Evaluation of bacterial hosts for conversion of lignin-derived p-coumaric acid to 4-vinylphenol.

Hydroxycinnamic acids such as p-coumaric acid (CA) are chemically linked to lignin in grassy biomass with fairly labile ester bonds and therefore represent a straightforward opportunity to extract and valorize lignin components. In this work, we investigated the enzymatic conversion of CA extracted from lignocellulose to 4-vinylphenol (4VP) by expressing a microbial phenolic acid decarboxylase in Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis. The performance of the recombinant strains was evaluated in response to the substrate concentration in rich medium or a lignin liquor and the addition of an organic overlay to perform a continuous product extraction in batch cultures. We found that using undecanol as an overlay enhanced the 4VP titers under high substrate concentrations, while extracting > 97% of the product from the aqueous phase. C. glutamicum showed the highest tolerance to CA and resulted in the accumulation of up to 187 g/L of 4VP from pure CA in the overlay with a 90% yield when using rich media, or 17 g/L of 4VP with a 73% yield from CA extracted from lignin. These results indicate that C. glutamicum is a suitable host for the high-level production of 4VP and that further bioprocess engineering strategies should be explored to optimize the production, extraction, and purification of 4VP from lignin with this organism.

Modelling of growth kinetics of isolated Pseudomonas sp. and optimisation of parameters for enhancement of xanthine oxidoreductase production by statistical design of experiments.

This report presents the substrate inhibitory effect of xanthine (XN) on microbial growth and optimisation of effective parameters to achieve high enzyme activity of xanthine oxidoreductase (XOR) through statistical design. Three efficient isolated strains (Pseudomonas aeruginosa CEBP1 and CEBP2, Pseudomonas sp. CEB1G) were screened for growth kinetic studies. Substrate inhibitory models (eg. Aiba, Edward) could explain the growth kinetics of CEBP1, CEBP2 and CEB1G very well with various initial [XN] (S0), e.g., 0.1-35 g L-1. Highest XOR activity was obtained at stationary phase when biomass yield was high. Highest catalytic efficiency (kcat/KM) of XOR was obtained by CEBP1 at optimum specific growth rate of 0.082 h-1 and biomass yield of 0.196 g g-1 at S0 = 5 g L-1. The effects of S0, pH and temperature were studied by Box-Behnken experimental design to evaluate the interactive effects of the significant variables influencing XOR production by CEBP1. ANOVA with high correlation coefficient (R2 > 0.99) and lower 'Prob > F'value (< 0.05) validated the second order polynomial model for the enzyme production. The highest XOR activity of 31.2 KU min-1 mg-1 was achieved by CEBP1 under optimised conditions (35 °C; S0=5 g L-1; pH = 7.0) as compared to any report in literature. A sevenfold substrate affinity of the enzyme was observed after purification.

Potential of metabolic engineering in bacterial nanosilver synthesis.

The widespread applications of silver nanoparticles in present days demand an industrial-scale production process. The ability of bacteria to synthesise silver nanoparticles can be exploited to overcome many shortcomings associated with conventional production processes, such as high cost and nanoparticle toxicity. However, lack of a standardised protocol and suboptimal yield remain a major obstacle for bacterial synthesis route. A potential, yet unexplored, solution to this problem could be envisioned through rewiring of the metabolic network to direct cellular resources towards the product of interest. Mathematical modelling of metabolic pathway is the key to understand and manipulate the cellular metabolism for enhanced production of desired metabolite(s). The present study provides a perspective on the scope of metabolic engineering approaches to enhance bacterial synthesis of silver nanoparticles.

Scientific discovery as a combinatorial optimisation problem: how best to navigate the landscape of possible experiments?

A considerable number of areas of bioscience, including gene and drug discovery, metabolic engineering for the biotechnological improvement of organisms, and the processes of natural and directed evolution, are best viewed in terms of a 'landscape' representing a large search space of possible solutions or experiments populated by a considerably smaller number of actual solutions that then emerge. This is what makes these problems 'hard', but as such these are to be seen as combinatorial optimisation problems that are best attacked by heuristic methods known from that field. Such landscapes, which may also represent or include multiple objectives, are effectively modelled in silico, with modern active learning algorithms such as those based on Darwinian evolution providing guidance, using existing knowledge, as to what is the 'best' experiment to do next. An awareness, and the application, of these methods can thereby enhance the scientific discovery process considerably. This analysis fits comfortably with an emerging epistemology that sees scientific reasoning, the search for solutions, and scientific discovery as Bayesian processes.

Physiological limitations and opportunities in microbial metabolic engineering.

Metabolic engineering can have a pivotal role in increasing the environmental sustainability of the transportation and chemical manufacturing sectors. The field has already developed engineered microorganisms that are currently being used in industrial-scale processes. However, it is often challenging to achieve the titres, yields and productivities required for commercial viability. The efficiency of microbial chemical production is usually dependent on the physiological traits of the host organism, which may either impose limitations on engineered biosynthetic pathways or, conversely, boost their performance. In this Review, we discuss different aspects of microbial physiology that often create obstacles for metabolic engineering, and present solutions to overcome them. We also describe various instances in which natural or engineered physiological traits in host organisms have been harnessed to benefit engineered metabolic pathways for chemical production.

Inducible Population Quality Control of Engineered Bacillus subtilis for Improved N-Acetylneuraminic Acid Biosynthesis.

Biosynthesis by microorganisms using renewable feedstocks is an important approach for realizing sustainable chemical manufacturing. However, cell-to-cell variation in biosynthesis capability during fermentation restricts the robustness and efficiency of bioproduction, hampering the industrialization of biosynthesis. Herein, we developed an inducible population quality control system (iPopQC) for dynamically modulating the producing and nonproducing subpopulations of engineered Bacillus subtilis, which was constructed via inducible promoter- and metabolite-responsive biosensor-based genetic circuit for regulating essential genes. Moreover, iPopQC achieved a 1.97-fold increase in N-acetylneuraminic acid (NeuAc) titer by enriching producing cell subpopulation during cultivation, representing 52% higher than that of previous PopQC. Strains with double-output iPopQC cocoupling the expression of double essential genes with NeuAc production improved production robustness further, retaining NeuAc production throughout 96 h of fermentation, upon which the strains cocoupling one essential gene expression with NeuAc production abolished the production ability.

Corynebacterium Cell Factory Design and Culture Process Optimization for Muconic Acid Biosynthesis.

Muconic acid (MA) is a valuable compound for adipic acid production, which is a precursor for the synthesis of various polymers such as plastics, coatings, and nylons. Although MA biosynthesis has been previously reported in several bacteria, the engineered strains were not satisfactory owing to low MA titers. Here, we generated an engineered Corynebacterium cell factory to produce a high titer of MA through 3-dehydroshikimate (DHS) conversion to MA, with heterologous expression of foreign protocatechuate (PCA) decarboxylase genes. To accumulate key intermediates in the MA biosynthetic pathway, aroE (shikimate dehydrogenase gene), pcaG/H (PCA dioxygenase alpha/beta subunit genes) and catB (chloromuconate cycloisomerase gene) were disrupted. To accomplish the conversion of PCA to catechol (CA), a step that is absent in Corynebacterium, a codon-optimized heterologous PCA decarboxylase gene was expressed as a single operon under the strong promoter in a aroE-pcaG/H-catB triple knock-out Corynebacterium strain. This redesigned Corynebacterium, grown in an optimized medium, produced about 38 g/L MA and 54 g/L MA in 7-L and 50-L fed-batch fermentations, respectively. These results show highest levels of MA production demonstrated in Corynebacterium, suggesting that the rational cell factory design of MA biosynthesis could be an alternative way to complement petrochemical-based chemical processes.

Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol.

Human PrimPol is a DNA primase/polymerase involved in DNA damage tolerance and prevents nuclear genome instability. PrimPol is also localized to the mitochondria, but its precise function in mitochondrial DNA maintenance has remained elusive. PrimPol works both as a translesion (TLS) polymerase and as the primase that restarts DNA replication after a lesion. However, the observed biochemical activities of PrimPol vary considerably between studies as a result of different reaction conditions used. To reveal the effects of reaction composition on PrimPol DNA polymerase activity, we tested the polymerase activity in the presence of various buffer agents, salt concentrations, pH values and metal cofactors. Additionally, the enzyme stability was analyzed under various conditions. We demonstrate that the reaction buffer with pH 6-6.5, low salt concentrations and 3 mM Mg2+ or 0.3-3 mM Mn2+ cofactor ions supports the highest DNA polymerase activity of human PrimPol in vitro. The DNA polymerase activity of PrimPol was found to be stable after multiple freeze-thaw cycles and prolonged protein incubation on ice. However, rapid heat-inactivation of the enzyme was observed at 37ºC. We also for the first time describe the purification of human PrimPol from a human cell line and compare the benefits of this approach to the expression in Escherichia coli and in Saccharomyces cerevisiae cells. Our results show that active PrimPol can be purified from E. coli and human suspension cell line in high quantities and that the activity of the purified enzyme is similar in both expression systems. Conversely, the yield of full-length protein expressed in S. cerevisiae was considerably lower and this system is therefore not recommended for expression of full-length recombinant human PrimPol.

Synthetic biology and regulatory networks: where metabolic systems biology meets control engineering.

Metabolic pathways can be engineered to maximize the synthesis of various products of interest. With the advent of computational systems biology, this endeavour is usually carried out through in silico theoretical studies with the aim to guide and complement further in vitro and in vivo experimental efforts. Clearly, what counts is the result in vivo, not only in terms of maximal productivity but also robustness against environmental perturbations. Engineering an organism towards an increased production flux, however, often compromises that robustness. In this contribution, we review and investigate how various analytical approaches used in metabolic engineering and synthetic biology are related to concepts developed by systems and control engineering. While trade-offs between production optimality and cellular robustness have already been studied diagnostically and statically, the dynamics also matter. Integration of the dynamic design aspects of control engineering with the more diagnostic aspects of metabolic, hierarchical control and regulation analysis is leading to the new, conceptual and operational framework required for the design of robust and productive dynamic pathways.

Caution required for handling genome editing technology.

Genome-editing technology, although a robust tool for genetic engineering, is creating indistinct regulatory boundaries between naturally occurring and modified organisms. However, researchers must act with caution in research and development to avoid misleading society. Furthermore, appropriate regulations should be proactively discussed and established for handling genome-editing technology.

Metabolic engineering for acetate control in large scale fermentation.

Escherichia coli is the most commonly used microorganism for production of recombinant proteins for different applications. Acetate accumulation during aerobic growth on glucose has significant negative impact on recombinant protein production in Escherichia coli. Various strategies, such as process and genetic approaches have been developed to limit acetate formation to increase the productivity of recombinant proteins. We developed a strategy to combine inactivation of pyruvate oxidase (poxB) and over-expression of acety-CoA synthetase (acs) in E. coli K strain for controlling acetate accumulation. A recombinant peptide was expressed and produced in the engineered strains with a very low acetate -formation in a 10-L fermentation process.

Engineering of glucosinolate biosynthesis: candidate gene identification and validation.

The diverse biological roles of glucosinolates as plant defense metabolites and anticancer compounds have spurred a strong interest in their biosynthetic pathways. Since the completion of the Arabidopsis genome, functional genomics approaches have enabled significant progress on the elucidation of glucosinolate biosynthesis, although in planta validation of candidate gene function often is hampered by time-consuming generation of knockout and overexpression lines in Arabidopsis. To better exploit the increasing amount of data available from genomic sequencing, microarray database and RNAseq, time-efficient methods for identification and validation of candidate genes are needed. This chapter covers the methodology we are using for gene discovery in glucosinolate engineering, namely, guilt-by-association-based in silico methods and fast proof-of-function screens by transient expression in Nicotiana benthamiana. Moreover, the lessons learned in the rapid, transient tobacco system are readily translated to our robust, versatile yeast expression platform, where additional genes critical for large-scale microbial production of glucosinolates can be identified. We anticipate that the methodology presented here will be beneficial to elucidate and engineer other plant biosynthetic pathways.