DTLVardef@1d40eb4org.highwire.dtl.DTLVardef@1115597org.highwire.dtl.DTLVardef@10a50f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

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

Modulating Bacterial Nanocellulose Crystallinity through Post-Transcriptional Repression in Komagataeibacter xylinus

Bacterial nanocellulose (BC), a versatile and biodegradable polymer, has been extensively studied as an alternative biomaterial for various applications. For biomedical and packaging uses, where precise control over nanocellulose structure is essential, existing literature describes BC structural modification processes that involve additives or additional steps. With the aim to develop a programmable method to control the bulk microstructure, we developed sRNA-based post-transcriptional repression cassettes that allows precise regulation of the crystalline phase of BC. Before investigating the effects of post-transcriptional repression of bcsD, bcsZ, and ccpA genes, known to influence BC crystallinity, we validated the specificity of the sRNA repression cassette by targeting a genome-integrated red fluorescent protein, mRFP1. The observed growth inhibition (>80%) caused by overexpressed Hfq RNA chaperone in Komagataeibacter xylinus was alleviated ([≤] 23%) by its removal, resulting in a 43% reduction in relative mRFP1 expression. By varying the design of the repression cassette and the inducer concentrations, we successfully modulated the repression of the target genes [with relative expression reductions of 6%-34% for bcsD, 8%-24% for bcsZ, and 2%-20% for ccpA, as confirmed by qRT-PCR]. These gene repression levels led to statistically significant changes in the amorphous content of the BC microstructure, as demonstrated by X-ray diffraction and wide-angle X-ray scattering analysis.

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↗

bioRxiv2021

Characterization of highly ferulate-tolerant Acinetobacter baylyi ADP1 isolates by a rapid reverse-engineering method

Adaptive laboratory evolution (ALE) is a powerful approach for improving phenotypes of microbial hosts. Evolved strains typically contain numerous mutations that can be revealed by whole-genome sequencing. However, determining the contribution of specific mutations to new phenotypes is typically challenging and laborious. This task is complicated by factors such as the mutation type, the genomic context, and the interplay between different mutations. Here, a novel approach was developed to identify the significance of mutations in strains derived from Acinetobacter baylyi ADP1. This method, termed Rapid Advantageous Mutation ScrEening and Selection (RAMSES), was used to analyze mutants that emerged from stepwise adaptation to, and consumption of, high levels of ferulate, a common lignin-derived aromatic compound. After whole-genome sequence analysis, RAMSES allowed both rapid determination of effective mutations and seamless introduction of the beneficial mutations into the chromosomes of new strains with different genetic backgrounds. This simple approach to reverse-engineering exploits the natural competence and high recombination efficiency of ADP1. The growth advantage of transformants under selective pressure revealed key mutations in genes related to aromatic transport, including hcaE, hcaK, and vanK, and a gene, ACIAD0482, which is associated with lipopolysaccharide synthesis. This study provides insights into enhanced utilization of industrially relevant aromatic substrates and demonstrates the use of A. baylyi ADP1 as a convenient platform for strain development and evolution studies. ImportanceMicrobial conversion of lignin-enriched streams is a promising approach for lignin valorization. However, the lignin-derived aromatic compounds are toxic to cells at relevant concentrations. Adaptive laboratory evolution is a powerful approach to develop more tolerant strains, but revealing the underlying mechanisms behind phenotypic improvement typically involves laborious processes. We employed Acinetobacter baylyi ADP1, an aromatic compound degrading strain that may be useful for biotechnology. The natural competence and high recombination efficiency of strain ADP1 can be exploited for critical applications such as the breakdown of lignin and plastics, abundant polymers composed of aromatic subunits. The natural transformability of this bacterium enabled us to develop a novel approach that allows rapid screening of advantageous mutations from ALE-derived aromatic-tolerant ADP1 strains. We clarified the mechanisms and genetic targets for improved tolerance towards common lignin-derived aromatic compounds. This study facilitates metabolic engineering for lignin valorization.

microbiology↗

bioRxiv2021

A two-staged bacterial process coupling methanotrophic and heterotrophic bacteria for 1-alkene production from methane

Methane (CH4) is a sustainable carbon feedstock source for aerobic CH4-oxidizing bacteria (methanotrophs) to produce value-added chemicals. Under substrate-limited (e.g., CH4, oxygen and nitrogen) conditions, CH4 oxidation results in the production of various short-chain organic acids and platform chemicals. These CH4-derived products could be broadened by utilizing them as a feedstock for heterotrophic bacteria. As a proof of concept, a two-stage system for CH4 abatement and 1-alkene production was developed in this study. Types I and II methanotrophs, i.e., Methylobacter tundripaludum SV96 and Methylocystis rosea SV97, respectively, were investigated in batch tests under different CH4 and air supplementation schemes. CH4 oxidation under either microaerobic or aerobic conditions induced the production of formate, acetate, succinate, and malate in M. tundripaludum SV96, accounting for 4.8-7.0% of consumed CH4-carbon while M. rosea SV97 produced the same compounds except for malate, and with lower efficiency than M. tundripaludum SV96, accounting for 0.7-1.8% of consumed CH4-carbon For the first time, the organic acids-rich spent media of methanotrophs were successfully used for 1-alkene production using engineered Acinetobacter baylyi ADP1 tesA-undA cells. The highest yield of 1-undecene was obtained from spent medium of M. tundripaludum SV96 at 68.9 {+/-} 11.6 mol mol Csubstrate-1.

microbiology↗

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↗

bioRxiv2019

Enhanced Population Control in Synthetic Bacterial Consortium by Interconnected Carbon Cross-Feeding

Engineered microbial consortia can provide several advantages over monocultures in terms of utilization of mixed substrates, resistance to perturbations, and division of labor in complex tasks. However, maintaining stability, reproducibility, and control over population levels in variable conditions can be challenging in multi-species cultures. In our study, we modeled and constructed a synthetic symbiotic consortium with a genetically encoded carbon cross-feeding system. The system is based on strains of Escherichia coli and Acinetobacter baylyi ADP1, both engineered to be incapable of growing on glucose on their own. In a culture supplemented with glucose as the sole carbon source, growth of the two strains is afforded by the exchange of gluconate and acetate, resulting in inherent control over carbon availability and population balance. We investigated the system robustness in terms of stability and population control under different inoculum ratios, substrate concentrations, and cultivation scales, both experimentally and by modeling. To illustrate how the system might facilitate division of genetic circuits among synthetic microbial consortia, a green fluorescent protein sensitive to pH and a slowly-maturing red fluorescent protein were expressed in the consortium as measures of a circuits susceptibility to external and internal variability, respectively. The symbiotic consortium maintained stable and linear growth and circuit performance regardless of the initial substrate concentration or inoculum ratios. The developed cross-feeding system provides simple and reliable means for population control without expression of non-native elements or external inducer addition, being potentially exploitable in consortia applications involving precisely defined cell tasks or division of labor.

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

Production of alkanes from CO2 by engineered bacteria

BackgroundMicrobial biosynthesis of alkanes is considered a promising method for the sustainable production of drop-in fuels and chemicals. Carbon dioxide would be an ideal carbon source for these production systems, but efficient production of long carbon chains from CO2 is difficult to achieve in a single organism. A potential solution is to employ acetogenic bacteria for the reduction of CO2 to acetate, and engineer a second organism to convert the acetate into long-chain hydrocarbons.\n\nResultsIn this study, we demonstrate alkane production from CO2 by a system combining the acetogen Acetobacterium woodii and a non-native alkane producer Acinetobacter baylyi ADP1 engineered for alkane production. Nine synthetic two-step alkane biosynthesis pathways consisting of different aldehyde- and alkane-producing enzymes were combinatorically constructed and expressed in A. baylyi. The aldehyde-producing enzymes studied were AAR from Synechococcus elongatus, Acr1 from A. baylyi, and Ramo, a putative dehydrogenase, from Nevskia ramosa. The alkane-producing enzymes were ADOs from S. elongatus and Nostoc punctiforme, and CER1 from Arabidopsis thaliana. The performance of the pathways was evaluated with a twin-layer biosensor, which allowed the monitoring of both the intermediate, fatty aldehyde, as well as the alkane production. The highest alkane production, as indicated by the biosensor, was achieved with a pathway consisting of AAR and ADO from S. elongatus. The performance of this pathway was further improved by balancing the relative expression levels of the enzymes in order to limit the accumulation of the intermediate fatty aldehyde. Finally, the acetogen A. woodii was used to produce acetate from CO2 and H2, and the acetate was used for alkane production by the engineered A. baylyi, thereby leading to the net production of long-chain alkanes from CO2.\n\nConclusionsA modular system for the production of drop-in liquid fuels from CO2 was demonstrated. Among the studied synthetic pathways, the combination of ADO and AAR from S. elongatus was found to be the most efficient in heterologous alkane production in A. baylyi. Furthermore, limiting the accumulation of the fatty aldehyde intermediate was found to be beneficial for the alkane production.

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↗