Biology subjects

Benites, V.

Publications and source records attributed to Benites, V..

4 recordsLinked to original sources

bioRxiv2020

A bimodular PKS platform that expands the biological design space

Traditionally engineered to produce novel bioactive molecules, Type I modular polyketide synthases (PKSs) could be engineered as a new biosynthetic platform for the production of de novo fuels, commodity chemicals, and specialty chemicals. Previously, our investigations manipulated the first module of the lipomycin PKS to produce short chain ketones, 3-hydroxy acids, and saturated, branched carboxylic acids. Building upon this work, we have expanded to multi-modular systems by engineering the first two modules of lipomycin to generate unnatural polyketides as potential biofuels and specialty chemicals in Streptomyces albus. First, we produce 20.6 mg/L of the ethyl ketone, 4,6 dimethylheptanone through a reductive loop exchange in LipPKS1 and a ketoreductase knockouts in LipPKS2. We then show that an AT swap in LipPKS1 and a reductive loop exchange in LipPKS2 can produce the potential fragrance 3-isopropyl-6-methyltetrahydropyranone. Highlighting the challenge of maintaining product fidelity, in both bimodular systems we observed side products from premature hydrolysis in the engineered first module and stalled dehydration in reductive loop exchanges. Collectively, our work expands the biological design space and moves the field closer to the production of “designer” biomolecules.HighlightsEngineered lipomycin module 1 and module 2 to produce unnatural polyketides as valuable bio-based chemicalsA reductive loop swap and ketoreductase knockout used to produce 20 mg/mL of a novel ethyl ketone, a gasoline replacementAn acyltransferase swap and reductive loop swap successfully produced δ-lactone, a potential fragrant compoundIncomplete reduction and premature hydrolysis observed in engineered modulesCompeting Interest StatementJ.D.K. has a financial interest in Amyris, Lygos, Demetrix, Napigen, Maple Bio, Berkeley Brewing Sciences, Ansa Biotech and Apertor Labs.View Full Text

synthetic biology↗

bioRxiv2019

Chemoinformatic-guided engineering of polyketide synthases

Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A significant challenge in PKS design is engineering a partially reductive module to produce a saturated {beta}-carbon through a reductive loop exchange. In this work, we sought to establish that chemoinformatics, a field traditionally used in drug discovery, could provide a viable strategy to reductive loop exchanges. We first introduced a set of donor reductive loops of diverse genetic origin and chemical substrate structures into the first extension module of the lipomycin PKS (LipPKS1). Product titers of these engineered unimodular PKSs correlated with atom pair chemical similarity between the substrate of the donor reductive loops and recipient LipPKS1, reaching a titer of 165 mg/L of short chain fatty acids produced by Streptomyces albus J1074 harboring these engineered PKSs. Expanding this method to larger intermediates requiring bimodular communication, we introduced reductive loops of divergent chemosimilarity into LipPKS2 and determined triketide lactone production. Collectively, we observed a statistically significant correlation between atom pair chemosimilarity and production, establishing a new chemoinformatic method that may aid in the engineering of PKSs to produce desired, unnatural products.

biochemistry↗

bioRxiv2019

The response of Pseudomonas putida to a complex lignolysate

There is strong interest in the valorization of lignin derived from plant biomass to produce valuable products; however, the structural complexity of this biopolymer has been a major bottleneck to conversion. Chemical pretreatment liberates soluble fractions of lignin that may be upgraded by biological conversion. Here, ionic liquid pretreatment was employed to obtain soluble aromatic-rich fractions from sorghum, which were converted by Pseudomonas putida KT2440, a promising host for bioconversion of aromatics derived from lignin. Growth studies and mutational analysis demonstrated that P. putida growth on these soluble lignin-derived fractions, referred to as lignolysate, was dependent on aromatic monomers derived from lignin (p-coumarate and ferulate), but other, unknown factors in the lignolysate contributed to growth. Proteomic and metabolomic analyses provided evidence that these unknown factors were amino acids and residual ionic liquid. Proteomic measurements indicated a coordinated response in which these substrates were catabolized simultaneously. A cholinium catabolic pathway was identified and deletion of five genes in the pathway abrogated the ability of P. putida to grow on cholinium as a sole carbon source. This work demonstrates that lignolysates obtained through biomass pretreatment contain multiple substrates and conversion strategies for lignin-derived should take this complexity into account.\n\nImportanceLignin is one of the most abundant biopolymers on Earth and is generated as a co-product in the processing of lignocellulosic biomass. Valorization of these residual lignin streams is a promising method to enhance the economic viability of modern lignocellulosic biorefineries. In this study we developed a process to couple chemical depolymerization of lignin and biological conversion using Pseudomonas putida KT2440. Water-soluble and bioavailable lignolysate was obtained from sorghum and further characterized as a growth substrate for P. putida. Proteomic and metabolomic analyses demonstrated that P. putida metabolized other components of the lignolysate beyond monoaromatic compounds, which illuminates how microbes can process complex lignolysates obtained from plants. Understanding the underlying microbial responses in lignolysates will enable the design of rational strategies for lignin valorization.

microbiology↗

bioRxiv2019

Host engineering for improved valerolactam production in Pseudomonas putida

Pseudomonas putida is a promising bacterial chassis for metabolic engineering given its ability to metabolize a wide array of carbon sources, especially aromatic compounds derived from lignin. However, this omnivorous metabolism can also be a hindrance when it can naturally metabolize products produced from engineered pathways. Herein we show that P. putida is able to use valerolactam as a sole carbon source, as well as degrade caprolactam. Lactams represent important nylon precursors, and are produced in quantities exceeding one million tons per year[1]. To better understand this metabolism we use a combination of Random Barcode Transposon Sequencing (RB-TnSeq) and shotgun proteomics to identify the oplBA locus as the likely responsible amide hydrolase that initiates valerolactam catabolism. Deletion of the oplBA genes prevented P. putida from growing on valerolactam, prevented the degradation of valerolactam in rich media, and dramatically reduced caprolactam degradation under the same conditions. Deletion of oplBA, as well as pathways that compete for precursors L-lysine or 5-aminovalerate, increased the titer of valerolactam from undetectable after 48 hours of production to ~90 mg/L. This work may serve as a template to rapidly eliminate undesirable metabolism in non-model hosts in future metabolic engineering efforts.

synthetic biology↗