Biology subjects

Eng, T. T.

Publications and source records attributed to Eng, T. T..

6 recordsLinked to original sources

bioRxiv2023

Sustainable production of 2,3,5,6-Tetramethylpyrazine at high titer in engineered Corynebacterium glutamicum using statistical design of experiments

BackgroundThe industrial amino acid production workhorse, Corynebacterium glutamicum naturally produces low levels of 2,3,5,6-tetramethylpyrazine (TMP), a valuable flavor, fragrance and commodity chemical. We have previously reported serendipitous production of TMP in C. glutamicum strains. In the present study, we demonstrate TMP production in C. glutamicum type strain ATCC13032 via the expression of a heterologous TMP pathway in a defined medium followed by statistical design of experiments to understand the effect of the media composition on TMP production. ResultsThe C. glutamicum strain engineered to overexpress acetolactate synthase and alpha-acetolactate decarboxylase from Lactococcus lactis produced [~]0.8 g/L TMP in CGXII minimal medium supplemented with 40 g/L glucose in 24-deep well plates. This engineered strain also demonstrated growth and TMP production when the minimal medium was supplemented with up to 40% (v/v) hydrolysates derived from ionic liquid pretreated sorghum biomass. A screen for improvements in media composition on TMP titer was conducted using fractional factorial design that identified glucose and urea as significant components affecting TMP production. These two components were further optimized using response surface methodology. In the optimized CGXII medium, the engineered strain could produce up to 3.56 g/L TMP (4-fold enhancement in titers and 2-fold enhancement in yield, mol/mol) from 80 g/L glucose and 11.9 g/L urea in shake flask batch cultivation. ConclusionsWe engineered the industrially relevant host, C. glutamicum for targeted production of TMP by heterologous expression of pathway proteins. We demonstrated the capability of the engineered strain for growth and TMP production utilizing real world carbon streams such as hydrolysates. We further identified glucose and urea as the key minimal media components significantly affecting TMP production using statistical media optimization.

synthetic biology↗

bioRxiv2023

Ensemble and Iterative Engineering for Maximized Bioconversion to the Blue Pigment, Indigoidine from Non-Canonical Sustainable Carbon Sources

While many heterologous molecules can be produced via microbial bioconversion processes, maximizing their titers, rates, and yields from lignin-derived carbon streams remains challenging. Growth coupling can not only increase titers and yields but also shift the production period from stationary phase to growth phase. These methods for designing growth-coupling strains however require multi-gene edits for implementation which may be perceived as impractical. Here, we computationally evaluated 4,114 potential solutions for growth coupling para-coumarate to indigoidine production and prototype two cut sets in Pseudomonas putida KT2440. We used adaptive laboratory evolution (ALE) on the initial triple deletion strain to restore growth on p-CA. Using X-ray tomography on this post-ALE strain we revealed increased cell density and decreased cell volume. Proteomics identified upregulated peroxidases that mitigate reactive oxygen species formation. Nine iterative stepwise modifications further informed by model-guided and rational approaches realized a growth coupled strain that produced 7.3 g/L indigoidine at 77% MTY in para-coumarate minimal medium. These ensemble strategies provide a blueprint for producing target molecules at high product titers, rates, and yields. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/532821v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@f0754aorg.highwire.dtl.DTLVardef@a45d24org.highwire.dtl.DTLVardef@df0596org.highwire.dtl.DTLVardef@1e7e140_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2021

Optimizing a High Performing Multiplex-CRISPRi P. putida strain with Integrated Metabolomics and 13C-Metabolic Flux Analyses

Microbial cell factory development often faces bottlenecks after initial rounds of design-build-test-learn (DBTL) cycles as engineered producers respond unpredictably to further genetic modifications. Thus, deciphering metabolic flux and correcting bottlenecks are key components of DBTL cycles. Here, a 14-gene edited Pseudomonas putida KT2440 strain for heterologous indigoidine production was examined using both 13C-metabolic flux analysis (13C-MFA) and metabolite measurements. The results indicated the conservation of the cyclic Entner-Doudoroff (ED)-EMP pathway flux, downregulation of the TCA cycle and pyruvate shunt, and glyoxylate shunt activation. At the metabolite level, the CRISPR/dCpf1-interference mediated multiplex repression decreased gluconate/2-ketogluconate secretion and altered several intracellular TCA metabolite concentrations, leading to succinate overflow. Further strain engineering based on the metabolic knowledge first employed an optimal ribosome binding site (RBS) to achieve stronger product-substrate growth coupling (1.6-fold increase). Then, deletion strains were constructed using ssDNA recombineering. Of the five strains tested, deletion of the PHA operon ({Delta}phaAZC-IID) resulted in a 2.2-fold increase in growth phase production compared to the optimal RBS construct. After 72 h of batch cultivation, the {Delta}phaAZC-IID strain had 1.5-fold and 1.8-fold increases of indigoidine titer compared to the improved RBS construct and the original strain, respectively. Overall, the findings provided actionable DBTL targets as well as insights into physiological responses and flux buffering when new recombineering tools were used for engineering P. putida KT2440.

synthetic biology↗

bioRxiv2021

High Throughput Fitness Profiling Reveals Loss Of GacS-GacA Regulation Improves Indigoidine Production In Pseudomonas putida

Pseudomonas putida KT2440 is an emerging industrial microbe amenable for use with renewable carbon streams including aromatics such as para-coumarate (pCA). We examined this microbe under common stirred-tank bioreactor parameters with quantitative fitness assays using a pooled transposon library containing nearly all (4,778) non-essential genes. Assessing differential fitness values by monitoring changes in mutant strain abundance over time identified 31 genes with improved fitness in multiple bioreactor-relevant parameters. Twenty-one genes from this subset were reconstructed, including GacA, a signaling protein, TtgB, an ABC transporter, and PP_0063, a lipid A acyltransferase. Twelve deletion strains with roles in varying cellular functions were evaluated for conversion of pCA, to a heterologous bioproduct, indigoidine. Several mutants, such as the {Delta}gacA strain improved both fitness in a bioreactor and showed an 8-fold improvement in indigoidine production (4.5 g/L, 0.29 g/g pCA, 23% MTY) from pCA as the carbon source.

systems biology↗

bioRxiv2020

Iron Supplementation Eliminates Antagonistic Interactions Between Root Associated Bacteria

The rhizosphere microbiome (rhizobiome) plays a critical role in plant health and development. However the processes by which the constituent microbes interact to form and maintain a community are not well understood. To investigate these molecular processes, we examined pairwise interactions between 11 different microbial isolates under selected nutrient-rich and nutrient-limited conditions. We observed that when grown with media supplemented with 56 mM glucose, 2 microbial isolates were able to inhibit the growth of 6 out of 11 other microbes tested. The interaction between microbes persisted even after the antagonistic microbe was removed, upon exposure to spent media. To probe the genetic basis for these antagonistic interactions, we used a barcoded transposon library in a proxy bacterium, Pseudomonas putida, to identify genes which showed enhanced sensitivity to the antagonistic factor(s) secreted by Acinetobacter sp. 02. Iron metabolism-related gene clusters in P. putida were implicated by this systems-level analysis. The supplementation of iron prevented the antagonistic interaction in the original microbial pair supporting the hypothesis that iron limitation drives antagonistic microbial interactions between rhizobionts. We conclude that rhizobiome community composition is influenced by competition for limiting nutrients with implications for growth and development of the plant.

microbiology↗

bioRxiv2020

Genome-scale metabolic rewiring to achieve predictable titers rates and yield of a non-native product at scale

Achieving high titer rates and yields (TRY) remains a bottleneck in the production of heterologous products through microbial systems, requiring elaborate engineering and many iterations. Reliable scaling of engineered strains is also rarely addressed in the first designs of the engineered strains. Both high TRY and scale are challenging metrics to achieve due to the inherent trade-off between cellular use of carbon towards growth vs. target metabolite production. We hypothesized that being able to strongly couple product formation with growth may lead to improvements across both metrics. In this study, we use elementary mode analysis to predict metabolic reactions that could be targeted to couple the production of indigoidine, a sustainable pigment, with the growth of the chosen host, Pseudomonas putida KT2440. We then filtered the set of 16 predicted reactions using -omics data. We implemented a total of 14 gene knockdowns using a CRISPRi method optimized for P. putida and show that the resulting engineered P. putida strain could achieve high TRY. The engineered pairing of product formation with carbon use also shifted production from stationary to exponential phase and the high TRY phenotype was maintained across scale. In one design cycle, we constructed an engineered P. putida strain that demonstrates close to 50% maximum theoretical yield (0.33 g indigoidine/g glucose consumed), reaching 25.6 g/L indigoidine and a rate of 0.22g/l/h in exponential phase. These desirable phenotypes were maintained from batch to fed-batch cultivation mode, and from 100ml shake flasks to 250 mL ambr(R) and 2 L bioreactors.

bioengineering↗